38. The use of 1,25[α] dihydroxyvitamin D3 as a cryosensitizing agent in a murine prostate cancer model

Vitamin D3 has been identified as a possible adjunctive treatment for cancer therapies due to its antiproliferative and antitumorigeneic properties. It has also been implicated in the inflammation process, which is critical to the initiation and progression of prostate cancer (CaP). Cryotherapy, the use of low temperatures to ablate tumorigenic tissues, has recently emerged as a primary treatment option for CaP. For these reasons we hypothesized that combining calcitriol with the sub lethal temperatures experienced in the periphery of the cryogenic lesion would result in an increase in apoptotic cell death as compared to either cryotherapy or calcitriol alone. This study aimed to identify the pathways activated when the biologically active metabolite of vitamin D3 (calcitriol) is combined with cryotherapy in a murine model of prostate cancer. The RM-9 cell line was treated with 1,25 α dihydroxyvitamin D3 [25–150 nM] for 24 h prior to exposure to low temperature in a temperature controlled bath. Ice nucleation was initiated by liquid nitrogen vapor and cell viability was assessed using alamarBlue for 3 days post-freeze. Data indicated that pre-treatment with calcitriol sensitizes RM-9 cells to enhanced cellular injury at low temperature. Treatment with calcitriol alone [25–400 nM] shows no effect on cell viability. Similarly, a single freeze between −5 °C and −15 °C alone had little effect on survival. However, when cells are pre-treated with calcitriol and then exposed to low temperature, there is a synergistic effect of increased efficacy of cell death. At −15 °C coupled with calcitriol [50–75 nM] cell death increased to 55% of day 1 controls following a single freeze exposure, compared to negligible cell death with freeze alone. Repeat exposure resulted in 90% cell death compared to 65% in dual freeze alone. Western blotting of signaling proteins during the calcitriol and cryotherapy sensitization regimes confirmed the activation of apoptosis. Specifically, pro-apoptotic Bid and pro-caspase-3 was found to decrease at 1 h following combination treatment, indicative of cleavage to the active form. The survival protein Akt was found to be upregulated at 4 h post-cryo at higher doses of calcitriol (150 nM), which could be indicative of pro-survival signaling through the PI3-K pathway. Additionally molecular analysis of in vivo tissue tumor samples treated with calcitriol and cryotherapy also indicated the involvement of apoptosis through caspase-3 and caspase-9 cleavage. Previous studies have elucidated the in vitro benefits of cryosensitization strategies including combining drugs such as 5-fluorouracil and taxotere with cryotherapy; however, in vivo chemotherapeutic drugs present with toxic side effects as well as the likelihood of developing resistance. The need for the development of novel, non-toxic treatments for prostate cancer is essential, as many current strategies have negative side effects. The development of an adjunctive therapy combining calcitriol and cryotherapy represents a potentially highly effective, less toxic minimally invasive alternative treatment option.