CHARACTERISTIC OF APOPTOSIS AND EXPRESSION OF GENES-RELATED APOPTOSIS (c-myc, c-erb AND c-fos ONCOGENE) IN HeLa CELL LINES AFTER EXPOSURE BY NEEM (Azadirachta indica A.Juss)

Authors

  • Dessy Arisanty
  • Zolkapli Eshak
  • Fauziah Othman
  • Asmah Rahmat
  • Abdah M.D. Akim
  • Nurmawaty -
  • Suhaida. M. Jalil

DOI:

https://doi.org/10.25077/jrk.v1i2.35

Abstract

 

ABSTRACT

 

Azadirachta indica A. Juss is a medicinal plant commonly known as neem. The effect of neem leaves extract on cervical cancer cells, however, has never been studied. Due to the lack of information, this study was conducted to determine the effect of neem leaves extract on cervical cancer (HeLa) cell growth. In vitro cytotoxicity effect of ethanolic neem extract indicated the presence of cytotoxicity activity of the extract against HeLa cells with IC50 of  30.0 μg/mL. The morphological changes under confocal laser scanning microscope (CLSM) on HeLa cells were cell shrinkage and membrane blebbing. There were also cells with condensed nucleus and few cells have fragmented nucleus, and finally formed apoptotic body. Control cells showed a clear cytoplasm and centrally placed nucleus and no cells exhibited any apoptotic features. Appearance of apoptotic cells under scanning electron microscope (SEM) are indentations, blebs and hole on cell surface and disintegration of cell. The controls remained morphologically normal. Apoptotic features of the cells are widely seen with longer incubation time while 24 hours incubation time, it is scarcely seen. The RT-PCR product showed that the c-erb gene expression was expressed in both treated and untreated HeLa cells. Contrary, the the c-myc and c-fos oncogenes on  HeLa cells which exposed to A. indica  EtOH extract were significantly decreased.  Thus, the results from this study strongly suggest that the ethanolic extract of A. indica may contain bioactive compound(s) that caused cervical cancer cells, HeLa cell death by apoptosis mechanism and lead to succession of discovering new alternative treatment for cervical cancer. 

 

Keywords : cytotoxic, apoptosis genes, oncogenes, neem

References

Coarret, M. D., and Mittnacht, S., 2005, Oncogene and cell proliferation : The genetic and Biology of cancer revealing avenues for therapy, Current Opinion in Genetic and Development, 15: 1-4.

Anonymous, 2004, MAKNA: Majlis Kanser Nasional.

Shiva, 2001, Neem: A Plant for All season.

At: www.vshiva.net/naturefact/neem/htm.

Schmutterer, H. S., 1995, Neem tree, Azadirachta indica A.Juss and other meliaceous plants, VCH, weinheim, Germany.

Khosla, P., Bhanwara, S., Singh, S., Seth, S., and Srivastava, R. K., 2000, A study of hypoglycemic effect of Azadirachta indica in normal and alloxan diabetic rabbits, J. of Physiol. Pharmacol., 44 (1): 69-74.

Khan, P. K., Awasthy, K. S., 2003, Cytogenetic toxicity of neem, Food and Chemical Toxicology, 41: 1325-1328.

Krishnaveni, P., Ajantha, S., Narayani, M., Suherman, J., Susi Endrini, Asmah, R., Fauziah, O., 2002, Confocal microscopy on the effect of neem leaf extract on MCF-7 breast cancer cell lines. Proc. Seminar Update on Microscopy and Microanal. UPM Press: Serdang, 48-49.

Fauziah, O., Fakurazi S., Asmah R., Ajantha, S., Krishnaveni, P., and Tengku Shahrul, T. M. Y., 2003, Azadirachta indica induces apoptosis in MCF-7 Breast cancer cells. Proceedings of the 12th Scientific Conference and 13th Annual General Meeting Electron Microscopy Society of Malaysia, Langkawi, Kedah, Malaysia, 512-516.

Wyllie, A. H., Kerr, J. F. R., and Currie, R., 1980, Cell death : The significance of apoptosis, Int. Rev. Cytol., 68: 251-306.

Steller, H., 1995, Mechanism and genes of ellular suicide, Science, 267: 1445-1449.

Yasuhara, S., Zhu, Y., Matsui, T., Tipirneni, N., Yasuhara, Y., Kaneki, M., Rosenzweig, A., Jeevendra, A.,, 2003, Comparison of Comet Assay, Electron Microscopy, and Flow Cytometry for Detection of Apoptosis, J. Histochem. Cytochem., 51: 873 - 885.

Kelly, K. J., Sandoval, M. R., Dunn, K. W., Molitoris, B. A., and Dagher, P. C., 2003, A novel method to determine Specificity sensitivity of the TUNELreaction in the quantitation of apoptosis, Am. J. Physiol Cell Physiol, 284: 1309 – 1318.

Nasi, S., Ciarapica, R., Jucker, R., Rosati, J., and Soucek, L., 2003, Making decision trough c-myc, FEBS letter., 490: 153-162.

Suzanne, D. C., Kathrin, G., Eugene, S. K., Pratiratibha, K., Andrew, J., Wagner, Maura, O., and Nissim, H., 2000, Induction of Cell Cycle Progression and Acceleration of apoptosis are two separable functions of c-myc: Transrepression correlates with acceleration of apoptosis, Molecular and Cellular Biology, 20: 6008–6018.

Grooteclaes, M., Vernimmen, D., Plaza, S., Pasleu, F., Hodzie, D., and Winker, R., 1999, A new cis element is involved in the HER-2 gene overexpression in Human breast cancer cells, Cancer Research, 59: 2527-2531.

Cunni, M., Kramar, A., Courjal, F., Johannas, D., et al., 2000, Relating genotype and phenotype in breast cancer an analysis of the prognosis significance of amplification at eight different genes or loci and p53 mutation, Cancer Research, 60: 1077-1083.

Robert, A. H. and Alfred, N., 1999, In Vitro transcriptional analysis of the human c-fos proto-oncogene, The Journal of Biological Chemistry, 266 : 19572-19582.

Nurulaini, R., 2004, A study on the cytoskeletal proteins of breast cancer cell lines treated with Neem (Azadirachta indica) leaf extract, Final Year thesis, University Putra Malaysia.

Ajantha, S., 2000, Evaluation of apoptosis inducing ability of Neem (Azadirachta indica) leaf extract on MCF-7 breast cancer cell lines, Final Year thesis, University Putra Malaysia.

Lu, Z., Zhang, C., and Zhai, Z., 2005, Nucleoplasmin regulates chromatin condensation during apoptosis, PNAS 102: 2778-2783.

Cohen, J. J., 1993, Overview : Mechanism of apoptosis, Immunology Today, 14: 126-130.

Kumar, V., Ramzi S., Cotran, Stanley L., Robbins, 2003, Basic Pathology 7th Edition. Saunders Elsevier Science, India.

McConkey, D. J., 1998, Biochemical determinants of apoptosis and necrosis, Toxicol. Lett., 99: 157–168.

Sluyser M., 1996, Apoptosis in Normal Development and Cancer, 2-86.

Fauziah, O., Purton, M. D., and Solomon, S. E., 1996, Scanning Electron Microscopy of the Respiratory Epithelium of Chicks Fumigated with Formaldehyde Vapour, British Poultry Science, 37: 563-570.

Willey, J. C., Crawford, E. L., Jackson, C. M., Weaver, D. A., Hoban, J. C., Khuder, S. A., and DeMuth, J. P., 1998, Expression measurement of many genes simultaneously by quantitative RT-PCR using standardized mixtures of competitive templates, American Journal Respiratory Cell Molecular Biology, 19: 6 - 17.

Loitsch, S. M., Kippenberger, S., Dauletbaev, N., Wagner, T. O., and Bargon, J., 1999, Reverse transcription-competitive multiplex PCR improves quantification of mRNA in clinical samples-Application to the low abundance CFTR mRNA, Clinical Chemistry, 45: 619–624.

Ramachandran, T., Habour, D. V., and Thompson, E. B., 1993, Suppression of c-myc is a critical step in glucocorticoid –induced human leukemia cell lysis, The Journal of Biological Chemistry, 28: 18306 – 18312.

Gauduchon, J., Gouilleux, F., Maillard, S., Marsaud, V., Renoir, J. M., and Brigitte, S., 2005, 4-Hydroxytamoxifen Inhibits Proliferation of Multiple Myeloma Cells In vitro through Down-Regulation of c-Myc, Up-Regulation of p27Kip1, and Modulation of Bcl-2 Family Members, Clinical Cancer Research, 11: 2345 - 2354.

Hsu, S. H., Hsu, J. W., Liu, M. C., Chen, L. Y., and Chang C-D., 2000, Retinoic Acid-Mediated G1 Arrest Is Associated with Induction of p27Kip1 and Inhibition of Cyclin-Dependent Kinase 3 in Human Lung Squamous Carcinoma CH27 Cells. Experimental Cell Research, 258: 322-331.

Dong, J. M., Naito, Y., and Tsuruo, T., 1997, C-myc plays a role in cellular susceptibility to death receptor-mediated and chemotherapy-induced apoptosis in human monocytic leukemia U937, Oncogene, 15: 639–647.

Adachi, S., Obaya, A.J., Han, Z., Desmone, N.R., Wyche, J.H., and Sedivy, J.M., 2001, C-myc is necessary for DNA damage induce apoptosis in G2 phase of the cell cycle. Molecular and cell Biology, 21: 4929 – 4937.

McClelland, R. A., Barrow, D., Tracie-Ann, M., Dutkowski, C. M., Pamment, J., Knowlden, J.M., Gee, J.M.W., and Nicholson, R.I., 2001, Enhanced Epidermal Growth Factor Receptor Signaling in MCF7 Breast Cancer Cells after Long-Term Culture in the Presence of the Pure Antiestrogen ICI 182,780 (Faslodex), Endocrinology, 142: 2776- 2789.

Kalra, N., and Kumar, V., 2004, C-fos Is a Mediator of the c-myc-induced Apoptotic Signaling in Serum-deprived hepatoma cells via the p38 mitogen-activated protein kinase pathway, Journal of Biological Chemistry, 279: 25313–25319.

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Published

2015-02-12

How to Cite

Arisanty, D., Eshak, Z., Othman, F., Rahmat, A., Akim, A. M., -, N., & M. Jalil, S. (2015). CHARACTERISTIC OF APOPTOSIS AND EXPRESSION OF GENES-RELATED APOPTOSIS (c-myc, c-erb AND c-fos ONCOGENE) IN HeLa CELL LINES AFTER EXPOSURE BY NEEM (Azadirachta indica A.Juss). Jurnal Riset Kimia, 1(2), 116. https://doi.org/10.25077/jrk.v1i2.35

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Vol. 1 No. 2 (2008): March

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