DOI: 10.31038/CST.2017231

Abstract

Background: Evidence of cancer cure or improvement of quality of life in cancer patients has been reported using prescriptions based on traditional knowledge, but not scientifically investigated. The objectives of the current study is to evaluate the anti-proliferative action of a poly herbal drug which is currently used in traditional medicine name “Le Pana Guliya (LPG)”

Methods: The aqueous extract of LPG was freeze dried. Total phenolic content was assayed using Folin Ciocalteu reagent. Antioxidant activity of the extract was evaluated using DPPH radical scavenging assay in vitro. Brine shrimp assay was conducted to determine the cytotoxicity (LD50) of the LPG. The cell viability was determined by MTT assay and cytotoxicity by LDH leakage assay after 24 and 48 hours incubation with LPG. Morphological Changes after the treatment of the drug was observed using an inverted light microscope. RD, MCF-7 cells and CC1 cells were used in all experiments.

Results: The TPC of the LPG was 5.31 ± 0.14 W/W% of GAE and antioxidant capacity is comparable to ascorbic acid. LPG exhibited strong cytotoxic activity against RD and MCF-7 cell lines with MTT assay. A 50% leakage of LDH was observed at concentrations less than 30μg/mL and 10 μg/mL for both RD and MCF-7 cells respectively after 24 hour exposure. While, LPG exhibited strong cytotoxic activity against RD and MCF-7 cells the brine shrimp and CC1 cells results (EC50>100μg/mL) suggest that the LPG have minimum cytotoxicity towards the normal healthy cells.

Conclusion: The present study provides evidence for potent anti cancer activity of LPG on RD and MCF-7 cells. The brine shrimp bio-assay and CC1 cells results suggests that the extract have minimum cytotoxicity towards the normal healthy cells.

Keywords

Anti-proliferative activity, Cytotoxicity, MTT assays, LDH assay, Brine shrimp assay, Light microscopy, RD cells, MCF-7, CC1, DPPH

Introduction

Natural products have played an imperative role in the lead-finding of candidates for the development of present-day cancer chemotherapy due to their low toxicity and side effects. They offer a valuable source of a wide variety of chemical structures with biological activities (lead molecules) for the development of novel drugs [1]. Approximately 60% of all drugs currently undergoing clinical trials for cancer treatment are natural products or compounds derived from natural products [2, 3]. These substances embrace some of the most exciting new chemotherapeutic agents currently available for use in a clinical setting [3].

Sri Lanka is gifted with many plants which have vast medicinal value. Use of plants for medicinal preparations is a primary part of the Sri Lankan cultural life and this is implausible to change in the years to come. The Sri Lankan traditional medicine system shows extensive use of plant products in cancer treatment. A significant surge of interest in chemoprevention research has thus led to the discovery of many phytochemicals as successful chemo preventive agents.

Many traditional healers and folklore medical practitioners in the country have been treating cancer patients for many years using various medicinal plant species. There is evidence of case reports on cancer cure using traditional knowledge, but they are not scientifically investigated. In addition to use of a single plant, poly herbal formulations of drugs are intensively used in Sri Lanka. The poly herbal drug ‘Le Pana Guliya’ (LPG) found in Ola leaf inscriptions is prescribed to treat different types of cancers by traditional doctors.This study was aimed at investigating of its cytotoxic effect against two different cancer cell lines compared with healthy CC1 cells.

Materials And Methods

Chemicals and equipment

Chemicals needed for cell culture and cytotoxicity studies were purchased from Sigma –Aldrich (St Louis, MO63178, USA). 1-Diphenyl-2-picrylhydrazyl (DPPH), TritonX-100, was purchased from Fluka. Tris base was purchased from Promega (Madison, WI 53711–5399, USA). All chemicals used were of analytical grade.
Shimadzu UV 1601 UV visible spectrophotometer (Kyoto, Japan) was used to measure the absorbance. LFT 600 EC freeze dryer was used to obtain the freeze dried powder of the poly herbal drug. Cells were incubated at 37°C in a humidified CO2 incubator (SHEL LAB/Sheldon manufacturing Inc. Cornelius, OR 97113, USA). Inverted fluorescence microscope (Olympus Optical Co. Ltd. 1X70-S1F2, Japan) for observation of cells, and photographs were taken using microscope digital camera (MDC200 2M PIXELS, 2.0 USB). Deionized water from UV ultra-filtered water system (Waterproplus LABCONCO Corporation, Kansas city, Missouri 64132–2696) and distilled water was used in all experiments.

Cell cultures

Human Rhabdomyosarcoma cell line (RD), and human breast adenocarcinoma cell line (MCF-7) were cultured in Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 10% heat inactivated fetal bovine serum (FBS), penicillin (100 U/mL) and streptomycin (100 U/mL). The cells were maintained in 25 cm2 plastic tissue culture flasks at 37oC in a humidified atmosphere containing 5% CO2 in air. Exponentially growing cells were used in all experiments. The normal rat fibroblast (CC1) cell line was employed as the control.

Preparation of poly-herbal extract

The poly herbal drug (5 g) soaked in distilled water (100 mL) was kept in the rotary shaker for 48 hours in an air tight dark bottle. The extract was then filtered through a layer of muslin cloth and filtrate was centrifuged at 3,000 rpm for 15 minutes at 4oC to remove any debris.

The supernatant was freeze dried, and stored at -20°C in an air tight vial until used. The freeze dried extract was reconstituted with distilled water for experimental purposes.

Antioxidant activity

The DPPH assay was used to determine the radical scavenging activity of the extract, as reported previously by Perera (2008) [4]. Briefly different concentrations of plant extract (100 µL) was mixed with DPPH reagent prepared in absolute ethanol (100 µM, 900 µL) and incubated for 30 minutes in darkness at 37oC, in a water bath. The percentage of de-colorization was obtained spectro-photometrically at 517 nm. The control was prepared as above without adding extract while L-ascorbic acid was used as the positive control.

At least three independent tests were performed for each sample. Percentage inhibition was calculated from the following formula:

The effective concentration of the sample required to scavenge the respective radical by 50% (EC50) was calculated using the linear segment of the curve obtained with percentage inhibition against concentration.

Total phenolic content (TPC)

The total phenolic content of the lyophilized sample (n = 6) of the poly herbal drug was determined using the Folin-Ciocalteu method [5].

Brine shrimp bioassay

Brine shrimp assay was conducted to determine the cytotoxicity (LD50) of the poly herbal drug as explained previously by Soysa (2014) and Middleton (2005) [6, 7].

Briefly, dried cysts of brine shrimps (10 mg) were allowed to hatch at room temperature in sterile sea water. After 24 hours the free nauplii were selected and 10 were added to each petridish containing different concentrations of the extract in sea water. After 24 and 48 hours, the petridishes were observed using a magnifying glass and the number of survived nauplii in each petridish was counted.

The mortality end-point of this bioassay was defined as the absence of controlled forward motion during 30 seconds of observation [7].

The percentage lethality was determined by comparing the surviving larvae of the test and the control. The percentage survival was calculated as:

Cell viability assay

The effect of aqueous extract of the drug on the cell viability was determined by MTT assay. The live cells reduce yellow MTT to purple formazan crystals by mitochondrial dehydrogenase enzyme [8]. Cells were suspended in the growth medium and seeded in 24-well plates at 2 x 105 cells/well for overnight. The cells were then treated with different concentrations of the drug at 37oC for 24 and 48 hours. A positive control with cyclohexamide (50 g/mL) and a negative control without the drug were simultaneously conducted.

Percentage viability of the cells treated with the drug was calculated comparing the viability of the un-treated cells.

After 24/48 hours, the utilized growth media was subsequently replaced with 1.0 mL of minimum essential media (MEM), and 100 µL of MTT (5 mg/mL in PBS). The cells were incubated at 37o C for 4 hours and the medium was carefully removed. The formazan product was dissolved in acidified isopropanol (0.05 M HCl in isopropyl alcohol (IPA); 750 µL) and absorbance was read at 570 nm. Cell survival was expressed as a percentage of viable cells of treated samples to that of untreated cells (negative control). The drug extracts were prepared in triplicate and each experiment was performed in triplicates to each preparation.

Lactate dehydrogenase (LDH) activity

Cytotoxicity induced by the LPG evaluated by lactate dehydrogenase (LDH) leakage into the culture medium, as explained in Fotakis and Timbrell (2006) [9]. Cells seeded in 24 well plates (2 x 105 cells/well) were exposed to different concentrations of the drug. After 24/48 hours incubation the culture medium was aspirated and centrifuged at 4000 rpm for 5 min in order to obtain a cell free supernatant. The cell lysate was prepared by treating the cells with Triton X 100 (0.1%; 1 mL) and sonicating the contents for 20 seconds. The final suspension was centrifuged at 4000 rpm for 5 minutes. Medium and the lysate were subjected to LDH assay. The activity of LDH in the medium was determined using a commercially available, LDH assay kit (HUMAN).

Negative control and positive control with cyclohexamide (50g/mL) were also carried out along with the experiment to measure the percentage LDH leakage.

The absorbance was measured at 340 nm at intervals of 15 seconds for 1.5 minutes using an air blank. The rate of decline (gradients of the graphs) of NADH concentration was used to calculate the LDH activity in the supernatant and the lysate.

The percentage LDH leakage to the medium was calculated using the following equation.

Where total LDH activity = LDH activity of supernatant + LDH activity of the lysate.

Light microscopy

RD, MCF-7 and CC1 cells grown at 70% confluence were treated with different concentrations of aqueous drug extracts for 24, 48 and 72 hours and observed under phase-contrast inverted fluorescence microscope (40X). The changes in morphology were compared with positive and negative controls.

Statistical analysis

All the results of the experiments were expressed as the mean ± standard deviation (Mean ± SD). The measurements were performed in triplicate and values shown are representatives of at least three independent experiments. Least square linear regression analysis was applied using Microsoft excel to determine the EC50/LD50 values and for the calibration curves.

R² > 0.99 was considered as linear for the calibration curves.
Significant differences of each test result were statistically analyzed using “Mann-Whitney U” test significances with 95% significance using SPSS version 16.

Results

Anti-oxidant capacity and total Phenolic content

The aqueous extracts of the poly herbal drug showed moderate free radical scavenging activity with a value of EC₅₀ = 68.0 ± 2.8 g/mL with compared to the positive control, L- ascorbic acid (3.4±0.9g/mL; n=6).
The percentage total phenolic content of the aqueous extract of the poly herbal drug was 5.3 ± 0.1 W/W % of Gallic Acid Equivalence (GAE).

Brine shrimp bioassay

LPG shows no lethality towards the brine shrimp even after 48 hours exposure up to a concentration of 10 to 2000 μg/mL. This results show that the aqueous extract either does not possess any significant (p>0.05) alterations in the cellular functions or no significant cytotoxity.

Cell viability assay

The effect of LPG on cell viability of RD, MCF-7 and CC1 cells was evaluated by tetrazolium assay (MTT).
Multiple concentrations of the aqueous extract of the poly herbal drug were used and effective concentrations were calculated for each cell line (EC₅₀) from dose response curve. The percentage cell viability of RD, MCF-7 and CC1 are shown in Figure 1.

Figure 1. The percentage cell viability of on RD, CC1 and MCF-7 cell lines as determined by MTT assay, (a) after 24 hours treatment (b) 48 hours and (c) 72 hours with aqueous extract of the poly herbal drug. The data are presented as mean ± SD of six independent experiments.

The aqueous extract of the LPG exhibited no significant cytotoxic activity (p>0.05) against CC1 cell line, achieving an EC₅₀ of 85.3 ± 9.8 µg/mL after 72 hour incubation.

On the contrary, the aqueous extract of the poly herbal drug exhibited significant cytotoxic activity (p<0.05) against RD and MCF-7 cell lines compared to the CC1 cells as shown in Table I.

Table I. EC₅₀ values obtained for MTT assay for the three different cell lines.

^*All results were mean of 6 independent measurements ± standard deviation. “Mann-Whitney U” test at 95% confidence level showed a significant difference (p < 0.05) in both RD and MCF-7 cells compared to CC1 cells.

After 24 and 48 hour incubation with Cyclohexamide (Positive control) at a concentration of 50µg/mL, the percentage viability showed by tested cells depicted in Table 2. The percentage cell viability obtained for MTT assay at a concentration of 50µg/mL of LPG was calculated using respective regression equations to compare the capacity to inhibit cell proliferation, with that of the positive control. The values are shown in Table 2.These results exhibited the significant cytotoxicity (p< 0.05) exert by the aqueous extract of LPG against cancer cells compared to CC1 cells as determined by MTT assay.

Table 2. Percentage cell viability obtained by MTT assay at 50 µg/mL Cyclohexamide (+ve Control) and 50 µg/mL LPG after 24 hours and 48 hours exposure

^*All results were mean n=6 measurements ± standard deviation in three independent experiments.

Lactate dehydrogenase (LDH) assay

Measurement of LDH activity is also an indicator of cell viability through evaluation of the cell membrane permeability. The enzyme activity is measured externally, as it leaks from dead cells which lose their membrane integrity. The LDH release curves for RD, MCF-7 and CC1 cell lines treated with different concentrations of the LPG suggested that the cytotoxic effect of the extract was concentration dependent (Figure 2).

Figure 2. Lactate Dehydrogenase (LDH) release from RD, MCF-7 and CC1 cells with aqueous extract of the poly herbal drug, (a) after 24 hour, (b) after 48 hour incubation. The data are presented as mean ± SD of six independent experiments.

Fifty percent leakage of LDH to the media was observed at 26.9± 1.6 and 30.5 ±2.8μg/mL for RD and MCF-7 cells after 24 hour incubation respectively. After 48 hours of incubation with LPG the EC₅₀ values further decreased and 50% LDH leakage arise at 8.2± 0.2 and 6.0± 0.2 μg/mL for RD and MCF-7 cells respectively. In contrast to RD and MCF-7 cells the CC1 cells showed 50% leakage of LDH at 159.3± 3.1 and 103.1 ±5.2μg/mL LPG concentrations after 24 and 48 hours respectively.

Light microscopy

Morphological alterations of RD, MCF-7 and CC1 cell lines upon exposure to the aqueous extract of the poly herbal drug was observed under the phase contrast inverted microscope. The microscopic observations revealed that the aqueous extract has a significant cytotoxic effect on RD and MCF-7 cells compared to the negative control. However CC1 cells treated with the drug did not show significant cell death (Figure 3).

Figure 3. Light microscopy images of RD, MCF-7 and CC1 cells treated with their respective EC50 values of LPG, with magnification of 40X. (Black arrow – indicates healthy spindle shape cells; Red arrow – dead and shrinkage cells due to the LPG treatment).

Discussion

Cancer has become the most leading cause of death worldwide. It has become an emerging health problem affecting both developing and developed countries. According to the World Health Organization reports published in 2014, 8.2 million patients have died from cancer in 2012 and the number of annual cancer cases reported in 2012 was 14 million. It has been also estimated that the number of annual cancer cases would have increased from 14 million in 2012 to 22 million within the next two decades [10]. The most effective treatment approaches in cancer are chemotherapy and radiotherapy. Nevertheless, the higher incidence in side effects, have made investigators engage in finding novel anticancer compounds with less adverse effects. As a result of this, plants and other natural sources have provided nearly 60% of anti cancer agents which are currently in use [11]. Especially the most novel chemotherapeutic agents more than ever before are botanical in kind [2].

Traditional folklore medicine which involves the use of natural elements uses either single or multiple herbs as a mixture (polyherbs) for treatment of diseases. The use of polyherbal formulation is found to be more effective than the use of a single herb as the active phytochemical constituents of the individual plants are insufficient to achieve the desirable therapeutic effect. But when combining the herbs in a particular ratio the synergistic effect produced by the active phytochemicals of several plants give a better therapeutic effect [12].

Polyherbal preparations are used worldwide for treatment of various disease conditions including cancer. As the polyherbal formulations have gained more attention, many studies have been conducted to identify the mechanism of action and the efficacy of these polyherbal preparations. A latest study [13] which gives a global perspective on polyherbal formulations reports that for the past six years, only 2 publications have been made regarding polyherbal formulations that show/have anticancer activity.

Zyflamend® used in China [14], Sho-saiko-to® used in Japan [15] and Arbudhcure® used in India [16] are few of the polyherbal preparations used for cancer treatment where attempts have been made to validate scientifically for their mechanism of action and therapeutic efficacy.

With compared to the aforementioned poly herbal drugs the polyherbal LPG is not commercialized and no scientific research has been carried out up to this study to identify its mechanism of action or evaluate its effectiveness on treatment of cancer even though it is being used to treat 32 different types of cancer in traditional medicinal system in Sri Lanka.

Secondary metabolites of plants including phenolic compounds are very important for their essential functions in reproduction, growth and in defense mechanisms [17]. Phenolic compounds also provide natural antioxidants for protection against many diseases including cancers. It has been reported that antioxidant effects of plants are mainly attributed to the radical scavenging activity of phenolic compounds such as flavonoids, polyphenols, tannins, and phenolic terpenes. The polyphenols have the capability to scavenge ROS as well as oxidatively generated free radicals which derive from bio-molecules [18].

As depicted in results the quantitative measurement of poly phenolic content obtained in the present study showed that the water extract of LPG has moderate levels of polyphenols. Similarly the EC50 values obtained for the different samples of LPG for DPPH assay showed higher values compared to EC50 of ascorbic acid which evidently indicate that the LPG has a lower anti-oxidant capacity.

It was reported by several authors, that the proliferation of cancer cells are inhibited by the high antioxidant and poly phenolic content present in plant extracts [14, 19]. This probable fact that presence of low polyphenolic content and low antioxidant potential was explained in Hamberger and Hastettman 1991[20], as possible changes occurring in the chemical composition of the plant materials during the processes of the drug manufacturing that could result in a reduced pharmacological activity in mixtures. Thus it can be suggested that even though the LPG exerts an effective antiproliferative activity and cytotoxicity, the mechanism of action must be in a different pathway compared to most of the other traditional medicinal drugs which exert antiproliferative activity via the phenolics and antioxidants.

Cytotoxic action in brine shrimp assay is brought about by the active ingredients present in the drug by disturbing the fundamental mechanisms associated with cell growth, mitotic activity, differentiation and function [21]. According to previous reports a crude plant extract is toxic (active) if it has an LD50< 1000 µg/mL while non-toxic (inactive) if LD50> 1000 µg/mL [22]. The present study evidently indicates that the aqueous extract of LPG shows no lethality towards the brine shrimp even after 48 hours of exposure within the concentrations tested ranging 10 to 2000 µg/mL. This results show that the aqueous extract either does not possess any significant (p>0.05) alterations in the cellular functions or no significant cytotoxicity. Similar results were observed in a previous study which shows anticancer activity of the plant extract of Flueggea leucopyrus against HEp 2 cells but no toxicity for brine shrimps [6].

In this study the cell viability of the tested cell lines were determined by the MTT reduction assay. Each cell type was treated with different concentration of LPG extract and incubated for 24 and 48 hours. The EC50 value was obtained from the graph between the percentages of cell viability versus concentrations of LPG extract (Figure 1). The values were used to describe the degree of cytotoxicity of the extract towards cell lines. The cell viability of CC1 cells were further determined after 72 hours due to the high viability of the cells at 48 hours of incubation (187.58 ± 9.59 µg/mL).

It was established by the American National Cancer Institute (NCI), that if a crude extract shows EC50< 30 µg/mL after an incubation of 72 hours it is considered as cytotoxic [23]. However crude extract with EC50 less than 20µg/mL after 48 hours incubation is supposed to be highly cytotoxic [24]. Present study shows a potent cytotoxic effect on RD and MCF-7 cells with the aqueous extract of the LPG even at 24 hours which is much lower than the value specified by the NCI. However, the extract was less cytotoxic towards normal mouse fibroblast cell line CC1.

After 24 and 48 hours of incubation with Cyclohexamide (Positive control) at a concentration of 50μg/mL, the percentage viabilities of the tested cells are showed in Table 2. In contrast, the percentage viability obtained for MTT assay at the same concentration of LPG (50 μg/mL) treatment was markedly lower for RD and MCF-7 cells (Table 2).The EC50values obtained for MTT assay indicate that the cytotoxicity of the LPG extract against the cancer cell lines is within the limit of cytotoxicity (EC50 < 30 μg/mL), as reported by the American National Cancer Institute (NCI) over 72 hour post exposure and it is beyond the limits for CC1 cells (Table 1) Thus LPG shows significant anti-proliferative activity on cancer cells studied compared to the standard anti cancer drug. Cyclohexamide showed a % cell viability of 69.93 ± 9.26% and 49.31 ± 3.88% for 24 and 48 hours respectively for CC1 cells, suggesting its high toxicity towards the normal cells compared to the LPG (Table 2).

It is reported that the poly herbal anticancer drug Zyflamend® shows an EC50 value of 200 µg/mL against prostate cancer cell line (PCC) after an incubation period of 96 hours [14]. Japanese poly herbal drug Sho-saiko-to® shows an EC50 value of 353.5 µg/mL against human hepatocellular carcinoma (HCC-KIM) at 72 hour incubation [15], while Arbudhcure® shows an EC50 of 33.0 µg/mL against HeLa cells for 48 hours post exposure to the respective drug [16]. The current study demonstrates that LPG shows higher anti-proliferative activity on four different cancer cell lines investigated in the present study in comparison with aforementioned polyherbal drugs.

Measurement of LDH activity is another indicator of cell viability through evaluation of the cell membrane permeability. Previous studies suggest that LDH is a more reliable and accurate marker of cytotoxicity, since damaged cells is fragmented completely during the course of prolonged incubation with substances [25]. The intracellular LDH release to the medium is a measure of irreversible cell death. Xia (2007) [26] reported that there is a correlation between the direct involvement of LDH up regulation and subsequent induction of apoptosis, which is ideal for an effective anti cancer drug.

Fifty percent leakage of LDH to the media was observed at 26.9± 1.6 and 30.5 ±2.8μg/mL for RD and MCF-7 cells after 24 hour incubation respectively. After 48 hours of incubation with LPG the EC50 values further decreased and 50% LDH leakage arise at 8.2± 0.2 and 6.0± 0.2 μg/mL for RD and MCF-7 cells respectively. In contrast to RD and MCF-7 cells the CC1 cells showed 50% leakage of LDH at 159.3± 3.1 and 103.1 ±5.2μg/mL LPG concentrations after 24 and 48 hours respectively. These results indicate that the LPG is having minimal cytotoxic effect on control cells while it possesses a remarkable cytotoxicity on two cancer cell types studied.

Morphological alterations of RD, MCF-7 and CC1 cell lines upon exposure to the aqueous extract of the LPG revealed that even at low concentrations the morphology of the RD and MCF-7 cells changed into an irregular shape with extremely condensed nuclear contents, signs of membrane blebbing, suggesting an autophagic mechanism of cell death. The untreated cells (negative control)of RD and MCF-7 cell lines show cells with normal morphology (Spindle shape/ elongated cells) that adhered to the culture plate with no signs of cells death and growth disorders.

The positive control also shows the typical morphological changes which indicate the cellular damage in RD and MCF-7 cells. In contrast to the RD and MCF-7 cells the CC1 cells do not show any significant morphological changes even after 72 hour incubation with the extract.

Conclusions

The present study provides strong evidence for potent anti-cancer activity of the poly herbal drug “Le Pana Guliya” on RD and MCF-7 cells compared to controls. The brine shrimp and CC1 cells results suggest that the extract has minimum cytotoxicity towards the normal healthy cells. This calls for further studies on the active components and the mechanism of action involved in cytotoxicity of the chemotherapeutic action of LPG.

Acknowledement: The financial support by World Class University Research Grant No. AP/3/2012/CG/10is highly acknowledged.

Conflict of Interest: No conflict of interest between authors.

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DOI: 10.31038/CST.2017222

Abstract

Background: Breast cancer (BC) and its treatments lead to numerous side effects that affect a person’s life for years after treatment has ended. Research shows that regular exercise limits many of these side effects.However, less than 30% of BC survivors regularly exercise due to many barriers at both the patient and health care professional level. The purpose of this pilot trial is to assess the feasibility and effectiveness of conducting a novel KT intervention using exercise and self-management versus usual care among BC survivors.

Methods: Study Design: Pilot randomized controlled trial. Eligibility: Women older than 18 years who are currently undergoing chemotherapy treatment for BC. Intervention: The intervention group includes an 8-session multi-component intervention with a structured aerobic exercise program plus SM supervised by a physiotherapist. Randomization: Participants will be randomly allocated using a 1: 1 allocation ratio to receive the intervention of structured exercise plus SM program or usual care. Outcomes: The primary feasibility outcomes include recruitment rate, retention rate, and adherence rate. The secondary outcomes include exercise knowledge and behavior, HRQoL and resource utilization. Analysis: A blinded assessor will assess outcomes at baseline, post intervention, at 2- and 4-month follow up. Intervention feasibility and effectiveness will be assessed using descriptive statistics and analysis of covariance for continuous outcomes.

Discussion: This study aims to assess the feasibility of a novel KT intervention to close the current KT gap and increase exercise awareness for women with BC. This project will assess process and resource variables before implementation of a larger scale intervention. The overall project goal is to promote sustainable exercise behaviour to help manage the burden of BC.

Trial Registration: This trial was registered on ClinicalTrials.gov on March 21, 2017 (Identifier: NCT03087461).

Keywords:

Breast neoplasms, exercise, translational medical research, pilot study, rehabilitation

Introduction

Breast cancer (BC) and its treatments lead to numerous side effects that affect a person’s quality of life (QOL) for years after treatment has ended [1-5]. Research has shown that regular exercise limits many of these side effects and can prevent disease recurrence [5-10]. However, less than 30% of survivors participate in regular exercise [11-13]. Previous research conducted by our team has shown that over 80% of BC survivors in southwestern Ontario are unaware of the benefits of exercise and are not educated on the need to stay physically active [11], health professionals face many institutional, personal, and patient-related barriers to promoting exercise [14], and there is a need for novel knowledge translation (KT) strategies within cancer institutions that focus on easy-to-access exercise interventions and education by physiotherapists (PTs) [15].

Moreover, the societal burden of this disease is projected to increase substantially over the next two decades. The Canadian Cancer Society’s 2015 Statistics report [16] suggests that the number of new cancer cases in women will increase by 74% by the year 2032 due to the aging Canadian population. The number of new cases of BC is projected to increase by more than 10,000 in this same time period [16]. Fortunately, due to improved screening and treatment techniques, survival rates for BC are increasing [3]. However, physical and functional sequelae prevent survivors from returning to their activities associated with work, leisure, and domestic roles [3]. Exercise is an effective, safe, and cost-efficient way to manage this burden and return women to their pre-cancer activity levels. Therefore KT research is needed to determine how to best translate and integrate this research knowledge into clinical practice in order to elicit sustainable behaviour change for this population. Pilot work completed for this project has shown that in order to change clinical practice, implementation strategies using accessible exercise options and education are needed within the institution to maximize women’s engagement with exercise information provided and to partake in this behaviour [15]. Along with this, self-management (SM) programs have been shown to improve QOL and physical side effects in BC survivors, however, the implementation of these programs in clinical practice is scarce [17]. The current knowledge to practice gap in the field of BC rehabilitation shows that novel KT strategies are needed.

A pilot study is an, “investigation designed to test the feasibility of methods and procedures for later use on a large scale or to search for possible effects and associations that may be worth following up in a subsequent larger study” [18]. For this project, a pilot study is needed as the first step in order to assess process and resource variables before implementation of a large scale intervention [19]. Process variables include measuring recruitment rate, retention rate, and adherence rates to the intervention provided [19]. Resource variables include determining the centers willingness and capacity to house a specific intervention, equipment availability, intervention location, and budget concerns [19]. There is currently a lack of pilot trials for a novel KT intervention of this sort and therefore this pilot study will aid in shaping and guiding a larger, phase III trial.

Methods & materials

Study Purpose:

The purpose of this study is to determine whether KT strategies, focusing on accessible exercise locations and SM education by PTs using technology, are feasible and impact exercise knowledge and behaviour, QOL, and need for additional health care services among women with BC. Specifically, the objectives of this project are to: (1) Determine the feasibility (through recruitment, retention and adherence rates) of providing a complex KT intervention designed specifically for women with BC using technology, and (2) Determine preliminary estimates of effects of the KT intervention on levels of exercise knowledge and behaviour, health related quality of life and, resource utilization, among BC survivors over a four month period.

Study Design & Participants:

This study is a pilot randomized controlled trial. Eligible participants will include community-dwelling, English-speaking women, over 18 years, who are currently undergoing chemotherapy for Stage 1-3 BC and have been cleared by their oncologist to participate in moderate intensity aerobic exercise. Participants will be excluded from the study if they have another chronic disease, cognitive impairment or injury that prevents them from participating independently in moderate intensity exercise.

Recruitment:

Medical oncologists and Primary Care Nurses at the Juravinski Cancer Centre (JCC) will recognize possible participants for this study within their patient caseload. For those they think are eligible, the health care professional will briefly discuss the study with their patient and get consent for the patient to be contacted by a member of the research team. Possible participants will be contacted by phone to discuss eligibility and potential study enrollment. The Hamilton Integrated Research Board approved this study (reference #: 3124) in April 2017. All participants will provide written informed consent on the approved consent forms prior to enrollment in this study.

Intervention:

This pilot project will implement a multi-dimensional KT intervention including an exercise and SM program. Refer to Figure 1 for study flow chart.

The exercise intervention will involve an evidence-based moderate intensity aerobic exercise program, using recumbent bikes, delivered within the cancer institution. Results from participants in our focus group run during the pilot work for this project suggest that exercise programs should be delivered in the institution where women are waiting for their chemotherapy. Delivering the program in this environment will increase the accessibility of the services and we anticipate this approach will increase exercise awareness. Participants will take part in the 30-minute, moderate intensity (50-70%HRmax or 4-6/10 on Rate of Perceived Exertion scale) aerobic exercise program for 8 sessions. The intervention will be supervised by a PT educated in cancer rehabilitation and who has been trained in the specific protocol used and in working with women with BC.

The SM component will include educational modules created by a PT. Participants will view these 30 minute modules prior to each exercise intervention, over the same 8 sessions. Content provided within the program will include information on the benefits of exercise during and after BC treatment, safe exercise prescription, how to self-monitor exercise levels, action planning for specific exercise strategies, and precautions related to exercise and BC. Refer to Table 1 for details of SM content for each week of the program. A variety of tools will be used in the SM program, including a mobile app and e-health resources for BC. Having numerous sessions will allow the PT to provide consultation in respect to exercise adaptation, parameters, and programming in order to facilitate long-term exercise engagement and participation. Adherence and fidelity to the specified exercise and self-management protocol will be monitored through random observation by study investigators. (Figure 1, Table 1).

Table 1. Self-Management Content

Session	Content
1	Introductions. What are the side effects of treatment? Why do they happen? Benefits of exercise for women with breast cancer. Types of exercise and safety precautions during exercise (how to monitor BP, HR, RPE) What is self-management? How to participate in effective self-management. Self-management and breast cancer. Introduction to goal setting/action planning.
2	Review of previous week goal/action plan. The importance of posture for women with breast cancer (common postural issues, how to assess posture, how to ensure optimal posture). Relaxation and breathing techniques to manage anxiety and stress. Set goal/action plan for week.
3	Review of previous week goal/action plan. Appropriate exercise techniques to maintain/increase endurance: –                      Description of aerobic exercise –                      Types of aerobic exercise –                      Parameters for aerobic exercise Set goal/action plan for week.
4	Review of previous week goal/action plan Appropriate exercise techniques to maintain/increase strength –                      Description of strengthening exercise –                      Types of strengthening exercise –                      Parameters for strengthening exercise Set goal/action plan for week.
5	Review of previous week goal/action plan Other forms of Exercise (flexibility, yoga, Tia Chi, etc) Appropriate exercise techniques to maintain/increase flexibility: –                      Description of flexibility exercises –                      Types of flexibility exercises –                      Parameters for flexibility exercise Description of other forms of exercise: –                      Types of other forms of exercise –                      Parameters of other forms exercise Set goal/action plan for week.
6	Review of previous week goal/action plan. Self-monitoring physical activity levels: –                      Introduction to Breast Cancer Physio Guide (App) –                      Introduction to Stanford Action Planning App –                      Other techniques to monitor physical activity levels Set goal/action plan for week.
7	Review of goal/action plan. Communicating with others (family, health professionals) about exercise and physical activity. Available exercise programs in the community. How to move forward: how to evaluate progress. Set goal/action plan for week.
8	Review of goal/action plan. Summary of self-management program. How did you use self-management information? Questions/comments.

Outcomes:

Primary Outcomes: The feasibility and effectiveness of the KT intervention will be assessed using quantitative outcomes. The primary outcomes of feasibility variables will be assessed at baseline and post intervention, where applicable. Feasibility will be assessed by measuring recruitment (percentage (%) of eligible patients recruited), retention (% of consented patients who complete the intervention), and adherence rates (% of sessions attended) to the intervention).

Secondary Outcomes: Secondary effectiveness outcomes will be assessed at four time points: baseline, post intervention, and at 2 and 4 month follow up. At baseline, participants will be instructed on how to complete each self-report measure by an assessor blinded to participant group allocation. All post-intervention, 2 and 4 month follow up, assessments will be mailed to participants to complete and return to study investigators using pre-paid postage envelops. No identifiers will be used on these assessments and therefore assessors performing the analysis of data will be blinded to participant group allocation. Hard copies of the completed outcome measures will be stored in a locked filing cabinet only accessible by the study investigators at McMaster University and data entered into statistical analysis software will be stored on a password protected computer.

Level of exercise knowledge and behaviour will be assessed using a Theory of Planned Behaviour (TPB) [20] based questionnaire. The TPB has been used extensively to determine levels of intention and behaviour for various health behaviours, including exercise [21,22].

Quality of life will be measured using the FACT-B [23], a selfreport measure designed to assess multi-dimensional QOL specifically for women with BC.

Need for additional health care services will be measured using the EQ-5D [24] and a piloted self-report questionnaire assessing health care facility visits, doctor visits, procedures received, support services used, loss of work, and prescription medications used.

Sample Size:

Debate exists as to whether sample size calculations for pilot studies are necessary. Some authors suggest that no calculation is needed as long as the pilot study is large enough to provide useful information about the aspects that are being examined for feasibility [19]. However other authors suggest using a percentage of the sample required for a full study [25,26], or to use a confidence interval (CI) to establish feasibility [19,26]. For this project we have decided to calculate sample size based on the proportion of success of the primary outcome of feasibility (using estimates for adherence rates) [19,25,26].

Therefore, using a Z value from the standard normal distribution to reflect a 95% confidence interval (1.96), E as the desired margin of error (0.2), and p as the proportion of successes in the population (0.75-estimated adherence rates), the sample size for this pilot study will be at least 18 participants (9/group). With an expected drop out rate of 25%, based on previous exercise based literature with this population, the final sample size for this project should be 23 participants. In order to ensure an even number of individuals can be randomized to each group, this will be rounded up to a total of 24 participants (12/group).

Randomization-Sequence Generation:

Prior to participant randomizations, all eligible participants will complete the following forms: (a) patient information form, (b) Godin leisure time exercise questionnaire, (c) consent form, and (d) baseline measures. All participants will be informed verbally in person and in writing that they have equal chance of being randomized into the intervention or control group. They will not be made aware of the study hypotheses. Randomization to intervention or control group will be completed by a member of the research team who is independent of the intervention on a record by record basis using a computer software program (STATA/MP v14). This researcher will remain blind to the identity of each treatment group (by randomizing only to group A or B) during the randomization process. Participants may withdraw from the study at any time. Investigators may withdraw a participant from the research study if circumstances arise which warrant doing so (for example, safety).

Allocation Concealment & Implementation:

Allocation of participant randomization will be concealed in sequentially numbered, opaque, sealed envelops. The envelops will be opened sequentially by the researchers only after participant details have been written on the envelop by the researcher who completed randomization. If the participant is allocated to the intervention group, they will receive a phone call to organize details of the first intervention session (such as time and location).

Blinding:

Due to the nature of this knowledge translation study, participants and persons administering the intervention will not be blinded to group assignment. However, the assessor receiving the self-reported outcome measures will be blind to group allocation and will not be involved in running of the intervention. A researcher blinded to the group allocation of the participants will conduct all statistical analysis.

Statistical Methods:

Participant characteristics will be analyzed at baseline to ensure no significant differences exist between groups. Means and standard deviations (SD) will be used to report continuous variables and t-tests will be used to assess differences between the two groups for these variables. Frequencies will be used to report categorical variables and Pearsons X2 test will be used to assess differences between groups for these variables. All statistical analysis will be completed using STATA/ MP 14. Refer to Table 2 for a summary table of study objective and methodology.

Research Questions 1: Descriptive statistics will be used to measure feasibility (recruitment rate, retention rate, and adherence rates). Recruitment rates will be calculated by determining the percentage of eligible patients that were actually enrolled in the study. A recruitment log will be kept, detailing reasons for non-participation of eligible patients. Retention rate will be defined by calculating the percentage of enrolled patients who complete the intervention. Adherence rates will be calculated as a percentage of total sessions attended. Attendance will be tracked on the feasibility data collection sheet and reasons for non-participation on scheduled intervention days will be documented using an adherence log.

Table 2. Summary Table

Objective	Hypothesis	Outcome	Analysis Method
Determine the feasibility (through recruitment, retention and adherence rates) of providing a complex KT intervention including accessible exercise programs delivered within the cancer institution and a SM program designed specifically for women with BC using technology	Implementing and providing a complex KT intervention for women with BC is feasible (recruitment, retention, and adherence rates are 50%, 75%, and 75% respectively).	Recruitment Rate	Descriptive statistics (percentage (%) of eligible patients recruited). Recruitment logs will be kept, detailing reasons for non-participation of eligible patients.
		Retention Rate	Descriptive statistics (% of consented patients who complete the intervention).
		Adherence Rate	Descriptive statistics (% of sessions attended). Adherence rates will be tracked using the data collection sheet. Reasons for non-participation on scheduled intervention days will be documented using adherence logs.
Determine preliminary estimates of effects of the KT intervention of exercise plus SM versus usual care among BC survivors over a four-month period.	Compared to the control group, BC survivors participating in the KT intervention will have higher levels of exercise knowledge and behaviours and QOL, and less need for additional health care services.	Levels of exercise knowledge and behaviour (using a Theory of Planned Behaviour  based questionnaire)	An analysis of covariance will be used to determine within and between group differences. An intention to treat analysis will be used for these analyses.
		Health related quality of life (using the FACT-B)
		Resource utilization (using the EQ-5D and a piloted self-report questionnaire assessing health care facility visits, doctor visits, procedures received, support services used, loss of work, and prescription medications used)

Research Question 2: Effectiveness outcomes will be assessed at four time points: baseline, 12 weeks (post intervention), and at 2 and 4 month follow. An analysis of covariance will be used to determine within and between group differences. An intention to treat analysis will be used for these analyses. (Table 2)

Discussion

There is a small risk of participant injury during the exercise intervention. While it has been well documented that exercise is safe for this population if proper screening and precautions are followed, minor injuries have the potential to occur in any active intervention. In order to ensure safety, all participants will need medical clearance to participate in moderate intensity exercise from their medical oncologists and will be supervised by a physiotherapist during each session. Heart rate, blood pressure, rate of perceived exertion and oxygen saturation measurement tools will be present and used during each exercise session. While uncommon, all side effects secondary to exercise will be tracked using Exercise logs. Type, intensity, duration, and management of any side effect will be documented using these logs.

The findings of this KT pilot study will help to determine the feasibility and preliminary effectiveness of a novel implementation strategy. This project will inform a larger intervention trial which has the potential to change the way rehabilitation services are provided in clinical practice and impact all levels of BC prevention; secondary and tertiary prevention of treatment-related side effects, and primary prevention of disease recurrence through sustained behaviour change. The results from this pilot project should be interpreted with an understanding of the potential threats to the generalizability of the results. Specifically, the results of this pilot study will only be relevant to the implementation of a larger intervention at sites comparable to the JCC and will be specific to the unique characteristics of women with BC. As the intervention process and management is more extensive with larger numbers of participants, the researcher team will have to take into consideration additional time and resource needs when implement the larger scale project.

Trial status

Protocol Version Number: 3. Date: April 11, 2017. Approximate recruitment start date: June, 2017. Approximate recruitment end date: August, 2017. Any trial modifications will be updated in a timely manner on ClinicalTrials.gov and sent via email to appropriate parties.

Funding

The Hamilton Division of the Ontario Physiotherapy Association funds this project. This funding body has no role in the design of the study, collection, analysis or interpretation of data, or in writing of this or future manuscripts.

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DOI: 10.31038/CST.2017221

Abstract

The objective of this study was to obtain preliminary information on which non-occupational risk factors are responsible for the excess of lung cancer deaths seen in a cohort of workers in poultry slaughtering & processing plants, and to investigate whether established non-occupational risk factors for lung cancer mortality can be replicated. We conducted a pilot case-cohort study within a cohort of 43,904 poultry and non-poultry plant workers alive in 1990 and followed up to the end of 2003. Cases were 125 lung cancer deaths that occurred between 1990-2003 for whom interviews were successfully obtained. Controls (N=152) were derived from a random sample of the cohort in 1990. Statistical analysis was by logistic and Cox proportional hazards regression. The study successfully identified many of the established risk factors for lung cancer, and a few new ones were identified. The study demonstrates that valid case-cohort studies in this occupational group are feasible, and also successfully identified non-occupational risk factors that may need to be adjusted for in future planned large-scale studies of this occupational group.

Keywords

Chickens; meat; lung cancer; diet; non-occupational

Introduction

Workers in poultry slaughtering/processing plants have high exposures to oncogenic viruses that naturally infect and cause cancer in poultry. They also have exposures to chemical carcinogens at work. We initially performed three cohort mortality studies of 30,411 poultry workers and 16,405 non-poultry workers who were members of the United Food and Commercial Workers unions in the United States (N=46,816). An excess of lung cancer was consistently observed in the poultry workers [1-4]. We next conducted a pilot case-cohort study of lung cancer within a subset of 43,904 subjects of the 46,816 subjects that were alive in 1990, and followed them up to the end of 2003. The findings for occupational exposures have been published, [5] and the corresponding literature reviewed [6]. Here we present the results for non-occupational exposures. The study provides a unique opportunity for examining whether established non-occupational risk factors for lung cancer can be replicated in this group of workers who are among the lowest paid workers in industry. Also, if established risk factors for lung cancers are confirmed in this study, this will be strong evidence that future planned large full-scale studies to investigate lung cancer occurrence in this occupational group will give valid results.

Material & Methods

Cases were the first 125 lung cancer deaths out of 552 (23%) that occurred in the cohort between 1990 and 2003, for whom telephone interviews were obtained. Controls were similarly the first 152 subjects (10%) for whom telephone interviews were obtained. They originate from a random sample of 1,516 persons in the cohort that were alive in 1990. A telephone questionnaire was administered to the next-of-kin of study subjects if they were deceased (all cases, and 13% of controls) or to live control subjects themselves. Statistical analyses were conducted using both logistic regression and Cox proportional hazards regression methods as previously described [5].

Ethics: The study was approved by the University of North Texas Health Science Center’s Institutional Review Board

Results

The results are summarized in Tables 1&2.

Table 1. Poultry associated non-occupational exposures: Associations with lung cancer mortality, 1990-2003

			Adjusted Logistic Regression ORs†	Adjusted Cox Proportional HRs‡
	Cases (n=125)	Controls (n=152)	OR (95% CI)	HR (95% CI)
LIFESTYLE
Ever smoked tobacco	113	135	7.1 (2.8-18.0)	3.7 (1.8-7.4)
Mostly prepared own food	118	147	0.5 (0.2-1.1)	0.5 (0.3-0.9)
Ever drunk wine	113	146	0.4 (0.2-0.9)	0.6 (0.4-0.9)
Swam at least once a month for more than one year	115	147	0.4 (0.1-0.9)	0.5 (0.3-0.9)
Regularly performed any exercise	117	147	0.3 (0.1-0.5)	0.5 (0.3-0.7)
MEDICAL HISTORY
Ever treated with radiation therapy	106	141	16.6 (6.8-40.8)	5.3 (3.5-8.1)
Ever treated with radiation therapy for cancer	67	21	1.6 (0.5-5.4)	1.1 (0.6-1.9)
Ever treated with radiation therapy for skin/scalp conditions	68	20	1.0 (0.1-11.9)	1.5 (0.6-3.9)
Ever treated with radiation therapy for arthritis	70	20	0.5 (0.1-1.9)	0.8 (0.4-1.5)
Tuberculosis	102	144	9.0 (0.3-304.9)	2.2 (0.9-5.4)
Cirrhosis	112	147	7.8 (0.7-82.6)	2.0 (0.8-5.0)
Pancreatic inflammation	111	146	6.2 (0.7-55.5)	1.2 (0.5-2.8)
Infectious mononucleosis	109	147	3.6 (0.3-51.5)	1.7 (0.4- 6.9)
Cold sores on lip	114	145	0.6 (0.3-1.2)	0.6 (0.4-0.9)
Diabetes	114	148	0.3 (0.1-0.8)	0.4 (0.2-0.9)
Allergic to pollen	113	145	0.2 (0.1-0.5)	0.3 (0.2-0.7)
Allergy to drug medications	110	144	0.1 (0.0-0.4)	0.2 (0.1-0.5)
FOOD CONSUMPTION
Ate beef once every two weeks for most of life	114	144	14.9 (1.8-123.0)	9.6 (1.3-69.0)
Ate bacon every week for most of life	112	141	12.1 (4.2-35.1)	5.1 (2.4-11.2)
Ate uncooked fish/shellfish every week for most of life	111	142	2.0 (0.4-10.0)	1.4 (0.5-3.8)
Ate spicy foods every week for most of life	110	142	1.7 (0.9-3.4)	1.3 (0.9-1.9)
Ate chicken once every two weeks for most of life	116	144	1.7 (0.4-7.4)	1.5 (0.6-3.6)
Ate pork once every two weeks for most of life	114	144	1.6 (0.7-3.6)	1.4 (0.8-2.4)
Ate lamb once every two weeks for most of life	115	144	1.6 0.5-5.2)	1.1 (0.6-2.2)
Ate turkey once every two weeks for most of life	115	144	1.0 (0.5-1.9)	1.0 (0.7-1.5)
Ate fruits every week for most of life	108	143	0.8 (0.3-2.0)	0.9 (0.5-1.5)
Ate freshwater fish at least once a month	115	142	0.7 (0.4-1.4)	0.8 (0.5-1.2)
Ate cheese every week for most of life	109	142	0.7 (0.3-1.7)	0.8 (0.5-1.4)
Ate raw eggs at least once every two weeks for most of life	114	143	0.6 (0.1-3.9)	0.7 (0.3-2.1)
Ate veggies every week for most of life	111	143	0.5 (0.3-1.1)	0.6 (0.2-1.7)
Ate seafood once every two weeks for most of life	111	144	0.5 (0.2-0.9)	0.7 (0.4-1.0)
Ever ingested herbal leaves, drinks, medications at least once a week for >1yr	111	140	0.3 (0.1-0.9)	0.4 (0.2-1.0)
Ever adhered to a vegetarian diet for more than a year	114	143	0.2 (0.0-3.8)	0.3 (0.0-2.1)
Method of cooking meats
Ate meat fried at least once every two weeks for most of life	113	144	2.6 (1.0-6.6)	1.9 (1.0-3.8)
Ate meat salted at least once every two weeks for most of life	109	144	2.4 (1.2-4.8)	1.5 (1.0-2.2)
Ate meat raw at least once every two weeks for most of life	111	144	1.6 (0.2-11.3)	1.9 (0.7-5.3)
Ate meat barbequed at least once every two weeks for most of life	112	144	1.5 (0.8-2.9)	1.1 (0.7-1.6)
Ate meat smoked at least once every two weeks for most of life	110	144	1.3 (0.6-2.8)	1.0 (0.6-1.6)
DRUG USE
Use vitamins at least every week for more than a year	103	140	0.4 (0.2-0.8)	0.6 (0.4-1.0)
Ever had general anesthesia during surgery	108	140	0.4 (0.2-0.7)	0.6 (0.4-0.9)
Ever used hormone replacement therapy continuously for at least a year	24	57	0.2 (0.0-0.8)	0.3 (0.1-0.9)
FAMILY HISTORY OF CONDITIONS
Reported cancer in children	114	139	1.7 (0.5-6.4)	1.1 (0.6-1.9)
Reported cancer in parents	107	135	0.8 (0.4-1.6)	0.8 (0.5-1.2)
Reported cancer in spouse	113	139	0.8 (0.3-2.0)	1.1 (0.7-1.8)
IMMUNIZATIONS
Typhoid	46	127	2.1 (0.8-5.5)	1.4 (0.7-2.7)
Pneumococcal infections	48	127	1.7 (0.6-4.3)	1.4 (0.7-2.7)
Yellow fever	41	127	1.7 (0.5-5.6)	0.9 (0.4-2.2)
Ever received gamma globulin	56	131	1.6 (0.3-8.7)	1.8 (0.6-6.0)
Measles	42	124	1.5 (0.6-3.5)	1.0 (0.5-2.0)
Small pox	64	128	1.4 (0.6-3.1)	1.0 (0.6-1.7)
Diphtheria	55	120	1.3 (0.6-3.2)	1.0 (0.5-1.8)
Mumps	43	123	1.3 (0.5-3.0)	0.9 (0.5-1.7)
OTHER
Ever owned a cell phone	115	141	0.2 (0.1-0.4)	0.3 (0.1-0.6)

† Odds ratios (OR) were adjusted for smoking, gender, and age by the Logistic Regression Method
‡ Hazard ratios (HR) were adjusted for smoking, gender, and”> age by the Cox Proportional Hazard Method
@ For ever smoked tobacco, adjustment was for age and gender.

Table 2. Lung cancer mortality associated with frequent consumption of Beef and Bacon, adjusted for occupational exposures, meat preparation type, tobacco smoking, age, gender, and union site – (1990-2003)

	Ate a lot of Beef		Ate a lot of Bacon
	Case/control	HR (95% CI)	Case/control	HR (95% CI)
OCCUPATIONAL EXPOSURES
History of working in stockyard	56/99	—	56/98	2.7 (1.1-6.7)
Ever killed chickens at work	105/143	8.9 (1.2-64.6)	103/140	4.7 (2.1-10.3)
History of working in deli department	66/99	—	66/98	3.1 (1.3-7.4)
History of working in meat department	66/99	—	66/98	3.1 (1.3-7.1)
MEAT PREPARATION
Ate a lot of meat raw	109/144	9.8 (1.3-71.1)	108/141	4.8 (2.2-10.5)
Ate a lot of meat fried	111/144	8.9 (1.2-64.8)	110/141	4.7 (2.1-10.3)
Ate a lot of meat BBQ	111/144	9.7 (1.3-70.4)	109/141	5.0 (2.3-10.9)
Ate a lot of meat smoked	109/144	9.5 (1.3-69.0)	109/141	5.1 (2.3-11.2)
Ate a lot of meat salted	107/144	8.5 (1.2-62.0)	106/141	4.5 (2.0-10.1)

*HR = hazard ratio; CI = confidence interval; NOTE: Questionnaire defined a lot as “once every two weeks for most of life”

Discussion

With regard to lifestyle, established associations in the literature with cigarette smoking, drinking of wine and exercise were confirmed [7-9]. The significant association with radiation exposure may be real and consistent with well-documented reports of this association [7,10]; but it is also likely that it reflects using radiation treatment for the lung cancer, since no significant associations were seen for treating other conditions with radiation. The elevated but not statistically significant risk for tuberculosis seen is consistent with the findings of a comprehensive review [11]. Protective effects were seen for history of cold sores, diabetes, and allergy to pollen and medications. These are consistent with reports of allergies being protective risk factors for the disease [12-14].

Frequent consumption of beef and bacon were significantly associated with increased lung cancer risk, and the risks persisted after adjusting for occupational exposures that were associated with increased risks [5] Table 2. The risks also persisted irrespective of whether the beef or bacon was eaten raw, fried, barbecued, smoked, or salted – Table 2. The method of preparation of any of the different meats (beef, pork, poultry, etc.) was not an independent risk factor for lung cancer (data not shown) except for salted chicken, turkey and lamb for which the hazard ratios were 1.4 (95% CI, 1.0-2.2), 1.5 (95% CI, 1.0-2.2) and 1.5 (95% CI, 1.0-2.2), respectively. These associations with beef and bacon and salted meats are well documented in the literature [7,15-18], and mere consumption of these meats seem to be the most important factor.

Risk estimates for eating of seafood, ingestion of herbal leaves, drinks or medications, adherence to vegetarian diet, vitamin intake, hormone replacement therapy, and history of diabetes and general anesthesia were all below the null. These protective associations have been previously reported [7,19-21], except for history of diabetes and general anesthesia for which we have no explanation. The results for cellphone use are likely due to systematic bias resulting from cases (all deceased) dying during earlier periods when cellphone use was not in existence or infrequent while controls most of whom were alive, lived long enough into the more recent period of popular use; moreover, this reduced risk was also seen for the other cancers (liver, pancreas, brain) investigated [22,23].

Conclusion

The findings in this study are important for three reasons: 1) in spite of its small size, the study remarkably was able to confirm many of the reported non-occupational risk factors in the literature for lung cancer in this low-income population. This indicates that future planned case-cohort studies nested within occupational cohorts of workers in poultry slaughtering and processing plants that are assembled to investigate cancer occurrence in this industry, are feasible and capable of giving valid results. Such studies can provide valuable information on both occupational and non-occupational risk factors for various cancers. Secondly, this study has successfully identified non-occupational exposures that are risk factors for lung cancer in this specific population of poultry workers. These constitute some important potential confounding factors that may need to be adjusted for when investigating the role of occupational carcinogenic exposures (especially oncogenic viruses) in the occurrence of lung cancer in poultry slaughtering & processing plant workers in future full-scale large case-cohort studies. Finally, this small study indicates that established risk factors for lung cancer are equally applicable for this group of minimum-wage workers who belong to the lowest socioeconomic group.

Acknowledgements

Our appreciation and thanks go to the United Food & Commercial Workers International Union for their continuing support and collaboration over the years without which this research would not have been possible.

Funding Source

The study was funded by a grant 1 RO1 OH008071 from the National Institute for Occupational Safety & Health.

Competing interests: None

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DOI: 10.31038/CST.2017215

Abstract

Laryngeal squamous carcinoma (LSC) represents one of the refractory malignant tumors of head and neck squamous cell carcinoma (HNSCC), leading to high patient mortality and unsatisfactory overall 5-year survival. New therapeutic targets and modalities for effective chemoprevention and treatment are required to improve the survival of patients with LSC. Evidence from previous studies has suggested that proteasome inhibitors (PIs) possess effective antitumor activity and have been approved by the United States Food and Drug Administration (FDA) to treat myeloma. However, resistance of myeloma to PIs has been reported, which prevents PIs from their clinical uses in cancer treatment. So far, the underlying mechanisms of tumor cells anti-PIs effects remain unclear. In the present study, we found that constitutive activation of signal transducer and activator of transcription 3 (STAT3) was associated with clinicopathlologic factors in LSC patients, and persistent activation of STAT3 was found in Hep-2 laryngeal carcinoma cells. Moreover, we demonstrated that proteasome inhibitor MG-132 induces apoptosis and cell cycle arrest in Hep-2 cells, accompanied by increased activation of STAT3; knockdown of STAT3 expression by shRNA or application of STAT3 inhibitor AG490 potentiates the antitumor effects of MG-132 in vitro, which is attributed to the down-regulation of cyclin D1 and Bcl-2. Taken together, we identified that STAT3 activation plays a great role in therapeutic resistance to PIs in LSC; blocking STAT3 activation could potentiate the tumor killing effects of PIs in LSC or other malignancies involving STAT3 activation. Combined application of PIs and STAT3 inhibitor may implicate a promising strategy for managing LSC or even other HNSCC.

Keywords

Signal transducer and activator of transcription 3, STAT3; proteasome inhibitor; STAT3 inhibitor; laryngeal carcinoma; apoptosis

Introduction

Laryngeal squamous carcinoma (LSC) is a kind of head and neck squamous cell carcinoma (HNSCC), the latter of which ranks the sixth most common malignant cancer, and accounts for 35,00000 newly diagnosed cases annually worldwide [1]. Despite of successive advancement in diagnosis and treatment, the overall survival of HNSCC has just been slightly improved in recent years. In addition to distant metastasis and relapse, resistance to traditional therapeutic modalities has been the dominant cause of treatment failure, especially in advanced cases. For combating the therapeutic resistance of cancers, some novel concepts and strategies for targeted therapies have been introduced, most of which mainly focused on some key modulators of cell proliferation and apoptosis pathways. Although some preliminary results have been achieved, they are not as effective as expected.

Signal transducer and activator of transcription 3 (STAT3), a member of STAT family, is a focus of many oncogenic receptor tyrosine kinase pathways, such as EGFR, IL-6/JAK and Src [2]. It has been demonstrated that STAT3 can induce up-regulation of prosurvival proteins including Bcl-2, Bcl-xL, survivin and Mcl-1, and promote expression of angiogenesis factors, such as VEGF [3]. In fact, STAT3 is constitutively activated in many tumors [4-7] and plays a great role in proliferation, cell cycle progression and apoptosis of tumor cells [8]. In addition, activation of STAT3 is also associated with chemoresistance in many malignancies [9, 10]. Results from our previous study demonstrate that knockdown of STAT3 with specific shRNA can enhance radiosensitivity in LSC Hep-2 cells both in vitro and in vivo [11, 12]. Therefore, targeting STAT3 is a potential therapeutic strategy for LSC.

As is known, due to rapid proliferation and insufficient blood supplies, the microenvironments of most solid cancers are accompanied by hypoxia, glucose starvation and low PH, leading to endoplasmic reticulum (ER) stress due to causal accumulations of incorrect proteins [13]. Sequently, cancer cells must adapt to these unfavorable conditions for their survival via inhibiting synthesis of proteins and degrading unfolded proteins accumulated in ER by proteasome [14]. Therefore, targeting proteasome becomes a promising strategy for cancer therapy.

Early studies demonstrated that tumor-inhibiting effects of proteasome inhibitors (PIs) are attributed to repression of nuclear factor-κB (NF-κB) signaling and regulation of proteins associated with both cell cycle and apoptosis, such as p27^Kip1 [15], p21^cip/WAF1 [16], p53 [17], Bcl-2 [18, 19], Bax [17], Bim and Bik [20]. It is now believed that PIs induce ER stress-related cell death through unfolded protein response (UPR) [21] and confer preferential cytotoxicity towards hypoxic tumor cells [22]. However, cases of resistance to PIs are emerging, whereas the related mechanisms are not systematically elucidated [23]. Interestingly, a recent study has demonstrated that a proteasome inhibitor Bortezomib promotes STAT3 activation in HNSCC, suggestive of potential roles of STAT3 in the resistance of HNSCC to the proteasome inhibitor [24]. Therefore, it would be of great interest to elucidate whether the STAT3 plays a role in resistance LSC to PIs.

In the present study, we first assessed the associations between constitutive activation of STAT3 and clinicopathlological factors in LSC. We next investigated the role of STAT3 activation by protesome inhibitor and its impacts on proteasome-induced cell apoptosis in LSC.

Materials and Methods

Patients’ data

50 LSC patients (36 males and 14 females) who received surgical resection at Bethune International Peace Hospital were recruited, and their complete detailed clinicopathologic data were collected. Of the 50 cases, there were 17 cases of supraglottic and 34 cases of glottic cancers. The age of the present series ranged from 36 to 86 years, with a mean age of 58 years. All patients had undergone neither chemotherapy nor radiotherapy prior to surgery. According to UICC cancer staging system (2002), there were 7 cases of stage I, 6 cases of stage II, 17 cases of stage III and 20 cases of stage IV tumors. With regard to the histological grading, 6 cases were well differentiated, 13 cases were moderately differentiated and 31 cases were poorly differentiated squamous cell carcinomas (SCC). Twenty-two cases had neck node metastasis as proven by postoperative pathologic examinations.

Tumor tissue collection

With the informed consent of all patients, tissue specimens were collected immediately after surgical removal. Paired tumor-free paracancerous tissues (PCT) were taken at least 0.5 cm beyond the tumor margins. Each sample was cut in halves. One half was promptly frozen at -70 ℃ for subsequent Western blot, while the other was fixed immediately with 10% neutrally buffered formalin for histopathologic evaluation and immunohistochemistry. The acquisition of paraffin tissues and frozen specimens were approved by the Institutional Review Boards at Bethune International Peace Hospital. Neck dissection specimens were evaluated by two pathologists to confirm the status of neck node metastasis in these patients.

Immunohistochemistry and immunocytochemistry

Immunohistochemical and immunohistocytochemical studies were performed as described previously [25] using SP-9002 kit (including 0.3% hydrogen peroxide, 10% goat serum, biotinated secondary antibodies and horseradish peroxidase marked strepto- antibiotin, Zhong Shan Jin Qiao, Beijing) with some modifications. Briefly, for immunohistochemistry, consecutive sections were deparaffinized and hydrated, and subjected to 0.3% hydrogen peroxide for elimination of endogenous peroxidase at room temperature for 10 min. Slides were then heated for the retrieval of antigens in an autoclave sterilizer for 2 min. Goat serum (10%) was added to block nonspecific protein staining for 10 min. The sections were incubated with anti-STAT3 and anti-p-STAT3 (Santa Cruz Biotechnology Inc.) at 4 ℃overnight. Afterwards, they were rinsed by PBS and incubated at room temperature with biotinated secondary antibodies for 15 min under a humid condition followed by 15-min incubation with horseradish peroxidase-marked strepto- antibiotin at room temperature. Finally, they were detected with DAB kit (Zhong Shan Jin Qiao, Beijing). The immunoreactivity of STAT3 or p-STAT3 was evaluated as described previously [31]. Brown coloration was identified as positive staining. The distribution of positive staining cells was classified in 4 categories: weakly positive (+), positive (++), strongly positive (+++), and negative. For immunocytochemical study, Hep-2 cells were seeded in 6-well plates with a small cover slide in each well. When confluence reached 70% to 80%, cells grown on the cover slide were fixed with cold acetone at room temperature for 15 min. Cover slides were then subjected to 0.3% hydrogen peroxide for elimination of endogenous peroxidase at room temperature for 30 min, washed with PBS and permeabilized with 0.1% TritonX-100 on ice for 2min. The immunostaining of Hep-2 cells was performed as in immunohistocehmistry.

Cell line and culture

Hep-2 human LSC cell line (National Cancer Institute, USA) were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS) under the condition of 37℃, 20% O₂, 5%CO₂, 95% humidity in a CO₂ incubator. Cells were allowed for attachment overnight and those grown in logarithmic phase were used for subsequent experiments. All experimental groups came from the same parental cells. Cells were passaged every 2 to 3 days to maintain exponential growth.

Transfection of plasmids

The construction of STAT3-targeting shRNA expression vector and the transfection of plasmids were performed as our previous publications [12]. Briefly, the double strands of shRNA targeting gene of STAT3 (sense: 5′-CACCGCAGCAGCTGAACAACATGTTCAAGAGACATGTTGTTCAGCTGCTGCTTTTTTG-3′, the corresponding mRNA coding region is underlined) and the double strands of negative control gene (sense: 5′-CACCGTTCTCCGAACGTGTCACGTCAAGAGATTACGTGACACGTTCGGAGAATTTTTTG-3′, underline shows the target sequence) were inserted into pGPU6/GFP/Neo vector (Gene Pharma Co., Ltd), and named pGPU6/GFP/Neo-shSTAT3 and pGPU6/GFP/Neo-shNC, respectively. The procedure of transient transfection was performed according to Lipofectamine^TM2000 (Invitrogen, Corporation) manufacturer’s instructions in 96-well plates or/and 6-well plates. The thansfection efficiency was monitored by observation under a fluorescent microscope and by flow cytometry. Sequent assays were performed 24 hours after transfection.

MTT assay

The inhibition of hep-2 cells by chemicals was determined by MTT assay as described previously [26]. Transfected and untransfected hep-2 cells were subjected to MTT assay. After treatments with PIs (MG-132) (MERCK Corporation, Germany) and STAT3 inhibitor (AG940) (Sigma, USA) in different combinations and cisplatin (DDP, Shan Dong Qi Lu Pharmaceutics, China), 20μl 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT, 5mg/ml, Sigma, USA) were added into each well of 96-well plates. After incubation at 37 ℃ for 4h, culture medium was replaced by DMSO to dissolve formazan for coloration. Absorbance was measured at 490 nm using Enzyme-linked Immunosorbent Detector (Model 550, Bio-Rad, USA).

Flow cytometry

The transfection efficiency, apoptosis rate and cell cycle distributions were determined by a FACScan analyzer (Becton Dickinson FAC Sort, USA). Hep-2 cells, with or without treatments, were collected after trypsinization. Fresh cells were used for detection of the efficacy of transfection by flow cytometry. To determine apoptosis rate and cell cycle distribution, cells were washed by PBS for 3 times, and then were fixed with 75% ethanol overnight at 4 ℃. Prior to analysis, ethanol was discarded and 80μl RNase (final concentration: 50mg/L) and 150μl propidium iodide (final concentration: 50mg/L, Sigmar, USA) were used for staining of DNA for 30 min at 4℃. Apoptotic cells appeared as a magnitude of the sub-G1 peak due to nuclear fragmentation and loss of DNA.

Western blot

Western blot was carried out as described previously [25] with minor modifications. For tissue specimens, lysates were obtained from 100mg sample of each frozen tissue by a homogenizer. Subjected Hep-2 cells were harvested following trypsinization and centrifugalization. The cell pellets were resuspended in cell lysis buffer containing 500mM HEPES 25ml (0.1mol/L)，100mM NaCl 5ml (1mol/L)，2mM EDTA 0.2 ml (0.5mol/ L)，sodium deoxycholate 0.25g (0.5%)，NP-400 5ml (1%)，10%SDS 0.5ml (0.1%)，2×10^-3 mol/L PMSF and 2×10^-6 g/L Lupeptin to collect lysates. Subsequently, lysates originating from tissues or hep-2 cells were acquired in the same way. After determination of protein concentrations in the lysates, equivalent protein (100mg from tissues or 30mg from cells) were separated by electrophoresis on 4% to 10% SDS-PAGE gels. The subjected gels were then transferred onto nitrocellulose filters, and blocked in confining liquid containing 1% nonfat dry milk, 0.02% Tween-20 (Sigmar, USA), 10mmol/LTris-Cl，0.15mmol/L NaCl at 4℃ overnight. The blocked filters were incubated with primary antibodies (Santa Cruz Biotechnology int and Cell Signaling Technology, USA) at 4℃ overnight followed by rinse in TBST. The filters were incubated with secondary antibodies marked by HRE (Zhong Shan Jin Qiao, BeiJing) at room temperature for 1 hour followed by rinse in TBST two times. Finally, the blots were visualized by ECL kit (Pierce, USA).

Statistical analysis

Results were analyzed for statistical significance with the use of SPSS 13.0. Data were presented as mean values ± SD. The method of statistical analysis included student’s t-test for comparision of two groups, one-way ANOVA for comparison of more than two groups combined with or without least significant difference (LSD) followed for comparison of each two groups, and Pearson correlation analysis for correlation evaluation of different factors. P < 0.05 was considered significant.

Results

Constitutive activation of STAT3 is associated with clinicopathlological factors in LSC, including clinical stage, pathological grading and cervical lymph node metastasis. It has been reported that STAT3 is aberrantly activated in many tumors in comparison with its transient activation in normal tissues [27-30]. To determine activation status and expression distribution pattern of p-STAT3 in LSC, we compared levels of p-STAT3 protein of primary LSC tissues with corresponding tumor-free PCT by immunohistochemistry. As can be seen in Fig 1A-D, p-STAT3 was constitutively expressed in LSC compared with PCT. In positive samples, weakly positive staining dominates PCTs, whereas strong positive coloration occupies most of cancer tissues; it was also noted that the staining of p-STAT3 in poorly differentiated LSC is stronger than that in well differentiated LSC.

Figure 1. The expression of phosphorylated STAT3 (p-STAT3) in human laryngeal squamous cell carcinoma (SP×400). Tissue specimens were collected and p-STAT3 protein expression levels were assessed by immunohistochemistry and western blot. A. Expression of STAT3 in well-differentiated laryngeal squamous carcinoma. B. The expression of p-STAT3 protein in moderately-defferentiated laryngeal squamous cell carcinoma (SP×400). C. The expression of p-STAT3 protein in poorly differentiated laryngeal squamous cell carcinoma (SP×400). D. The expression of p-STAT3 protein in tumor-free paracancerous laryngeal epithelial tissues (SP×200). Yellow arrow indicates representative staining cells. E. Expression of Stat3, p-STAT3, and cyclin Dl in human laryngeal carcinoma by western blot. Elevated levels of p-STAT3 and cyclin Dl in tumor tissues (T) compared to adjacent normal laryngeal mucosa (N) were further confirmed.

These findings suggest that STAT3 is activated constitutively and persistently in LSC rather than in PCT, and its activated form, p-STAT3, is likely to function in nucleus as in other malignancies. Moreover, expression of p-STAT3 is closely related to the expression of cyclin D1. Results from western blot analysis showed the same expression pattern for each protein (Fig 1E) in that there is a correlation between p-STAT3 and the expression of cyclin D1, suggesting that cyclin D1 is regulated by p-STAT3 in LSC tissues.

It has been reported in many studies that activation of STAT3 is related to poor prognosis of various cancers [31-33]. To test whether STAT3 activation is pertinent to clinicopathologic factors of LSC, we analyzed the relationship between p-STAT3 and critical prognostic factors. Results in Table 1 indicates that there is a significant correlation between p-STAT3 expression and clinical stage, pathologic grade and neck node metastasis rather than age, sex, T stage and even the site of primary tumors.

Table 1. The relationship between the expression of Stat3 and phosphorylated Stat3 protein and clinicophalogical features in laryngeal squamous cell carcinoma

n			Stat3		p-Stat3
			M number (rate%)	P value	M number (rate%)	P value
Age	≥60	28	23(82.14)	0.0715	17(60.71)	0.6609
	＜60	22	13(59.09)		12(54.54)
Sex	M	36	27(75.00)	0.6841	23(63.89)	0.1761
	F	14	9 (64.29)		6 (42.86)
Tumor position	Supra-glottis	17	10(58.82)	0.2473	10(58.82)	0.9325
	glottis	33	26(78.78)		19(57.58)
Patho- grade	Well to moderately differentiated	19	10(52.63)	0.0169	5 (26.32)	0.0004
	Poorly differentiated	31	26(83.87)		24(77.42)
T stage	T1-T2	17	12(70.58)	0.0685	7 (41.18)	0.0836
	T3-T4	33	27(81.81)		22(66.67)
Clinical stage	Ⅰ-Ⅱ	13	5 (38.46)	0.0056	4 (30.77)	0.0208
	Ⅲ-Ⅳ	37	31(83.78)		25(67.57)
Lymph node metastasis	Yes	22	20(90.90)	0.0083	17(77.27)	0.0144
	No	28	16(57.14)		12(42.86)

Activation of STAT3 is in constant existence in Hep-2 laryngeal carcinoma cells (Hep-2 cells). To test whether STATA3 is also constitutively activated in Hep-2 cells, we examined the expression of STAT3 and its related proteins by immunocytochemistry and western blot. By immunocytochemistry, STAT3 was distributed in cytoplasm of cancer cells, while p-STAT3 was observed mostly within nucleus of Hep-2 cells, which resembles the expression pattern of STAT3 and p-STAT3 in LSC tissues (Fig 2A, B, C). To examine if these proteins were constantly and pervasively expressed, we detected expression of the two proteins using western blot at different time points after the setting of cell culture. The sequent 24 h, 48 h, and 72 h were chosen as time spots of analysis. The results of western blot showed that STAT3 and p-STAT3 (Fig 2D) can be detected at different time points, suggesting that constitutive activation of STAT3 is also present in Hep-2 cells as in primary LSC.

Figure 2. TExpression of STAT3 and p-STAT3 in human laryngeal carcinoma Hep-2 cells. Immunocytochemistry was used to demonstrate the intracellular distribution of STAT3 and p-STAT. PBS was used as a negative control. Besides, western blot was used to show the expression patterns of STAT3 and p-STAT3 overtimes (0h, 24h, 48h, 72h). β-actin represents the internal protein control. Constitutive expression and activation of STAT3 in human laryngeal carcinoma Hep-2 cells was noted. A. Cytoplasmic staining of STAT3 in Hep-2 cells (SP × 400). B. Nuclear staining of p-STAT3 in Hep-2 cells (SP × 400). C. Negative control (SP × 400). D. Expression of Stat3 and p-STAT3 in Hep-2 cells as demonstrated by western blot.

Proteasome inhibitor MG-132 induces apoptosis in Hep-2 cells, which is accompanied by additional activation of STAT3 and changes in the expression of several proteins related to cell cycle and apoptosis regulation. PIs have been approved by the United States Food and Drug Administration (FDA) for the treatments of multiple myeloma. In addition, PIs have been investigated for the treatments of many cancers. Herein, we aimed to test the therapeutic effects of PIs on Hep-2 cells. First, we tested the inhibition of proliferation in Hep-2 cells by a PI, MG-132, using MTT assay. Medium containing different concentrations of MG-132 was add into each well of treatment groups and maintained for different times. As seen in Fig 3A, the inhibition of Hep-2 cells by MG-132 displayed a concentration-dependent manner (24h：r=0.925，p<0.01；48h：r=0.944，p<0.01). Second, we attempted to confirm if MG-132 inhibits proliferation of Hep-2 cells in a time-dependent manner. For doing this, we selected the 2.5μM (IC50) of MG-132 to stimulate Hep-2 cells for 48 h. The results of Fig 3B show that the effects of MG-132 in proliferation inhibition is enhanced with prolongation of treatment time (r=0.945，p<0.01). Third, using flow cytometry, we determined apoptosis rate and cycle distribution of Hep-2 cells after treatment with MG-132. Similar with the inhibition of proliferation, MG-132 induces apoptosis in time- (r=0.888，p<0.01) and dose-dependent (r=0.842，p<0.01) manners (Fig 3C, 3D ).

Figure 3. Effects of a proteasome inhibitor, MG-132, on Hep-2 cells. Hep-2 cells were treated with different concentrations of MG-132 for 24 hours. A concentration of 2.5μM was chosen for stimulation of Hep-2 cells for different times. Alterations in cell apoptosis, cell cycle and protein expression of STAT3 and p-STAT3 were assessed simultaneously. A. The dosage and effect curve of MG-132 (n = 3). B. The time and effect curve of MG-132 (n = 3). C. Apoptosis rate of Hep-2 cells treated with MG-132 for different time (n = 3). D. Apoptosis rate of Hep-2 cells treated with MG-132 at different concentrations (*P < 0.05, versus untreated groups (0μM) (n = 3). E. The correlated expression of cell cycle regulation proteins in Hep-2 cells treated with MG-132 for different time. F. Variation of cell cycle of Hep-2 cells treated with MG-132 for different time (n = 3). G. MG-132-induced STAT3 activation and Bcl-2 expression in Hep-2 cells.

To confirm the mechanisms underlying the cell cycle changes induced by MG-132, we used western blot to check the expression levels of some cell cycle regulators. As seen in Fig 3E, the expression of P21 is elevated significantly compared with the decreased levels of the expression of cyclinD1, suggesting that the changes of cell cycle induced by MG-132 were associated with upregulation of P21 and downregulation of cyclinD1. Simultaneously, cell cycle arrest was noted at G0/G1 phase and/or G2/M phase overtimes after treatment with MG-132 (Fig 3F), the former of which is more prominent than the latter.

From the results of western blot in Fig 3G, we can confirm that STAT3 and its activated form, p-STAT3, are expressed at all times in cultured Hep-2 cells, even in the absence of MG-132 treatment, and the level of its expression was relatively constant and stable. In the presence of MG-132, the expression of p-STAT3 was elevated within short period of time and remained at a stable level; however, the increased expression of p-STAT3 was not related to the duration of MG-132 treatment. Bcl-2 was identified as a downstream of STAT3, and facilitates anti-apoptosis activity in cancer cells [34]. Expression of Bcl-2 protein increased overtime after MG-132 treatment, which may contributed to the MG-132-induced STAT3 activation and resistance of Hep-2 cells to MG-132-induced apoptosis.

Knockdown of STAT3 by shRNA, abrogates the expression and thus activation of STAT3, and potentiates the apoptosis-inducing effects of MG-132. To further confirm the role of the STAT3 activation in inducing resistance of Hep-2 cells to MG-132, we used RNAi to knockout the expression of STAT3, and thus the “constitutive” (inherent) and “PI-induced” (acquired) activation of STAT3. The transfection efficiency was 85.76% by flow cytometry (data not shown). The results of Fig 4A showed that combined group has a similar expression level of p-STAT3 with pshSTAT3 group, with a higher inhibition rate than any other groups (all p<0.01). Results from apoptosis detection Fig 4B showed a more striking apoptosis rate 55.80±3.12% in combined group than in any other group (all p<0.01). In pshSTAT3 group, moderate apoptosis and proliferation inhibition rates were induced. Therefore, blockade of STAT3 by shRNA, abrogates the expression and thus both “inherent” and “acquired” activation of STAT3, and potentiates the MG-132-induced apoptosis in Hep-2 cells.

Figure 4. Effects of MG-132 combined with STAT3 RNAi on proliferation inhibition and apoptosis in Hep-2 cells, as assessed by MTT assay and flow cytometry. Hep-2 cells were seeded into 96-well plates for MTT assay, or 6-well plates for flow cytometry. Subjected Hep-2 cells were categorized as five groups: normal control group (untreated Hep-2 cells), negative control group (Hep-2 cells transfected with negative plasmid, pshNeg group), MG-132 group (Hep-2 cells treated with 2.5μM MG-132 only), combined group (Hep-2 cells transfected with positive plasmid combined with 2.5μM MG-132 treatment), pshSTAT3 group (Hep-2 cells transfected with positive plasmid without any other treatment). A. The proliferation inhibition rate of Hep-2 cells in different groups (n=3) (versus pshNeg group, all *P<0.01; pshSTAT3 group versus MG-132 group, #P<0.01). B. The apoptosis rate of Hep-2 cells in different groups (n=3) (control group versus pshNeg group, *P<0.01; pshSTAT3 group versus MG-132 group, #P<0.01). C. Expression of p-STAT3, cyclin D1 and Bcl-2 in Hep-2 cells in different groups was analyzed by Western blotting.. Lane 1: control group; lane 2: pshNeg group; lane 3: MG-132 group; lane 4: combined group; lane 5: pshSTAT3 group.

With regard to the activation of STAT3 at different situations, as seen in Fig 4C, p-STAT3 was expressed at the highest level in MG-132 group. By contrast, p-STAT3, cyclin D1, and Bcl-2 were expressed at lower levels in combined group, compared with each of the normal control group, negative control group and MG-132 group. The similar expression and activation patterns of STAT3 noticed in combined group and pshSTAT3 group indicated that p-STAT3 may participate in the formation of resistance of Hep-2 cells to MG-132 at a transcriptional level. Taken together, activation of STAT3 is responsible for the resistance of Hep-2 cells to MG-132 at least in part by regulating the anti-apoptosis factor Bcl-2 expression.

Combined treatment with STAT3 inhibitor AG490 and proteasome inhibitor MG-132 induces apoptosis more effectively in Hep-2 cells. In view of the fact that activation of STAT3 played a role in the therapeutic resistance to MG-132 in Hep-2 cells, which can be sensitized by knockdown of STAT3, we studied the effects of combined using STAT3 inhibitor AG490 and proteasome inhibitor MG-132 on proliferation and apoptosis. As seen in Fig 5 A, B, combination treatment by AG490 and MG-132 can induce significant increase in cell proliferation inhibition and apoptosis, indicative of effective synergism.

Figure 5. Effects of AG-490 combined with MG-132 on Hep-2 cells. Hep-2 cells were seeded into 96-well or 6-well plates followed by treatment with 1μM of MG-132 for 48h with or without presence of different concentrations (10, 20, 40, 80μM) of AG490. A concentration of 10μmol/L was for analysis of AG490-induced apoptosis. A. The proliferation inhibition rate of Hep-2 cells treated with the different concentrations AG490 alone and in combination with MG-132 for 48h (n=3). B. The apoptosis rate of Hep-2 cells treated with AG490 alone and in combination with MG-132(1μM) for 48h (n=3) (compared with control group,*P<0.01; compared with AG490 group and MG-132 group, all #P<0.01).

Discussion

Like other malignancies in HNSCC, LSC often exhibits resistance to various therapeutic strategies including chemoradiation and targeted therapies, especially in advanced cases. This difficult situation is often attributed to aberrant expression and activation of some specific genes, such as STAT3, which are utilized by cancer cells to maintain their biological behaviors. In this regard, cancer cells may become vulnerable to the assigned treatment modalities secondary to abrogating the functions of major responsible oncogenes, the so-called “Achilles heel”, which constitutes the fundamental rationale for targeted therapy [35].

Previous studies have demonstrated that activation of STAT3 is associated with chemo- and/or radioresistance in Hep-2 cells, and knockdown of STAT3 by siRNA or blockade of STAT3 activation by its inhibitors sensitizes chemo- and/or radiotherapy both in vitro or in vivo [11, 12, 36, 37]. Importantly, we also demonstrated in the present investigation that constitutive activation of STAT3 was associated with clinicopathlologic factors in LSC, which include clinical stage, pathological grading and cervical lymph node metastasis. This is consistent with the findings in the study of Rosen et al, in which they demonstrated that p-STAT3 was associated with poor prognosis of ovarian cancer [38]. Moreover, results of Kusaba’s study [33] also demonstrated that p-STAT3 is correlated with invasion of vein, nodal metastasis and Dukes grades instead of pathological grading in ovarian cancer. In the present study, we found expression of cyclin D1, one of the important factors downstream of STAT3, was also elevated. Taken together, correlations between activation of STAT3 and clinicopathlological factors of different cancers may depend on cell types from which they originate.

In addition to intrinsic elements, cancer cells in solid tumors also depends on extrinsic factors that form surrounding microenvironment (also called “niche”) for their proliferation and survival. The feature of the niche is characterized by low PH, hypoxia and glucose-deprived starvation as a result of rapid proliferation by tumor cells and insufficient delivery by intra-tumor capillary [39]. Under the stressful conditions, a mass of misfolded or unfolded proteins accumulate in endocytoplasmic reticulum (ER). Tumor cells must activate UPR to inhibit synthesis of protein and mRNA and degrade excessive protein by proteasome to accommodate ER-stress, which is rarely experienced by normal tissue cells [40]. It has been demonstrated that severe ER-stress caused by accumulated misfolded proteins can activate programmed death in tumor cells [39]. Therefore, proteasome can serve as a potential target for cancer therapy.

Recently, PIs was introduced into clinical treatment of cancers, either as a single agent or a combined therapy to sensitize traditional chemo- and/or radiotherapy. However, the resistance to PIs remains a problem to solve, of which underlying mechanisms have not been explained pertinently [23]. The prerequisite for us to select PIs as an anti-LSC agent resides on the fact that PIs is effective against cyclin D1 highly-expressed cancers [41]. However, it remains to be elucidated whether other HNSCC cells were dependent or independent on cyclin D1 expression for their proliferation, survival and progression.

Given the fact that activation of STAT3 is associated with clinicopathologic behaviors and chemoradiation resistance in LSC, STAT3 is definitely a potential target for therapeutic purposes. The pro-survival and –proliferation effects of p-STAT3 let us to postulate that the constitutive (inherent) along with PI-induced (acquired) activation of STAT3 may constitute a major cause of resistance to PIs in LSC. Abrogation of both existed and acquired STAT3 activation may sensitize the LSC cells to PI treatment. In present investigation, we found for the first time that activation of STAT3 contributed to resistance of LSC to MG-132 at least in part by upregulating expression of antiapoptotic protein Bcl-2, which is partly consistent with results of a recent study [24]. Our results showed that PIs plus AG490 displayed a more powerful killing effect to LSC cells in a synergetic manner. However, such a combination may not be applied to all cancers, because overexpression of Bcl-2 is associated with a good prognosis in some other malignancies [42].

In the present study, we also demonstrated that MG-132 can induce G₀/G₁ cell cycle arrest, via downregulation of cyclin D1 in LSC, which is also observed in some other cancers [43-45]. It can be inferred that an ideal local control is to be achieved by PIs through inducing quiescence in LSC cells. However, a fact that cannot be ignored is that G₀/G₁ cycle arrest also constitutes a cause of therapeutic resistance in some tumors.

Although inhibiting STAT3 is capable of inducing quiescence and potentiating PI-induced apoptosis in LSC cells, there has been no effective STAT3 inhibitor that can be safely used for humans in clinic for treatment of HNSCC. We recently defined dihydroartemisinin as putative STAT3 inhibitor, which might be safely used in clinic for cancer treatment [46]. Much work has to be done for developing new STAT3-inhibiting agents and exploring new treatment strategies and modalities based on the synergized apoptosis-inducing effects of PIs with STAT3 inhibitors.

Acknowledgements: The research work in the present investigation is supported by Key Basic Research Project Fund of Hebei Province, China (Fund No. 14967721D) and in part by Scientific Research Fund of Bethune International Peace Hospital, China.

Conflict of Interest: None

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DOI: 10.31038/CST.2017214

Abstract

Purpose: To assess the relationship between IFN-γR expression and clinical radiotherapy outcome in nasopharyngeal carcinoma.

Methods: IFN-γR expression was examined by immunohistochemistry from 70 nasopharyngeal carcinoma patients. Then, statistical analysis was conducted to explore the correlation between IFN-γR expression and tumor clinicopathological charateristics. Finally, CNE-1 cells were employed to detect the effects of IFN-γR expression on radiotherapy outcomes by in vitro assays.

Results: IFN-γR was upregulated dramatically in nasopharyngeal carcinoma tissues. Elevated expression of IFN-γR correlated significantly with tumor stage, positive lymph node metastasis and poor prognosis in patients with nasopharyngeal carcinoma. In vitro assays demonstrated that overexpression of IFN-γR promoted the radio-resistance ability in CNE-1 cells. Knockdown of IFN-γR facilitated CNE-1 cells more sensitive to radiation, and a wobble mutant of IFN-γR could restore this effect.

Conclusions: Overexpression of IFN-γR may correlate positively with radiotherapy resistance of nasopharyngeal carcinoma.

Keywords

IFN-γR; overexpression; prognosis; radiation resistance; NPC

Introduction

Nasopharyngeal carcinoma is one of the most common malignancies and occupies the seventh leading cause of cancer-related deaths in China [1]. Particularly in South China, nasopharyngeal carcinoma is the most prevalent. Locally, advanced nasopharyngeal carcinoma patients are generally treated with radiotherapy, and the prognosis is related with radiotherapy [2]. Both local-regional failure and early systemic dissemination of the disease always contribute to treatment failure, which makes radio-sensitization promising in improving the outcome of therapeutic irradiation [3].

IFN-γ plays a pleiotropic role in modulating immunity and antiviral activity. It stimulates macrophages and NK cells to produce MHC class I and II molecules, while IFN-γR is not required for the development of the immune system [4]. Researches showed that IFN-γR was overexpressed in breast cancer and its amplification was closely related with lymph node metastasis and poor prognosis [5-7]. Until now, there are still no reports about whether IFN-γR has an effect on the radiotherapy for NPC. In the present study, we found for the first time that IFN-γR expression correlated with the prognosis of NPC patients who received radiotherapy. High IFN-γR levels improved the radiation resistance ability of NPC cells.

Patients and methods

Tissue samples

Seventy tissues of nasopharyngeal carcinoma were freshly collected by nasopharyngoscope and procured by the Department of Pathology, Cancer Center of Wuhan Union Hospital, China. The collection of tumor samples was approved by the institutional ethics committee. Informed consent forms had been signed for sample collection before. Tumor regions were technically separated by experienced pathologists and promptly stored at liquid nitrogen until needed. All patients included underwent concurrent chemotherapy (Cisplatin, 40mg/m2, weekly) with radiation therapy for the first time. They received irradiation with daily fraction doses of 2.12Gy for a total dose 70 Gy, which was administered with linear accelerators. The clinicopathologic features of patients were showed in Table 1.

Immunohistochemical staining

Immunohistochemical staining was conducted as previously described. After rehydration, the paraffin-embedded nasopharyngeal carcinoma tissues were immersed in 3% hydrogen peroxide solution for 10 min, and then heated in EDTA buffer (pH 8.0) for 25 min. The slides were blocked with 10% normal rabbit serum at 37°C for 30 min, and then incubated with rabbit polyclonal antibody against primary antibody (IFN-γR, 1:200, ABCAM) overnight at 37°C. The slides were washed with PBS, and then incubated with biotinylated second antibody (diluted 1:200) for another 30 min at 37°C. Finally, streptavidin-peroxidase reactivity was detected with DAB solution. Cell nucleus was counterstained with hematoxylin. The immunohistochemical slides were graded according to the ratio of tumor cells with nuclear labeling. The level of IFN-γR- positive staining was scored into three grades: 0 for < 10%, positive for 10–30%, strong positive for > 30%.

Plasmid constructs and small interfering RNA synthesis

Double-stranded oligonucleotide with the following sequence was synthesized into the pSilencer 3.1- H1 neo small interfering RNA (siRNA) expression vector, and the scrambled siRNA was applied as control. Mutant DNA-IFN-γR (BOSTER, Wuhan, China) was used to resist the effect of IFN-γR-RNAi.

Cell culture and transfection

The CNE-1 cells (one of the esophageal squamous carcinoma cell lines) were cultured in RPMI 1640 (Invitrogen) with 10% fetal bovine serum. Cell transfection was carried out using Lipofectamine 2000 (Invitrogen) following the manufacturer’s instruction. Cells were harvested after transient transfection for 48 h. For stable transfection, cells were selected with 200 μg/ml G418, and passaged by serial dilution. During the next two weeks, colonies were screened by selective medium. The positive colonies with IFN-γR overexpression and IFN-γR knockdown were chosen for experiments.

Western blot analysis

Total proteins were isolated from patient tissues and cultured cells with lysis buffer [10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP40] containing protease inhibitors. After centrifugation (15,000 RCF, 30 min, 4°C), supernatants were recovered for immunoblot analysis. The proteins were subjected to SDS-PAGE and then transferred onto polyvinylidenedifluoride (PVDF) membranes (Millipore). Next, the membranes were blocked and then incubated with primary antibody against IFN-γR (1:500, ABCAM) at 4°C overnight, then incubated with horseradish peroxidase-conjugated accordingly secondary antibody for 1 h at room temperature and developed using ECL detection reagent (BOSTER).

Ionizing radiation

CNE-1 cell line was exposed to IR in a JL Shepherd Model 143 137Cesium gamma and irradiated at a rate of 2.4 Gy/min.

Colony formation assay

A total of 400 cells were aliquoted into 6- well plate and exposed to γ rays in a dose of 5 Gy in triplicate. After incubation for 10 days, visible colonies were fixed in methanol and stained with Giemsa. Colonies were counted in each well. Each experiment was performed three times independently.

Results

Overexpression of IFN-γR in esophageal squamous cell carcinoma

IFN-γR expression was examined in 70 NPC samples and adjacent normal tissue by immunohistochemistry. Results showed that IFN-γR was hardly expressed or revealed weak expression in normal epithelial cells. While, in 36 cases of 70 NPC samples, IFN-γR expression exhibited significantly enhanced trend compared to the normal epithelium [Table 1]. Statistical analysis indicated that elevated expression of IFN-γR was significantly related to tumor stage, clinical stage and lymph node metastasis (p < 0.05) [Table 2].

Next, the effect of IFN-γR expression on patient’s survival was analyzed. IFN-γR expression is positive in some patients and negative positive in others [Figure 1A]. After the included patients underwent chemotherapy combined with radiation therapy (55 out of 70), compared to NPC patients with low IFN-γR expression in cancerous tissue was significantly higher, patients with cancerous tissue of high IFN-γR expression showed significantly lower survival rate (p = 0.001) [Figure 1B].

Effects of IFN-γR on the radio-sensitivity of CNE-1 cells

To confirm the clinical data, CNE-1 cells were radiated with a dose of 5 Gy for 0, 2, 4, 8 and 12 h to know IFN-γR expression change. IFN-γR expression was increased after radiation in a time-dependent manner, which implied that IFN-γR may be associated with the radiation response of CNE-1 cells [Figure 2].

Colony formation assay was conducted to detect the effects of IFN-γR on the radio-sensitivity of CNE-1 cells [8]. IFN-γR knock-down cells showed a significantly lower survival fraction than control groups [Figures 3A and B]. Conversely, the survival fraction of cells with forced IFN-γR expression was higher [Figures 3C and D]. Therefore, results indicated a positive relationship between IFN-γR expression and radio-sensitivity of CNE-1 cells.

Discussion

Overexpression of IFN-γR has been observed in various types of human tumors, and elevated expression of IFN-γR indicated poor prognoses for patients, such as gastric carcinoma, prostate cancer, oral squamous cell carcinoma and non-small cell lung cancer. Particularly, IFN-γR hyperexpression was found to be associated with poor prognosis and reduced p27 expression in gastric carcinoma. Some studies have revealed that IFN-γR was amplified and upregulated in breast cancer. Moreover, its overexpression indicated worse tumor stage and lymphatic metastasis. However, there have been no reports on IFN-γR expression in NPC until now. Our results showed for the first time that IFN-γR expression increased in NPC, which significantly related with tumor stage and positive lymphatic metastasis. Coincidently, other studies have demonstrated that elevated IFN-γR level was correlated with poor prognosis in colorectal carcinoma [9,10]. However, no significance was shown between IFN-γR level and overall survival of NPC patients in the present study. We could not help putting forward the speculation whether IFN-γR expression affects radiotherapy outcome. Survival analysis unveiled that the overall survival rate of patients received radiation therapy was indeed affected by IFN-γR expression. Histological detection also showed a strong association betweeen IFN-γR hyperexpression and T-stage and lymphatic metastasis. Besides, in vitro assay further confirmed this point. This was the first report about the correlation between IFN-γR expression and radiation resistant of carcinoma.

Some studies reported that downregulation of IFN-γR suppressed prostate cancer cells proliferation, anchorage-independent growth and migration 11,12], while knockdown of Cks2 induced apoptosis [13,14]. Decreased IFN-γR promoted G2/M phase arrest and programmed cell death in human lung cancer cells [15]. Some study also found that overexpressin of IFN-γR in breast cancer cells repressed cell apoptosis through the MEK-Erk pathway [16]. However, no reports have described the apoptotic effects of IFN-γR expression in radiation-treated cells. In our next study, we may find out that overexpression of IFN-γR could induce apoptosis in CNE-1 cells after radiation.

To conclude, this study identified that overexpression of IFN-γR correlated with the poor prognosis of NPC patients receiving radiotherapy and elevated IFN-γR expression promoted the radiation resistance ability of NPC cells.

Conflict of interest: None declared.

Source of funding: None.

Consent: Written informed consent was obtained from the patients.

Acknowledgments: The Authors thank all the people involved in the project.

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DOI: 10.31038/CST.2017213

Abstract

Purpose: Several studies provided evidence for the efficacy of dose-escalation on biochemical control (BC) of prostate cancer and High-dose-rate brachytherapy (HDR) is one method for it.

Materials and Methods: Patients with histological diagnosis Gleason scored (GS), clinical stage T1 to T3a, no evidence of metastatic disease, prostate volume

Results: From 1997 to 2005 there were 273 patients treated with this treatment combination at AC Camargo Cancer Center, Sao Paulo, Brazil. The median age and FU time were 64.7 and 10.3 years, respectively. Two hundred thirteen (78.0%) patients had FU longer than 5 years. Actuarial 10-year overall survival (OS), Clinical Specific Survival (CSS) and BC were 89.8%, 63.6% and 71.8%, respectively. On univariate analysis GS<7, clinical stage<T2b, low risk group (LR), absence adjuvant androgen deprivation (ADT), age>65, PSAi<10, localized EBRT and 3D-HDR plan were associated with improved CSS and BC, excluding PSAi, age for the last one. Multivariate Cox regression analysis confirmed LR, GS<7, PSAi<10, absence of ADT, age

Conclusion: The present data represents a unique uni-institutional study at long FU for the given technique. A comparison with the current literature confirms the excellent results achieved with this treatment modality. HDR has also the advantage of treatment time reduction and increasing in the capability of work load of the linear accelerators, especially in developing countries, where waiting lists and lack of radiation oncology facilities are a reality.

Keywords

prostate cancer, radiotherapy, high-dose rate brachythrerapy, biochemical control, PSA

Abbreviations and Acronyms

ADT: Adjuvant Androgen Deprivation
AJCC: American Joint Committee on Cancer
BC: Biochemical Control
CSS: Clinical Specific Survival
EBRT: External Beam Radiotherapy
FU: Fallow Up
GS: Gleason Score
HDR: High-Dose-Rate Brachytherapy
LR: Low Risk Group
NAAD: Neoadjuvant Hormonal Therapy
OS: Overall Survival
PSAi: Initial Prostate-Specific Antigen
TRUS: Trans Rectal Ultrasound

Introduction

More than 62% of Prostate Cancers (PCa) are diagnosed in men over 65 years. It has become a public health and socioeconomic problem with increasing incidence, in special due to a rapidly aging population worldwide [1]. In Brazil it was expected the diagnosis of 61,200 new cases of PCa in 2016, and the crude mortality for 2013 was around 14,000 deaths [2].

Management options for localized and locally advanced PCa are controversial and include active surveillance, radical prostatectomy, external beam radiotherapy (EBRT) and brachytherapy with low or high dose rate sources. [3]

Several studies provided evidence for the efficacy of dose-escalation on biochemical control (BC) of PCa. Mature results from randomized trials show a direct relation between increasing the radiation dose given to the prostate and/or seminal vesicles and BC [4-7].

High-dose-rate after loading brachytherapy (HDR) is one method that can deliver a high localized radiation dose to the tumor with excellent BC when combined to EBRT [8]. One prospective randomized trial with up to 10 years follow up has proved that HDR plus EBRT is more efficient than EBRT alone in terms of BC with less acute rectal toxicity and improved quality of life [9].

The aim of this retrospective study is to evaluate the mature results of patients with local and locally advanced PCa treated with combination of HDR and EBRT.

Materials and Methods

Patients with confirmed histological diagnosis Gleason scored (GS) of PCa, AJCC clinical stage T1 to T3a, with no evidence of metastatic disease and initial PSA <60mg/ml, prostate volume

This single-centre institutional protocol of treatment was performed in compliance with the Declaration of Helsinki and approved by the local research Ethics Committee. Written informed consent was mandatory.

External Beam Radiotherapy

The EBRT target volume was defined using diagnostics CT images on conventional two dimensional or 3D planning. The targets were the prostate gland and the proximal seminal vesicles with a 1 to 1.5 cm margin except to the posterior region, which margins were reduced to 0.5 to 1.0cm. The EBRT dose ranged from 45 to 54 Gy prescribed to the intersection point. Further details of the radiotherapy schedules have been published previously [10].

High Dose Rate Brachytherapy

HDR was done under spinal anesthesia. Using TRUS with a perineal template affixed to perineum the exact needles positions were determined intraoperatively. In a first moment treatments were planned based on semi-orthogonal X-rays – two dimensional planning (2D) – and after that we moved to three dimensional (3D) planning, based on CT images. The prostate gland, the rectum, and the urethral trajectory and length were countered and identified in both situations. Implant dosimetry geometric optimization was initially utilized, followed later by use of inverse planning. Treatment parameters and dose constraints changed minimally throughout the years. The patients considered low risk had 16 Gy given in 4 fractions BID, one single implant. Intermediate and high risk patients had 20 Gy given in the same treatment schedule. The dose-volume histogram constraints were as follows: the TRUS or CT-based prostate´s volume receiving 100% of the dose (V100) should be >95%, the uniformity index should be more than 50%, and the V150 less than 30%. The urethra maximum punctual and the maximal dose to 1cc of anterior rectal wall should not exceed 135% and 75% of prescribed doses, respectively.

Definition of end points and statistical analysis

BC was measured using PSA tests and assessed according to the Phoenix definitions [11]. Clinical Specific Survival (CSS) was calculated from the start of treatment to the lost of BC, diagnose of metastatic disease or death from PCa. The BC was evaluated from the date of start the treatment until date of first biochemical failure. The follow up (FU) program also included clinical investigation, digital rectal examination and image studies.

The statistical program SPSS (statistical package for the social sciences) Inc., released 2008, Statistics for Windows, version 20.0 (SPSS Inc., Chicago, IL) was used for all statistical analysis. The analysis of OS, CSS and BC was made using the Kaplan–Meier method. The log-rank test was used to test the significance when comparing different subgroups. Univariate and multivariate Cox regression analysis were also performed. The alpha level considered for statistically significant differences was 0.05.

Results

Between March, 1997 and March, 2005 there were 305 patients treated with combination of HDR and EBRT at the Department of Radiation Oncology, AC Camargo Cancer Center, Sao Paulo, Brazil. Thirty two patients were lost of FU and the data of 273 patients was available for analysis. Sixty four (27.1%) patients had pelvic EBRT and the remaining 209 (76.5%) localized EBRT. Clinical and treatments characteristics are depicted in Tables 1 and 2.

Table 1. Patients Characteristics

		Median	Range	Variable	n	%
	Age (years)	64.7	42-82
	Prostate Vol (cc)	36.3	19-72	<35	121	44.3
				>35	152	55.7
	PSAi (ng/ml)	10.3	1-52	<10	173	16.8
				10-20	54	19.8
				>20	46	63.4
	Gleason Score			<7	190	69.6
				=7	58	21.2
				>7	25	21.2
				Yes	47	17.2
				No	226	82.8
	Clinical Stage			<T2b	192	70.3
				T2b-c	47	17.2
				>T2c	34	12.5
	Risk Group			Low	133	48.7
				Interm	76	27.8
				High	64	23.4
ADT	NAAD			Yes	93	34.1
				No	180	65.9
	ADJ				91	33.3
	Salvage				37	13.6
	WO				145	53.2
EBRT	Pelvic				64	23.4
	Localized				209	76.6
	Comorbidities			No	146	53.5
				SAH	42	15.4
				Diabetes	19	7.0
				Other	39	14.3
TOTAL					273	100.0

Legend: ADJ (adjuvant hormonal therapy), ADT (Androgen deprivation therapy), BF (Bichemical failure, EBRT (External beam radiotherapy), NAAD (neoadjuvant hormonal therapy), SAH (Systemic arterial hypertension), Salvage (Salvage hormonal therapy)

Table 2. Treatment  Characteristics

		Median	Range	Variable	n	%
Dose	EBRT	50	40-54	< 50	149	54.6
				>50	124	45.4
	HDR			16	133	48.7
		18.3	16-20	20	140	51.3
	HDR plan			2D	167	61.2
				3D	106	38.8
	Interval	18.5	9-61	<18	173	63.4
				>18	100	36.6

Legend: EBRT (External beam radiotherapy), HDR (High-dose-rate brachytherapy), HDR plan 2D/3D (two or three dimensional planning)

The median age and FU time were 64.7 (range, 42-82) and 10.3 (range, 1-15) years, respectively. Two hundred thirteen (78.0%) patients had FU longer than 5 years, and of these 153 (56.1%) longer than 10 years.

Androgen deprivation therapy

Androgen deprivation therapy (ADT) in a short course neo-adjuvant ADT, was prescribed for less than 6 months. Neoadjuvant hormonal therapy (NAAD) was administered to 34.1% of the patients. Adjuvant hormonal therapy (ADJ) was observed, mostly, for intermediate and high risk patients (33.4%), generally for no more than 6 months for intermediate risk and up to 3-years in high risk patients. Salvage ADT was observed in 37 (90.2%) of 41 patients dead due PCa. The profile of hormonal therapy is shown in Tables 3 and 4.

Table 3. Neoadjuvant Hormonal therapy according to Risk Group – Risk NAAD Cross tabulation

		NAAD
Risk Group		WO %		YES  %		Total %
	Low	74	27.1	8	2.9	82	30.0
	Interm	65	23.8	35	12.8	100	36.6
	High	41	15.0	50	18.3	91	33.3
Total		180	65.9	93	34.1	273	100

Legend: ADJ (adjuvant hormonal therapy), Interm (intermediate), NAAD (neoadjuvant hormonal therapy), WO (without hormonal therapy)

Table 4. Adjuvant and Salvage Hormonal therapy according to Risk Group

	WO   %		ADJ   %		Salv    %		Total    %
Low	72	26.4	7	2.6	3	1.1	82	30.0
Interm	57	20.9	34	12.5	9	3.3	100	36.6
High	16	5.9	50	18.3	25	9.2	91	33.3
Total	145	53.2	91	33.4	37	13.6	273	100

Legend: ADJ (adjuvant hormonal therapy), Interm (intermediate), NAAD (neoadjuvant hormonal therapy), Salv (Salvage hormonal therapy), WO (without hormonal therapy)

The crude 10-year overall survival (OS) rate at was 52.7%. Actuarial 5- and 10-year OS, CSS and BC were 80.1%, 89.8%, 83.7%, 63.6%, 85.5% and 71.8%, respectively. (Figures 1-3)

Univariate and multivariate analysis

On univariate analysis GS <7, clinical stage <T2b, low risk group, absence adjuvant ADT, older age (>65-years), PSAi Univariate analysis failed to identify neoadjuvant androgen deprivation therapy (NAAD) as a predictor for BC in all group risks (p=ns). When we pooled the intermediate and high risk group into a unique denominated unfavorable risk group, NAAD also failed to predict improved CSS and BC.

Multivariate Cox regression analysis confirmed low risk group (HR 0.03, 95% CI 0.006-0.116, p<0.001) and intermediate risk (HR 0.09, 95% CI 0.042-0.216, p<0.001) compared to high risk, presence of ADT (HR 0.39, 95% CI 0.179-0.868, p=0.021) as favorable predictors for CSS. GS >7 (HR 3.24, 95% CI 1.279-8.220, p<0.001), PSA >10 (HR 6.19, 95% CI 2.015-19.041, p=0.001), age >65 years (HR 2.87, 95% CI 1.264-6.504, p=0.012) and EBRT dose >50 Gy (HR 14.50, 95% CI 1.874-112.164, p=0.010) were confirmed as adverse predictors for CSS. Tables 6-8, Figures 4-9.

Low risk group compared to intermediate, (HR 0.70, 95% CI 0.023-0.213, p<0.001) and high risk (HR 0.99, 95% CI 0.052-0.190, p<0.001) groups, was a favorable predictive factor for BC. GS >7 (HR 3.09, 95% CI 1.473-6.473, p=0.003) and PSAi >10 (HR 6.18, 95% CI 2.331-16.393, p<0.001) were negative predictive factor for BC.

Low risk group was confirmed as the only predictive factor for OS when compared to intermediate (HR 0.28, 95% CI 0.133-0.608, p=0.001) and high (HR 0.38, 95% CI 0.223-0.665, p=0.001) risk groups.

Table 6. Cox regression for CSS

	B	SE	Wald	df	Sig.	Exp(B)	95.0% CI for Exp(B)
							Lower	Upper
LR			44.294	2	.000
IR	-3.641	.761	22.910	1	.000	.026	.006	.116
HR	-2.351	.418	31.579	1	.000	.095	.042	.216
EBRT Pelvic x Local	-.110	.312	.124	1	.724	.896	.486	1.651
2D x 3D	-1.861	1.134	2.693	1	.101	.156	.017	1.436
ADT	-.931	.403	5.336	1	.021	.394	.179	.868
PSAi <10			10.858	2	.004
PSAi (>10<20)	1.925	.661	8.481	1	.004	6.856	1.877	25.048
PSAi (>20)	1.824	.573	10.133	1	.001	6.195	2.015	19.041
GS <7			7.328	2	.026
GS = 7	.335	.709	.223	1	.636	1.398	.349	5.608
GS >7	1.176	.475	6.145	1	.013	3.243	1.279	8.220
CS <T2b			3.350	2	.187
CS T2b/T2c	-.662	.551	1.443	1	.230	.516	.175	1.519
CS >T2c	.464	.818	.321	1	.571	1.590	.320	7.895
Age <65y	1.053	.418	6.352	1	.012	2.867	1.264	6.504
EBRT <50Gy	2.674	1.044	6.562	1	.010	14.498	1.874	112.164
HDR dose <20Gy	1.763	1.023	2.973	1	.085	5.830	.786	43.261
Interval EBRT to HDR	-.187	.303	.381	1	.537	.829	.458	1.502

Legend: 2D (two dimensional plan), 3D (tridimensional plan), ADT (Androgen deprivation therapy), BC (biochemical control), Comorb (Comorbidites), CS (Clinical stage), CSS (Clinical Specific Survival), EBRT (External beam radiotherapy), GS (Gleason score), IR (Intermediate risk group), HDR (High-dose-rate brachytherapy), HR (High risk group), LR (Low risk group), NAAD (neoadjuvant hormonal therapy), OS (overall survival), P vol. (Prostate volume cc)

Table 7. Cox regression for BC

	B	SE	Wald	df	Sig.	Exp(B)	95.0% CI for Exp(B)
							Lower	Upper
LR			62.053	2	.000
IR	-2.660	.568	21.948	1	.000	.070	.023	.213
HR	-2.309	.330	48.920	1	.000	.099	.052	.190
2D x 3D	-.789	.656	1.448	1	.229	.454	.126	1.642
ADT	-.455	.307	2.192	1	.139	.634	.347	1.159
PSAi <10			13.634	2	.001
PSAi (>10<20)	1.822	.498	13.403	1	.000	6.182	2.331	16.393
PSAi >20	1.346	.462	8.494	1	.004	3.843	1.554	9.502
GS <7			8.944	2	.011
GS =7	.957	.465	4.240	1	.039	2.603	1.047	6.469
GS >7	1.127	.378	8.915	1	.003	3.088	1.473	6.473
CS <T2b			2.825	2	.244
CS T2b/T2c	-.647	.410	2.493	1	.114	.523	.234	1.169
CS >T2c	-.306	.580	.278	1	.598	.737	.236	2.294
EBRT 50Gy	1.411	.550	6.590	1	.010	4.101	1.396	12.044
HDR dose <20Gy	-.470	.680	.477	1	.490	.625	.165	2.370
Interval EBRT to HDR	-.064	.230	.078	1	.780	.938	.597	1.473

Legend: 2D (two dimensional plan), 3D (tridimensional plan), ADT (Androgen deprivation therapy), BC (biochemical control, Comorb (Comorbidites), CS (Clinical stage), CSS (Clinical Specific Survival), EBRT (External beam radiotherapy), GS (Gleason score), IR (Intermediate risk group), HDR (High-dose-rate brachytherapy), HR (High risk group), LR (Low risk group), NAAD (neoadjuvant hormonal therapy), OS (overall survival), P vol. (Prostate volume)

Table 8. Cox regression for OS

	B	SE	Wald	df	Sig.	Exp(B)	95.0% CI for Exp(B)
							Lower	Upper
ADJ	.149	0.3	0.3	1	.580	1.161	.684	1.970
Age <65y	.223	0.2	1.0	1	.322	1.250	.804	1.942
Comorbidity	-.310	0.2	2.1	1	.149	.734	.482	1.117
EBRT Pelvic x Local	-.089	0.2	0.1	1	.722	.915	.560	1.494
LR			14.3	2	.001
IR	-1.259	0.4	10.5	1	.001	.284	.133	.608
HR	-.954	0.3	11.7	1	.001	.385	.223	.665

Legend: ADJ (adjuvant hormonal therapy, EBRT (External beam radiotherapy), IR (Intermediate risk group), HDR, HR (High risk group), LR (Low risk group), OS (overall survival)

Discussion

Conventional EBRT to treat PCa is securely limited to doses of 64–70 Gy in 1.8–2.0 Gy fractions. These levels of doses are determined by the risk of long-term toxic effects to the bladder and rectum. The clinical and biochemical relapse rates associated with these dose levels are around 33% within 5 years. Peeters et al 12 published the results of a randomized trial comparing total doses of 68 Gy and 78 Gy using EBRT alone. The 5-year OS in the higher dose arm of that study was 83% using ASTRO definition and the BC was significantly better in the 78-Gy arm compared to the 68-Gy arm, with an adjusted hazard ratio of 0.74 (p=0.02). Other published reports using dose-escalated photon beam EBRT alone also point in the same direction with respect to their long-term BC results [13-14].

HDR can escalate the dose given to the prostate by the combination with EBRT, and further more, in locally advanced disease has also the possibility of including the seminal vesicles when they needed to be encompassed. HDR has also a potential biological advantage through the delivery of high doses per fraction [10]. It is important to note that the comparisons between series published are difficult due differences in the techniques and planning for both, EBRT and HDR.

The combination of values of PSAi, GS and CS to identify a more or less aggressive disease has being extensively discussed in the literature, and was confirmed by this study. The most frequent challenge is to identify, for example the indication of prostate versus pelvic EBRT, varying risk categories, absence or use of NAAD, ADT and their length. Despite this, the combination of HDR and EBRT provides an optimal modulation of dose delivery. Results of this combination, in terms of BC and with more than 5 years of FU, range from 57% to 100% according to the risk group for biochemical failure (Table 9).

Table 9. Results of biochemical control by risk groups in series of HDR plus EBRT with more than 5-year follow up

Reference	n	Median FU  (months)	Biochemical control by risk groups (%) Low       Intermediate       High			Dose in Gy (HDR(n.fx)/EBRT)
13	344	61		84	74	19.5(3) / 46
14	121	63		91		10(1)/50
15	313	68	100	88	79	23(2)/46
16	229	61	95	90	57	21(3)/50.4
17	64	105		84	80	18(3)/45
18	90	95			80	16.5(3)/45
19	264	75	97			18(3)/45
20	100	62		84	82	10(1)/60
21	64	61		100	91	21(3)/50
22	196	66		86		18(3)/46
23	131	63		87	71	30(4)/45

The search for factors predicting BC and CSS is important on defining what patients should be treated more aggressively. We, as other authors [26-28] observed that PSAi <10 ng/ml was confirmed as a favorable predictive factor related to BC and CSS.

Age<65 years was found to be an adverse prognostic factor in our analysis for CSS, with a marginally statically significance impact on OS, not confirmed on multivariate analysis. Smolska-Ciszewska et al, conversely to our results, noted that younger age at time of treatment impacted only on OS, not explaining if there was any association between treatment and side effects or worsening of associated comorbidities. As in our analysis, they found that low risk group and association of HDR to EBRT correlated with improved BC [29].

As in our results, Kamrava et al found that T stage, GS, and use of ADT were significantly associated with CSS on univariate analysis, but on multivariate analysis only GS and use of ADT were significantly associated CSS [30]

It is expected that the grouping of the patients in risk groups for biochemical failure based on PSAi, GS and CS, to aggregate patients with adverse features in the intermediate and high risk, leading to worse CSS and BC for the two last one, what was confirmed in our analysis. This was also observed by Morris et al [31].

In our series, as in others, patients who received adjuvant ADT had significantly higher risk features suggesting patient selection bias for CSS in this group of patients, instead of a negative interaction between HDR and EBRT [31-32].

Mature data in the literature evaluated the 10-year outcomes of intermediate- and high-risk patients noting a clear dose response by increasing the dose escalation through HDR doses [27]. More recently the use of Intensity modulated radiation therapy combined to HDR has being investigated. Chen et al. published the results of 148 patients treated with HDR – 22 Gy in 4 fractions followed IMRT up to 50.4Gy. All patients with Gleason score of 8 or higher had ADT for 1 year. They noted a 4-year actuarial CSS of 96.8% and of 100%, 100% and 94% for low, intermediate and high risk, respectively [32].

The results of the first randomized prospective trial, which has addressed dose escalation using an HDR and EBRT is a trial with a relatively slow accrual rate. There are some critics that must be addressed as the changes in EBRT technique during the time of the study, and, by current standards, the control arm is a relatively low-dose treatment. Despite that, the study reported the results of 218 patients treated between 1997 and 2005. There were 108 patients assigned to EBRT alone and 110 patients treated by EBRT followed by HDR. They noted that CSS was significantly higher in patients treated with combined modality (p = 0.04). In multivariate analysis the category treatment modality and ADT were significant covariates for BC, but with no differences in OS, as observed in our study. After a median follow-up time of 10.5 year follow-up, an 18% increase in CSS was obtained relative to EBRT alone, reflecting a 31% reduction in the risk of recurrence (p = 0.01) and no evidence of an increase in long-term severe morbidity [9].

Surgically induced gland deformation is inevitable during brachytherapy procedures. We observed that the use more of advanced methods of images (3D plan based on CT, MR or TRUS) images to identify the target, organs at risk and needles is important and have already been reported to impact on BC and CSS [33]. In our analysis Multivariate Cox regression analysis confirmed EBRT dose >50 Gy as predictor for CSS and BC, but with no impact on OS. This may be explained by the relative low dose per fraction schedule used in both groups. Of importance is to note that the dose given by HDR was relative constant thorough the risk groups, leading to higher biological effective dose to intermediate and high risk patients, and even though, these patients had a worse outcome, showing that there is space for further studies of dose escalation or treatment combination. After 2005 with the introduction of real time TRUS image acquisition and planning we have changed our protocol and moved forward for a more intense dose escalation, increasing dose and reducing the number of fractions.

Other information of this study is that presence of NAAD had no impact on DSS, BC or OS in any risk group, showing that this treatment strategy should be reserved for downsizing the prostate prior to treatment, in special for low risk patients.

In conclusion, this report demonstrates that HDR combined with EBRT is an important and effective method in achieving dose escalation in the radical radiotherapy of PCa. This combination has also the advantage of treatment time reduction and in increasing in the capability of work load of the linear accelerators, especially in developing countries, where waiting lists and lack of radiation oncology facilities are a reality. The present data represents a unique uni-institutional study at long FU for the given technique, and a comparison with the current literature confirms the excellent results achieved with this treatment modality. The satisfactory BC, CSS and OS are probably result of improved LC achieved with dose escalation, showing that HDR is an optimal alternative method of local dose escalation when combined to EBRT.

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