Abstract 63: Navitoclax (ABT-263) induces apoptosis in colon cancer cells by upregulating caspase 3 through inhibition of Bcl-2 family members

Abstract

Abstract Colorectal cancer (CRC) is the third most common cancer in the United States. Most CRCs in patients receiving therapeutic agents are resistant to apoptotic stimuli. Two protein families, caspase enzymes and B-cell lymphoma-2 (Bcl-2) family proteins are involved in apoptosis. Caspase enzymes promote apoptosis, functioning in a cascade, of which caspase 3 is the pivotal protein. Members of the Bcl-2 family, Bcl-2, Bcl-xL, and Bax, are functionally opposed: Bcl-2 and Bcl-xL inhibit apoptosis, whereas Bax counteracts this effect. Only Bcl-2 family members that have the BH3-domain trigger apoptosis by binding to the pro-survival proteins, thereby neutralizing their functional activity. The approach of “BH-3 mimetics” was utilized for discovery and development of navitoclax (ABT-263), a small-molecule that mimics BH3-only proteins and thus induces apoptosis. Despite advances made in targeted therapy, the mechanism of action of ABT-263 is less studied, particularly for CRCs, which have over-expression of anti-apoptotic proteins, supporting avoidance of cell death, a hallmark of cancer. To identify the cellular mechanism of ABT-263 on apoptosis, we quantified the expression of anti-apoptotic Bcl-2-family members in CRC tumor samples relative to their corresponding controls and found several-fold higher expression of Bcl2, Bcl-xL, and Mcl-1 in the CRC tumor samples. Since ABT-263 is a classical drug for Bcl-2 inhibition, we studied its effect (2.5 µM for 24 hr) on RKO CRC cells, which have high endogenous expression of Bcl-2, by measuring gene expression profiles of Bcl-2 family members by qPCR, along with other proteins involved in apoptosis. ABT-263 treatment resulted in down-regulation of anti-apoptotic modulators, Bcl-2, Bcl-xL, and Bcl-w, by 27%, 29%, and 13%, respectively; these effects were concomitant with two-fold higher expression of caspase 3. Flow cytometry results with Annexin V/PI staining showed that ABT-263 induced apoptosis in RKO cells with 1.59-, 2.59-, and 2.76-fold elevations of early apoptotic, late apoptotic, and necrotic cells. Further, ABT-263 (2.5 µM) lowered cell proliferation by 15% after 24 hr. Similar lower gene expressions of Bcl-2 family members were evident for LS174T CRC cells, which also exhibit high endogenous Bcl-2 expression, when treated with ABT-263. These results highlight a previously unexplored effect of ABT-263-induced apoptosis in CRC cells, by upregulating caspase 3, through inhibition of Bcl-2 family members. These studies were supported by the Richard Elkus, MD, Eminent Scholars Program in Gastro-Intestinal Cancer Research at University of Alabama at Birmingham. Citation Format: Prachi Bajpai, Amr Elkholy, Sumit Agarwal, Hyung Gyoon Kim, Michael Behring, Upender Manne. Navitoclax (ABT-263) induces apoptosis in colon cancer cells by upregulating caspase 3 through inhibition of Bcl-2 family members [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 63.