P53-mediated cell cycle arrest and apoptosis through a caspase-3- independent, but caspase-9-dependent pathway in oridonin-treated MCF-7 human breast cancer cells.
OBJECTIVE
To study the caspase-3-independent mechanisms in oridonin-induced MCF-7 human breast cancer cell apoptosis in vitro.


METHODS
The viability of oridonin-treated MCF-7 cells was measured by MTT (thiazole blue) assay. Apoptotic cells with condensed nuclei were visualized by phase contrast microscopy. Nucleosomal DNA fragmentation was assayed by agarose gel electrophoresis. The apoptotic ratio was determined by lactate dehydrogenase assay. Cell cycle alternation and mitochondrial membrane potential were measured by flow cytometric analysis. Bax, Bcl-2, caspase-3, caspase-9, heat shock protein (Hsp)90, p53, p-p53, p21, Poly (ADP-ribose) polymerase (PARP), and the inhibitor of caspase-activated DNase (ICAD) protein expressions were detected by Western blot analysis.


RESULTS
Oridonin inhibited cell growth in a time- and dose-dependent manner. Cell cycle was altered through the upregulation of p53 and p21 protein expressions. Pancaspase inhibitor Z-VAD-fmk and calpain inhibitor II both decreased cell death ratio. Nucleosomal DNA fragmentation and the downregulation of DeltaPhimit were detected in oridonin-induced MCF-7 cell apoptosis, which was involved in a postmitochondrial caspase-9-dependent pathway. Decreased Bcl-2 and Hsp90 expression levels and increased Bax and p21 expression levels were positively correlated with elevated levels of phosphorylated p53 phosphorylation. Moreover, PARP was partially cleaved by calpain rather than by caspase-3.


CONCLUSIONS
DNA damage provoked alternations in the mitochondrial and caspase-9 pathways as well as p53-mediated cell cycle arrest, but was not related to caspase-3 activity in oridonin-induced MCF-7 cells.
Cui
Qiao
Q
China-Japan Research Institute of Medical Pharmaceutical Sciences, Shenyang Pharmaceutical University, Shenyang 110016, China.
Yu
Jing-hua
JH
Wu
Jin-nan
JN
Tashiro
Shin-ichi
S
Onodera
Satoshi
S
Minami
Mutsuhiko
M
Ikejima
Takashi
T
D000590:Amino Acid Chloromethyl Ketones / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D001943:Breast Neoplasms / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism*; D053453:Caspase 9 / Q000378:metabolism*; D002453:Cell Cycle / Q000187:drug effects*; D045744:Cell Line, Tumor; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000378:metabolism; D045786:Diterpenes, Kaurane / Q000494:pharmacology*; D004365:Drugs, Chinese Herbal; D005260:Female; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D008516:Medicine, Chinese Traditional; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects* / Q000502:physiology; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D051028:bcl-2-Associated X Protein / Q000378:metabolism
D000590:Amino Acid Chloromethyl Ketones; D051017:Apoptosis Regulatory Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D015853:Cysteine Proteinase Inhibitors; D045786:Diterpenes, Kaurane; D004365:Drugs, Chinese Herbal; D018841:HSP90 Heat-Shock Proteins; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C109664:caspase-activated DNase inhibitor; C011959:oridonin; D011065:Poly(ADP-ribose) Polymerases; D053148:Caspase 3; D053453:Caspase 9