Tumor Biol. DOI 10.1007/s13277-014-2869-x
RESEARCH ARTICLE
Induction of S phase arrest and apoptosis by ethyl acetate extract from Tetrastigma hemsleyanum in human hepatoma HepG2 cells Xin Peng & Ding-ding Zhuang & Qiao-sheng Guo
Received: 20 August 2014 / Accepted: 18 November 2014 # International Society of Oncology and BioMarkers (ISOBM) 2015
Abstract Tetrastigma hemsleyanum, a rare and endangered medicinal plant, has attracted much attention due to antitumor and immunomodulatory activities. In this study, the effect and mechanism of ethyl acetate extract from T. hemsleyanum (EET) on cell cycle and apoptosis in human hepatoma HepG2 cells were investigated. Twenty-five to 200 μg/mL of EET were found to have the antiproliferation effect toward HepG2 cells determined by MTT assay. The morphology of EETtreated HepG2 cells showed evidence of apoptosis that included blebbing and chromatin condensation, nucleic fragmentation, and so on. The DNA laddering assay confirmed that DNA fragmentation had occurred during late apoptosis. The cell-cycle analysis indicated that EET was able to induce S phase arrest and typical subdiploid peak in a dose- and timedependent manner. The apoptosis rate of 200 μg/mL treatment for 24 h was 42.24±4.90 %. The protein expression of Bax and P53 was increased after treatment, while that of Bcl2 was significantly decreased in a dose-dependent manner, which suggested that a high Bax/Bcl2 ratio and an upregulated P53 X. Peng : Q. petroleum ester > n-butanol extracts. Therefore, in the present study, we examined the effects of ethyl acetate extract of T. hemsleyanum (EET) on the cellcycle arrest and apoptosis induction in human hepatoma HepG2 cells. The results showed that EET was able to induce S phase arrest and typical subdiploid peak in a dose- and timedependent manner. The protein expression of Bax and P53 was increased after treatment, while Bcl2 was significantly decreased, which suggested that the cytotoxicity on HepG2
cells induced by EET is a result of both cell-cycle arrest and apoptosis. These results were a basis for the later study on the anticancer mechanism and the medicinal value development of T. hemsleyanum.
Materials and methods Antibodies and reagents HepG2 cells were purchased from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Rabbit polyclonal antibodies against BCl2, Bax, P53 (TransGen Biotech, China), CDK1 (Proteintech Biotech, USA), mouse monoclonal antibody against β-actin (TransGen Biotech, China), goat-antirabbit and goat-antimouse fluorescent markerconjugated secondary antibodies (Licor, USA), BCA protein assay kit (Beyotime Biotech, China), apoptotic DNA ladder detection kit (Beyotime Biotech, China), 3-[4,5-dimethy lthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) cell proliferation and cytotoxicity assay kit (Beyotime Biotech, China), fetal bovine serum (Sijiqing Biotech, China), RPMI1640 medium (Invitrogen, USA), and polyvinylidene fluoride (PVDF) membrane (Millipore Life Science, USA). Preparation of EET The commodities of T. hemsleyanum root were purchased from Yinzhou Medical Supplies Company in Zhejiang Province of China. One kilogram of the powdered T. hemsleyanum root (particle size
RESEARCH ARTICLE
Induction of S phase arrest and apoptosis by ethyl acetate extract from Tetrastigma hemsleyanum in human hepatoma HepG2 cells Xin Peng & Ding-ding Zhuang & Qiao-sheng Guo
Received: 20 August 2014 / Accepted: 18 November 2014 # International Society of Oncology and BioMarkers (ISOBM) 2015
Abstract Tetrastigma hemsleyanum, a rare and endangered medicinal plant, has attracted much attention due to antitumor and immunomodulatory activities. In this study, the effect and mechanism of ethyl acetate extract from T. hemsleyanum (EET) on cell cycle and apoptosis in human hepatoma HepG2 cells were investigated. Twenty-five to 200 μg/mL of EET were found to have the antiproliferation effect toward HepG2 cells determined by MTT assay. The morphology of EETtreated HepG2 cells showed evidence of apoptosis that included blebbing and chromatin condensation, nucleic fragmentation, and so on. The DNA laddering assay confirmed that DNA fragmentation had occurred during late apoptosis. The cell-cycle analysis indicated that EET was able to induce S phase arrest and typical subdiploid peak in a dose- and timedependent manner. The apoptosis rate of 200 μg/mL treatment for 24 h was 42.24±4.90 %. The protein expression of Bax and P53 was increased after treatment, while that of Bcl2 was significantly decreased in a dose-dependent manner, which suggested that a high Bax/Bcl2 ratio and an upregulated P53 X. Peng : Q. petroleum ester > n-butanol extracts. Therefore, in the present study, we examined the effects of ethyl acetate extract of T. hemsleyanum (EET) on the cellcycle arrest and apoptosis induction in human hepatoma HepG2 cells. The results showed that EET was able to induce S phase arrest and typical subdiploid peak in a dose- and timedependent manner. The protein expression of Bax and P53 was increased after treatment, while Bcl2 was significantly decreased, which suggested that the cytotoxicity on HepG2
cells induced by EET is a result of both cell-cycle arrest and apoptosis. These results were a basis for the later study on the anticancer mechanism and the medicinal value development of T. hemsleyanum.
Materials and methods Antibodies and reagents HepG2 cells were purchased from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Rabbit polyclonal antibodies against BCl2, Bax, P53 (TransGen Biotech, China), CDK1 (Proteintech Biotech, USA), mouse monoclonal antibody against β-actin (TransGen Biotech, China), goat-antirabbit and goat-antimouse fluorescent markerconjugated secondary antibodies (Licor, USA), BCA protein assay kit (Beyotime Biotech, China), apoptotic DNA ladder detection kit (Beyotime Biotech, China), 3-[4,5-dimethy lthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) cell proliferation and cytotoxicity assay kit (Beyotime Biotech, China), fetal bovine serum (Sijiqing Biotech, China), RPMI1640 medium (Invitrogen, USA), and polyvinylidene fluoride (PVDF) membrane (Millipore Life Science, USA). Preparation of EET The commodities of T. hemsleyanum root were purchased from Yinzhou Medical Supplies Company in Zhejiang Province of China. One kilogram of the powdered T. hemsleyanum root (particle size
