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Tumor Biology

, Volume 36, Issue 4, pp 2541–2550 | Cite as

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
Research Article

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 EET-treated 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 time-dependent 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 might contribute to the pro-apoptotic activity of EET via the mitochondria-dependent pathway. The protein expression of cyclin-dependent kinase 1 (CDK1) was decreased in EET-treated HepG2 cells, suggesting that EET evoked S phase arrest possibly through the downregulation of cyclin A-CDK1 complex. In conclusion, the cytotoxicity on HepG2 cells induced by EET is a result of both cell-cycle arrest and apoptosis. Thus, it may have therapeutic potential for the treatment of liver cancer.

Keywords

Tetrastigma hemsleyanum HepG2 cells Apoptosis Cells cycle arrest 

Notes

Acknowledgments

This work was supported by the traditional Chinese medical science technology research projects of Zhejiang Province (Grant No. 2013ZA119), the public welfare technology research projects of Zhejiang Province (Grant No. 2013C32103), and the agricultural research projects of Ningbo City (Grant No. 2014C10031).

References

  1. 1.
    Zhao J-L, Zhao J, Jiao H-J. Synergistic growth-suppressive effects of quercetin and cisplatin on HepG2 human hepatocellular carcinoma cells. Appl Biochem Biotechnol. 2014;172:784–91.CrossRefPubMedGoogle Scholar
  2. 2.
    Fulda S, Debatin KM. Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy. Oncogene. 2006;25:4798–811.CrossRefPubMedGoogle Scholar
  3. 3.
    Dziul DB, Jakubowicz-Gil J, Paduch R, Głowniak K. Combined treatment with quercetin and imperatorin as a potent strategy for killing HeLa and Hep2 cells. Mol Cell Biochem. 2014;392:213–27.CrossRefGoogle Scholar
  4. 4.
    Ghate NB, Hazra B, Sarkar R, Chaudhuri D, Mandal N. Alteration of Bax/Bcl2 ratio contributes to Terminalia belerica-induced apoptosis in human lung and breast carcinoma. In Vitro Cell Dev Biol Anim. 2014;50:527–37.CrossRefPubMedGoogle Scholar
  5. 5.
    Miao Y, Xiao B, Jiang Z, Guo Y, Mao F. Growth inhibition and cell-cycle arrest of human gastric cancer cells by Lycium barbarum polysaccharide. Med Oncol. 2010;27:785–90.CrossRefPubMedGoogle Scholar
  6. 6.
    Li J, Zhang F, Wang S. A polysaccharide from pomegranate peels induces the apoptosis of human osteosarcoma cells via the mitochondrial apoptotic pathway. Tumor Biol. 2014. doi: 10.1007/s13277-014-1983-0.Google Scholar
  7. 7.
    Dai YJ, Shen ZG, Liu Y, Wang LL, Hannaway D, Lu HF. Effects of shade treatments on the photosynthetic capacity, chlorophyll fluorescence, and chlorophyll content of Tetrastigma Hemsleyanum Diels et Gilg. Environ Exp Bot. 2009;65:177–82.CrossRefGoogle Scholar
  8. 8.
    Xu CJ, Ding GQ, Fu JY, Meng J, Zhang RH, Lou XM. Immunoregulatory effects of ethyl-acetate fraction of extracts from Tetrastigma Hemsleyanum Diels et. Gilg on immune functions of ICR mice. Biomed Environ Sci. 2008;21:325–31.CrossRefPubMedGoogle Scholar
  9. 9.
    He FG. Research progress in anticancer effect of Tetrastigma hemsleyanum Diels et Gilg and its mechanism. J Oncol. 2010;16:75–7 (in Chinese).Google Scholar
  10. 10.
    Yuming Y, Yili W, Yeling T, Guanhai D. Comparative study on anti-tumor effects of different processed Tetrastigma hemsleyanum Diels et Gilg on Lewis lung cancer and H22 liver cancer in mice. Chin Arch Tradit Chin Med. 2013;31:2674–6 (in Chinese).Google Scholar
  11. 11.
    Feng Z-Q, Ni K-F, He Y, Ding Z-S. Experimental study on effect of Tetrastigma hemsleyanum Diels et Gilg flavone on inducing apoptosis of SGC-7901 cell line in vitro. Chin J Clin Pharmacol Ther. 2006;11:669–72. in Chinese.Google Scholar
  12. 12.
    Ding G-Q, Zhen J-X, Wei K-M, Pu J-B. Toxicological effects of the extract of Tetrastigma hemsleyanum Diels et. Gllg on hepatocellular carcinoma cell line HepG2 and primary rat hepatocytes in vitro. Zhejiang Prev Med. 2005;17:1–5. in Chinese.Google Scholar
  13. 13.
    Xin P, Shuang-Lin Z, Jun-Yao H, Li D. Influence of rare earth elements on metabolism and related enzyme activity and isozyme expression in Tetrastigma hemsleyanum cell suspension cultures. Biol Trace Elem Res. 2013;152:82–90.CrossRefPubMedGoogle Scholar
  14. 14.
    Lee S-T, Lu M-H, Chien L-H. Suppression of urinary bladder urothelial carcinoma cell by the ethanol extract of pomegranate fruit through cell cycle arrest and apoptosis. BMC Complement Alternat Med. 2013;13:364–75.CrossRefGoogle Scholar
  15. 15.
    Bendris N, Lemmers B, Blanchard JM, Arsic N. Cyclin A2 mutagenesis analysis: a new insight into CDK activation and cellular localization requirements. PLoS One. 2011;6:e22879.CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Miao Y, Xiao B, Jiang Z, Guo Y, Mao F, Zhao J, et al. Growth inhibition and cell-cycle arrest of human gastric cancer cells by Lycium barbarum polysaccharide. Med Oncol. 2010;27:785–90.CrossRefPubMedGoogle Scholar
  17. 17.
    Lee S-T, Lu M-H, Chien L-H, Wu T-F, Huang L-C, Liao G-I. Suppression of urinary bladder urothelial carcinoma cell by the ethanol extract of pomegranate fruit through cell cycle arrest and apoptosis. BMC Complement Alternat Med. 2013;13:364–74.CrossRefGoogle Scholar
  18. 18.
    Liu D, Ju JH, Lin G, Xu XD, Yang JS, Tu GZ. New C- glycosylflavones from Tetrastigma hemsleyanum (Vitaceae). Acta Bot Sin. 2002;44:227–9.Google Scholar
  19. 19.
    Peng X, He J-Y. The inhibitory effect of Ca2+ on the flavonoid production of Tetrastigma hemsleyanum suspension cells induced by metal elicitors. In Vitro Cell Dev Biol Plant. 2013;49:550–9.CrossRefGoogle Scholar
  20. 20.
    Dziul D, Jakubowicz-Gil J, Paduch R, Głowniak K, Gawron A. Combined treatment with quercetin and imperatorin as a potent strategy for killing HeLa and Hep2 cells. Mol Cell Biochem. 2014;392:213–27.CrossRefGoogle Scholar
  21. 21.
    Ramos S. Effects of dietary flavonoids on apoptotic pathways related to cancer chemoprevention. J Nutr Biochem. 2007;18:427–42.CrossRefPubMedGoogle Scholar
  22. 22.
    Murakami A, Ashida H, Terao J. Multitargeted cancer prevention by quercetin. Cancer Lett. 2008;269:315–25.CrossRefPubMedGoogle Scholar
  23. 23.
    Auyeung KK, Ko JK. Novel herbal flavonoids promote apoptosis but differentially induce cell cycle arrest in human colon cancer cell. Invest New Drugs. 2010;28:1–13.CrossRefPubMedGoogle Scholar
  24. 24.
    Ola MS, Nawaz M, Ahsan H. Role of Bcl2 family proteins and caspases in the regulation of apoptosis. Mol Cell Biochem. 2011;351:41–58.CrossRefPubMedGoogle Scholar
  25. 25.
    Antonsson B. Mitochondria and the Bcl2 family proteins in apoptosis signaling pathways. Mol Cell Biochem. 2004;256/257:141–55.CrossRefGoogle Scholar
  26. 26.
    Lin W, Tongyi S. Role of Bax/Bcl-2 family members in green tea polyphenol induced necroptosis of p53-deficient Hep3B cells. Tumor Biol. 2014. doi: 10.1007/s13277-014-2064-0.Google Scholar
  27. 27.
    Yan F, Liu Y, Wang W. Matrine inhibited the growth of rat osteosarcoma UMR-108 cells by inducing apoptosis in a mitochondrial–caspase-dependent pathway. Tumor Biol. 2013;34:2135–40.CrossRefGoogle Scholar
  28. 28.
    Miyashita T, Krajewski S, Krajewska M, Wang HG, Lin HK, Liebermann DA, et al. Tumor suppressor p53 is a regulator of bcl-2 and bax gene expression in vitro and in vivo. Oncogene. 1994;9:1799–805.PubMedGoogle Scholar
  29. 29.
    Lee TL, Yeh J, Friedman J, Yan B, Yang X, Yeh NT, et al. A signal network involving coactivated NF-kappaB and STAT3 and altered p53 modulates BAX/BCL-XL expression and promotes cell survival of head and neck squamous cell carcinomas. Int J Cancer. 2008;122:1987–98.CrossRefPubMedGoogle Scholar
  30. 30.
    Yu Q. Restoring p53-mediated apoptosis in cancer cells: new opportunities for cancer therapy. Drug Resist Updat. 2006;9:19–25.CrossRefPubMedGoogle Scholar
  31. 31.
    Chipuk JE, Kuwana T, Bouchier-Hayes L, et al. Direct activation of Bax by p53 mediates mitochondrial membrane permeabilization and apoptosis. Science. 2004;303:1010–4.CrossRefPubMedGoogle Scholar
  32. 32.
    Zakaria Y, Rahmat A, Pihie AHL, Abdullah NR, Houghton PJ. Eurycomanone induce apoptosis in HepG2 cells via up-regulation of p53. Cancer Cell Int. 2009;9:1–21. doi: 10.1186/1475-2867-9-16.CrossRefGoogle Scholar
  33. 33.
    Chou C-C, Yang J-S, Lu H-F, Ip S-W, Lo C. Quercetin-mediated cell cycle arrest and apoptosis involving activation of a caspase cascade through the mitochondrial pathway in human breast cancer MCF-7 cells. Arch Pharm Res. 2010;33:1181–91.CrossRefPubMedGoogle Scholar
  34. 34.
    Lai PBS, Chi T-Y, Chen GG. Different levels of p53 induced either apoptosis or cell cycle arrest in a doxycycline-regulated hepatocellular carcinoma cell line in vitro. Apoptosis. 2007;12:387–93.CrossRefPubMedGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2015

Authors and Affiliations

  1. 1.Institute and Department of Chinese Medicinal MaterialsNanjing Agricultural UniversityNanjingChina
  2. 2.Institute of BiopharmaceuticalZhejiang Pharmaceutical CollegeNingboChina
  3. 3.Ningbo Institute of Microcirculation and HenbaneNingboChina

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