Advertisement

Tumor Biology

, Volume 37, Issue 1, pp 865–876 | Cite as

Ethylacetate extract from Tetrastigma hemsleyanum induces apoptosis via the mitochondrial caspase-dependent intrinsic pathway in HepG2 cells

  • Xin Peng
  • Yuan-yuan Zhang
  • Jin Wang
  • Qingyong Ji
Research Article

Abstract

Ethylacetate extract of Tetrastigma hemsleyanum (EET) has a potent antitumor activity in vitro and in vivo. However, the molecular mechanism underlying EET-induced apoptosis remains elusive. As part of our continuing studies, we investigated the apoptosis mechanism of HepG2 cells exposed to different concentrations of EET in vitro. Confocal laser scanning was used to detect the apoptotic morphological changes. Flow cytometer and inverted fluorescence microscope were used to detect the mitochondrial membrane potential and cytosolic Ca2+ level. Western blotting analysis was used to evaluate the expression of the apoptosis-related proteins. Annexin V/PI staining was used to investigate cell apoptosis. Spectrophotometry was used to detect the activity of caspase family. The results showed that distinct apoptotic morphological changes occurred in HepG2 cells treated by EET. EET caused collapse of mitochondrial membrane potential, elevation of cytosolic Ca2+ level, and evoked release of cytochrome c from mitochondria in a concentration-dependent manner. The apoptosis was accompanied by a significant activation of caspase-3, caspase-9, and the cleavage of poly (ADP-ribose) polymerase, but there was no significant change in either the activity or the expression level of caspase-8. Furthermore, EET-induced apoptosis could be inhibited by caspase-9 inhibitor Z-LEHD–FMK but not by caspase-8 inhibitor Z-IETD–FMK. Taken together, these overall results demonstrated that EET-induced apoptosis of HepG2 cells was mediated by the mitochondrial caspase-dependent intrinsic pathway rather than the death receptor/caspase-8-mediated signaling route.

Keywords

Tetrastigma hemsleyanum HepG2 cells Apoptosis Caspase Ca2+ 

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).

Conflicts of interest

None

Authors’ contributions

Xin Peng contributed for cell culture, western blotting, and statistical analysis and wrote the manuscript. Yuan-yuan Zhang contributed for the detection of cell apoptosis ratio. Jin Wang contributed for the detection of the apoptotic morphological changes. Qingyong Ji was responsible for the preparation of EET.

References

  1. 1.
    Zhao JL, Zhao J, Jiao HJ. 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.
    Fei HR, Chen HL, Xiao T, et al. Caudatin induces cell cycle arrest and caspase-dependent apoptosis in HepG2 cell. Mol Biol Rep. 2012;39:131–8.CrossRefPubMedGoogle Scholar
  4. 4.
    Xu CJ, Ding GQ, Fu JY, et al. 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
  5. 5.
    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
  6. 6.
    Yuming Y, Yili W, Yeling T, et al. 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
  7. 7.
    Peng X, Zhuang DD, Guo QS. Induction of S phase arrest and apoptosis by ethylacetate extract from Tetrastigma hemsleyanum in human hepatoma HepG2 cells. Tumor Biol. 2015. doi: 10.1007/s13277-014 -2869-x.Google Scholar
  8. 8.
    Nagata S. Apoptosis by death factor. Cell. 1997;88:355–65.CrossRefPubMedGoogle Scholar
  9. 9.
    Li P, Zhao QL, Wu LH, et al. Isofraxidin, a potent reactive oxygen species (ROS) scavenger, protects human leukemia cells from radiation-induced apoptosis via ROS/mitochondria pathway in p53-independent manner. Apoptosis. 2014;19:1043–53.CrossRefPubMedGoogle Scholar
  10. 10.
    Wen XX, Jian Z, Dan Z, et al. Denatonium inhibits growth and induces apoptosis of airway epithelial cells through mitochondrial signaling pathways. Respir Res. 2015;16:13–22.CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Byczkowska A, Kunikowska A, Kaźmierczak A. Determination of ACC-induced cell-programmed death in roots of Vicia faba ssp. minor seedlings by acridine orange and ethidium bromide staining. Protoplasma. 2013;250:121–8.CrossRefPubMedGoogle Scholar
  12. 12.
    Kim MS, Bak Y, Park YS, et al. Wogonin induces apoptosis by suppressing E6 and E7 expressions and activating intrinsic signaling pathways in HPV-16 cervical cancer cells. Cell Biol Toxicol. 2013;29:259–72.CrossRefPubMedGoogle Scholar
  13. 13.
    Looi CY, Moharram B, Paydar M, et al. Induction of apoptosis in melanoma A375 cells by a chloroform fraction of Centratherum anthelminticum (L.) seeds involves NF-kappaB, p53 and Bcl-2-controlled mitochondrial signaling pathways. BMC Complem Altern Med. 2013;13:166–79.CrossRefGoogle Scholar
  14. 14.
    Zhang Y, Zhao L, Li X, et al. V8, a newly synthetic flavonoid, induces apoptosis through ROS mediated ER stress pathway in hepatocellular carcinoma. Arch Toxicol. 2014;88:97–107.CrossRefPubMedGoogle Scholar
  15. 15.
    Banfalvi G. Apoptotic agents inducing genotoxicity-specific chromatin changes. Apoptosis. 2014;19:1301–16.CrossRefPubMedGoogle Scholar
  16. 16.
    Frenzel A, Grespi F, Chmelewskij W, et al. Bcl2 family proteins in carcinogenesis and the treatment of cancer. Apoptosis. 2009;14:584–96.CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Sun SL, Guo L, Ren YC, et al. Anti-apoptosis effect of polysaccharide isolated from the seeds of Cuscuta chinensis Lam on cardiomyocytes in aging rats. Mol Biol Rep. 2014;41:6117–24.CrossRefPubMedGoogle Scholar
  18. 18.
    Yan F, Liu Y, Wang WB. 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
  19. 19.
    Li JX, Shen YQ, Cai BZ, et al. Arsenic trioxide induces the apoptosis in vascular smooth muscle cells via increasing intracellular calcium and ROS formation. Mol Biol Rep. 2010;37:1569–76.CrossRefPubMedGoogle Scholar
  20. 20.
    Lazebnik YA, Kaufmann SH, Desnoyers S, et al. Cleavage of poly (ADP-ribose) polymerase by a proteinase with properties like ICE. Nature. 1994;371:346–7.CrossRefPubMedGoogle Scholar
  21. 21.
    Wang XB, Gao HY, Hou BL, et al. Nanoparticle realgar powders induce apoptosis in U937 cells through caspase MAPK and mitochondrial pathways. Arch Pharm Res. 2007;30:653–8.CrossRefPubMedGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2015

Authors and Affiliations

  • Xin Peng
    • 1
  • Yuan-yuan Zhang
    • 2
  • Jin Wang
    • 3
  • Qingyong Ji
    • 1
  1. 1.Institute of BiopharmaceuticalZhejiang Pharmaceutical CollegeNingboChina
  2. 2.Nanjing General Hospital of Nanjing Military CommandNanjingChina
  3. 3.Nanjing Agriculture UniversityNanjingChina

Personalised recommendations