Cellular Oncology

, Volume 38, Issue 3, pp 195–204 | Cite as

Anti-proliferative, apoptotic and signal transduction effects of hesperidin in non-small cell lung cancer cells

  • Zeynep Birsu Cincin
  • Miray Unlu
  • Bayram Kiran
  • Elif Sinem Bireller
  • Yusuf Baran
  • Bedia CakmakogluEmail author
Original Paper



Hesperidin, a glycoside flavonoid, is thought to act as an anti-cancer agent, since it has been found to exhibit both pro-apoptotic and anti-proliferative effects in several cancer cell types. The mechanisms underlying hesperidin-induced growth arrest and apoptosis are, however, not well understood. Here, we aimed to investigate the anti-proliferative and apoptotic effects of hesperidin on non-small cell lung cancer (NSCLC) cells and to investigate the mechanisms involved.


The anti-proliferative and apoptotic effects of hesperidin on two NSCLC-derived cell lines, A549 and NCI-H358, were determined using a WST-1 colorimetric assay, a LDH cytotoxicity assay, a Cell Death Detection assay, an AnnexinV-FITC assay, a caspase-3 assay and a JC-1 assay, respectively, all in a time- and dose-dependent manner. As a control, non-cancerous MRC-5 lung fibroblasts were included. Changes in whole genome gene expression profiles were assessed using an Illumina Human HT-12v4 beadchip microarray platform, and subsequent data analyses were performed using an Illumina Genome Studio and Ingenuity Pathway Analyser (IPA).


We found that after hesperidin treatment, A549 and NCI-H358 cells exhibited decreasing cell proliferation and increasing caspase-3 and other apoptosis-related activities, in conjunction with decreasing mitochondrial membrane potential activities, in a dose- and time-dependent manner. Through a GO analysis, by which changes in gene expression profiles were compared, we found that the FGF and NF-κB signal transduction pathways were most significantly affected in the hesperidin treated NCI-H358 and A549 NSCLC cells.


Our results indicate that hesperidin elicits an in vitro growth inhibitory effect on NSCLC cells by modulating immune response-related pathways that affect apoptosis. When confirmed in vivo, hesperidin may serve as a novel anti-proliferative agent for non-small cell lung cancer.


Non-small cell lung cancer Hesperidin Anti-proliferative effect Apoptosis Gene expression profile 



Nucleosomal enrichment factor


Fetal bovine serum


Fibroblast growth factor


Fluorescein ısothiocyanate


Ingenuity pathway analysis


Lactate dehydrogenase


Eagle’s minimum essential medium


Nuclear factor kappa B


Non-small cell lung cancer




Roswell Park Memorial Institute-1640



This work was funded by Istanbul University Scientific Research Project number 9205. We would like to thank Mr. David Chapman for editing the manuscript.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

13402_2015_222_Fig7_ESM.gif (33 kb)
Supplemental Fig. 7 FGF signaling network. FGF network of 50 μM hesperidin-stimulated genes in A549 (a), NCI-H358(b) and MRC-5(c) cells. Ingenuity pathways analysis (IPA) software was used to identify genes involved in the FGF signaling that were differentially expressed in A549, NCI-H358 and MRC-5 cells. Genes labeled in red and green were identified as up- and down-regulated, respectively, whereas other genes were identified on the basis of the network analysis. (GIF 32 kb)
13402_2015_222_MOESM1_ESM.tif (1.7 mb)
Fig. 7 High Resolution Image (TIFF 1736 kb)
13402_2015_222_Fig8_ESM.jpg (132 kb)
Supplemental Fig. 8 NF-κB signaling network. NF-κB network of 50 μM hesperidin-stimulated genes in NCI-H358 cells. Ingenuity pathways analysis (IPA) software was used to identify genes involved in the NF-κB signaling that were differentially expressed in NCI-H358 cells. Genes labeled in red and green were identified as up- and down-regulated, respectively, whereas other genes were identified on the basis of the network analysis. (JPEG 131 kb)
13402_2015_222_MOESM2_ESM.docx (15 kb)
ESM 1 (DOCX 14 kb)
13402_2015_222_MOESM3_ESM.docx (15 kb)
ESM 2 (DOCX 14 kb)
13402_2015_222_MOESM4_ESM.doc (31 kb)
ESM 3 (DOC 31 kb)
13402_2015_222_MOESM5_ESM.docx (16 kb)
ESM 4 (DOCX 15 kb)


  1. 1.
    W. Pao, N. Girard, New driver mutations in non-small-cell lung cancer. Lancet Oncol. 12, 175–180 (2011)CrossRefPubMedGoogle Scholar
  2. 2.
    W.D. Travis, Classification of lung cancer. Semin. Roentgenol. 46, 178–186 (2011)CrossRefPubMedGoogle Scholar
  3. 3.
    Q. Wu, Y.F. Chen, J. Fu, Q.H. You, S.M. Wang, X. Huang, X.J. Feng, S.H. Zhang, Short hairpin RNA-mediated down-regulation of CENP-A attenuates the aggressive phenotype of lung adenocarcinoma cells. Cell. Oncol. 37, 399–407 (2014)CrossRefGoogle Scholar
  4. 4.
    A. Koren, H. Motaln, T. Cufer, Lung cancer stem cells: a biological and clinical perspective. Cell. Oncol. 36, 265–275 (2013)CrossRefGoogle Scholar
  5. 5.
    N. Peled, M.W. Wynes, N. Ikeda, T. Ohira, K. Yoshida, J. Qian, M. Ilouze, R. Brenner, Y. Kato, C. Mascaux, F.R. Hirsch, Insulin-like growth factor-1 receptor (IGF-1R) as a biomarker for resistance to the tyrosine kinase inhibitor gefitinib in non-small cell lung cancer. Cell. Oncol. 36, 277–288 (2013)CrossRefGoogle Scholar
  6. 6.
    A. Maier, AL. Peille, V. Vuaroqueaux, M. Lahn. Anti-tumor activity of the TGF-β receptor kinase inhibitor galunisertib (LY2157299 monohydrate) in patient-derived tumor xenografts. Cell. Oncol. 2015 Jan 9. [Epub ahead of print] DOI  10.1007/s13402-014-0210-8
  7. 7.
    P. Ulivi, R. Silvestrini, Role of quantitative and qualitative characteristics of free circulating DNA in the management of patients with non-small cell lung cancer. Cell. Oncol. 36, 439–448 (2013)CrossRefGoogle Scholar
  8. 8.
    G. Giaccone, Twenty-five years of treating advanced NSCLC: what have we achieved? Ann. Oncol. 15 Suppl 4: iv81–83 (2004)Google Scholar
  9. 9.
    J.W. Neal, L.V. Sequist, Exciting new targets in lung cancer therapy: ALK, IGF-1R, HDAC, and Hh. Curr. Treat. Options in Oncol. 11, 36–44 (2010)CrossRefGoogle Scholar
  10. 10.
    R. Sangha, P.N. Lara, P.C. Mack, D.R. Gandara, Beyond antiepidermal growth factor receptors and antiangiogenesis strategies for nonsmall cell lung cancer: exploring a new frontier. Curr. Opin. Oncol. 21, 116–123 (2009)CrossRefPubMedGoogle Scholar
  11. 11.
    S. Mateen, K. Raina, R. Agarwal, Chemopreventive and anti-cancer efficacy of silibinin against growth and progression of lung cancer. Nutr. Cancer J. 65, 3–11 (2013)CrossRefGoogle Scholar
  12. 12.
    J. Nones, T.C.E. Spohr, F.C. Gomes, Hesperidin, a flavone glycoside, as mediator of neuronal survival. Neurochem. Res. 36, 1776–1784 (2011)CrossRefPubMedGoogle Scholar
  13. 13.
    T. Tanaka, R. Takahashi, Flavonoids and asthma. Nutrients 10, 2128–2143 (2013)CrossRefGoogle Scholar
  14. 14.
    J.A. Manthey, K. Grohmann, N. Guthrie, Biological properties of citrus flavonoids pertaining to cancer and inflammation. Curr. Med. Chem. 8, 135–153 (2001)CrossRefPubMedGoogle Scholar
  15. 15.
    J. Yu, L. Wang, R.L. Walzem, E.G. Miller, L.M. Pike, B.S. Patil, Antioxidant activity of citrus limonoids, flavonoids, and coumarins. J. Agric. Food Chem. 53, 2009–2014 (2005)CrossRefPubMedGoogle Scholar
  16. 16.
    S.L. Hwang, P.H. Shih, G.C. Yen, Neuroprotective effects of citrus flavonoids. J. Agric. Food Chem. 60, 877–885 (2012)CrossRefPubMedGoogle Scholar
  17. 17.
    E. Meiyanto, A. Hermawan, Anindyajati: natural products for cancer-targeted therapy: citrus flavonoids as potent chemopreventive agents. Asian. Pac. J. Cancer Prev. 13, 427–1436 (2012)CrossRefPubMedGoogle Scholar
  18. 18.
    J.A. Manthey, N. Guthrie, Antiproliferative activities of citrus flavonoids against six human cancer cell lines. J. Agric. Food Chem. 50, 5837–5843 (2002)CrossRefPubMedGoogle Scholar
  19. 19.
    G. Saiprasad, P. Chitra, R. Manikandan, G. Sudhandiran, Hesperidin alleviates oxidative stress and downregulates the expressions of proliferative and inflammatory markers in azoxymethane-induced experimental colon carcinogenesis in mice. Inflamm. Res. 62, 425–440 (2013)CrossRefPubMedGoogle Scholar
  20. 20.
    O.G. Benavente, J. Castillo, M. Alcaraz, V. Vicente, J.A. Del, A. Ortuno, Beneficial action of Citrus flavonoids on multiple cancer-related biological pathways. Curr. Cancer Drug Targets 7, 795–809 (2007)CrossRefGoogle Scholar
  21. 21.
    J. Nones, T.C.E. Spohr, F.C. Gomes, Hesperidin, a flavone glycoside, as mediator of neuronal survival. Neurochem. Res. 36, 1776–1784 (2011)CrossRefPubMedGoogle Scholar
  22. 22.
    V. Gaur, A. Kumar, Hesperidin pre-treatment attenuates NO-mediated cerebral ischemic reperfusion injury and memory dysfunction. Pharmacol. Rep. 62, 635–648 (2010)CrossRefPubMedGoogle Scholar
  23. 23.
    S. Ou, Pharmacological action of hesperidin. Zhong. Yao. Cai. 25, 531–533 (2002)PubMedGoogle Scholar
  24. 24.
    O. Benavente-Garcia, J. Castillo, Update on uses and properties of citrus flavonoids: new findings in anticancer, cardiovascular, and anti-inflammatory activity. J. Agric. Food Chem. 56, 6185–6205 (2008)CrossRefPubMedGoogle Scholar
  25. 25.
    A. Chanet, D. Milenkovic, C. Manach, A. Mazur, C. Morand, Citrus flavanones: what is their role in cardiovascular protection? J. Agric. Food Chem. 60, 8809–8822 (2012)CrossRefPubMedGoogle Scholar
  26. 26.
    B.D. Sahu, M. Kuncha, G.J. Sindhura, R. Sistla, Hesperidin attenuates cisplatin-induced acute renal injury by decreasing oxidative stress, inflammation and DNA damage. Phytomedicine 20, 453–460 (2013)CrossRefPubMedGoogle Scholar
  27. 27.
    J.R. Patil, K.N. Chidambara Murthy, G.K. Jayaprakasha, M.B. Chetti, B.S. Patil, Bioactive compounds from Mexican lime [Citrus aurantifolia] juice induce apoptosis in human pancreatic cells. J. Agric. Food Chem 57, 10933–10942 (2009)CrossRefPubMedGoogle Scholar
  28. 28.
    M. Galleano, O. Pechanova, C.G. Fraga, Hypertension, nitric oxide, oxidants, and dietary plant polyphenols. Curr. Pharm. Biotechnol. 11, 837–848 (2010)CrossRefPubMedGoogle Scholar
  29. 29.
    H.J. Park, M.J. Kim, E. Ha, J.H. Chung, Apoptotic effect of hesperidin through caspase3 activation in human colon cancer cells, SNU-C4. Phytomedicine 15, 147–151 (2008)CrossRefPubMedGoogle Scholar
  30. 30.
    A. Ghorban, M. Nazari, M. Jeddi-Tehrani, H. Zand, The citrus flavonoid hesperidin induces p53 and inhibits NF-κB activation in order to trigger apoptosis in NALM-6 cells: involvement of PPARγ-dependent mechanism. Eur. J. Nutr. 51, 39–46 (2012)CrossRefGoogle Scholar
  31. 31.
    M. Nazari, A. Ghorbani, A. Hekmat-Doost, M. Jeddi-Tehrani, H. Zand, Inactivation of nuclear factor-κB by citrus flavanone hesperidin contributes to apoptosis and chemo-sensitizing effect in Ramos cells. Eur. J. Pharmacol. 650, 526–533 (2011)CrossRefPubMedGoogle Scholar
  32. 32.
    K. Mohankumar, S. Pajaniradje, S. Sridharan, V.K. Singh, L. Ronsard, A.C. Banerjea, B.C. Selvanesan, M.S. Coumar, L. Periyasamy, R. Rajagopalan, Apoptosis induction by an analog of curcumin (BDMC-A) in human laryngeal carcinoma cells through intrinsic and extrinsic pathways. Cell. Oncol. 37, 439–454 (2014)CrossRefGoogle Scholar
  33. 33.
    S. Aranganathan, N. Nalini, Efficacy of the potential chemopreventive agent, hesperetin [citrus flavanone], on 1,2-dimethylhydrazine induced colon carcinogenesis. Food Chem. Toxicol. 47, 2594–2600 (2009)CrossRefPubMedGoogle Scholar
  34. 34.
    C.J. Lee, L. Wilson, M.A. Jordan, V. Nguyen, J. Tang, G. Smiyun, Hesperidin suppressed proliferations of both human breast cancer and androgen-dependent prostate cancer cells. Phytother. Res. Suppl 1, S15–S19 (2010)CrossRefGoogle Scholar
  35. 35.
    G. Saiprasad, P. Chitra, R. Manikandan, G. Sudhandiran, Hesperidin induces apoptosis and triggers autophagic markers through inhibition of Aurora-A mediated phosphoinositide-3-kinase/Akt/mammalian target of rapamycin and glycogen synthase kinase-3 beta signalling cascades in experimental colon carcinogenesis. Eur. J. Cancer 50, 2489–2507 (2014)CrossRefPubMedGoogle Scholar
  36. 36.
    S. Yumnam, HS. Park, MK. Kim, A. Nagappan, GE. et al, Hesperidin induces paraptosis like cell death in hepatoblatoma, HepG2 cells: involvement of ERK1/2 MAPK. PLoS One 30;9(6):e101321 (2014)Google Scholar

Copyright information

© International Society for Cellular Oncology 2015

Authors and Affiliations

  • Zeynep Birsu Cincin
    • 1
  • Miray Unlu
    • 2
  • Bayram Kiran
    • 3
  • Elif Sinem Bireller
    • 1
  • Yusuf Baran
    • 2
  • Bedia Cakmakoglu
    • 1
    Email author
  1. 1.Institute of Experimental Medical Research, Department of Molecular MedicineIstanbul UniversityCapaTurkey
  2. 2.Department of Molecular Biology and GeneticsIzmir Institute of TechnologyUrlaTurkey
  3. 3.Department of BiologyKastamonu UniversityKastamonuTurkey

Personalised recommendations