PCa is the most common solid tumor in US males and the second leading cause of cancer related deaths . A common treatment option for localized PCa is RT, which may possess similar survival rates at 5, 10, and 15-years to radical prostatectomy. Though radiation dose increase has associated with higher cancer control rates, high dose radiation can cause important side effects, such as urinary dysfunction, impotence, and rectal symptoms. Several reports have demonstrated that the addition of androgen deprivation to RT may improved the results of patients with intermediate- and high-risk PCa. However in high-risk patients treated with RT, 5-year recurrence free survival is approximately 50%.
Thus, new molecular targets for the enhancement of RT and regression of RT adverse event are needed for some patients with localized prostate cancer.
NSAIDs have been demonstrated to potentiate radiosensitivity of cancer cells, and recent studies reported the radiosensitizing effect of selective COX-2 inhibitors on tumor cells [23–25, 33].
The cells overexpressing COX-2 tend to be resistant to apoptosis, and COX-2 inhibitors have been shown to induce apoptosis in these types of cells. Therefore, we hypothesized that the COX inhibitor, diclofenac could enhance the effect of radiation on prostate cancer cells that constitutively express COX-2.
Although many of recent studies reported about the relation between COX inhibitor and radiosensitizing effect, conducted chiefly in vitro experiments study. We further attempted to show the potentiation of RT in vivo experiments.
This study is the first evidence that treatment with diclofenac increased the radiosensitivity of prostate cancer cells by suppression of COX-2 up-regulation and induction of TRAIL in vitro, and that topical treatment with diclofenac gel enhanced antitumor potential of RT in vivo.
The underlying mechanism responsible for the antitumor effect of COX inhibitor has not been clearly elucidated, although several possibilities have been proposed, i.e. modulation of angiogenesis, regulation of cell cycle, and reverse of PGs-induced immunosuppression . PGs, which were produced by COX-2, have been proposed to promote the proliferation and metastasis of cancer cells and secondarily encourage the growth of cancer cells by immunosuppression. Furthermore, PGE1 and PGE2 are known to induce angiogenesis [9, 18]. Milas and his colleagues demonstrated that the NSAID indomethacin prolongs tumor growth delay and increases the tumor cure rate after radiotherapy [22, 24]. It has been proposed that NSAIDs increased tumor radiation response by lowering the level of PGs in the tumor . Based on recent findings that PGs and COX-2 are radioprotective factors, it is reasonable to assume that diclofenac inhibited tumor cell proliferation and increased tumor radiosensitivity by reducing tumor-producing PGs [34, 35]. Recent experiments using selective or not selective COX-2 inhibitor, including celecoxib and NS-398, demonstrated that COX-2 overexpression might be responsible for radioresistance [6, 25], and that suppression of COX-2 overexpression render cells susceptible to RT.
NSAIDs have been demonstrated to potentiate the effect of RT on cancer cells in vitro and in vivo, however, no study has reported on the efficacy of topical diclofenac gel.
In this study, we investigated the antitumor potential and the radiosensitizing effect of diclofenac on prostate cancer cells. In LNCaP-COX-2 cells, diclofenac significantly reduced the cell viability and sensitized to RT. COX-2 overexpression is responsible for radioresistance in clonogenic assay. LNCaP-COX-2 cells were more susceptible to diclofenac than LNCaP-neo cells. These findings suggested that LNCaP-COX-2 might be addictive to COX-2 than LNCaP-Neo cells, and that COX-2 might be one of survival factors for LNCaP-COX-2 cells. We first showed that the combination of RT and topical treatment with diclofenac gel significantly induced the tumor growth delay than either treatment alone in prostate cancer tumor in vivo. The radiosensitizing effect of diclofenac was not observed in LNCaP-Neo cells that lacked COX-2 expression.
In real-time PCR analysis, RT induced TRAIL and combination with diclofenac enhanced the RT-induced TRAIL.
We first suggested that additional aspects of the COX inhibitor, diclofenac-induced potentiation of tumor radioresponse were caused by the induction of TRAIL.
TRAIL is an identified member of the TNF ligand family that can induce a rapid caspase-dependent apoptosis with high specificity for malignant cells.
Recent study has been shown that significant release of TNF-related apoptosis inducing ligand was observed in response to ionizing radiation(IR) in lung cancer cells , and that IR up-regulates TRAIL-Receptor surface expression . TNF-α and TRAIL are directly involved in apoptosis and are induced by IR . Irradiation induced release of tumor necrosis factor-α, or TRAIL .
Our findings that RT significantly induced TRAIL in real-time PCR analysis are consistent with these results. In addition, combination of RT and diclofenac significantly increased the induction of TRAIL compared with either alone. The enhancement of TRAIL can explained the observed increase of apoptosis.
Apoptosis is controlled via two major pathways, including one that originates at the cell membrane and another that involves the mitochondria. The membrane death receptor (DR) pathway involves DRs such as Fas, TNF-R1, DR-3, DR-4, and DR-5, that are activates by their respective ligands and engages the intracellular apoptotic machinery. In addition, caspase pathway is involved in TRAIL-induced cell death, and IR-induced TRAIL contributed to cell death via increase of caspase-8, caspase-9, and caspase-3 activation. COX-2 expression has shown to significantly attenuate TRAIL-induced caspase-8, caspase-9, and caspase-3 . NSAIDs, sulindac sulfide, increased both DR-4 and DR-5 mRNA levels. Modulation of the level of DR-5 regulated the apoptotic response to TRAIL . RT induced TRAIL in both LNCaP-COX-2 and LNCaP-Neo cells (data not shown) in real time PCR analysis. COX-2 overexpression was shown to attenuate the effect of TRAIL-induced apoptosis and reduce Fas-mediated apoptosis [31, 39].