JCT  Vol.6 No.8 , August 2015
Chemotherapeutic Effect of Withaferin A in Human Oral Cancer Cells
Abstract: Withaferin A (WA) is a bioactive compound derived from a medicinal plant Withania somnifera and has potential therapeutic effects against various types of cancers. The purpose of this study is to investigate an apoptotic effect of WA and identify its molecular target in HSC-3 and HSC-4 human oral cancer cell lines using Trypan blue exclusion assay, DAPI staining and western blotting. WA inhibited cell viability and induced apoptosis in a concentration- or time-dependent manner, as evidenced by induction of nuclear condensation and fragmentation, activation of caspase 3 and poly (ADP-ribose) polymerase (PARP) cleavage. WA-induced apoptosis was partly diminished by Z-VAD, a pancaspase inhibitor. WA also increased Bim and Bax protein in HSC-3 and HSC-4 cells, respectively. These results suggest that WA may be a potential chemotherapeutic drug candidate against human oral cancer.
Cite this paper: Yang, I. , Kim, L. , Shin, J. and Cho, S. (2015) Chemotherapeutic Effect of Withaferin A in Human Oral Cancer Cells. Journal of Cancer Therapy, 6, 735-742. doi: 10.4236/jct.2015.68080.

[1]   Gibson, M.K., Li, Y., Murphy, B., Hussain, M.H., DeConti, R.C., Ensley, J., et al. (2005) Randomized Phase III Evaluation of Cisplatin plus Fluorouracil versus Cisplatin plus Paclitaxel in Advanced Head and Neck Cancer (E1395): An Intergroup Trial of the Eastern Cooperative Oncology Group. Journal of Clinical Oncology, 23, 3562-3567.

[2]   Zheng, G., Peng, C., Jia, X., Gu, Y., Zhang, Z., Deng, Y., et al. (2015) ZEB1 Transcriptionally Regulated Carbonic Anhydrase 9 Mediates the Chemoresistance of Tongue Cancer via Maintaining Intracellular pH. Molecular Cancer, 14, 84.

[3]   Casagrande, N., Celegato, M., Borghese, C., Mongiat, M., Colombatti, A. and Aldinucci, D. (2014) Preclinical Activity of the Liposomal Cisplatin Lipoplatin in Ovarian Cancer. Clinical Cancer Research, 20, 5496-5506.

[4]   Berruti, A., Terzolo, M., Sperone, P., Pia, A., Della Casa, S., Gross, D.J., et al. (2005) Etoposide, Doxorubicin and Cisplatin plus Mitotane in the Treatment of Advanced Adrenocortical Carcinoma: A Large Prospective Phase II Trial. Endocrine-Related Cancer, 12, 657-666.

[5]   von Schwarzenberg, K. and Vollmar, A.M. (2013) Targeting Apoptosis Pathways by Natural Compounds in Cancer: Marine Compounds as Lead Structures and Chemical Tools for Cancer Therapy. Cancer Letters, 332, 295-303.

[6]   Choi, E.S., Kim, J.S., Kwon, K.H., Kim, H.S., Cho, N.P. and Cho, S.D. (2012) Methanol Extract of Sanguisorba officinalis L. with Cytotoxic Activity against PC3 Human Prostate Cancer Cells. Molecular Medicine Reports, 6, 670-674.

[7]   Leeman-Neill, R.J., Wheeler, S.E., Singh, S.V., Thomas, S.M., Seethala, R.R., Neill, D.B., et al. (2009) Guggulsterone Enhances Head and Neck Cancer Therapies via Inhibition of Signal Transducer and Activator of Transcription-3. Carcinogenesis, 30, 1848-1856.

[8]   Sathya, S., Sudhagar, S., Vidhya Priya, M., Bharathi Raja, R., Muthusamy, V.S., Niranjali Devaraj, S., et al. (2010) 3beta-hydroxylup-20(29)-ene-27,28-dioic Acid Dimethyl Ester, a Novel Natural Product from Plumbago zeylanica Inhibits the Proliferation and Migration of MDA-MB-231 Cells. Chemico-Biological Interactions, 188, 412-420.

[9]   Woo, S.M., Min, K.J., Kim, S., Park, J.W., Kim, D.E., Kim, S.H., et al. (2014) Axl Is a Novel Target of Withaferin A in the Induction of Apoptosis and the Suppression of Invasion. Biochemical and Biophysical Research Communications, 451, 455-460.

[10]   Rasool, M. and Varalakshmi, P. (2006) Immunomodulatory Role of Withania somnifera Root Powder on Experimental Induced Inflammation: An in Vivo and in Vitro Study. Vascular Pharmacology, 44, 406-410.

[11]   Mohan, R., Hammers, H.J., Bargagna-Mohan, P., Zhan, X.H., Herbstritt, C.J., Ruiz, A., et al. (2004) Withaferin A Is a Potent Inhibitor of Angiogenesis. Angiogenesis, 7, 115-122.

[12]   Stan, S.D., Hahm, E.R., Warin, R. and Singh, S.V. (2008) Withaferin A Causes FOXO3a- and Bim-Dependent Apoptosis and Inhibits Growth of Human Breast Cancer Cells in Vivo. Cancer Research, 68, 7661-7669.

[13]   Mayola, E., Gallerne, C., Esposti, D.D., Martel, C., Pervaiz, S., Larue, L., et al. (2011) Withaferin A Induces Apoptosis in Human Melanoma Cells through Generation of Reactive Oxygen Species and Down-Regulation of Bcl-2. Apoptosis, 16, 1014-1027.

[14]   Cory, S. and Adams, J.M. (2002) The Bcl2 Family: Regulators of the Cellular Life-or-Death Switch. Nature Reviews Cancer, 2, 647-656.

[15]   Taylor, R.C., Cullen, S.P. and Martin, S.J. (2008) Apoptosis: Controlled Demolition at the Cellular Level. Nature Reviews Molecular Cell Biology, 9, 231-241.

[16]   Lomonosova, E. and Chinnadurai, G. (2008) BH3-Only Proteins in Apoptosis and Beyond: An Overview. Oncogene, 27, S2-S19.

[17]   Kuwana, T., Bouchier-Hayes, L., Chipuk, J.E., Bonzon, C., Sullivan, B.A., Green, D.R., et al. (2005) BH3 Domains of BH3-Only Proteins Differentially Regulate Bax-Mediated Mitochondrial Membrane Permeabilization Both Directly and Indirectly. Molecular Cell, 17, 525-535.

[18]   Sehrawat, A. and Singh, S.V. (2015) Short-Form RON Overexpression Augments Benzyl Isothiocyanate-Induced Apoptosis in Human Breast Cancer Cells. Molecular Carcinogenesis. (In Press)

[19]   Rah, B., Rasool, R.U., Nayak, D., Yousuf, S.K., Mukherjee, D., Kumar, L.D., et al. (2015) PAWR-Mediated Suppression of BCL2 Promotes Switching of 3-Azido Withaferin A (3-AWA)-Induced Autophagy to Apoptosis in Prostate Cancer Cells. Autophagy, 11, 314-331.

[20]   Li, X., Zhu, F., Jiang, J., Sun, C., Wang, X., Shen, M., et al. (2015) Synergistic Antitumor Activity of Withaferin A Combined with Oxaliplatin Triggers Reactive Oxygen Species-Mediated Inactivation of the PI3K/AKT Pathway in Human Pancreatic Cancer Cells. Cancer Letters, 357, 219-230.

[21]   Zhang, X., Samadi, A.K., Roby, K.F., Timmermann, B. and Cohen, M.S. (2012) Inhibition of Cell Growth and Induction of Apoptosis in Ovarian Carcinoma Cell Lines CaOV3 and SKOV3 by Natural Withanolide Withaferin A. Gynecologic Oncology, 124, 606-612.

[22]   Cai, Y., Sheng, Z.Y., Chen, Y. and Bai, C. (2014) Effect of Withaferin A on A549 Cellular Proliferation and Apoptosis in Non-Small Cell Lung Cancer. Asian Pacific Journal of Cancer Prevention, 15, 1711-1714.

[23]   Samadi, A.K., Tong, X., Mukerji, R., Zhang, H., Timmermann, B.N. and Cohen, M.S. (2010) Withaferin A, a Cytotoxic Steroid from Vassobia breviflora, Induces Apoptosis in Human Head and Neck Squamous Cell Carcinoma. Journal of Natural Products, 73, 1476-1481.

[24]   Wu, J., Yi, W., Jin, L., Hu, D. and Song, B. (2012) Antiproliferative and Cell Apoptosis-Inducing Activities of Compounds from Buddleja Davidii in Mgc-803 Cells. Cell Division, 7, 20.

[25]   Badmus, J.A., Ekpo, O.E., Hussein, A.A., Meyer, M. and Hiss, D.C. (2015) Antiproliferative and Apoptosis Induction Potential of the Methanolic Leaf Extract of Holarrhena floribunda (G. Don). Evidence-Based Complementary and Alternative Medicine, 2015, Article ID: 756482.

[26]   Zhao, F., Hu, Y., Chong, C., Lu, M., Chen, L., Kan, W., et al. (2014) Ardisiphenol D, a Resorcinol Derivative Identified from Ardisia brevicaulis, Exerts Antitumor Effect through Inducing Apoptosis in Human Non-Small-Cell Lung Cancer A549 Cells. Pharmaceutical Biology, 52, 797-803.

[27]   Tang, X., Shen, C., Cheema, S.A., Chen, L., Xiao, X., Zhang, C., et al. (2010) Levels and Distributions of Polycyclic Aromatic Hydrocarbons in Agricultural Soils in an Emerging E-Waste Recycling Town in Taizhou Area, China. Journal of Environmental Science and Health, Part A: Toxic/Hazardous Substances and Environmental Engineering, 45, 1076-1084.

[28]   Jaganathan, S.K., Mazumdar, A., Mondhe, D. and Mandal, M. (2011) Apoptotic Effect of Eugenol in Human Colon Cancer Cell Lines. Cell Biology International, 35, 607-615.

[29]   Shin, J.A., Kim, J.S., Hong, I.S. and Cho, S.D. (2012) Bak Is a Key Molecule in Apoptosis Induced by Methanol Extracts of Codonopsis lanceolata and Tricholoma matsutake in HSC-2 Human Oral Cancer Cells. Oncology Letters, 4, 1379-1383.

[30]   Patil, D., Gautam, M., Mishra, S., Karupothula, S., Gairola, S., Jadhav, S., et al. (2013) Determination of Withaferin A and Withanolide A in Mice Plasma Using High-Performance Liquid Chromatography-Tandem Mass Spectrometry: Application to Pharmacokinetics after Oral Administration of Withania somnifera Aqueous Extract. Journal of Pharmaceutical and Biomedical Analysis, 80, 203-212.

[31]   Panjamurthy, K., Manoharan, S., Nirmal, M.R. and Vellaichamy, L. (2009) Protective Role of Withaferin-A on Immunoexpression of p53 and Bcl-2 in 7,12-Dimethylbenz(a)Anthracene-Induced Experimental Oral Carcinogenesis. Investigational New Drugs, 27, 447-452.

[32]   Subramanian, C., Zhang, H., Gallagher, R., Hammer, G., Timmermann, B. and Cohen, M. (2014) Withanolides Are Potent Novel Targeted Therapeutic Agents against Adrenocortical Carcinomas. World Journal of Surgery, 38, 1343-1352.

[33]   Ghobrial, I.M., Witzig, T.E. and Adjei, A.A. (2005) Targeting Apoptosis Pathways in Cancer Therapy. CA: A Cancer Journal for Clinicians, 55, 178-194.

[34]   Danial, N.N. and Korsmeyer, S.J. (2004) Cell Death: Critical Control Points. Cell, 116, 205-219.

[35]   Zhao, L., He, F., Liu, H., Zhu, Y., Tian, W., Gao, P., et al. (2012) Natural Diterpenoid Compound Elevates Expression of Bim Protein, Which Interacts with Antiapoptotic Protein Bcl-2, Converting It to Proapoptotic Bax-Like Molecule. Journal of Biological Chemistry, 287, 1054-1065.

[36]   Yang, P.Y., Hu, D.N. and Liu, F.S. (2013) Cytotoxic Effect and Induction of Apoptosis in Human Cervical Cancer Cells by Antrodia camphorata. The American Journal of Chinese Medicine, 41, 1169-1180.

[37]   Wang, J., Liu, S., Yin, Y., Li, M., Wang, B., Yang, L., et al. (2015) FOXO3-Mediated Up-Regulation of Bim Contributes to Rhein-Induced Cancer Cell Apoptosis. Apoptosis, 20, 399-409.

[38]   Yu, H.J., Shin, J.A., Nam, J.S., Kang, B.S. and Cho, S.D. (2013) Apoptotic Effect of Dibenzylideneacetone on Oral Cancer Cells via Modulation of Specificity Protein 1 and Bax. Oral Diseases, 19, 767-774.