Abstract
Prostate cancer is the second most diagnosed cancer worldwide and accounts for 15% of all cancer in men. Localised prostate cancer, contained within the capsule of the prostate gland, is effectively treated with surgery, radiotherapy and hormone therapy, however, the cancer can advance to a much more aggressive state, known as castrate-resistant prostate cancer (CRPC). At this stage, the prostate cancer has developed mechanisms to thrive in a low androgen environment therefore resisting a common therapeutic strategy. Often these cancers have metastasised beyond the prostate gland and can result in mortality within 12 months of diagnosis, as therapeutic options to combat this cancer stage are extremely limited. Previous literature has indicated both curcumin analogues and selective oestrogen receptors (SERMs) play a role in reducing prostate cancer in vitro. This study aimed to determine therapeutic use of the SERM, raloxifene, and curcumin analogue, RL91, for a combinatorial approach against CRPC. These compounds have previously shown superior cytotoxic efficacy in combination in vitro however their effect in a murine model of CRPC is not known.
The mechanism of cell death was determined in vitro using a human prostate cancer cell line, PC3, which represented CRPC. Cells were exposed to RL91 and raloxifene for up to 72 h, at concentrations similar to previously obtained IC50 values, before flow cytometry was completed to determine drug effect on cell cycle distribution and apoptosis. RL91 (4 µM) elicited a G2/M phase arrest responsible for significant induction of apoptosis, with 9% of total cells apoptotic. In contrast, raloxifene (10 μM) elicited a G1 phase arrest that was not sufficient to induce apoptosis. The combination of the two compounds elicited a greater G2/M phase arrest and apoptosis, with 28% of total cells apoptotic.
The efficacy of RL91 and raloxifene was then determined in a murine model of CRPC. PC3 cells (2x105) were inoculated directly to the ventral prostate lobe of SCID mice to create an orthotopic tumour. Animals were orally dosed with raloxifene (8.5 mg/kg), RL91 (8.5 mg/kg) daily or the combination of the two at their equivalent doses, for six weeks. At necropsy prostates and surrounding lymph nodes were collected to examine drug effect on primary tumour growth and metastasis. Lungs were also collected. Prostate size was measured using digital calipers and lymph nodes were stained for human mitochondria to detect the presence of PC3 cells. RL91 elicited a 61% reduction in primary tumour size but had no effect on metastasis. In contrast, raloxifene elicited a 79% reduction in primary tumour size as well as significantly reduced the area of lymph nodes and the extent of PC3 infiltration to this tissue. Surprisingly, the combination of RL91 and raloxifene behaved poorly in vivo and combination-treated animals had primary tumours and metastasis similar to those of vehicle treated animals. No lung metastases were identified in any animals.
These results indicate that raloxifene, as a single agent, is effective at reducing prostate tumour growth, likely due to an increase in apoptotic cells and reduction in cellular proliferation caused by cell cycle arrest. Overall, this study highlights the potential use of raloxifene for the treatment of CRPC, however not in combination with the curcumin analogue, RL91.