title: Centchroman induces G0/G1 arrest and caspase-dependent apoptosis involving mitochondrial membrane depolarization in MCF-7 and MDA MB-231 human breast cancer cells.
abstract: Studies with Centchroman (CC) as a candidate anti-breast cancer agent are into phase III multicentric clinical trial in stage III/IV breast cancer. We have previously demonstrated its anti-neoplastic activity in Estrogen Receptor positive (ER+ve) MCF-7 Human Breast Cancer Cells (HBCCs). We now present the basis for anti-neoplastic activity of CC, mediated through apoptosis in both ER+ve/-ve MCF-7 and MDA MB-231 HBCCs respectively, compared to Tamoxifen (TAM) as a positive control. All the experiments were performed with 48 h estrogen-deprived cells exposed to CC/TAM for the subsequent 48 h. Cytotoxic potential of CC was assessed through SRB assay. Cell-cycle analysis, Time-dependent cytotoxicity, Reactive Oxygen Species (ROS) and Mitochondrial Membrane Permeability were investigated by Flow Cytometry. Early-stage apoptosis was detected by Annexin-PI staining. Caspases were assayed colorimetrically whereas nuclear derangements were assessed morphologically through PI staining and finally by DNA fragmentation analysis. Cell viability studies confirmed the IC50 of CC in MCF-7 and MDA MB-231 cells to be 10 and 20 microM (P < 0.001) respectively, suggesting enhanced susceptibility of the former cell type to CC. FACS data reveals CC mediated G0/G1 arrest (P < 0.01) along with the presence of prominent sub-G0/G1 peak (P < 0.001) in both the cell types suggesting ongoing apoptosis. Phosphatidylserine externalization, mitochondrial events, caspase evaluation and nuclear morphology changes reveal initiation/progression of caspase-dependent apoptosis even at a dose of 1 microM which eventually leads to DNA fragmentation in both the cell types. Results demonstrate that CC induces caspase-dependent apoptosis in MCF-7 and MDA MB-231 cells irrespective of ER status similar to TAM in terms of anti-neoplastic activity.
authors.affiliation: Tissue and Cell Culture Unit (TCCU), Central Drug Research Institute, Lucknow, 226001, India. manisha992001@yahoo.com
authors.forename: Manisha
authors.initials: M
authors.lastname: Nigam
authors.forename: Vishal
authors.initials: V
authors.lastname: Ranjan
authors.forename: Swasti
authors.initials: S
authors.lastname: Srivastava
authors.forename: Ramesh
authors.initials: R
authors.lastname: Sharma
authors.forename: Anil K
authors.initials: AK
authors.lastname: Balapure
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D001943:Breast Neoplasms; D020169:Caspases / Q000378:metabolism*; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000648:ultrastructure; D048430:Cell Shape; D002470:Cell Survival / Q000187:drug effects; D002486:Centchroman / Q000494:pharmacology*; D002986:Clinical Trials as Topic; D053938:DNA Fragmentation; D004965:Estrogen Antagonists / Q000494:pharmacology*; D005260:Female; D016193:G1 Phase / Q000187:drug effects*; D006801:Humans; D008564:Membrane Potentials / Q000502:physiology; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D016192:Resting Phase, Cell Cycle / Q000187:drug effects*; D013629:Tamoxifen / Q000494:pharmacology
chemical_list: D000970:Antineoplastic Agents; D004965:Estrogen Antagonists; D013629:Tamoxifen; D002486:Centchroman; D020169:Caspases