Molecular Mechanism Underlying Hesperetin-induced Apoptosis by in silico Analysis and in Prostate Cancer PC-3 Cells

  • Sambantham, Shanmugam (Department of Genetics, Dr.ALMPGIBMS, University of Madras) ;
  • Radha, Mahendran (Department of Genetics, Dr.ALMPGIBMS, University of Madras) ;
  • Paramasivam, Arumugam (Department of Genetics, Dr.ALMPGIBMS, University of Madras) ;
  • Anandan, Balakrishnan (Department of Genetics, Dr.ALMPGIBMS, University of Madras) ;
  • Malathi, Ragunathan (Department of Genetics, Dr.ALMPGIBMS, University of Madras) ;
  • Chandra, Samuel Rajkumar (Department of Genetics, Dr.ALMPGIBMS, University of Madras) ;
  • Jayaraman, Gopalswamy (Department of Genetics, Dr.ALMPGIBMS, University of Madras)
  • Published : 2013.07.30


Aim: To investigate the molecular mechanisms underlying triggering of apoptosis by hesperetin using in silico and in vitro methods. Methods: The mechanism of binding of hesperetin with NF-${\kappa}B$ and other apoptotic proteins like BAX, BAD, $BCL_2$ and $BCL_{XL}$ was analysed in silico using Schrodinger suite 2009. In vitro studies were also carried out to evaluate the potency of hesperetin in inducing apoptosis using the human prostate cancer PC-3 cell line. Results: Hesperetin was found to exhibit high-affinity binding resulting from greater intermolecular forces between the ligand and its receptor NF-${\kappa}B$ (-7.48 Glide score). In vitro analysis using MTT assay confirmed that hesperetin reduced cell proliferation ($IC_{50}$ values of 90 and $40{\mu}M$ at 24 and 48h respectively) in PC-3 cells. Hesperetin also downregulated expression of the anti-apoptotic gene $BCL_{XL}$ at both mRNA and protein levels and increased the expression of pro-apoptotic genes like BAD at mRNA level and BAX at mRNA as well as protein levels. Conclusion: The results suggest that hesperetin can induce apoptosis by inhibiting NF-${\kappa}B$.




  1. Yamanaka K, Rocchi P, Miyake H, et al (2005). A novel antisense oligonucleotide inhibiting several antiapoptotic Bcl-2 family members induces apoptosis and enhances chemosensitivity in androgen-independent human prostate cancer PC3 cells. Mol Cancer Ther, 4, 1689-98.
  2. Ghosh G, van Duyne G, Ghosh S, Sigler PB (1995). Structure of $NF-{\kappa}B$ p50 homodimer bound to a kB site. Nature, 373, 303-10.
  3. Jemal A, Bray F, Center MM, et al (2011). Global Cancer Statistics. CA Cancer J Clin, 61, 69-90.
  4. Leelavinothan P, Kalist S (2011). Beneficial effect of hesperetin on cadmium induced oxidative stress in rats: an in vivo and in vitro study. Eur Rev Med Pharmacol Sci, 15, 992-1002
  5. Liu D, Huang Z (2011). Synthetic peptides and non-peptidic molecules as probes of structure and function of Bcl-2 family proteins and modulators of apoptosis. Apoptosis, 6, 453-62.
  6. Lowry OH, Rosebrough NJ, Farr AL, Randall RY (1951). Protein measurement with the Folin-phenol reagent. J Biol Chem, 193, 265-75.
  7. Mosmann T (1983). Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods, 65, 55-63.
  8. Nalini N, Aranganathan S, Kabalimurthy J (2012). Chemopreventive efficacy of hesperetin (citrus flavonone) against 1,2-dimethylhydrazine-induced rat colon carcinogenesis. Toxicol Mech Methods, 22, 397-408.
  9. Orallo F, Alvarez E, Basaran H, Lugnier C (2004). Comparative study of the vasorelaxant activity, superoxide-scavenging ability and cyclic nucleotide phosphodiesterase-inhibitory effects of hesperitin and hesperidin. Nauyn-Schmeideberg's Arch Pharmacol, 370, 452-63.
  10. Perkins ND (2012). The diverse and complex roles of $NF-{\kappa}B$ subunits in cancer. Nat Rev Cancer, 12, 121-32.
  11. Ririe KM, Rasmussen RP, Wittwer CT (1997). Product differentiation by analysis of DNA melting curves during polymerase chain reaction. Anal Biochem, 245, 154-60.
  12. Schmittgen TD and Livak KJ (2008). Analyzing real-time PCR data by the comparative CT method. Nature Protocols, 3, 1101-8.
  13. So FV, Guthrie N, Chambers AF, Carroll KK (1997). Inhibition of proliferation of estrogen receptor-positive MCF-7 human reast cancer cells by flavonoids in the presence and absence of excess estrogen. Cancer Lett, 112, 127-33.
  14. Tanaka T, Makita H, Kawabata K, et al (1997). Chemoprevention of azoxymethane-induced rat colon carcinogenesis by the naturally occurring flavonoids, diosmin and hesperidin. Carcinogenesis, 18, 957-65.
  15. van de Loosdrecht AA, Beelen RH, Ossenkoppele GJ, Broekhoven MG, Langenhuijsen MM (1994). A tetrazoliumbased colorimetric MTT assay to quantitate human monocyte mediated cytotoxicity against leukemic cells from cell lines and patients with acute myeloid leukemia. J Immunol Methods, 174, 311-20.
  16. Vladic-Stjernholm Y, Vladic T, Blesson CS, Ekman-Ordeberg G, Sahlin L (2009). Prostaglandin treatment is associated with a withdrawal of progesterone and androgen at the receptor level in the uterine cervix. Reprod Biol Endocrinol, 7, 116.
  17. Xiao G, Fu J (2011). $NF-{\kappa}B$ and cancer: a paradigm of Yin-Yang. Am J Cancer Res, 1, 192-221.
  18. Billen LP, Kokoski CL, Lovell JF, Leber B, Andrews DW (2008). Bcl-XL inhibits membrane permeabilization by competing with Bax. PLoS Biol, 6, 147.
  19. Borradaile NM, Carroll KK, Kurowska EM (1999). Regulation of HepG2 cell apolipoprotein B metabolism by the citrus flavanones hesperetin and naringenin. Lipids, 34, 591-8.
  20. Choi EJ (2007). Hesperetin induced G1-phase cell cycle arrest in human breast cancer MCF-7 cells: involvement of CDK4 and p21. Nutr Cancer, 59, 115-9.
  21. Choi EJ (2008). Antioxidative effects of hesperetin against 7, 12-dimethylbenz(a)anthracene-induced oxidative stress in mice. Life Sciences, 82, 1059-64.
  22. Dolcet X, Llobet D, Pallares J, Matias-Guiu X (2005). $NF-{\kappa}B$ in development and progression of human cancer. Virchows Arch, 446, 475-82.
  23. Elmore S (2007). Apoptosis: a review of programmed cell death. Toxicol Pathol, 35, 495-516.
  24. Elumalai P, Gunadharini DN, Senthilkumar K, et al (2012). Induction of apoptosis in human breast cancer cells by nimbolide through extrinsic and intrinsic pathway. Toxicol Lett, 215, 131-42.

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