Abstract
Abstract Introduction/Purpose: Misregulation of BCL-2 family of proteins renders a survival signal to withstand cytotoxic anticancer drugs and is often found in drug resistant cells. The drug resistance phenotype often accompanies activation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, as well as an enhancement of cancer stem cell-like (CSC) characteristics. Thus, inhibition of anti-apoptotic BCL-2 family proteins has been proposed as a possible antineoplastic strategy. However, the effects of BCL-2 inhibitors on drug resistant breast cancer have not yet been elucidated. In the present study, the effect of sabutoclax, a pan-active BCL-2 protein family antagonist, on two chemoresistant breast cancer cell lines was assessed. Methods: MTT assays and drug resistance clonogenic assays were performed to evaluate the cell viability in response to drug treatments. Apoptosis was determined by flow cytometry after annexin V staining, by caspase 3/7 and caspase 9 activity assays and the levels of a series of apoptosis-related proteins by real-time PCR and western blot.The expression levels of AKT and pAKT were assessed by western blot to exam the effect of sabutoclax on the activity of PI3K/AKT pathway. The expression of breast cancer stem cells (BCSC) related markers (CD44, CD24 and ALDH1) were detected by flow cytometry. Mammosphere formation assays and soft agar colony formation assay were performed to test the effect of sabutoclax on the proportion of stem-like cell population. To validate the results showed in breast cancer cell lines, we also tested the ex vivo effect of sabutoclax on nine fresh human breast tumor samples by IHC. The effect of sabutoclax on tumor growth was also studied in mouse xenografts. Results We found that sabutoclax showed a significant cytotoxic activity on chemoresistant breast cancer cells both in vitro and in vivo. When chemotherapeutic agents were combined with sabutoclax, strong synergistic antiproliferative effects were observed. Inhibition of BCL-2, MCL-1and BCL-xL leads to caspase-3/7 and caspase-9 activation, sabutoclax modulated Bax, Bim, PUMA and survivin expression. Furthermore, sabutoclax effectively eliminated the CSC subpopulation and reduced sphere formation of drug-resistant cells through down-regulation of the PI3K/AKT signaling pathway. A similar effect was observed in a panel of nine breast tumors ex vivo. Conclusions: Our findings indicate that sabutoclax partially overcomes drug resistance of breast cancer cells by reactivation of apoptosis, mediates by the inhibition of several anti-apoptotic BCL-2 family proteins such as BCL-2, MCL-1, BCL-XL and BFL-1, and, by abolition of PI3K/AKT pathway and crosstalk between AKT and BCL-2 family proteins is indispensable for maintaining stemness and chemoresistance in BCSCs. This suggests a strong rationale to explore the therapeutic strategy of using sabutoclax alone or in combination for chemotherapy-nonresponsive breast cancer patients. Citation Format: Zhang J. Sabutoclax, pan-active BCL-2 protein family antagonist, eliminates cancer stem cells through inhibiting PI3K/Akt pathway in breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-03-17.
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