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Journal of Integrative Medicine ›› 2015, Vol. 13 ›› Issue (1): 34-44.doi: 10.1016/S2095-4964(15)60156-X

• Research Article • Previous Articles     Next Articles

Evaluation of chemopreventive potentials of ethanolic extract of Ruta graveolens against A375 skin melanoma cells in vitro and induced skin cancer in mice in vivo

Samrat Ghosh, Sourav Sikdar, vinaba Mukherjee, Anisur Rahman Khuda-Bukhsh   

  1. Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani-741235, West Bengal, India
  • Received:2014-07-26 Accepted:2014-11-21 Online:2015-01-10 Published:2018-09-25

Chemopreventive approach with natural products, particularly plants and plant-derived ones, is receiving increasing attention for their effective role against cancer without any palpable side effects. In this study, efficacy of ethanolic extract of Ruta graveolens (RG) on skin melanoma cells (A375) in vitro and on 7,12-dimethylbenz(a)anthracene (DMBA)-induced skin cancer in vivo has been tested in Swiss albino mice.
Studies on cell viability, apoptosis and autophagy induction were conducted in vitro. To check apoptosis, assays like alteration in mitochondrial membrane potential, annexin V-fluorescein isothiocyanate/propidium iodide assay and immunoblot were performed. Fluorescence microscopic and immunoblot assays were performed to confirm autophagy induction. The effects of RG were determined by evaluating body weight, tumor incidence, tumor volume and tumor burden in mice. Enzymatic and non-enzymatic antioxidant status was assessed. The role of some relevant signaling proteins was also analyzed.
RG caused death of A375 cells through induction of caspase 3-mediated apoptosis and Beclin-1-associated autophagy. Moreover, RG administration (75 mg/kg body weight) which showed no acute or chronic toxicity, showed significant reduction in the skin tumor burden of DMBA-painted mice. RG also demonstrated potent anti-lipid peroxidative and antioxidant functions during the course of skin cancer induction by DMBA.
Chemopreventive potential of RG was demonstrated from overall results of this study, indicating its possible use in therapeutic formulation of an effective drug to treat skin cancer.

Key words: Ruta graveolens, Plant extracts, Chemoprevention, Skin neoplasms, Apoptosis, Autophagy

[1] Eickhorst K , DeLeo V, Csaposs J.Rue the herb: Ruta graveolens ― associated phytophototoxicity. Dermatitis, 2007,18(1):52-57
doi: 10.2310/6620.2007.06033 pmid: 17303046
[2] Oliva A, Meepagala KM, Wedge DE, Harries D, Hale AL, Aliotta G, Duke SO . Natural fungicides from Ruta graveolens L. leaves, including a new quinolone alkaloid. J Agric Food Chem, 2003,51(4):890-896
doi: 10.1021/jf0259361
[3] Wessner D, Hofmann H, Ring J . Phytophotodermatitis due to Ruta graveolens applied as protection against evil spells. Contact Dermatitis, 1999,41(4):232
doi: 10.1111/j.1600-0536.1999.tb06145.x pmid: 10515113
[4] International Program on Chemical Safety. Chemical safety information from intergovernmental organizations. 1989.
[5] Lefranc F, Facchini V, Kiss R . Proautophagic drugs: a novel means to combat apoptosis-resistant cancers, with a special emphasis on glioblastomas. Oncologist, 2007,12(12):1395-1403
doi: 10.1634/theoncologist.12-12-1395
[6] Ghosh S, Bishayee K, Khuda-Bukhsh AR . Graveoline isolated from ethanolic extract of Ruta graveolens triggers apoptosis and autophagy in skin melanoma cells: A novel apoptosis-independent autophagic signalling pathway. Phytother Res, 2014,28(8):1153-1162
doi: 10.1002/ptr.v28.8
[7] Naithani R, Huma L, Moriarty RM , McCormick DL, Mehta RG.Comprehensive review of cancer chemopreventive agents evaluated in experimental chemoprevention models and clinical trials. Curr Med Chem, 2008,15(11):1044-1071
doi: 10.2174/092986708784221403
[8] Renju GL, Manoharan S, Balakrishnan S, Senthil N . Chemopreventive and antilipidperoxidative potential of Clerodendron inerme(L) Gaertn in 7,12-dimethylbenz(a)anthracene induced skin carcinogenesis in Swiss albino mice. Pak J Biol Sci, 2007,10(9):1465-1470
doi: 10.3923/pjbs.2007.1465.1470
[9] Alias LM, Manoharan S, Vellaichamy L, Balakrishnan S, Ramachandran CR . Protective effect of ferulic acid on 7,12-dimethylbenz(a)anthracene-induced skin carcinogenesis in Swiss albino mice. Exp Toxicol Pathol, 2009,61(3):205-214
doi: 10.1016/j.etp.2008.09.001
[10] Ruitenberg JJ, Mulder CB, Maino VC, Landay AL, Ghanekar SA . VACUTAINER CPT and Ficoll density gradient separation perform equivalently in maintaining the quality and function of PBMC from HIV seropositive blood samples. BMC Immunol, 2006,7:11
doi: 10.1186/1471-2172-7-11
[11] Ghosh S, Bishayee K, Paul A, Mukherjee A, Sikdar S, Chakraborty D, Boujedaini N, Khuda-Bukhsh AR . Homeopathic mother tincture of Phytolacca decandra induces apoptosis in skin melanoma cells by activating caspase-mediated signaling via reactive oxygen species elevation. J Integr Med, 2013,11(2):116-124
doi: 10.3736/jintegrmed2013014
[12] Ghosh S, Bishayee K, Khuda-Bukhsh AR . Oleanolic acid isolated from ethanolic extract of Phytolacca decandra induces apoptosis in A375 skin melanoma cells: drug-DNA interaction and signaling cascade. J Integr Med, 2014,12(2):102-114
doi: 10.1016/S2095-4964(14)60015-7
[13] Kanzawa T, Kondo Y, Ito H, Kondo S, Germano I . Induction of autophagic cell death in malignant glioma cells by arsenic trioxide. Cancer Res, 2003,63(9):2103-2108
doi: 10.1038/sj.onc.1208095 pmid: 12727826
[14] Chakraborty D, Bishayee K, Ghosh S, Biswas R, Mandal SK, Khuda-Bukhsh AR .[ 6]-Gingerol induces caspase 3 dependent apoptosis and autophagy in cancer cells: drug-DNA interaction and expression of certain signal genes in HeLa cells. Eur J Pharmacol, 2012,694(1-3):20-29
doi: 10.1016/j.ejphar.2012.08.001
[15] Azuine MA, Bhide SV . Chemopreventive effect of turmeric against stomach and skin tumors induced by chemical carcinogens in Swiss mice. Nutr Cancer, 1992,17(1):77-83
doi: 10.1080/01635589209514174
[16] Bradford MM . A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, 1976,72:248-254
doi: 10.1016/0003-2697(76)90527-3
[17] Chance B, Maehly AC . Assay of catalase and peroxidase. Methods Enzymol, 1955,2:764-775
doi: 10.1016/S0076-6879(55)02300-8
[18] Kakkar P, Das B, Viswanathan PN . A modified spectrophotometric assay of superoxide dismutase. Indian J Biochem Biophys, 1984,21(2):130-132
doi: 10.1515/bchm2.1984.365.2.1489 pmid: 6490072
[19] Ellman GL . Tissue sulfhydryl groups. Arch Biochem Biophys, 1959,82(1):70-77
doi: 10.1016/0003-9861(59)90090-6
[20] Buege JA, Aust SD . Microsomal lipid peroxidation. Methods Enzymol, 1984,105:302-310
[21] Habig WH, Pabst MJ, Jakoby WB . Glutathione-S-transferase, the first enzymatic step in mercapturic acid formation. J Biol Chem, 1974,249(22):7130-7139
pmid: 4436300
[22] Paglia DE, Valentine WN . Studies on the quantitative and qualitative characterisation of erythrocyte glutathione peroxidase. J Lab Clin Med, 1967,70(1):158-169
[23] Gozuacik D, Kimchi A . Autophagy as a cell death and tumor suppressor mechanism. Oncogene, 2004,23(16):2891-2906
doi: 10.1038/sj.onc.1207521
[24] Tsujimoto Y, Shimizu S . Another way to die: autophagic programmed cell death. Cell Death Differ, 2005,12(Suppl 2):1528-1534
doi: 10.1038/sj.cdd.4401777 pmid: 16247500
[25] Mizushima N . Methods for monitoring autophagy. Int J Biochem Cell Biol, 2004,36(12):2491-2502
doi: 10.1016/j.biocel.2004.02.005
[26] Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, Kominami E, Ohsumi Y, Yoshimori T . LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J, 2000,19(21):5720-5728
doi: 10.1093/emboj/19.21.5720
[27] Kung CP, Budina A, Balaburski G, Bergenstock MK, Murphy M . Autophagy in tumor suppression and cancer therapy. Crit Rev Eukaryot Gene Expr, 2011,21(1):71-100
doi: 10.1615/CritRevEukarGeneExpr.v21.i1.50 pmid: 3187613
[28] Pattingre S, Tassa A, Qu X, Garuti R, Liang XH, Mizushima N, Packer M, Schneider MD, Levine B . Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagy. Cell, 2005,122(6):927-939
doi: 10.1016/j.cell.2005.07.002 pmid: 16179260
[29] Levine B, Sinha S, Kroemer G . Bcl-2 family members: dual regulators of apoptosis and autophagy. Autophagy, 2008,4(5):600-606
doi: 10.4161/auto.6260 pmid: 2749577
[30] Green DR, Kroemer G . The central executioner of apoptosis: mitochondria or caspases? Trends Cell Biol. 1998; 8(7):267-271.
doi: 10.1016/S0962-8924(98)01273-2 pmid: 9714597
[31] Oltvai ZN, Milliman CL, Korsmeyer SJ . Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell, 1993,74(4):609-619
[32] Antonsson B, Conti F, Ciavatta A, Montessuit S, Lewis S, Martinou I, Bernasconi L, Bernard A, Mermod JJ, Mazzei G, Maundrell K, Gambale F, Sadoul R, Martinou JC . Inhibition of Bax channel-forming activity by Bcl-2. Science, 1997,277(5324):370-372
doi: 10.1126/science.277.5324.370
[33] Dejean LM, Martinez-Caballero S, Guo L, Hughes C, Teijido O, Ducret T, Ichas F, Korsmeyer SJ, Antonsson B, Jonas EA, Kinnally KW . Oligomeric Bax is a component of the putative cytochrome c release channel MAC, mitochondrial apoptosis-induced channel. Mol Biol Cell, 2005,16(5):2424-2432
doi: 10.1091/mbc.e04-12-1111
[34] Xu N, Lao Y, Zhang Y, Gillespie DA . Akt: a double-edged sword in cell proliferation and genome stability. J Oncol, 2012,2012:951724
[35] Keeney S , McKenna H, Fleming P, McIlfatrick S.Attitudes, knowledge and behaviours with regard to skin cancer: a literature review. Eur J Oncol Nurs, 2009,13(1):29-35
doi: 10.1016/j.ejon.2008.11.004 pmid: 19153059
[36] Meijerman I, Beijnen JH, Schellens JH . Combined action and regulation of phase II enzymes and multidrug resistance proteins in multidrug resistance in cancer. Cancer Treat Rev, 2008,34(6):505-520
doi: 10.1016/j.ctrv.2008.03.002
[37] Choi SC, Yun KJ, Kim TH, Kim HJ, Park SG, Oh GJ, Chae SC, Oh GJ, Nah YH, Kim JJ, Chung HT . Prognostic potential of glutathione S-transferase M1 and T1 null genotypes for gastric cancer progression. Cancer Lett, 2003,195(2):169-175
doi: 10.1016/S0304-3835(03)00158-7
[38] Das I, Saha T . Effect of garlic on lipid peroxidation and antioxidation enzymes in DMBA-induced skin carcinoma. Nutrition, 2009,25(4):459-471
doi: 10.1016/j.nut.2008.10.014 pmid: 19084378
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