Oridonin induces apoptosis of HeLa cells via altering expression of Bcl-2/Bax and activating caspase-3/ICAD pathway.
OBJECTIVE
To study the mechanisms by which oridonin inhibited HeLa cell growth in vitro.


METHODS
Viability of oridonin-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. Caspase activity was assayed using fluorometric protease assay. ICAD, Bcl-2, and Bax proteins expression were detected by Western blot analysis.


RESULTS
Oridonin induced oligonucleosomal fragmentation of DNA and increased caspase-3 activity, on the other hand, reduced the expression of inhibitor of caspase-3-activated DNase (ICAD), a caspase-3 substrate, at 12 h in HeLa cells. Oridonin-induced DNA fragmentation, caspase-3 activation and down-regulation of ICAD expression were effectively inhibited by a caspase-3 inhibitor, z-DEVD-fmk (z-Asp-Glu-Val-Asp-fmk). However, pretreatment with an inhibitor of poly (ADP-ribose) polymerase (PARP), 3, 4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone (DPQ), did not suppress oridonin-induced HeLa cell death. In addition, oridonin-induced apoptosis was associated with an increase in the expression of the apoptosis inducer Bax, and a significant reduction in expression of the apoptosis suppressor Bcl-2 in mitochondria.


CONCLUSIONS
Oridonin induces HeLa cells apoptosis by altering balance of Bcl-2 and Bax protein expression and activation of caspase-3/ICAD pathway.
Zhang
Chun-Ling
CL
China-Japan Research Institute of Medical and Pharmaceutical Sciences, Shenyang Pharmaceutical University, Shenyang 110016, China.
Wu
Li-Jun
LJ
Tashiro
Shin-Ichi
S
Onodera
Satoshi
S
Ikejima
Takashi
T
D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D016923:Cell Death / Q000187:drug effects; D053938:DNA Fragmentation; D004224:Diterpenes / Q000302:isolation & purification / Q000494:pharmacology*; D045786:Diterpenes, Kaurane; D006367:HeLa Cells; D006801:Humans; D037602:Isodon / Q000737:chemistry; D010946:Plants, Medicinal / Q000737:chemistry; D011506:Proteins / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D051028:bcl-2-Associated X Protein
D051017:Apoptosis Regulatory Proteins; C494826:BAX protein, human; D004224:Diterpenes; D045786:Diterpenes, Kaurane; D011506:Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; C109664:caspase-activated DNase inhibitor; C011959:oridonin; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases