Abstract A075: Bcl-2 family proteins as drug targets; Can prostate cancer be primed for apoptosis?

Abstract

Abstract Cancer is a disease of accumulated genetic mutations that alter a cell’s function. One of the common cellular functions affected in cancer cells is the apoptotic pathway. Apoptosis, also known as programmed cell death, is a regulatory biologic function controlled by protein interactions within the BCL-2 protein family. Normally, cells use this pathway to induce cell death when they detect an error in the cell’s functioning that cannot be fixed. In multiple types of cancers, including prostate cancer, there is often an excess of pro-survival BCL-2 proteins. Consequently, these cells have an inability to induce apoptosis, leading to an increase in cancer cell survival, sometimes even in the presence of chemotherapeutic chemicals. This research investigates the potential difference in BCL-2 proteins between metastatic prostate cancer cells and premetastatic prostate cancer cells. Specifically, changes in cancer cell response to chemotherapy after inhibiting antiapoptotic BCL-2 proteins via small-molecule inhibitors are being evaluated. We hypothesize that chemoresistant cancer cells are sensitized to BCL-2 inhibitors by first priming their mitochondria with a traditional chemotherapeutic agent. “Mitochondrial priming” is bringing the antiapoptotic BCL-2 proteins to their apoptotic threshold (1). To accomplish these aims, metastatic prostate cancer cell lines PC3, LnCap C4/2, and DU145 as well as a normal prostate cancer cell line, PNT2, were used. We first sought to compare the level of BCL-2 family proteins among the different cell types. PC3 cells have significantly higher levels of BCL-2, BCL-xL, and BAD as detected by Western blot and quantified by densitometry, corroborating findings from other labs. Next, cells were treated with 5uM etoposide or 100nM docetaxel for 24 hours or 48 hours, and viability was assessed by MTT and apoptosis via Annexin V-FITC. We found that cell viability was significantly decreased in LnCap and DU145 cells at 24 hours, which can be attributed specifically to apoptosis. Moreover, increasing amounts of apoptosis occurred after 48 hours of treatment. Comparatively, PNT2 and PC3 cells have significantly less apoptosis induced by either etoposide or docetaxel treatment. Apoptosis was further confirmed by caspase-3 activation as well as the ability to block apoptosis by pretreatment with the caspase inhibitor Boc-D-fmk in LnCap cells. Additionally, overexpression of BCL-xL in LnCap cells abrogated etoposide-induced apoptosis. Therefore BCL-xL is a potential target for priming. Next, we focused on enhancing the apoptotic effects of etoposide and docetaxel by combing their use with ABT-737, which is a pan BCL-2 antiapoptotic inhibitor. Evaluating the effects of these drugs on various prostate cancerous cell lines in comparison to the effects on a normal prostate cell line may provide insight into their clinical usefulness and relevance in combination treatments. Cells were treated with 0, 100nM, 500nM, 1uM, 2.5uM, or 5uM ABT-737 for 24 hours and apoptosis was assessed via Annexin V-FITC. Preliminary results suggest that on its own, ABT-737 has little effect on LnCap cells (~10%) and no effect on PC3, DU-145, or PNT2 cells. However, pretreating LnCap cells for 24 hours with etoposide as low as 100nM followed by 5 hours exposure to 500nM ABT-737 can induce twice as much apoptosis. These results suggest that etoposide “primes” cells and then the BCL-2 inhibitor pushes them over the edge towards apoptosis. Since less etoposide (100nM) in the combined treatment was effective, as opposed to 500nM when given alone, it may be possible to administer lower amounts of clinically established chemotherapeutics. However, preliminary results also show a ~30% increase in apoptosis in DU145 cells and 50% increase in the normal prostate cell line (PNT2) after combination treatment of 50nM docetaxel for 48 hours followed by 5 hours exposure to 1uM ABT-737. These results suggest that even though lower levels of chemotherapeutics may be effective at killing prostate cancer cells when paired with ABT-737, this combination treatment may be just as harmful to healthy prostate cells. Ongoing studies are also focused on combination treatments with ABT-199, which is selective for antiapoptotic BCL-xL. Reference: 1. Chonghalie TN, et al. Pretreatment mitochondrial priming correlates with clinical response to cytotoxic chemotherapy. Science 2011:334(6059):1129-33. Citation Format: Kaitlyn Walsh, Rachel Wilson, Mary E. McManamy, Victoria Del Gaizo Moore. Bcl-2 family proteins as drug targets; Can prostate cancer be primed for apoptosis? [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A075.