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Evaluation of Caspase Activation to Assess Innate Immune Cell Death
Published on: January 20, 2023
Centchroman mediated apoptosis involves cross-talk between extrinsic/intrinsic pathways and oxidative regulation
Manisha Nigam1, Neetu Singh, Vishal Ranjan
1Tissue and Cell Culture Unit (TCCU), Central Drug Research Institute, Council of Scientific & Industrial Research, Lucknow, India-226001.
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.
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Cell death was observed in the early 19th century, but there was no experimental evidence to prove it. In 1842, Carl Vogt first discovered cell death in a metamorphic toad; however, it was not termed ‘cell death.’ Scientists discovered different cell death pathways only in the 20th century...

