Open AccessInvolvement of mitochondria and caspase pathways in N ‐demethyl‐clarithromycin‐induced apoptosis in human cervical cancer HeLa cell
Author(s) -
QIAO Aimin,
IKEJIMA Takashi,
TASHIRO Shinichi,
ONODERA Satoshi,
ZHANG Weige,
WU Yingliang
Publication year - 2006
Publication title -
acta pharmacologica sinica
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.514
H-Index - 90
eISSN - 1745-7254
pISSN - 1671-4083
DOI - 10.1111/j.1745-7254.2006.00444.x
Subject(s) - hela , apoptosis , microbiology and biotechnology , viability assay , cytochrome c , poly adp ribose polymerase , biology , caspase 3 , dna fragmentation , programmed cell death , caspase , cell , biochemistry , polymerase , dna
Aim: To study the mechanisms by which N ‐demethyl‐clarithromycin (NDC) induces human cervical cancer HeLa cell apoptosis in vitro. Methods : The viability of N ‐demethyl‐clarithromycin‐induced HeLa cells was measured by MTT assay. Apoptotic cells with condensed nuclei were visualized by phase contrast microscopy. Nucleosomal DNA fragmentation was assayed by agarose gel electrophoresis. Measurement of mitochondrial transmembrane potential was analyzed by a FACScan flowcytometer. Caspase‐3, poly‐(ADP‐ribose) polymerase (PARP), caspase‐activated DNase (ICAD), Bcl‐2, Bax, p53, and SIRT1 protein expression and the release of cytochrome c were detected by Western blot analysis. Results : N ‐demethyl‐clarithromycin, an anti‐inflammatory substance, inhibited HeLa cell growth in a dose‐ and time‐dependent manner. N ‐demethyl‐clarithromycin induced HeLa cell death through the apoptotic pathways. The pancaspase inhibitor (z‐VAD‐fmk), caspase‐3 inhibitor (z‐DEVD‐fmk) andthecaspase‐9 inhibitor (z‐LEHD‐fmk) partially enhanced cell viability induced by N ‐demethyl‐clarithromycin, but the caspase‐8 inhibitor (z‐IETD‐fmk) had almost no effect. Caspase‐3 was activated then followed by the degradation of caspase‐3 substrates, the inhibitor of ICAD and PARP. Simultaneously, mitochondrial transmembrane potential was markedly reduced and the release of cytochrome c in the cytosol was increased. N ‐demethyl‐clarithromycin upregulated the expression ratio of mitochondrial Bax/Bcl‐2, and significantly increased the expression of the p53 protein. It also downregulated anti‐apoptotic protein SIRT1 expression. Conclusion: N ‐demethyl‐clarithromycin induced apoptosis in HeLa cells via the mitochondrial pathway.