Etoposide is a chemotherapy drug derived from the natural lignin podophyllotoxin. Our novel generated Aza-podophyllotoxin compounds (AZP 8a & AZP 9a) are analogues of podophyllotoxin and were previously screened for anti-cancer activity through the NCI 60 cell line screening panel showing activity on various cell types including colon cancer. This study expands the toxicological screening by studying apoptosis and various hallmark events as part of the mechanism of action of these compounds on colon cancer cells. The COLO 205 cell line was selected and exposed to AZP to determine the IC50 doses at 24 hours treatment. Apoptosis hallmark events such as migration of phosphatidylserine (PS) to the cell membrane, DNA fragmentation, cell cycle effects, mitochondrial membrane permeabilization and caspase activation were included. Experiments were performed in triplicates for all tested compounds including AZP 8a, AZP 9a, camptothecin as positive control and vehicle as negative control. Our results present contrasting apoptotic activity between the experimental compounds. Compound 8a presented migration of PS (annexin V assay), DNA fragmentation and cell cycle arrest at S phase. Compound 9a presented PS migration with fragmented DNA, cell cycle arrest at S phase, mitochondrial membrane permeabilization and activation of caspase 3, 8 and 9. Compound 8a without the oxygen atoms in ring A appears to cause effects similarly to autophagy as induced by etoposide, a cancer drug analogue of our heterocyclic compounds. Compound 9a with the oxygen atoms in expanded ring A presented induction of cell death following activation of a classical apoptosis pathway. Our results suggest that minor structural differences among these AZP
References
[1]
Nagar, N., Jat, R., Saharan, R., Verma, S., Sharma, D. and Bansal, K. (2011) Podophyllotoxin and Their Glicosidic Derivatives. Pharmacophore, 2, 124-134.
[2]
Kaplan, I.W. (1942) Codylomata acuminata. The New Orleans Medical and Surgical Journal, 94, 388.
[3]
Liu, Y.Q., Yang, L.M. and Tian, X. (2007) Podophyllotoxin: Current Perspectives. Current Bioactive Compounds, 3 37-66. http://dx.doi.org/10.2174/157340707780126499
[4]
Wang, R.W., Rebhum, L.I., Kupchan, S.M. (1977) Anti Mitotic and Antitubulin Activity of the Tumor Inhibitor Steganacin. Cancer Research, 37, 3071-3079.
[5]
Stähelin, H.F., Von Wartburg, A. and Stã, H.F. (1991) The Chemical and Biological Route from Podophyllotoxin Glucoside to Etoposide: Ninth Cain Memorial Award Lecture The Chemical and Biological Route from Podophyllotoxin Glucoside to Etoposide: Ninth Cain Memorial Award Lecture1, 5-15.
[6]
Baldwin, E.L. and Osheroff, N. (2005) Etoposide, Topoisomerase II and Cancer. Current Medicinal Chemistry—Anti-Cancer Agents, 5, 363-372.
[7]
Liu, J., Cao, B., Gao, Y., Bai, M., Mei, X., Chen, H., Jiang, Y.G. and Huang, D.J. (2013) Design, Synthesis, and Antitumor Activity of Novel Podophyllotoxin Derivatives as Potent Anticancer Agents. Journal of Asian Natural Products Research, 15, 982-992.
[8]
Ma, Y., Fang, S., Li, H., Han, C., Lu, Y., Zhao, Y., Liu, Y. and Zhao, C. (2013) Biological Evaluation and Molecular Modelling Study Of podophyllotoxin Derivatives as Potent Inhibitors of Tubulin Polymerization. Chemical Biology & Drug Design, 82, 12-21. http://dx.doi.org/10.1111/cbdd.12130
[9]
Reynolds, A.J., Scott, A.J., Turner, C.I. and Sherburn, M.S. (2003) The Intramolecular Carboxyarylation Approach to Podophyllotoxin. Journal of the American Chemical Society, 125, 12108-12109.
[10]
Larsen, A., Petersson, I. and Svensson, B. (1989) Podophyllum Derivatives (CPH 82) Compared with Placebo in the Treatment of Rheumatoid Arthritis. British Journal of Rheumatology, 28, 124-127. http://dx.doi.org/10.1093/rheumatology/28.2.124
[11]
Wantke, F., Fleischl, G., Götz, M. and Jarisch, R. (1993) Topical Podophyllotoxin in Psoriasis Vulgaris. Dermatology, 186, 79. http://dx.doi.org/10.1159/000247309
[12]
Kumar, A., Kumar, V., Alegria, A. and Malhotra, S. (2011) N-Hydroxyethyl-4-aza-didehydropodophyllo Toxin Derivatives as Potential Antitumor Agents. European Journal of Pharmaceutical Sciences, 44, 21-26.
http://dx.doi.org/10.1016/j.ejps.2011.04.013
[13]
Kumar, A. and Alegria, A.E. (2010) Synthesis of Novel Functionalized 4-aza-2,3-didehydropodophyllotoxin Derivatives with Potential Anti-Tumor Activity. Journal of Heterocyclic Chemistry, 47, 1275-1282.
http://dx.doi.org/10.1002/jhet.467
[14]
Hitotsuyanagi, Y., Fukuyo, M., Tsuda, K., Kobayashi, M., Ozeki, A., Itokawa, H. and Takeya, K. (2000) 4-Aza-2,3- dehydro-4-deoxypodophyllotoxins: Simple Aza-Podophyllotoxin Analogues Possessing Potent Cytotoxicity. Bioorganic & Medicinal Chemistry Letters, 10, 315-317. http://dx.doi.org/10.1016/S0960-894X(99)00693-9
[15]
Kravtsov, V.D., Daniel, T.O. and Koury, M.J. (1999) Comparative Analysis of Different Methodological Approaches to the in Vitro Study of Drug-Induced Apoptosis. The American Journal of Pathology, 155, 1327-139. http://dx.doi.org/10.1016/S0002-9440(10)65235-2
[16]
Huang, W.W., Ko, S.W., Tsai, H.Y., Chung, J.G., Chiang, J.H., Chen, K.T., Chen, Y.C., Chen, H.Y., Chen, Y.F. and Yang, J.S. (2011) Cantharidin Induces G2/M Phase Arrest and Apoptosis in Human Colorectal Cancer COLO 205 Cells through Inhibition of CDK1 Activity and Caspase-Dependent Signaling Pathways. International Journal of Oncology, 38, 1067-1073. http://dx.doi.org/10.3892/ijo.2011.922.
[17]
Mukherjee, A., Dutta, S., Shanmugavel, M., Mondhe, D.M., Sharma, P.R., Singh, S.K., Saxena, A.K. and Sanyal, U. (2010) 6-Nitro-2-(3-hydroxypropyl)-1H-benz[de]isoquinol ine-1,3-dione, a Potent Antitumor Agent, Induces Cell Cycle Arrest and Apoptosis. Journal of Experimental & Clinical Cancer Research, 29, 175. http://dx.doi.org/10.1186/1756-9966-29-175
[18]
Schutters, K. and Reutelingsperger, C. (2010) Phosphatidylserine Targeting for Diagnosis and Treatment of Human Diseases. Apoptosis: An International Journal on Programmed Cell Death, 15, 1072-1082. http://dx.doi.org/10.1007/s10495-010-0503-y
[19]
Kumar, N.D.R., George, V.C., Suresh, P.K. and Kumar, R.A. (2012) Cytotoxicity, Apoptosis Induction and Anti-Metastatic Potential of Oroxylum indicum in Human Breast Cancer Cells. Asian Pacific Journal of Cancer Prevention: APJCP, 13, 2729-2734. http://dx.doi.org/10.7314/APJCP.2012.13.6.2729
[20]
Sharma, M., Agrawal, S.K., Sharma, P.R., Chadha, B.S., Khosla, M.K. and Saxena, K. (2010) Cytotoxic and Apoptotic Activity of Essential Oil from Ocimumviride towards COLO 205 Cells. Food and Chemical Toxicology: An International Journal Published for the British Industrial Biological Research Association, 48, 336-344. http://dx.doi.org/10.1016/j.fct.2009.10.021
[21]
Wei, P.-L., Tu, S.-H., Lien, H.-M., Chen, L.-C., Chen, C.-S., Wu, C.-H., Huang, C.-S., et al. (2012) The in Vivo Anti-tumor Effects On human COLO 205 Cancer Cells of the 4,7-Dimethoxy-5-(2-propen-1-yl)-1,3-benzodioxole (apiole) Derivative of 5-Substituted 4,7-dimethoxy-5-methyl-l,3-benzodioxole (SY-1) Isolated from the Fruiting Body of Antrodia Camphorate. Journal of Cancer Research and Therapeutics, 8, 532-536. http://dx.doi.org/10.4103/0973-1482.106529
[22]
Lin, J.-C., Ho, Y.-S., Lee, J.-J., Liu, C.-L., Yang, T.-L. and Wu, C.-H. (2007) Induction of Apoptosis and Cell-Cycle Arrest in Human Colon Cancer Cells by Meclizine. Food and Chemical Toxicology: An International Journal Published for the British Industrial Biological Research Association, 45, 935-944. http://dx.doi.org/10.1016/j.fct.2006.11.016
[23]
Ho, Y.-S., Wu, C.-H., Chou, H.-M., Wang, Y.-J., Tseng, H., Chen, C.-H., Chen, L.-C., et al. (2005) Molecular Mechanisms of Econazole-Induced Toxicity on Human Colon Cancer Cells: G0/G1 Cell Cycle Arrest and Caspase 8-Independent Apoptotic Signaling Pathways. Food and Chemical Toxicology: An International Journal Published for the British Industrial Biological Research Association, 43, 1483-1495. http://dx.doi.org/10.1016/j.fct.2005.04.002
[24]
Watanapokasin, R., Jarinthanan, F., Nakamura, Y., Sawasjirakij, N., Jaratrungtawee, A. and Suksamrarn, S. (2011) Effects of α-Mangostin on Apoptosis Induction of Human Colon Cancer. World Journal of Gastroenterology: WJG, 17, 2086-2095. http://dx.doi.org/10.3748/wjg.v17.i16.2086
[25]
Yi, C.H. and Yuan, J. (2009) The Jekyll and Hyde Functions of Caspases. Developmental Cell, 16, 21-34. http://dx.doi.org/10.1016/j.devcel.2008.12.012
[26]
Ory, B., Blanchard, F., Battaglia, S., Gouin, F., Re, F. and Heymann, D. (2007) Zoledronic Acid Activates the DNA S-Phase Checkpoint and Induces Osteosarcoma Cell Death Characterized by Apoptosis-Inducing Factor and Endonuclease-G Translocation Independently of p53 and Retinoblastoma Status. Molecular Pharmacology, 71, 333-343.
[27]
Chen, Q., Xie, W., Kuhn, D.J., Voorhees, P.M., Lopez-Girona, A., Mendy, D., Corral, L.G., et al. (2008) Targeting the p27 E3 Ligase SCF(Skp2) Results in p27- and Skp2-Mediated Cell-Cycle Arrest and Activation of Autophagy. Blood, 111, 4690-4699. http://dx.doi.org/10.1182/blood-2007-09-112904
[28]
Komata, T., Kanzawa, T., Takeuchi, H., Germano, I. M., Schreiber, M., Kondo, Y., & Kondo, S. (2003) Antitumour Effect of Cyclin-Dependent Kinase Inhibitors (p16(INK4A), p18(INK4C), p19(INK4D), p21(WAF1/CIP1) and p27(KIP1)) on Malignant Glioma Cells. British Journal of Cancer, 88, 1277-1280. http://dx.doi.org/10.1038/sj.bjc.6600862
[29]
Liang, J., Shao, S.H., Xu, Z.X., Hennessy, B., Ding, Z., Larrea, M., Kondo, S., Dumont, D.J., Gutterman, J.U., Walker, C.L., Slingerland, J.M. and Mills, G.B. (2007) The Energy Sensing LKB1-AMPK Pathway Regulates p27(kip1) Phosphorylation Mediating the Decision to Enter Autophagy or Apoptosis. Nature Cell Biology, 9, 218-224.
[30]
Liu, Y.-P., Chen, H.-L., Tzeng, C.-C., Lu, P.-J., Lo, C.-W., Lee, Y.-C., Tseng, C.-H., et al. (2013) TCH-1030 Targeting on Topoisomerase I Induces S-Phase Arrest, DNA Fragmentation, and Cell Death Of Breast Cancer Cells. Breast Cancer Research and Treatment, 138, 383-393. http://dx.doi.org/10.1007/s10549-013-2441-1
[31]
Yoo, S.H., Yoon, Y.G., Lee, J.S., Song, Y.S., Oh, J.S., Park, B.S. and Yoo, Y.H. (2012) Etoposide Induces a Mixed Type of Programmed Cell Death and Overcomes the Resistance Conferred by Bcl-2 in Hep3B Hepatoma Cells. International Journal of Oncology, 41, 1443-1454.
[32]
Schleich, K., Krammer, P.H. and Lavrik, I.N. (2013) The Chains of Death: A New View on Caspase-8 Activation at the DISC. Cell Cycle, 12, 193-194. http://dx.doi.org/10.4161/cc.23464
[33]
Ehrhardt, H., Häcker, S., Wittmann, S., Maurer, M., Borkhardt, A., Toloczko, A., Debatin, K.M., Fulda, S. and Jeremias, I. (2008) Cytotoxic Drug-Induced, p53-Mediated Upregulation of Caspase-8 in Tumor Cells. Oncogene, 27, 783-793. http://dx.doi.org/10.1038/sj.onc.1210666
[34]
Shawgo, M.E., Shelton, S.N. and Robertson, J.D. (2008) Caspase-Mediated Bak Activation and Cytochrome C Release during Intrinsic Apoptotic Cell Death in Jurkat Cells. The Journal of Biological Chemistry, 283, 35532-35538. http://dx.doi.org/10.1074/jbc.M807656200
[35]
Enari, M., Sakahira, H., Yokoyama, H., Okawa, K., Iwamatsu, A. and Nagata, S. (1998) A Caspase-Activated DNase that Degrades DNA during Apoptosis, and Its Inhibitor ICAD. Nature, 391, 43-50. http://dx.doi.org/10.1038/34112
[36]
Alenzi, F.Q., Lotfy, M. and Wyse, R. (2010) Swords of Cell Death: Caspase Activation and Regulation. Asian Pacific Journal of Cancer Prevention: APJCP, 11, 271-80. http://www.ncbi.nlm.nih.gov/pubmed/20843101
[37]
Liu, J., Uematsu, H., Tsuchida, N. and Ikeda, M.-A. (2011) Essential Role of Caspase-8 in p53/p73-Dependent Apoptosis Induced by Etopo Side in Head and Neck Carcinoma Cells. Molecular Cancer, 10, 95. http://dx.doi.org/10.1186/1476-4598-10-95
[38]
Hitotsuyanagi, Y., Kobayashi, M., Morita, H., Itokawa, H. and Takeya, K. (1999) Synthesis of (-)-4-Aza-4-deoxypodophyllotoxin from (-)-Podophyllotoxin. Tetrahedron Letters, 40, 9107- 9110.
http://dx.doi.org/10.1016/S0040-4039(99)01932-2
[39]
Hitotsuyanagi, Y., Fukuyo, M., Tsuda, K., Kobayashi, M., Ozeki, A., Itokawa, H. and Takeya, K. (2000) 4-Aza-2,3-dehydro-4-deoxypodophyllotoxins: Simple Aza-Podophyllotoxin Analogues Possessing Potent Cytotoxicity. Bioorganic & Medicinal Chemistry Letters, 10, 315-317. http://dx.doi.org/10.1016/S0960-894X(99)00693-9
[40]
Raimond, B.G., Ravelli, B.G., Patrick, A.C., Isabelle, J., Sylvie, L., André, S. and Marcel, K. (2004) Insight into Tubulin Regulation from a Complex with Colchicine and a Stathmin-Like Domain, Nature, 428, 198-202.
http://dx.doi.org/10.1038/nature02393
