, Volume 70, Issue 2, pp 831–842 | Cite as

Melilotus indicus extract induces apoptosis in hepatocellular carcinoma cells via a mechanism involving mitochondria-mediated pathways

  • Amer Ali Abd El-Hafeez
  • Hazim O. Khalifa
  • Rania Abdelrahman Elgawish
  • Samia A. Shouman
  • Magdy Hussein Abd El-Twab
  • Seiji Kawamoto
Original Article


Melilotus indicus, is a traditional medicine used as analgesic and emollient. Although Melilotus indicus extract (MIE) has recently been shown to suppress growth of several tumor cell lines, information regarding its antitumor mechanism is completely unknown. Here, we report the mechanism underlying the effects of MIE on human hepatocellular carcinoma cells, specifically HepG2, and SNU-182 cells. Methanolic MIE impaired the proliferation, and induced cell death in both HepG2 and SNU-182 cells but not in normal hepatic L-02 cells. Mechanistically, flow cytometric analysis revealed that MIE induces apoptosis in HepG2, and SNU-182 cells. However, MIE-induced apoptosis were not affected by a pan caspase inhibitor z-VAD-fmk as well as MIE did not stimulate caspase activation. Furthermore we found that MIE-induced apoptosis could be attributed to a mechanism involving mitochondria-mediated pathways evidenced by decrease in the mitochondrial membrane potential (ΔΨm), increase in the Bax/Bcl-2 ratio, and translocation of apoptosis inducing factor (AIF) from the mitochondria to the nucleus. Suppression in AIF expression by siRNA reduced MIE-induced apoptosis which suggested the dependency of MIE on AIF to induce apoptosis in hepatocellular carcinoma cells. To the best of our knowledge this is the first report elucidating the anticancer mechanism of MIE. Our findings suggested that MIE might be a good extract for developing anticancer drugs for human hepatocellular carcinoma treatment.


Melilotus indicus Cancer Apoptosis AIF 


Compliance with ethical standards

Conflict of interest

All authors declare that they have no conflicts of interest.

Supplementary material

10616_2018_195_MOESM1_ESM.pptx (59 kb)
Supplementary material 1 (PPTX 58 kb)


  1. Abd El-Hafeez AA, Rakha OM (2017) Paederus alfieri extract induces apoptosis in human myeloid leukemia K562 cells. Asian J Pharm Clin Res 10:1–4Google Scholar
  2. Abd El-Hafeez AA, Fujimura T, Kamei R, Hirakawa N, Baba K, Ono K, Kawamoto S (2017a) A methoxyflavanone derivative from the Asian medicinal herb (Perilla frutescens) induces p53-mediated G2/M cell cycle arrest and apoptosis in A549 human lung adenocarcinoma. Cytotechnology. Google Scholar
  3. Abd El-Hafeez AA, Fujimura T, Kamei R, Hirakawa N, Baba K, Ono K, Kawamoto S (2017b) Synergistic tumor suppression by a Perilla frutescens-derived methoxyflavanone and anti-cancer tyrosine kinase inhibitors in A549 human lung adenocarcinoma. Cytotechnology. Google Scholar
  4. Abu-Dahab R, Afifi F (2007) Antiproliferative activity of selected medicinal plants of Jordan against a breast adenocarcinoma cell line (MCF7). Sci Pharm 75:121–146CrossRefGoogle Scholar
  5. Ahmed SAK, Al-Refai M (2014) Chemical constituents and cytotoxic activities of the extracts of Melilotus indicus. Eur J Chem 5:503–506CrossRefGoogle Scholar
  6. Ahmed D, Baig H, Zara S (2012) Seasonal variation of phenolics, flavonoids, antioxidant and lipid peroxidation inhibitory activity of methanolic extract of Melilotus indicus and its sub-fractions in different solvents. Int J Phytomed 4:326–332Google Scholar
  7. Al Sherif EA (2009) Melilotus indicus (L.) All., a salt-tolerant wild leguminous herb with high potential for use as a forage crop in salt-affected soils. Flora Morphol Distrib Funct Ecol Plants 204:737–746CrossRefGoogle Scholar
  8. Basañez G, Soane L, Hardwick JM (2012) A new view of the lethal apoptotic pore. PLoS Biol 10:e1001399CrossRefGoogle Scholar
  9. Bleicken S, Landeta O, Landajuela A, Basañez G, García-Sáez AJ (2013) Proapoptotic Bax and Bak proteins form stable protein-permeable pores of tunable size. J Biol Chem 288:33241–33252CrossRefGoogle Scholar
  10. Cabon L, Galán-Malo P, Bouharrour A, Delavallée L, Brunelle-Navas MN, Lorenzo HK, Gross A, Susin SA (2012) BID regulates AIF-mediated caspase-independent necroptosis by promoting BAX activation. Cell Death Differ 19:245–256CrossRefGoogle Scholar
  11. Cohen GM (1997) Caspases: the executioners of apoptosis. Biochem J 326:1–16CrossRefGoogle Scholar
  12. Dai L, Ren P, Liu M, Imai H, Tan EM, Zhang J-Y (2014) Using immunomic approach to enhance tumor-associated autoantibody detection in diagnosis of hepatocellular carcinoma. Clin Immunol 152:127–139CrossRefGoogle Scholar
  13. Diederich M, Noworyta K (2012) Natural compounds as inducers of cell death, vol 1. Springer, BerlinCrossRefGoogle Scholar
  14. Dong QM, Ling C, Chen X, Zhao L (2015) Inhibition of tumor necrosis factor-α enhances apoptosis induced by nuclear factor-κB inhibition in leukemia cells. Oncol Lett 10:3793–3798CrossRefGoogle Scholar
  15. El-Ghani MMA, El-Sawaf N (2004) Diversity and distribution of plant species in agro-ecosystems of Egypt. Syst Geogr Plants 74:319–336Google Scholar
  16. Elmore S (2007) Apoptosis: a review of programmed cell death. Toxicol Pathol 35:495–516CrossRefGoogle Scholar
  17. Fulda S, Debatin K (2006) Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy. Oncogene 25:4798–4811CrossRefGoogle Scholar
  18. García-Sáez AJ, Fuertes G, Suckale J, Salgado J (2010) Permeabilization of the outer mitochondrial membrane by Bcl-2 proteins. In: Anderluh G, Lakey J (eds) Proteins membrane binding and pore formation. Advances in experimental medicine and biology, vol 677. Springer, New York, NY, pp 91–105CrossRefGoogle Scholar
  19. Hengartner MO (2000) The biochemistry of apoptosis. Nature 407:770–776CrossRefGoogle Scholar
  20. Horinaka M, Yoshida T, Shiraishi T, Nakata S, Wakada M, Nakanishi R, Nishino H, Matsui H, Sakai T (2005) Luteolin induces apoptosis via death receptor 5 upregulation in human malignant tumor cells. Oncogene 24:7180–7189CrossRefGoogle Scholar
  21. Hussain K, Shahazad A, Zia-ul-Hussnain S (2008) An ethnobotanical survey of important wild medicinal plants of Hattar district Haripur, Pakistan. Ethnobot Leafl 12:29–35Google Scholar
  22. Joza N, Susin SA, Daugas E, Stanford WL (2001) Essential role of the mitochondrial apoptosis-inducing factor in programmed cell death. Nature 410:549–554CrossRefGoogle Scholar
  23. Karakaş FP, Yildirim A, Türker A (2012) Biological screening of various medicinal plant extracts for antibacterial and antitumor activities. Turk J Biol 36:641–652Google Scholar
  24. Kim MJ, Woo JS, Kwon CH, Kim JH, Kim YK, Kim KH (2012) Luteolin induces apoptotic cell death through AIF nuclear translocation mediated by activation of ERK and p38 in human breast cancer cell lines. Cell Biol Int 36:339–344CrossRefGoogle Scholar
  25. Kundu T, Dey S, Roy M, Siddiqi M, Bhattacharya R (2005) Induction of apoptosis in human leukemia cells by black tea and its polyphenol theaflavin. Cancer Lett 230:111–121CrossRefGoogle Scholar
  26. Lipton SA, Bossy-Wetzel E (2002) Dueling activities of AIF in cell death versus survival: DNA binding and redox activity. Cell 111:147–150CrossRefGoogle Scholar
  27. Loeffler M, Daugas E, Susin SA, Zamzami N, Metivier D, Nieminen AL, Brothers G, Penninger JM, Kroemer G (2001) Dominant cell death induction by extramitochondrially targeted apoptosis-inducing factor. FASEB J 15:758–767CrossRefGoogle Scholar
  28. Mahyar-Roemer M, Katsen A, Mestres P, Roemer K (2001) Resveratrol induces colon tumor cell apoptosis independently of p53 and precede by epithelial differentiation, mitochondrial proliferation and membrane potential collapse. Int J Cancer 94:615–622CrossRefGoogle Scholar
  29. Martinou J-C, Green DR (2001) Opinion: breaking the mitochondrial barrier. Nat Rev Mol Cell Biol 2:63–67CrossRefGoogle Scholar
  30. Marzo I, Perez-Galan P, Giraldo P, Rubio-Felix D, Alberto A, Naval J (2001) Cladribine induces apoptosis in human leukaemia cells by caspase-dependent and-independent pathways acting on mitochondria. Biochem J 359:537–546CrossRefGoogle Scholar
  31. Mukhtar E, Mustafa Adhami V, Khan N, Mukhtar H (2012) Apoptosis and autophagy induction as mechanism of cancer prevention by naturally occurring dietary agents. Curr Drug Targets 13:1831–1841CrossRefGoogle Scholar
  32. Nagata S, Nagase H, Kawane K, Mukae N, Fukuyama H (2003) Degradation of chromosomal DNA during apoptosis. Cell Death Differ 10:108–116CrossRefGoogle Scholar
  33. Parrish AB, Freel CD, Kornbluth S (2013) Cellular mechanisms controlling caspase activation and function. Cold Spring Harb Perspect Biol 5:a008672CrossRefGoogle Scholar
  34. Pistritto G, Trisciuoglio D, Ceci C, Garufi A, D’Orazi G (2016) Apoptosis as anticancer mechanism: function and dysfunction of its modulators and targeted therapeutic strategies. Aging 8:603–619CrossRefGoogle Scholar
  35. Pleşca-Manea L, Pârvu AE, Pârvu M, Taaˇmaş M, Buia R, Puia M (2002) Effects of Melilotus officinalis on acute inflammation. Phytother Res 16:316–319CrossRefGoogle Scholar
  36. Quattrocchi U (2012) CRC world dictionary of medicinal and poisonous plants: common names, scientific names, eponyms, synonyms, and etymology (5 volume set). CRC Press, Boca Raton, FL, USACrossRefGoogle Scholar
  37. Rizk A-F (1990) Poisonous plant contamination of edible plants. CRC Press, Boca Raton, FL, USAGoogle Scholar
  38. Rogers ME, Colmer TD, Frost K, Henry D, Cornwall D, Hulm E, Deretic J, Hughes SR, Craig AD (2008) Diversity in the genus Melilotus for tolerance to salinity and waterlogging. Plant Soil 304:89–101CrossRefGoogle Scholar
  39. Ruffa M, Ferraro G, Wagner M, Calcagno M, Campos R, Cavallaro L (2002) Cytotoxic effect of Argentine medicinal plant extracts on human hepatocellular carcinoma cell line. J Ethnopharmacol 79:335–339CrossRefGoogle Scholar
  40. Shun-jiu ZJ-wZ, Li QS-hL (2007) Treatment of deep venous thrombosis of lower extremities by low molecular weight heparin sodium and melilotus extract tablet. Chin J Clin Pharm 3:018Google Scholar
  41. Stefanović OD, Tešić JD, Čomić LR (2015) Melilotus albus and Dorycnium herbaceum extracts as source of phenolic compounds and their antimicrobial, antibiofilm, and antioxidant potentials. J Food Drug Anal 23:417–424CrossRefGoogle Scholar
  42. Susin SA, Lorenzo HK, Zamzami N, Marzo I (1999) Molecular characterization of mitochondrial apoptosis-inducing factor. Nature 397:441–446CrossRefGoogle Scholar
  43. Tait SW, Green DR (2008) Caspase-independent cell death: leaving the set without the final cut. Oncogene 27:6452–6461CrossRefGoogle Scholar
  44. Wang X, Yuan S, Wang J, Lin P, Liu G, Lu Y, Zhang J, Wang W, Wei Y (2006) Anticancer activity of litchi fruit pericarp extract against human breast cancer in vitro and in vivo. Toxicol Appl Pharmacol 215:168–178CrossRefGoogle Scholar
  45. Xu F, Zeng W, Mao X, Fan G-K (2008) The efficacy of melilotus extract in the management of postoperative ecchymosis and edema after simultaneous rhinoplasty and blepharoplasty. Aesthet Plast Surg 32:599–603CrossRefGoogle Scholar
  46. Yadava R, Jain S (2005) A new bioactive flavone glycoside from the seeds of Melilotus indica All. J Asian Nat Prod Res 7:595–599CrossRefGoogle Scholar
  47. Zamzami N, Marchetti P, Castedo M, Zanin C, Vayssiere J-L, Petit PX, Kroemer G (1995) Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo. J Exp Med 181:1661–1672CrossRefGoogle Scholar
  48. Zhang XY, Tao JY, Zhao L, Huang ZJ, Xiong FL, Zhang SL, Li CM, Xiao F (2007) In vitro anti-inflammatory effects of different solution fractions of ethanol extract from Melilotus suaveolens Ledeb. Chin Med J 120:1992–1998Google Scholar
  49. Zhao L, Tao J-Y, Zhang S-L, Pang R, Jin F, Dong J-H, Guo Y-J (2007) Inner anti-inflammatory mechanisms of petroleum ether extract from Melilotus suaveolens Ledeb. Inflammation 30:213–223CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  1. 1.Pharmacoloy and Experimental Oncology Unit, Cancer Biology Department, National Cancer InstituteCairo UniversityCairoEgypt
  2. 2.Hiroshima Research Center for Healthy Aging (HiHA), Department of Molecular Biotechnology, Graduate School of Advanced Sciences of MatterHiroshima UniversityHigashihiroshimaJapan
  3. 3.Department of Pharmacology, Faculty of Veterinary MedicineKafrelsheikh UniversityKafrelsheikhEgypt
  4. 4.Department of Forensic Medicine and Toxicology, Faculty of Veterinary MedicineSuez Canal UniversityIsmailiaEgypt
  5. 5.Graduate School of Biosphere ScienceHiroshima UniversityHigashihiroshimaJapan
  6. 6.Department of Botany and Microbiology, Faculty of ScienceMinia UniversityEl-Minia CityEgypt

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