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Anonna squamosa leaves ethanolic extract mediates apoptosis through heat shock protein mitigation and G2/M cell cycle arrest in Human Hepatocellular carcinoma (SK‐Hep1) cells
Author(s) -
Lumbera Wenchie Marie Lara,
Vergara Emil Joseph Sanvictores,
Kim Yeon Soo,
Cruz Joseph Flores,
Hwang Seong Gu
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.1090.3
Subject(s) - cell cycle , viability assay , cyclin dependent kinase 1 , apoptosis , cell cycle checkpoint , cell growth , biology , western blot , cell , heat shock protein , microbiology and biotechnology , cyclin b1 , chemistry , biochemistry , gene
The current study aimed to evaluate the chemotherapeutic property of Anonna squamosa (AS) leaves ethanolic extract in human hepatocellular carcinoma (HHC) SK‐Hep1 cells and identify its specific mechanism of action on the molecular level. Powdered AS leaves were soaked in 80% ethanol, subjected to rotary evaporator for extraction then used as treatment for SK‐Hep1 cells in the cell viability assay, cell cycle analysis, RT‐PCR and Western blot analysis. The cell viability assay demonstrated an increase in the cell proliferation among normal liver cell (CCL‐13) and cytotoxicity among SK‐Hep1 cells incubated with different AS concentration (0, 12.5, 25, 50, 100 and 200μg/ml) as treatment for both 24 and 48 hours. These results were further evaluated in the flow cytometry analysis which exhibited cell accumulation in the Sub/G1 and G2/M phase thereby cell cycle arrest identified at these points. Apoptotic and G2/M cell cycle arrest related genes and proteins expression were examined with RT‐PCR and Western blot to determine the specific molecular mechanism by which AS ethanolic extract causes HHC cell death. Both revealed a downstream expression of heat shock protein 70 (HSP70) and 27 (HSP27) unblocking apoptosis exhibited by an upstream expression of CASPASE‐3. G2/M cell cycle arrest was further verified by the down regulation of cell division cycle 25c (CDC25C) inhibiting the Cyclin B‐cyclin dependent kinase 1 (CDK1) complex exhibited by their decreased mRNA and protein expressions. Therefore, AS leaves ethanolic extract have a great chemotherapeutic potential against HHC (SK‐Hep1 cells) through HSP70 and HSP27 inhibition allowing programmed cell death through CASPASE‐3enhancement together with G2/M phase cell cycle arrest through Cyclin B‐CDK1 complex interference by CDC25C inactivation.