Anticancer Medicinal Plant, Epipremnum pinnatum (L.) Engl. Chloroform Extracts Elicited Both Apoptotic and Non-Apoptotic Cell Deaths in T-47D Mammary Carcinoma Cells

Main Article Content

Tan Mei Lan* Shaida Fariza Sulaiman Tengku Sifzizul Tengku Muhammad


Epipremnum pinnatum (L.) Engl. Chloroform extract produced significant growth inhibition against T-47D breast carcinoma cells and analysis of cell death mechanism indicated that the extract elicited both apoptotic and non-apoptotic programmed cell deaths. T-47D cells exposed to the extract produced a significant up-regulation of c-myc and caspase-3 mRNA expression levels as compared to untreated cells. The up-regulation of caspase-3 mRNA expression appeared to be mediated mainly via both protein kinase C and tyrosine kinases pathways. T-47D cells exposed to the extract at EC50 concentration (72 h) for 24 h demonstrated typical DNA fragmentation associated with apoptosis, as carried out using a DNA fragmentation detection assay. However, ultrastructural analysis using transmission electron microscope demonstrated distinct vacuolated cells, which indicated a Type II non-apoptotic cell death although the presence of cell and nuclear blebbing, apoptotic bodies and chromatin changes associated with apoptosis were also detected. The presence of non-apoptotic programmed cell death was also detected with annexin-V and propidium iodide staining. These findings suggested that up-regulation of caspase-3 and c-myc mRNA expression may have contributed to both apoptotic and non-apoptotic programmed cell death, respectively in the Epipremnum pinnatum (L.) Engl. Chloroform extract-treated T-47D cells.

Keywords: Medicinal Plant, Epipremnum pinnatum (L.) Engl., apoptotic, non-apoptotic

Corresponding author: E-mail:



Download data is not yet available.

Article Details

Research Articles


[1] Wong, K.T., Tan, B.K.H. 1996. In-vitro cytotoxicity and immunomodulating property of Rhaphidora korthalsii, Journal of Ethnopharmacology, 52, 53-7.
[2] Wong, K.T., Tan, B.K.H., Sim, K.Y., Goh, S.H. 1996. A cytotoxic melanin precursor, 5,6-Dihydroxyindole, from the folkloric anti-cancer plant; Rhaphidophora korthalsii, Natural Product Letters, 9(2), 137-140.
[3] Chan, M.J., Turner, I.M. 1998. The use of Epipremnum pinnatum (Araceae) in Singapore in the treatmeant of cancer: an unreported application of a herbal medicine, Economic Botany, 52(1), 108.
[4] Goh, S.H. 1999. Bioactive principles from folkloric anti-neoplastic plants and from bio-prospecting the Malaysian Forest. In: The International Conference on Biodiversity and Bioresources: Conservation and Utilization, Phuket, Thailand, November, 23-27 (
[5] Tan, M.L.,Najimudin, N., Sulaiman, S.F., Tengku Muhammad, T.S. 2005. Growth arrest and non-apoptotic programmed cell death associated with the up-regulation of c-myc mRNA expression in T-47D breast tumor cells following exposure to Epipremnum pinnatum (L.) Engl. Hexane extract, Journal of Ethnopharmacology., 96, 375-383.
[6] Raskin, I., Ribnicky, D.M., Komarnytsky, S., Ilic, N., Poulev, A., Borisjuk, N., Brinker, A., Moreno, D.A., Ripoll, C., Yakoby, N., O’ Neal, J.M., Cornwell, T., Pastor, I., Fridlender, B. 2002. Plants and human health in the twenty first century, Trends in Biotechnology, 20(12), 522-531.
[7] Kibertis, P., Roberts, L. 2002. It’s not just the genes. Science, 296, 685.
[8] Gryfe, R., Swallow, C., Bapat, B., Redston, M., Gallinger, S., Couture, J. 1997. Molecular biology of colorectal cancer, Current Problems in Cancer, 21, 233-300.
[9] Potten, C.S. 1997. Epithelial cell growth and differentiation. American Journal of Physiology, 36, G253-G257.
[10] Kamesaki, H. 1998. Mechanisms involved in chemotherapy-induced apoptosis and their implications in cancer chemotherapy, International Journal of Hematology, 68, 29-43.
[11] Arends, M.J., Wyllie, A.H. 1991. Apoptosis mechanisms and roles in pathology, International Review of Experimental Pathology, 32, 223-54.
[12] Kerr, J.F.R.,Winterford, C.M., Harmon, B.V. 1994. Apoptosis; its significance in cancer and cancer therapy, Cancer, 73(8), 2013-26.
[13] Gavrieli, Y., Sherman, Y., Ben-Sasson, S.A. 1992. Identification of programmed cell death in-situ via specific labeling of nuclear DNA fragmentation, Journal of Cell Biology, 119(3), 493-501.
[14] Fadok, V.A., Savill, J.S., Haslett, C., Bratton, D.L., Doherty, D.E., Campbell, P.A., Henson, P.M. 1992(a). Different populations of macrophages use either the vitronectin receptor or the phosphatidylserine receptor to recognize and remove apoptosis, Journal of Immunology, 149(12), 4029-35.
[15] Fadok, V.A., Voelker, D.R., Campbell, P.A., Cohen, J.J., Bratton, D.L., Henson, P.M. 1992b. Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages, Journal of Immunology, 148, 2207-16.
[16] Verhoven, B., Schlegel, A.R., Williamson, P. 1995. Mechanism of phosphatidylserine exposure, a phagocyte recognition signal, on apoptotic T-lymphocytes, Journal of Experimental Medicine, 182, 1597-1601.
[17] Vermes, I., Haanem, C., Steffens-Nakken, H., Reutelingsperger, C. 1995. A novel assay for apoptotic cells using fluorescein labelled annexin V, Journal of Immunological Methods, 184, 39-51.
[18] Adams, J.M., Cory, S 2002. Apoptosomes: engines for caspase activation, Current Opinion in Cell Biological, 14, 715-720.
[19] Beauparlant, P., Shore, G.C. 2003. Therapeutic activation of caspases in cancer: a question of selectivity, Current Opinion in Drug Discovery and Development, 6, 179-187.
[20] Kastan, M.B., Onyekwere, O., Sidransky, D., Vogelstein, B., Craig, R.W. 1991. Participation of p53 protein in the cellular response to DNA damage, Cancer Research, 51(23), 6304-11.
[21] Nelson, W.G., Kastan, M.B. 1994. DNA strand breaks: the DNA template alterations that trigger p53-dependent DNA damage response pathways, Molecular Cell Biology, 14(3), 1815-23.
[22] Kim, R., Ohi, Y., Inoue, H., Toge, T. 1999. Taxotere activates transcription factor AP-1 in association with apoptotic cell death in gastric cancer cell lines, Anticancer Research, 19(16B), 5399-405.
[23] Janus, F., Albrechtsen, N., Dornreiter, I., Wiesmuller, L., Grosse, F., Deppert, W. 1999. The dual role model for p53 in maintaining genomic intergrity, Cellular and Molecular Life Sciences, 55, 12-27.
[24] Wurm, F.M., Gwinn, K.A., Kingston, R.E. 1986. Inducible overproduction of mouse c-myc protein in mammalian cells, Proceedings of the National Academy of Sciences USA, 83, 5414-8.
[25] Wyllie, A.H., Rose, K.A., Morris, R.G., Steel, C.M., Foster, E., Spandidos, D.A. 1987. Rodent fibroblast tumours expressing human myc and ras genes; growth, metastasis and endogenous oncogene expression, British Journal of Cancer, 56, 251-9.
[26] Evan, G.I., Wyllie, A.H., Gilbert, C.S., Littlewood, T.D., Land, H., Brooks, M., Waters, C.M., Penn, L.Z., Hancock, D.C. 1992. Induction of apoptosis in fibroblasts by c-myc protein, Cell, 69, 119-28.
[27] Askew, O.S., Ashmun, R.A., Simmons, B.C., Cleveland, J.L. 1991. Constitutive c-myc expression in an IL-3-dependent myeloid cell line suppresses cell cycle arrest and accelerates apoptosis, Oncogene, 6, 1915-22.
[28] Kuida, K., Zheng, T.S., Na, S.Q., Kuan, C.Y., Yang, D., Karasuyama, H., Rakic, P., Flavell, R.A. 1996. Decreased apoptosis in the brain and premature lethality in CPP32-deficient mice, Nature, 384, 368-72.
[29] Schlegel, J., Peter, I., Orrenius, S., Miller, D.K., Thornberry, N.A., Yamin, T.T., Nicholson, D.W. 1996. CPP-32/apopain is the key interleukin-1β-converting enzyme-like protease involved in Fas-mediated apoptosis, Journal Biological Chemistry, 271(4), 1841-4.
[30] Janicke, R.U., Sprengart, M.L., Wati, M.R., Porter, A.G. 1998. Caspase-3 is required for DNA fragmentation and morphological changes associated with apoptosis, Journal of Biological Chemistry, 273(16), 9357-60.
[31] Kousteni, S., Tura-Kockar, F., Ramji, D.P. 1999. Sequence and expression analysis of a novel Xenpus laevis cDNA that encodes a protein similar to bacterial and chloroplast ribosomal protein L24, Gene, 235, 13-18.
[32] Tengku Muhammad, T.S., Hughes, T.R., Ranki, H., Cryer, A., Ramji, D.P. 2000. Differential regulation of macrophage CCAAT-Enhancer binding protein isoforms by lipopolysaccharide and cytokines, Cytokine, 12, 1430-1436.
[33] Ahn, C.H., Kong, J.Y., Choi, W.C., Hwang, M.S. 1996. Selective inhibition of the effects of phorbol ester on doxorubicin resistance and p-glycoprotein by the protein kinase C inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7) in multidrug-resistant MCF-7/Dox human breast carcinoma cells, Biochemical Pharmacology, 52, 393-9.
[34] Lin, C.H., Kuan, I.H., Wang, C.H., Lee, H.M., Lee, W.S., Sheu, J.R., Hsiao, G., Wu, C.H., Kuo, H.P. 2002. Lipoteichoic acid-induced cyclooxygenase-2 expression requires activations of p44/42 and p38 mitogen-activated protein kinase signal pathways, European Journal of Pharmacology, 450, 1-9.
[35] Fujihara, M., Muroi, M., Muroi, Y., Ito, N., Suzuki, T. 1993. Mechanism of lipopolysaccharide-triggered junB activation in a mouse macrophage-like cell line (J774), Journal of Biological Chemistry, 268(20), 14898-905.
[36] Eason, S., Martin, W. 1995. Involvement of tyrosine kinase and protein kinase C in the induction of nitric oxide synthase by lipopolysaccharide and interferon-gamma in J774 macrophages, Archives International Pharmacodynamic Therapy, 330(2), 225-40.
[37] Glass, D.B. 1983. Differential responses of cyclic GMP-dependent and cyclic AMP-dependent protein kinases to synthetic peptide inhibitors, Biochemical Journal, 213, 159-64.
[38] Tengku-Muhammad, T.S., Hughes, T.R., Cryer, A., Ramji, D.P. 1999. Involvement of both the tyrosine kinase and the phosphatidylinositol-3’ kinase signal transduction pathways in the regulation of lipoprotein lipase expression in J774.2 macrophages by cytokines and lipopolysaccharide, Cytokine, 11(7), 463-8.
[39] Geran, R.I., Greenberg, N.H., Macdonald, M.M., Schumacher, A.M., Abbott, B.J. 1972. Protocols for screening chemical agents and natural products against animal tumours and other biological systems, Cancer Chemotherapy Reports, 3, 59-61.
[40] Hoffman, B., Liebermann, D.A. 1998. The proto-oncogene c-myc and apoptosis, Oncogene, 17, 3351-7.
[41] Leng, Y., Gu, Z.P., Cao, L. 2000. Apoptosis induced by droloxifene and c-myc, bax and bcl-2 mRNA expression in cultured luteal cells of rats, European Journal of Pharmacology, 409(2), 123-31.
[42] Thornberry, N.A., Lazebnik, Y. 1998. Caspases: enemies within, Science, 281, 1312-1316.
[43] Budihardjo, I., Oliver, H., Lutter, M., Luo, X., Wang, X. 1999. Biochemical pathways of caspase activation during apoptosis, Annual Review of Cell and Development Biology, 15, 260-290.
[44] Wolf, B.B., Green, D.R. 1999. Suicidal tendencies: apoptotic cell death by caspase family proteinases, Journal Biological Chemistry, 274, 20049-20052.
[45] Earnshaw, W.C., Martins, L.M., Kaufmann, S.H. 1999. Mammalian caspases: Structure, activation, substates and functions during apoptosis, Annual Review of Biochemistry, 68, 383-424.
[46] Blatt, N.B., Glick, G.D. 2001. Signaling pathways and effector mechanisms pre-programmed cell death, Bioorganic and Medicinal Chemistry, 9, 1371-84.
[47] Chang, Q., Tepperman, B.L. 2001. The role of protein kinase C isozymes in TNF-alpha-induced cytotoxicity to a rat intestinal cell line, American Journal of Physiology – Gastrointestinal and Liver Physiology, 280(4), G572-83.
[48] Basu, A., Akkaraju, G.R. 1999. Regulation of caspase activation and cis-diammine-dichloroplatinum(II)-induced cell death by protein kinase C, Biochemical Journal, 38(14), 4245-51.
[49] Park, I.C., Park, M.J., Rhee, C.H., Lee, J.I., Choe, T.B., Jang, J.J., Lee, S.H., Hong, S.I. 2001. Protein kinase C activation by PMA rapidly induces apoptosis through caspase-3/CPP32 and serine protease(s) in a gastric cancer cell line, International Journal of Oncology, 18(5), 1077-83.
[50] Cataldi, A., Miscia, S., Centurione, L., Rapino, M., Bosco, D., Grifone, G., Valerio, V.D., Garaci, F., Rana, R. 2002. Role of nuclear PKC delta inmediating caspase-3 up-regulation in Jurkat T leukemic cells exposed to ionizing radiation, Journal of Cellular Biochemistry, 86(3), 553-60.
[51] Koriyama, H., Kouchi, Z., Umeda, T., Saido, T.C., Momoi, T., Ishiura, S., Suzuki, K. 1999. Proteolytic activation of protein kinase C δ and ε by caspase-3 in U937 cells during chemotherapeutic agent-induced apoptosis, Cell Signaling, 11(11), 831-38.
[52] Sato, N., Sakamaki, K., Terada, N., Arai, K., Miyajima, A. 1993. Signal transduction by the high-affinity GM-CSF receptor: two distinct cytoplasmic regions of the common beta subunit responsible for different signaling, EMBO Journal, 12(11), 4181-9.
[53] Bowen, I.D., Bowen, S.M. 1990. Programmed cell death in tumours and tissues, 1st edition, Chapman and Hall, London.
[54] Bowen, I.D., Mullarkey, K., Morgan, S.M. 1996. Programmed cell death in the salivary glands of blow fly Calliphora vomitoria, Microscopy Research and Techniques, 34, 202-7.
[55] Bowen, I.D., Bowen, S.M., Jones, A.H. 1998. Mitosis and Apoptosis: Matters of life and death, Chapman and Hall, London.
[56] Bowen, I.D., Amin, F. 2000. Tumor cell death. In: Programmed cell death in animals and plants (eds Bryant et al), BIOS Scientific Publishers, United Kingdom.
[57] Amin, F., Bowen, I.D., Szegedi, Z., Mihalik, R., Szende, B. 2000. Apoptotic and non-apoptotic modes of programmed cell death in MCF-7 human breast carcinoma cells, Cell Biology International, 24(4), 253-60.
[58] Szende, B., Keri, G.Y., Szegedi, Z.S., Benedeczky, I., Csikos, A., Orfi, L., Gazit, A. 1995. Tryphostin induces non-apoptotic programmed cell death in colon tumor cells, Cell Biology International, 19, 903-11.
[59] Ou, D., Bonomi, P., Jao, W., Jadko, S., Harris, J.E., Anderson, K.M. 2001. The mode of cell death in H-358 lung cancer cells cultured with inhibitors of 5-lipooxygenase or the free radical spin trap, NTBN. Cancer Letters, 166(2), 223-31.
[60] Ellington, A.A., Berhow, M., Singletary, K.W. 2005. Induction of macroautophagy in human colon cancer cells by soybean B-group triterpenoid saponins, Carcinogenesis, 26(1), 159-67.
[61] Tanida, I., Ueno, T., Kominami, E. 2004. LC3 conjugation system in mammalian autophagy, The International Journal of Biochemistry and Cell Biology, 36, 2503-2518.
[62] Meijer, A.J., Dubbelhuis, P.F. 2004. Amino acid signaling and the integration of metabolism, Biochemical and Biophysical Research Communications, 313, 397-403.
[63] Gozuacik, D., Kimchi, A. 2004. Autophagy as a cell death and tumor suppressor mechanism, Oncogene, 23(16), 2891-2906.
[64] Marino, G., Lopez-Otin, C. 2004. Autophagy: molecular mechanisms, physiological functions and relevance in human pathology, Cellular and Molecular Life Sciences, 61(12), 1439-1454.
[65] Meijer, A.J., Codogno, P. 2004. Regulation and role of autophagy in mammalian cells, International Journal Biochemistry and Cell Biology, 36(12), 2445-2462.
[66] Gutierrez, M.G., Munafo, D.B., Beron, W., Colombo, M.I. 2004. Rab 7 is required for the normal progression of the autophagic pathway in mammalian cells, Journal Cell Science, 117(13), 2687-2697.