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Radiation therapy (XRT) for treatment of localized prostate cancer (PCA) has outcomes similar to surgery and medical therapy. Toxicities of XRT and the relative radioresistance of PCA limit the effectiveness of this treatment method. Safe and effective radiosensitizing agents are lacking to enhance the effectiveness for XRT for PCA. In this study, the effect of XRT in combination with the radiosensitizing agent resveratrol (RSV) was investigated in a radioresistant PCA cell line, PC-3. Our results show the addition of RSV to XRT (XRT/RSV) synergistically enhanced XRT-induced apoptosis and inhibition of PC-3 proliferation. The antiproliferative effect of XRT/RSV treatment correlated with increased expression of p15, p21, and mutant p53 and decreased expression of cyclin B, cyclin D, and cdk2. Increased apoptosis correlated with increased expression of Fas and TRAILR1. Furthermore, XRT/RSV had little effect on the expression of p-AKT, whereas it increased the expression level of p-H2A.X, a marker for senescence. These data highlight the potential of RSV as a radiation sensitizer for PCA treatment and warrant further investigation. (Cancer Sci 2012; 103: 1090–1098)
Prostate cancer (PCA) is the most common non-cutaneous malignancy and the second leading cause of cancer mortality in elderly men in the USA. Approximately 240 890 new cases of PCA and 33 720 deaths were projected to occur in the USA in 2011. In addition to surgery, chemotherapy, and hormonal therapy, radiation therapy (XRT) is an established therapeutic method for PCA treatment. Radiation therapy is used to treat localized PCA to decrease tumor burden and ameliorate tumor-related symptoms. The efficacy of XRT largely depends on the radiosensitivity of the tumor. Unfortunately, PCA is among the more radioresistant malignant tumors. The high radiation dose associated with XRT for PCA may have severe side-effects, such as impotence, urinary dysfunction, and rectal symptoms; low dose XRT has little effect on PCA. A safe and effective radiosensitising agent is needed to allow a decrease in the radiation dose and side-effects associated with XRT for PCA.
Resveratrol (trans-3,4′,5-trihydroxystilbene, RSV) is a polyphenolic compound that occurs naturally in grapes (such as in red wine) and peanuts, as well as in other plant species such as Polygonum cuspidatum and Yucca schidigera.[3-9] The biological function of RSV is very complex. Multiple studies have shown neuroprotective, immunomodulatory, anti-inflammatory, antioxidant, and antitumor functions. In recent years, RSV has been recognized as a promising anticancer agent. Its antitumor functions have been investigated in breast cancer, thyroid cancer, squamous cell carcinoma, HL-60 leukemia, colon cancer, ovarian carcinoma, and PCA cell lines.[10, 11] Incubation of the PCA cell line, DU145, with RSV resulted in decreased growth and increased apoptosis of cancer cells.
Several studies implicate RSV as a chemotherapy sensitizer, thus, it is reasonable to hypothesize that the combination of RSV with radiation might potentiate the destruction of cancer cells.[13-16] Until now, there was only one study suggesting that RSV can sensitize the DU145 PCA cell line to radiation. Confirmation of the radiosensitizing effects of RSV in other PCA cell lines and the detailed molecular mechanisms of this phenomenon have not been investigated. The current study was designed to test the hypothesis that RSV enhances radiation sensitivity in the PC-3 PCA cell line by altering cell proliferation and apoptosis. Additionally, we report the mechanisms underlying the cellular changes observed when RSV is used in combination with XRT.
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In this study, we show that RSV synergizes with XRT to inhibit the proliferation of a PCA cell line by promoting apoptosis and senescence. The antiproliferative effect of XRT/RSV correlated with increased expression of antiproliferative molecules p15, p21, and mutant p53 (mp53) and decreased expression of pro-proliferative molecules cyclin B, cyclin D, and cdk2. Increases in apoptosis correlated with increased expression of pro-apoptotic molecules Fas and TRAILR1. Furthermore, we showed that XRT/RSV promoted senescence as evidenced by increased expression of p-H2A.X, but had little effect on the expression level of p-AKT. To our knowledge, this is the first study to indicate the synergistic effect of XRT and RSV on proliferation and survival of PC-3 PCA cells. This is also the first study to investigate the detailed molecular mechanisms by which XRT/RSV inhibits the survival of PCA cells.
Consistent with previous studies, our study further confirmed that PC-3 cells are relatively radioresistant.[2, 12, 18, 38, 39] The percentage of surviving PC-3 colonies after radiation at 2 Gy was comparable to that of non-radiated controls. Even at 8 Gy, proliferation was reduced only by half, revealing the resistance of PC-3 cells to radiation. Interestingly, the percentage of PC-3 colonies after XRT/RSV decreased to 5 ± 2% at the dose of 8 Gy. At the dose of 2 Gy, the percentage of colonies of PC-3 cells decreased from 91 ± 4% (without RSV) to 15 ± 4% (with RSV). These results strongly suggest a radiosensitizing role for RSV. Radiosensitization of PCA may allow for reduction of effective radiation dose and side-effects associated with XRT. Whether RSV functions in the same capacity in the in vivo setting awaits the results of further animal studies and clinical trials.
The eukaryotic cell cycle is tightly regulated.[23-27] The balance between pro- and antiproliferative molecules plays an important role in cell proliferation. Cyclin B, D, and cyclin E, as well as cdk2, play major roles in proliferation. Downregulation of cyclin D delays or inhibits entry to the S phase and overexpression of cyclin D shortens the G1 phase.[23-27] Cyclin E is active in late G1 phase and is maximal at the G1–S transition. The important antiproliferative molecules p15, p18, p21, p27, and p53 exert their effect through inhibition of cyclin-dependent kinases.[23-27] In this study, we found that the antiproliferative effect of XRT/RSV correlated with increased expression of p15, p21, and p53 and decreased expression of cyclin B, cyclin D, and cdk2. Decreased expression of cyclin D and cdk2 is consistent with studies using DU145 PCA cells treated with RSV. Our data indicated that XRT/RSV disrupted the pro-proliferative mechanism and induced antiproliferative regulatory molecules in PC-3 to inhibit cell proliferation in treated cells. Previous reports showed variable expression of p53 and/or mp53 in PC-3 cells[40-44] and mp53 upregulated 15-lipoxygenase-1 in murine and human PCA.[43, 45] In our study, we detected mp53 mRNA and protein expression in PC-3 cells.[40, 43] Although the detailed functionality of mp53 protein in PC-3 is unknown, the upregulation of mp53 by the XRT/RSV correlated with an antiproliferative effect on PC-3 cells.
Apoptosis is mediated through the sequential activation of a series of caspases induced either through a receptor-mediated or mitochondrial-mediated pathway.[28, 29] Fas, FasL, TRAILR1, and TRAIL belong to tumor necrosis factor receptor family and have a pro-apoptotic function in the receptor-mediated pathway of apoptosis. FLIP inhibits death receptor-mediated apoptosis by blocking activation of caspase-8. Bcl-2 inhibits apoptosis, and Bcl-2 family protein Bax promotes apoptosis through regulation of mitochondrial voltage-dependent anion channels.[29, 31] As early as 1 day after XRT, mRNA expression of Fas and TRAILR1 was increased in PC-3 cells treated with XRT/RSV as compared to cells treated with XRT alone. In this same time period, few TUNEL+ apoptotic cells were detected in any treatment group, suggesting that XRT/RSV induction of apoptosis occurs more slowly than changes in the mechanisms governing inhibition of proliferation. When apoptosis was evaluated by TUNEL staining 3 days after XRT, there were still few TUNEL+ cells in the XRT group, whereas more than two-thirds of the XRT/RSV treated PC-3 cells were TUNEL+. The difference in apoptosis between treatment groups was confirmed by measuring caspase-3 activity. Thus, XRT/RSV increases apoptotic cell death in PC-3 cells through the upregulation of Fas and TRAILR1.
In the analysis of cell cycle molecules, we unexpectedly found the addition of RSV to PC-3 cells without XRT increased the expression of cyclin D. We believe the increase in cyclin D in this scenario is part of an adaptative response to cell injury to prevent cells from further damage. As well, the mRNA expression of p18 was increased in cells treated with XRT compared to that in controls, but its expression was decreased in cells treated with XRT/RSV compared to that of cells treated with XRT alone, although its expression was still higher than in control cells. When analyzing the mRNA expression of other pro-and antiproliferative molecules, consistent with studies by others,[17, 46-48] we also found that RSV alone increased the expression of pro-apoptotic molecule FasL, TRAIL, and Bax, as well as anti-apoptotic molecule Bcl-2, but the addition of XRT partially or fully abolished this effect. Furthermore, FLIP contributed little to the synergistic effect of RSV with radiation. It is possible that there might be some other unexamined and/or unidentified pro- and antiproliferative and/or pro- and anti-apoptotic molecules that also play roles in PC-3 cell proliferation and/or apoptosis. Thus, it is reasonable to argue that it is not a specific pro- or antiproliferative and/or pro- and anti-apoptotic molecule, but it is the balance between pro- and antiproliferative molecules and the balance between pro- and anti-apoptotic molecules that dictates the fate of PC-3 cells for their proliferation, quiescence, or apoptosis.
Cellular senescence occurs by irreversible growth arrest. Alongside apoptosis, senescence is a critical anticancer mechanism. We have established a role for senescence in the XRT/RSV inhibition of PC-3 PCA cells as evidenced by increased expression of p-H2A.X in cells treated with XRT/RSV compared to cells treated with XRT alone. p21, p27, and p53 are involved in the process of senescence.[49, 50] We have shown increased expression of p21 in XRT/RSV treated cells (Figs 2, 3) and this also might contribute to senescence. Further studies are needed to examine the role of mp53 in this process.
Although RSV is thought to be safe for humans, optimal dosing has yet to be established and the side-effects at therapeutic levels are unknown. An oral dosage above 2.5 g per day may be associated with gastrointestinal discomfort or diarrhea, whereas side-effects are much less common at lower doses. Studies are still ongoing for the evaluation of RSV dosing, side-effects, and therapeutic benefits in humans.
In summary, RSV enhances radiation sensitivity in PCA by inhibiting cell proliferation and promoting cell senescence and apoptosis in vitro. Our data highlight the potential of RSV as a radiation sensitizer for PCA. Further in vivo studies and potentially clinical trials using XRT/RSV in PCA treatment are warranted to address the true therapeutic potential of this combination.