Induction of apoptosis in human hepatocarcinoma SMMC-7721 cells in vitro by flavonoids from Astragalus complanatus.
OBJECTIVE
Flavonoids extracted from the seeds of Astragalus complanatus R.Br. reduce the proliferation of many cancer cells. The present study was carried out to evaluate the effects of these flavonoids from Astragalus complanatus (FAC) on human hepatocarcinoma cell viability and apoptosis and to investigate its mechanisms of action in SMMC-7721 cells.


METHODS
Cell viability was measured using the MTT assay. To detect apoptotic cells, SMMC-7721 cells treated with FAC were stained with Hoechst 33258 and subjected to agarose gel electrophoresis. Quantitative detection of apoptotic cells was performed by flow cytometry. The effects of FAC on apoptosis and cell cycle regulatory genes and proteins in SMMC-7721 cells were examined using an S series apoptosis and cell cycle gene array and Western blot analysis.


RESULTS
The growth of SMMC-7721 and HepG2 cells was inhibited by treatment with FAC. Cell death induced by FAC was characterized by nuclear condensation and DNA fragmentation. Moreover, the cell cycle was arrested in the G0/G1 and S phases in FAC-treated SMMC-7721 cells. A sub-G1 peak with reduced DNA content was also formed. The activity of caspase-3 was significantly increased following FAC treatment. Microarray data indicated that the expression levels of 76 genes were changed in SMMC-7721 cells treated with FAC: 35 genes were up-regulated and 41 were down-regulated. Western blot analysis showed that caspase-3, caspase-8, Bax, P21, and P27 protein levels in SMMC-7721 cells were increased after 48 h of FAC treatment, while cyclinB1, cyclinD1, CDK1, and CDK4 protein levels were decreased.


CONCLUSIONS
These results suggest that FAC may play an important role in tumor growth suppression by inducing apoptosis in human hepatocarcinoma cells via mitochondria-dependent and death receptor-dependent apoptotic pathways.
Department of Pharmacology, Medical College of Soochow University, 199 RenAi Road, Suzhou 215123, PR China.
Yan-Wei
YW
Hu
Chun-Yu
CY
Liu
Chong-Min
CM
Du
Jian
J
Zhang
Wen-Qian
WQ
Wu
Zhen-Lun
ZL
Gu
D000972:Antineoplastic Agents, Phytogenic / Q000302:isolation & purification / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D027883:Astragalus Plant / Q000737:chemistry*; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy / Q000503:physiopathology; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D053938:DNA Fragmentation / Q000187:drug effects; D005419:Flavonoids / Q000302:isolation & purification / Q000494:pharmacology*; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D008113:Liver Neoplasms / Q000188:drug therapy* / Q000503:physiopathology; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D012639:Seeds
D000972:Antineoplastic Agents, Phytogenic; D005419:Flavonoids