Abstract
Hesperetin, a flavonoid from citrus fruits, has been proved to possess biological activity on various types of human cancers. However, few related studies on hepatocellular carcinoma are available. In this study, we aimed to investigate the effect of hesperetin on hepatocellular carcinoma cells in vitro and in vivo and clarify its potentially specific mechanism. Compared with the control group, the proliferations of hepatocellular carcinoma cells in hesperetin groups were significantly inhibited (P < 0.05), and a dose- and time-dependent inhibition of cell viability was observed. When pretreated with H2O2 (1 mM) or N-acetyl-l-cysteine (5 mM), the inhibition of cell viability by hesperetin was enhanced or reduced, respectively (P < 0.05). Similarly, the levels of intracellular ROS, ATP and Ca2+ changed in different groups (P < 0.05). The results of Hoechst 33258 staining showed that the percentages of apoptotic cells in hesperetin groups are remarkably higher than that in control group (P < 0.05). And the results of Western blot showed that hesperetin caused an increase in the levels of cytosolic AIF, cytosolic Apaf-1, cytosolic Cyt C, caspase-3, caspase-9 and Bax and a decrease in that of Bcl-2, mitochondrial AIF, mitochondrial Apaf-1 and mitochondrial Cyt C (P < 0.05). Meanwhile, hesperetin significantly inhibited the growth of xenograft tumors. Our study suggests that hesperetin could inhibit the proliferation and induce the apoptosis of hepatocellular carcinoma via triggering the activation of the mitochondrial pathway by increasing the levels of intracellular ROS, ATP and Ca2+.
Similar content being viewed by others
References
Siegel R, Ma J, Zou Z, Jemal A. Cancer statistics, 2014. CA Cancer J Clin. 2014;64:9–29.
Forner A, Llovet JM, Bruix J. Hepatocellular carcinoma. Lancet. 2012;379:1245–55.
Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, Parkin DM, Forman D, Bray F. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer. 2015;136:E359–86.
Monteil M, Migianu-Griffoni E, Sainte-Catherine O, Di Benedetto M, Lecouvey M. Bisphosphonate prodrugs: synthesis and biological evaluation in HuH7 hepatocarcinoma cells. Eur J Med Chem. 2014;77:56–64.
Tang TC, Man S, Lee CR, Xu P, Kerbel RS. Impact of metronomic UFT/cyclophosphamide chemotherapy and antiangiogenic drug assessed in a new preclinical model of locally advanced orthotopic hepatocellular carcinoma. Neoplasia. 2010;12:264–74.
Gil-Izquierdo A, Gil MI, Ferreres F, Tomás-Barberán FA. In vitro availability of flavonoids and other phenolics in orange juice. J Agric Food Chem. 2001;49:1035–41.
Garg A, Garg S, Zaneveld LJ, Singla AK. Chemistry and pharmacology of the citrus bioflavonoid hesperidin. Phytother Res. 2001;15:655–69.
Choi EJ. Hesperetin induced G1-phase cell cycle arrest in human breast cancer MCF-7 cells: involvement of CDK4 and p21. Nutr Cancer. 2007;59:115–9.
Cai Y, Luo Q, Sun M, Corke H. Antioxidant activity and phenolic compounds of 112 traditional Chinese medicinal plants associated with anticancer. Life Sci. 2004;12(74):2157–84.
Sambantham S, Radha M, Paramasivam A, Anandan B, Malathi R, Chandra SR, Jayaraman G. Molecular mechanism underlying hesperetin-induced apoptosis by in silico analysis and in prostate cancer PC-3 cells. Asian Pac J Cancer Prev. 2013;14:4347–52.
Ye L, Chan FL, Chen S, Leung LK. The citrus flavonone hesperetin inhibits growth of aromatase-expressing MCF-7 tumor in ovariectomized athymic mice. J Nutr Biochem. 2012;23:1230–7.
Alshatwi AA, Ramesh E, Periasamy VS, Subash-Babu P. The apoptotic effect of hesperetin on human cervical cancer cells is mediated through cell cycle arrest, death receptor, and mitochondrial pathways. Fundam Clin Pharmacol. 2013;27:581–92.
Aranganathan S, Nalini N. Antiproliferative efficacy of hesperetin (citrus flavanoid) in 1,2-dimethylhydrazine-induced colon cancer. Phytother Res. 2013;27:999–1005.
O’Prey J, Brown J, Fleming J, Harrison PR. Effects of dietary flavonoids on major signal transduction pathways in human epithelial cells. Biochem Pharmacol. 2003;66:2075–88.
Yang HL, Chen SC, Senthil Kumar KJ, Yu KN, Lee Chao PD, Tsai SY, Hou YC, Hseu YC. Antioxidant and anti-inflammatory potential of hesperetin metabolites obtained from hesperetin-administered rat serum: an ex vivo approach. J Agric Food Chem. 2012;60:522–32.
Haidari F, Ali Keshavarz S, Reza Rashidi M, Shahi M. Orange juice and hesperetin supplementation to hyperuricemic rats alter oxidative stress markers and xanthine oxidoreductase activity. J Clin Biochem Nutr. 2009;45:285–91.
Sivagami G, Vinothkumar R, Bernini R, Preethy CP, Riyasdeen A, Akbarsha MA, Menon VP, Nalini N. Role of hesperetin (a natural flavonoid) and its analogue on apoptosis in HT-29 human colon adenocarcinoma cell line—a comparative study. Food Chem Toxicol. 2012;50:660–71.
Bae YS, Oh H, Rhee SG, Yoo YD. Regulation of reactive oxygen species generation in cell signaling. Mol Cells. 2011;32:491–509.
Alfadda AA, Sallam RM. Reactive oxygen species in health and disease. J Biomed Biotechnol. 2012;2012:936486.
Matsuzawa A, Ichijo H. Stress-responsive protein kinases in redox-regulated apoptosis signaling. Antioxid Redox Signal. 2005;7:472–81.
Tarasov AI, Griffiths EJ, Rutter GA. Regulation of ATP production by mitochondrial Ca(2 +). Cell Calcium. 2012;52:28–35.
Naranjo JR, Mellström B. Ca2+-dependent transcriptional control of Ca2+ homeostasis. J Biol Chem. 2012;287:31674–80.
Lewis A, Hayashi T, Su TP, Betenbaugh MJ. Bcl-2 family in inter-organelle modulation of calcium signaling; roles in bioenergetics and cell survival. J Bioenerg Biomembr. 2014;46:1–15.
Berridge MJ, Bootman MD, Roderick HL. Calcium signalling: dynamics, homeostasis and remodelling. Nat Rev Mol Cell Biol. 2003;4:517–29.
Felsenfeld A, Rodriguez M, Levine B. New insights in regulation of calcium homeostasis. Curr Opin Nephrol Hypertens. 2013;22:371–6.
Brini M, Calì T, Ottolini D, Carafoli E. The plasma membrane calcium pump in health and disease. FEBS J. 2013;280:5385–97.
Samanta K, Douglas S, Parekh AB. Mitochondrial calcium uniporter MCU supports cytoplasmic Ca2+ oscillations, store-operated Ca2+ entry and Ca2+-dependent gene expression in response to receptor stimulation. PLoS ONE. 2014;9:e101188.
Brookes PS, Yoon Y, Robotham JL, Anders MW, Sheu SS. Calcium, ATP, and ROS: a mitochondrial love-hate triangle. Am J Physiol Cell Physiol. 2004;287:C817–33.
Brinkkoetter PT, Song H, Lösel R, Schnetzke U, Gottmann U, Feng Y, Hanusch C, Beck GC, Schnuelle P, Wehling M, van der Woude FJ, Yard BA. Hypothermic injury: the mitochondrial calcium, ATP and ROS love-hate triangle out of balance. Cell Physiol Biochem. 2008;22:195–204.
Voronina S, Okeke E, Parker T, Tepikin A. How to win ATP and influence Ca(2 +) signaling. Cell Calcium. 2014;55:131–8.
Yang Y, Wolfram J, Shen H, Fang X, Ferrari M. Hesperetin: an inhibitor of the transforming growth factor-β (TGF-β) signaling pathway. Eur J Med Chem. 2012;58:390–5.
Yang Y, Wolfram J, Boom K, Fang X, Shen H, Ferrari M. Hesperetin impairs glucose uptake and inhibits proliferation of breast cancer cells. Cell Biochem Funct. 2013;31:374–9.
Green DR, Kroemer G. The pathophysiology of mitochondrial cell death. Science. 2004;305:626–9.
Chen Q, Lesnefsky EJ. Blockade of electron transport during ischemia preserves bcl-2 and inhibits opening of the mitochondrial permeability transition pore. FEBS Lett. 2011;585:921–6.
Acknowledgments
The study was supported by research grants from the Natural Science Foundation of Hubei Province (No. 2014CKB494).
Conflict of interest
None.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Zhang, J., Song, J., Wu, D. et al. Hesperetin induces the apoptosis of hepatocellular carcinoma cells via mitochondrial pathway mediated by the increased intracellular reactive oxygen species, ATP and calcium. Med Oncol 32, 101 (2015). https://doi.org/10.1007/s12032-015-0516-z
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s12032-015-0516-z