Summary
We investigated the cytotoxic effects of nimbolide, a limonoid present in leaves and flowers of the neem tree (Azadirachta indica) on human choriocarcinoma (BeWo) cells. Treatment with nimbolide resulted in dose- and time-dependent inhibition of growth of BeWo cells with IC50 values of 2.01 and 1.19 μM for 7 and 24 h respectively, accompanied by downregulation of proliferating cell nuclear antigen. Examination of nuclear morphology revealed fragmentation and condensation indicating apoptosis. Increase in the generation of reactive oxygen species (ROS) that was reversed by addition of reduced glutathione suggested ROS involvement in the cytotoxicity of nimbolide. A decrease in Bcl-2/Bax ratio with increased expression of Apaf-1 and caspase-3, and cleavage of poly(ADP-ribose) polymerase provide compelling evidence that nimbolide-induced apoptosis is mediated by the mitochondrial pathway. The results of the present study suggest that nimbolide has immense potential in cancer prevention and therapy based on its antiproliferative and apoptosis inducing effects.
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References
Hanahan D, Weinberg RA (2000) The hallmarks of cancer. Cell 100:57–70 doi:10.1016/S0092-8674(00)81683-9
Lowe SW, Lin AW (2000) Apoptosis in cancer. Carcinogenesis 21:485–495 doi:10.1093/carcin/21.3.485
Reed JC (1999) Mechanisms of apoptosis avoidance in cancer. Curr Opin Oncol 11:68–75 doi:10.1097/00001622-199901000-00014
Vermeulen K, Van Bockstaele DR, Berneman ZN (2005) Apoptosis: mechanisms and relevance in cancer. Ann Hematol 84:627–639 doi:10.1007/s00277-005-1065-x
Sun S-Y, Hail N, Lotan R Jr (2004) Apoptosis as a novel target for cancer chemoprevention. J Natl Cancer Inst 96:662–672
Hsu S, Singh B, Schuster G (2004) Induction of apoptosis in oral cancer cells: agents and mechanisms for potential therapy and prevention. Oral Oncol 40:461–473 doi:10.1016/j.oraloncology.2003.09.012
Khan N, Afaq F, Mukhtar H (2008) Cancer chemoprevention through dietary antioxidants: progress and promise. Antioxid Redox Signal 10:475–510 doi:10.1089/ars.2007.1740
Davis CD (2007) Nutritional interactions: Credentialing of molecular targets for cancer prevention. Exp Biol Med 232:176–183
Deorukhkar A, Krishnan S, Sethi G et al (2007) Back to basics: how natural products can provide the basis for new therapeutics. Expert Opin Investig Drugs 16:1753–1773 doi:10.1517/13543784.16.11.1753
Kang K, Lee HJ, Kim CY (2007) The chemopreventive effects of Saussurea salicifolia through induction of apoptosis and phase II detoxification enzyme. Biol Pharm Bull 30:2352–2359 doi:10.1248/bpb.30.2352
Biswas K, Chattopadhyay I, Banerjee RK (2002) Biological activities and medicinal properties of neem (Azadirachta indica). Curr Sci 82:1336–1345
Subapriya R, Nagini S (2005) Medicinal properties of neem leaves: A review. Curr Med Chem Anticancer Agents 5:149–156 doi:10.2174/1568011053174828
Subapriya R, Kumaraguruparan R, Nagini S (2006) Expression of PCNA, cytokeratin, Bcl-2 and p53 during chemoprevention of hamster buccal pouch carcinogenesis by ethanolic neem (Azadirachta indica) leaf extract. Clin Biochem 39:1080–1087 doi:10.1016/j.clinbiochem.2006.06.013
Subapriya R, Velmurugan B, Nagini S (2005) Modulation of xenobiotic-metabolizing enzymes by ethanolic neem leaf extract during hamster buccal pouch carcinogenesis. J Exp Clin Cancer Res 24:207–214
Subapriya R, Nagini S (2003) Ethanolic neem leaf extract protects against N-methyl-N’-nitro-N-nitrosoguanidine-induced gastric carcinogenesis in Wistar rats. Asian Pac J Cancer Prev 4:215–223
Manikandan P, Vidjaya Letchoumy P, Gopalakrishnan M et al (2008) Evaluation of Azadirachta indica leaf fractions for in vitro antioxidant potential and in vivo modulation of biomarkers of chemoprevention in the hamster buccal pouch carcinogenesis model. Food Chem Toxicol 46:2332–2343 doi:10.1016/j.fct.2008.03.013
Roy MK, Kobori M, Takenaka M et al (2007) Antiproliferative effect on human cancer cell lines after treatment with nimbolide extracted from an edible part of the neem tree (Azadirachta indica). Phytother Res 21:245–250 doi:10.1002/ptr.2058
Sastry BS, Suresh Babu K, Hari Babu T et al (2006) Synthesis and biological activity of amide derivatives of nimbolide. Bioorg Med Chem Lett 16:4391–4394 doi:10.1016/j.bmcl.2006.05.105
Cohen E, Quistad GB, Casida JE (1996) Cytotoxicity of nimbolide, epoxyazadiradione and other limonoids from neem insecticide. Life Sci 58:1075–1081 doi:10.1016/0024-3205(96)00061-6
Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63 doi:10.1016/0022-1759(83)90303-4
Keum YS, Kim J, Lee KH et al (2002) Induction of apoptosis and caspase-3 activation by chemopreventive [6]-paradol and structurally related compounds in KB cells. Cancer Lett 177:41–47 doi:10.1016/S0304-3835(01)00781-9
Wang IK, Lin-Shiau SY, Lin JK (1999) Induction of apoptosis by apigenin and related flavonoids through cytochrome c release and activation of caspase-9 and caspase-3 in leukaemia HL-60 cells. Eur J Cancer 35:1517–1525 doi:10.1016/S0959-8049(99)00168-9
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254 doi:10.1016/0003-2697(76)90527-3
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685 doi:10.1038/227680a0
Setzer WN, Setzer MC (2003) Plant-derived triterpenoids as potential antineoplastic agents. Mini Rev Med Chem 3:540–556 doi:10.2174/1389557033487854
Mandal-Ghosh I, Chattopadhyay U, Baral R (2007) Neem leaf preparation enhances Th1 type immune response and anti-tumor immunity against breast tumor associated antigen. Cancer Immun 7:8
Bose A, Haque E, Baral R (2007) Neem leaf preparation induces apoptosis of tumour cells by releasing cytotoxic cytokines from human peripheral mononuclear cells. Phytother Res 21:914–920 doi:10.1002/ptr.2185
Suresh K, Suresh PK, Vijayababu MR et al (2006) Anticancer effects of ethanolic neem leaf extract on prostate cancer cell line (PC-3). J Ethnopharmacol 105:246–250 doi:10.1016/j.jep.2005.11.006
Akudugu J, Gade G, Bohm L (2001) Cytotoxicity of azadirachtin A in human glioblastoma cell lines. Life Sci 68:1153–1160 doi:10.1016/S0024-3205(00)01017-1
Nanduri S, Thunuguntla SSR, Nyavanandi VK et al (2003) Biological investigation and structure-activity relationship studies on azadirone from Azadirachta indica A. Juss. Bioorg Med Chem Lett 13:4111–4115 doi:10.1016/j.bmcl.2003.08.030
Kigodi PG, Blasko G, Thebtaranonth Y et al (1989) Spectroscopic and biological investigation of nimbolide and 28-deoxonimbolide from Azadirachta indica. J Nat Prod 52:1246–1251 doi:10.1021/np50066a008
Cohen E, Quisted GB, Jefferies PR (1996) Nimbolide is the principle cytotoxic component of neem seed insecticide preparations. Pestic Sci 48:135–140 doi: 10.1002/(SICI)1096-9063(199610)48:2>135::AID-PS451<3.0.CO;2-J
Moldovan GL, Pfander B, Jentsch S (2007) PCNA, the maestro of the replication fork. Cell 129:665–679 doi:10.1016/j.cell.2007.05.003
Maga G, Hubscher U (2003) Proliferating cell nuclear antigen (PCNA): a dancer with many partners. J Cell Sci 116:3051–3060 doi:10.1242/jcs.00653
Jeong S-Y, Seol D-W (2008) The role of mitochondria in apoptosis. BMB Rep 41:11–22
Youle RJ, Strasser A (2008) The Bcl-2 protein family: opposing activities that mediate cell death. Nat Rev Mol Cell Biol 9:47–59 doi:10.1038/nrm2308
Mackey TJ, Borkowski A, Amin P et al (1998) Bcl-2/bax ratio as a predictive marker for therapeutic response to radiotherapy in patients with prostate cancer. Urology 52:1085–1090 doi:10.1016/S0090-4295(98)00360-4
Roy MK, Kobori M, Takenaka M et al (2006) Inhibition of colon cancer (HT-29) cell proliferation by a triterpenoid isolated from Azadirachta indica is accompanied by cell cycle arrest and upregulation of p21. Planta Med 72:917–923 doi:10.1055/s-2006-946694
Ralph SJ, Low P, Dong L et al (2006) Mitocans: mitochondrial targeted anti-cancer drugs as improved therapies and related patent documents. Recent Patents Anticancer Drug Discov 1:327–346
Acknowledgements
This work was supported by a grant from the Department of Science and Technology, New Delhi, India. AJ Rao is a recipient of the Ramanna Fellowship from the Department of Science and Technology, Govt. of India.
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An erratum to this article can be found at http://dx.doi.org/10.1007/s10637-012-9816-8
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Harish Kumar, G., Chandra Mohan, K., Jagannadha Rao, A. et al. Nimbolide a limonoid from Azadirachta indica inhibits proliferation and induces apoptosis of human choriocarcinoma (BeWo) cells. Invest New Drugs 27, 246–252 (2009). https://doi.org/10.1007/s10637-008-9170-z
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DOI: https://doi.org/10.1007/s10637-008-9170-z