Activation of ATM-Chk2 by 16-dehydropregnenolone induces G1 phase arrest and apoptosis in HeLa cells.
The natural pregnane steroid 16-dehydropregnenolone (16-DHP) showed the growth inhibitory activity against human tumor cells; however, the mechanisms of actions of 16-DHP were not clarified. In this study, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to investigate the growth inhibitory effect of 16-DHP. Cell cycle distribution was analyzed using flow cytometry. Hoechst 33258 staining and DNA agarose gel electrophoresis were used to detect apoptosis. The levels of proteins were probed by Western blotting, and caspase activities were analyzed using Caspase Activity Kit. We found that 16-DHP inhibited the growth of human cervical carcinoma cells (HeLa cells) in a time- and dose-dependent manner. The growth inhibitory effect of 16-DHP was associated with G1 arrest mediated by ataxia telangiectasia mutated (ATM)-checkpoint kinase 2 (Chk2)-p53 signaling, as demonstrated by induction of the phosphorylations of ATM, Chk2, and p53 proteins. Followed by G1 arrest, 16-DHP-treated HeLa cells underwent caspase-dependent apoptosis. The inhibitors of caspase-3 and caspase-9 but not caspase-8 inhibitor blocked 16-DHP-induced apoptosis. Moreover, 16-DHP increased the level of Bax protein and the release of cytochrome c from mitochondria, but had no effect on the level of Bcl-2. These results suggested that 16-DHP inhibited the growth of HeLa cells via inducing ATM-Chk2-p53 activation-mediated G1 arrest and mitochondrial cell apoptosis.
Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang, China.
En-Long
EL
Ma
Dong-Mei
DM
Zhao
Yan-Chun
YC
Li
Hong
H
Cao
Qiu-Yu
QY
Zhao
Jian-Chun
JC
Li
Li-Xin
LX
Sun
D017209:Apoptosis / Q000187:drug effects*; D064007:Ataxia Telangiectasia Mutated Proteins; D053148:Caspase 3 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000187:drug effects / Q000378:metabolism; D064447:Checkpoint Kinase 2; D004268:DNA-Binding Proteins / Q000187:drug effects / Q000378:metabolism; D016193:G1 Phase / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D015394:Molecular Structure; D011284:Pregnenolone / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000187:drug effects / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000187:drug effects / Q000378:metabolism
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; C003592:16-dehydropregnenolone; D011284:Pregnenolone; D064447:Checkpoint Kinase 2; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C578634:CHEK2 protein, human; D017346:Protein Serine-Threonine Kinases; D053148:Caspase 3; D053453:Caspase 9