Involvement of mitochondria and caspase pathways in N-demethyl-clarithromycin-induced apoptosis in human cervical cancer HeLa cell.
OBJECTIVE
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-clarithro-mycin induced HeLa cell death through the apoptotic pathways. The pan-caspase inhibitor (z-VAD-fmk), caspase-3 inhibitor (z-DEVD-fmk) and the caspase-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.


CONCLUSIONS
N-demethyl-clarithromycin induced apoptosis in HeLa cells via the mitochondrial pathway.
Qiao
Ai-min
AM
Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang 110016, China.
Ikejima
Takashi
T
Tashiro
Shin-ichi
S
Onodera
Satoshi
S
Zhang
Wei-ge
WG
Wu
Ying-liang
YL
D000893:Anti-Inflammatory Agents / Q000008:administration & dosage / Q000737:chemistry / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3 / Q000378:metabolism*; D049109:Cell Proliferation / Q000187:drug effects; D017291:Clarithromycin / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology*; D053938:DNA Fragmentation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D006367:HeLa Cells; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000378:metabolism*; D015394:Molecular Structure; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D015398:Signal Transduction; D056564:Sirtuin 1; D037761:Sirtuins / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000378:metabolism
D000893:Anti-Inflammatory Agents; D019253:Proto-Oncogene Proteins c-bcl-2; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; D053148:Caspase 3; C447939:SIRT1 protein, human; D056564:Sirtuin 1; D037761:Sirtuins; D017291:Clarithromycin