DOI QR코드

DOI QR Code

Induction of Human Hepatocellular Carcinoma HepG2 Cell Apoptosis by Naringin

  • Banjerdpongchai, Ratana (Department of Biochemistry, Faculty of Medicine, Chiang Mai University) ;
  • Wudtiwai, Benjawan (Department of Biochemistry, Faculty of Medicine, Chiang Mai University) ;
  • Khaw-on, Patompong (Department of Biochemistry, Faculty of Medicine, Chiang Mai University)
  • Published : 2016.07.01

Abstract

Naringin, a bioflavonoid found in Citrus seeds, inhibits proliferation of cancer cells. The objectives of this study were to investigate the mode and mechanism(s) of hepatocellular carcinoma HepG2 cell death induced by naringin. The cytotoxicity of naringin towards HepG2 cells proved dose-dependent, measured by MTT assay. Naringin-treated HepG2 cells underwent apoptosis also in a concentration related manner, determined by annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) employing flow cytometry. Mitochondrial transmembrane potential (MTP) measured using 3,3'-dihexyloxacarbocyanine iodide ($DiOC_6$) and flow cytometer was reduced concentration-dependently, which indicated influence on the mitochondrial signaling pathway. Caspase-3, -8 and -9 activities were enhanced as evidenced by colorimetric detection of para-nitroaniline tagged with a substrate for each caspase. Thus, the extrinsic and intrinsic pathways were linked in human naringin-treated HepG2 cell apoptosis. The expression levels of pro-apoptotic Bax and Bak proteins were increased whereas that of the anti-apoptotic Bcl-xL protein was decreased, confirming the involvement of the mitochondrial pathway by immunoblotting. There was an increased expression of truncated Bid (tBid), which indicated caspase-8 proteolysis activity in Bid cleavage as its substrate in the extrinsic pathway. In conclusion, naringin induces human hepatocellular carcinoma HepG2 cell apoptosis via mitochondria-mediated activation of caspase-9 and caspase-8-mediated proteolysis of Bid. Naringin anticancer activity warrants further investigation for application in medical treatment.

Keywords

Naringin;hepatocellular carcinoma cells;HepG2 cells;apoptosis;caspase-9;caspase-8

Acknowledgement

Supported by : Thailand Research Fund (TRF)

References

  1. Arts MJ, Haenen GR, Wilms LC, et al (2002). Interactions between flavonoids and proteins: effect on the total antioxidant capacity. J Agric Food Chem, 50, 1184-7. https://doi.org/10.1021/jf010855a
  2. Arul D, Subramanian P (2013). Naringenin (Citrus flavonone) induces growth inhibition, cell cycle arrest and apoptosis in human hepatocellular carcinoma cells. Pathol Oncol Res, 19, 763-70. https://doi.org/10.1007/s12253-013-9641-1
  3. Banjerdpongchai R and Wilairat P (2005). Effects of watersoluble antioxidants and MAPKK/MEK inhibitor on curcumin-induced apoptosis in HL-60 human leukemic cells. Asian Pac J Cancer Prev, 6, 282-5.
  4. Banjerdpongchai R, Wudtiwai B, Khaw-On P, et al (2016). Hesperidin from Citrus seed induces human hepatocellular carcinoma HepG2 cell apoptosis via both mitochondrial and death receptor pathways. Tumour Biol, 37, 227-37. https://doi.org/10.1007/s13277-015-3774-7
  5. Banjerdpongchai R, Wudtiwai B, Pompimon W (2014). Stigmalactam from Orophea enterocarpa induces human cancer cell apoptosis via a mitochondrial pathway. Asian Pac J Cancer Prev, 15, 10397-400.
  6. Banjerdpongchai R, Wudtiwai B, Pompimon W (2015). Enterocarpam-III induces human liver and breast cancer cell apoptosis via mitochondrial and caspase-9 activation. Asian Pac J Cancer Prev, 16, 1833-7. https://doi.org/10.7314/APJCP.2015.16.5.1833
  7. Banjerdpongchai R, Wudtiwai B, Sringarm K (2013). Cytotoxic and apoptotic-inducing effects of purple rice extracts and chemotherapeutic drugs on human cancer cell lines. Asian Pac J Cancer Prev, 14, 6541-8. https://doi.org/10.7314/APJCP.2013.14.11.6541
  8. Cao G, Sofic E, Prior RL (1997). Antioxidant and prooxidant behavior of flavonoids: structure-activity relationships. Free Radic Biol Med, 22, 749-60. https://doi.org/10.1016/S0891-5849(96)00351-6
  9. Doostdar H, Burke MD, Mayer RT (2000). Bioflavonoids: selective substrates and inhibitors for cytochrome P450 CYP1A and CYP1B1. Toxicology, 144, 31-8. https://doi.org/10.1016/S0300-483X(99)00215-2
  10. Gee JM, DuPont MS, Rhodes MJ, et al (1998). Quercetin glucosides interact with the intestinal glucose transport pathway. Free Radic Biol Med, 25, 19-25. https://doi.org/10.1016/S0891-5849(98)00020-3
  11. Heim KE, Tagliaferro AR, Bobilya DJ (2002). Flavonoid antioxidants: chemistry, metabolism and structure-activity relationships. J Nutr Biochem, 13, 572-84. https://doi.org/10.1016/S0955-2863(02)00208-5
  12. Kanno S, Shouji A, Asou K, et al (2003). Effects of naringin on hydrogen peroxide-induced cytotoxicity and apoptosis in P388 cells. J Pharmacol Sci, 92, 166-70. https://doi.org/10.1254/jphs.92.166
  13. Kanno S, Shouji A, Hirata R, et al (2004). Effects of naringin on cytosine arabinoside (Ara-C)-induced cytotoxicity and apoptosis in P388 cells. Life Sci, 75, 353-65. https://doi.org/10.1016/j.lfs.2003.12.019
  14. Kanno S, Tomizawa A, Hiura T, et al (2005). Inhibitory effects of naringenin on tumor growth in human cancer cell lines and sarcoma S-180-implanted mice. Biol Pharm Bull, 28, 527-30. https://doi.org/10.1248/bpb.28.527
  15. Larsen AK, Moller MT, Blankson H, et al (2002). Naringinsensitive phosphorylation of plectin, a cytoskeletal crosslinking protein, in isolated rat hepatocytes. J Biol Chem, 277, 34826-35. https://doi.org/10.1074/jbc.M205028200
  16. Lewinska A, Siwak J, Rzeszutek I, Wnuk, M. (2015). Diosmin induces genotoxicity and apoptosis in DU145 prostate cancer cell line. Toxicol In Vitro, 29, 417-25. https://doi.org/10.1016/j.tiv.2014.12.005
  17. Li H, Yang B, Huang J, et al (2013). Naringin inhibits growth potential of human triple-negative breast cancer cells by targeting beta-catenin signaling pathway. Toxicol Lett, 220, 219-28. https://doi.org/10.1016/j.toxlet.2013.05.006
  18. Lu JV, Walsh CM (2012). Programmed necrosis and autophagy in immune function. Immunol Rev, 249, 205-17. https://doi.org/10.1111/j.1600-065X.2012.01147.x
  19. Raha S, Yumnam S, Hong GE, et al (2015). Naringin induces autophagy-mediated growth inhibition by downregulating the PI3K/Akt/mTOR cascade via activation of MAPK pathways in AGS cancer cells. Int J Oncol, 47, 1061-9. https://doi.org/10.3892/ijo.2015.3095
  20. Ramesh E, Alshatwi AA (2013). Naringin induces death receptor and mitochondria-mediated apoptosis in human cervical cancer (SiHa) cells. Food Chem Toxicol, 51, 97-105. https://doi.org/10.1016/j.fct.2012.07.033
  21. Rice-Evans CA, Miller NJ (1996). Antioxidant activities of flavonoids as bioactive components of food. Biochem Soc Transact, 24, 790-5. https://doi.org/10.1042/bst0240790
  22. Rice-Evans CA, Miller NJ, Paganga G (1996). Structureantioxidant activity relationships of flavonoids and phenolic acids. Free Radic Biol Med, 20, 933-56. https://doi.org/10.1016/0891-5849(95)02227-9
  23. Safa AR (2013). Roles of c-FLIP in apoptosis, necroptosis, and autophagy. J Carcinog Mutagen, 6, Pii003.
  24. Shalini S, Dorstyn L, Dawar S, Kumar S (2015). Old, new and emerging functions of caspases. Cell Death Differ, 22, 526-39. https://doi.org/10.1038/cdd.2014.216
  25. Spencer JP, Chowrimootoo G, Choudhury R, et al (1999). The small intestine can both absorb and glucuronidate luminal flavonoids. FEBS Lett, 458, 224-30. https://doi.org/10.1016/S0014-5793(99)01160-6
  26. Su Z, Yang Z, Xu Y, et al (2015). MicroRNAs in apoptosis, autophagy and necroptosis. Oncotarget, 6, 8474-90. https://doi.org/10.18632/oncotarget.3523
  27. Tan TW, Chou YE, Yang WH, et al (2014). Naringin suppress chondrosarcoma migration through inhibition vascular adhesion molecule-1 expression by modulating miR-126. Int Immunopharmacol, 22, 107-14. https://doi.org/10.1016/j.intimp.2014.06.029
  28. Ugocsai K, Varga A, Molnar P, et al (2005). Effects of selected flavonoids and carotenoids on drug accumulation and apoptosis induction in multidrug-resistant colon cancer cells expressing MDR1/LRP. In Vivo, 19, 433-8.
  29. Vanamala J, Leonardi T, Patil BS, et al (2006). Suppression of colon carcinogenesis by bioactive compounds in grapefruit. Carcinogenesis, 27, 1257-65. https://doi.org/10.1093/carcin/bgi318
  30. Vela L, Marzo I (2015). Bcl-2 family of proteins as drug targets for cancer chemotherapy: the long way of BH3 mimetics from bench to bedside. Curr Opin Pharmacol, 23, 74-81. https://doi.org/10.1016/j.coph.2015.05.014
  31. Zeng LX, Tao J, Liu HL, et al (2014). beta-Arrestin2 encourages inflammation-induced epithelial apoptosis through ER stress/PUMA in colitis. Mucosal Immunol, 35, 1932-43.