Induction of ER stress-mediated apoptosis in breast cancer cell line by the powerful alkylating agent bendamustine and insights into its molecular mechanisms.

Abstract

Bendamustine, an alkylating agent used in treating hematological cancers like non-Hodgkin's lymphoma (NHL) and chronic lymphocytic leukemia (CLL), has recently garnered attention for its potential in breast cancer therapy. This study explores its anticancer effects and molecular mechanisms in breast cancer cells. MDA-MB-231 cells were exposed to various concentrations of bendamustine (0-50 µM), and cytotoxicity was assessed using Alamar Blue and LDH assays, revealing a dose-dependent reduction in cell viability with an IC₅₀ value of 16.98 μM at 24 h (p < 0.001). Intracellular reactive oxygen species (ROS) were quantified by DCF-DA flow cytometry, showing a significant elevation following treatment (p < 0.01). Downregulation of major antioxidant enzymes (SOD, CAT, GPx1, GST; p < 0.01) and upregulation of TRPC6 (1.8-fold at 10 μM, 2.5-fold at 20 μM; p < 0.01) were detected by qPCR. Markers of apoptosis were evaluated by Annexin V/PI staining, revealing significant increases in both early and late apoptotic cell populations (p < 0.01), and by gene expression analysis showing increased BAX:BCL-2 ratio (1.5-fold at 10 μM, 7.8-fold at 20 μM; p < 0.01). CHOP mRNA, an ER stress-related pro-apoptotic gene, was significantly upregulated (2.5-fold at 20 μM; p < 0.01), supporting activation of the ER stress pathway. The results demonstrated that bendamustine exerted a dose-dependent cytotoxic effect, decreasing cell viability and increasing LDH release. It significantly increased ROS levels and altered the expression of apoptotic genes, promoting apoptosis in breast cancer cells. Furthermore, bendamustine-induced ER stress, shown by upregulated Chop expression, suggests that ER stress plays a role in its anticancer activity. These findings highlight bendamustine as a promising therapeutic agent for breast cancer treatment.