Induction of apoptosis and cell cycle arrest in human HCC MHCC97H cells with Chrysanthemum indicum extract.
OBJECTIVE
To investigate the effects of Chrysanthemum indicum extract (CIE) on inhibition of proliferation and on apoptosis, and the underlying mechanisms, in a human hepatocellular carcinoma (HCC) MHCC97H cell line.


METHODS
Viable rat hepatocytes and human endothelial ECV304 cells were examined by trypan blue exclusion and MTT assay, respectively, as normal controls. The proliferation of MHCC97H cells was determined by MTT assay. The cellular morphology of MHCC97H cells was observed by phase contrast microscopy. Flow cytometry was performed to analyze cell apoptosis with annexin V/propidium iodide (PI), mitochondrial membrane potential with rhodamine 123 and cell cycle with PI in MHCC97H cells. Apoptotic proteins such as cytochrome C, caspase-9, caspase-3 and cell cycle proteins, including P21 and CDK4, were measured by Western blotting.


RESULTS
CIE inhibited proliferation of MHCC97H cells in a time- and dose-dependent manner without cytotoxicity in rat hepatocytes and human endothelial cells. CIE induced apoptosis of MHCC97H cells in a concentration-dependent manner, as determined by flow cytometry. The apoptosis was accompanied by a decrease in mitochondrial membrane potential, release of cytochrome C and activation of caspase-9 and caspase-3. CIE arrested the cell cycle in the S phase by increasing P21 and decreasing CDK4 protein expression.


CONCLUSIONS
CIE exerted a significant apoptotic effect through a mitochondrial pathway and arrested the cell cycle by regulation of cell cycle-related proteins in MHCC97H cells without an effect on normal cells. The cancer-specific selectivity shown in this study suggests that the plant extract could be a promising novel treatment for human cancer.
Li
Zong-Fang
ZF
Department of General Surgery, The Second Affiliated Hospital, School of Medicine, Xi'an Jiaotong University, Xi'an 710004, Shaanxi Province, China. lzf2568@gmail.com
Wang
Zhi-Dong
ZD
Ji
Yuan-Yuan
YY
Zhang
Shu
S
Huang
Chen
C
Li
Jun
J
Xia
Xian-Ming
XM
D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000627:therapeutic use*; D017209:Apoptosis / Q000187:drug effects*; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy* / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D031202:Chrysanthemum; D051358:Cyclin-Dependent Kinase 4 / Q000378:metabolism; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D045304:Cytochromes c / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D008113:Liver Neoplasms / Q000188:drug therapy* / Q000378:metabolism; D008517:Phytotherapy; D010936:Plant Extracts / Q000627:therapeutic use*; D051381:Rats; D017207:Rats, Sprague-Dawley
D000972:Antineoplastic Agents, Phytogenic; C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D010936:Plant Extracts; D045304:Cytochromes c; C495900:CDK4 protein, human; D051358:Cyclin-Dependent Kinase 4; D053148:Caspase 3; D053453:Caspase 9