Centchroman mediated apoptosis involves cross-talk between extrinsic/intrinsic pathways and oxidative regulation

Abstract

Aims Centchroman (CC) has been established as a potent antineoplastic agent in MCF-7 (ER + ve) and MDA MB-231 (ER-ve) Human Breast Cancer Cells (HBCCs) previously by us. To elucidate its antineoplastic action, we investigated the factors involved in cell-cycle progression and apoptosis. Main methods Tamoxifen (TAM), a widely used antiestrogen was employed as a positive control. Role of Cycloheximide (CHX), Actinomycin-D (Act-D) and caspases were explored using specific inhibitors. Involvement of cell-cycle and apoptosis related factors were explored using western blotting and immunoprecipitation. Key findings Metabolic inhibitors viz. CHX, Act-D and pan-Caspase inhibitor, Z-VAD-FMK attenuated CC-induced apoptosis. The upregulation of both p21 Waf1/Cip1 and p27 Kip1 along with p21-CDK6 (Cyclin Dependent Kinase 6) and p21-PCNA (Proliferating Cell Nuclear Antigen) interaction suggests their role in CC-induced cell-cycle arrest. The downregulation of Cyclin-D 1 and -E levels further confirms the antiestrogenic profile of CC. Unlike MDA MB-231, in MCF-7 cells, CC upregulates the level of phospho-p53 (Ser-15) and FasL, suggesting the involvement of extrinsic pathway. CC altered the intracytosolic balance of members of Bcl-2 family along with the cleavage of Poly (ADP-ribose) polymerase (PARP), Bcl-X L, Bid and AIF (Apoptosis Inducing Factor). The evaluation of Mitogen Activated Protein Kinases (MAPKs) using specific inhibitors and Western blotting confirms CC-induced the upregulation of phospho-c-Jun and phospho-p38. Additionally elevated SOD (Superoxide Dismutase) and unaltered CAT (Catalase) expression further suggest the involvement of oxidative stress. Significance These results confirm that the antineoplasticity of CC in MCF-7 and MDA MB-231 cells involves the extrinsic and intrinsic pathways of apoptosis along with oxidative stress.