siRNA as a tool to delineate pathway channelization in H(2)O(2) induced apoptosis of primary Leydig cells in vitro

• Published in: Apoptosis (London) 2012-11, Vol.17 (11), p.1131-1143
• Main Authors: Anand, Himani, Misro, M M, Sharma, S B, Prakash, Sant
• Format: Article
• Language: English
• Subjects: Animals; Annexin A5 - metabolism; Apoptosis - drug effects; Biomarkers - metabolism; Caspase 3 - metabolism; Caspase 8 - metabolism; Cell Survival - drug effects; Cells, Cultured; Flow Cytometry; Fluorescein-5-isothiocyanate - metabolism; Gene Expression Regulation - drug effects; Gene Knockdown Techniques; Hydrogen Peroxide - pharmacology; Leydig Cells - cytology; Leydig Cells - drug effects; Leydig Cells - metabolism; Male; Protein Binding - drug effects; Rats; Rats, Wistar; RNA, Small Interfering - metabolism; Signal Transduction - drug effects; Transfection; Up-Regulation - drug effects
• ISSN: 1573-675X
• DOI: 10.1007/s10495-012-0749-7

Using siRNA as a tool, the channelization of pathway in H(2)O(2) induced apoptosis of primary Leydig cells was investigated in vitro. Exposure (4 h) to H(2)O(2) (250 μM) induced maximum apoptosis but affected Leydig cell viability significantly. Therefore, expression of apoptotic marker genes, caspase-8, -9, -3 and polyadenosine ribose polymerase was subsequently investigated using the same concentration post 1 h exposure. Incubation with siRNA (20 nM) either for caspase-8 or -9, inhibited their individual expressions by 55-60 % and activity, 50-55 %. The inhibition efficiency using siRNA was comparable with post- or pre-H(2)O(2) treatment of cells. Like siRNA, Eugenia jambolana (100 μg/ml) plant extract too, effectively countered over-expression of all apoptotic marker proteins. Silencing expressions of caspase 8 but not 9 through siRNA leads to a profound inhibition of caspase 3 implying that H(2)O(2) induced Leydig cell apoptosis is preferably channeled through extrinsic and later extending to other pathways.