القای آپوپتوز ناشی از اثر هارمین در رده سلولی سرطان کولون انسانی HT29 و تغییرات بیان ژن‌های آپوپتوتیک P53، Bax و Bcl-2

نوع مقاله : مقاله پژوهشی

نویسندگان

1 گروه زیست شناسی، واحد مشهد، دانشگاه آزاد اسلامی، مشهد، ایران

2 گروه زیست‌شناسی و مرکز تحقیقات بیولوژی کاربردی تکوین جانوری، واحد مشهد، دانشگاه آزاد اسلامی، مشهد، ایران

چکیده

آلکالوئیدها دارای اثرات ضدتکثیری و محرک آپوپتوز در سلول­های سرطانی هستند. هارمین نیز یکی از آلکالوئیدهای مهم در بذر اسپند است که اثرات ضدسرطانی آن گزارش شده است. هدف این مطالعه، بررسی اثر آلکالوئید هارمین بر القای آپوپتوز در رده سلولی سرطان کولون انسانی HT29 و تغییرات بیان ژن­های آپوپتوتیک P53، Bax و Bcl-2 است. به این منظور سلول­های سرطانی کولون HT29 با غلظت­های مختلف هارمین (35، 45، 55، 65 میکروگرم بر میلی­لیتر) به مدت 24 ساعت تیمار شدند. زیست­پذیری سلول­ها با استفاده از آزمون  MTTبررسی شد. تغییرات مرفولوژی هسته با رنگ­آمیزی DAPI بررسی گردید. همچنین، القای آپوپتوز با تست انکسین و تغییرات بیان ژن­های P53، Bax و Bcl-2 با استفاده از Real-time PCR  تعیین شد. نتایج این بررسی نشان داد که هارمین به صورت وابسته به غلظت باعث کاهش زیست­پذیری در سلول­های سرطانی کولون HT29 شد. همچنین، تیمار سلول­های سرطانی کولون HT29 با هارمین باعث تغییرات مرفولوژیک هسته سلول از جمله تراکم کروماتین هسته، تشکیل اجسام آپوپتوزی، و چروکیدگی غشای سلول شد. از طرفی، تیمار سلول­های سرطانی HT29 با هارمین، باعث کاهش بیان Bcl-2 و همزمان افزایش بیان P53 و Bax و در مجموع، کاهش نسبت Bcl-2 به Bax شد. همچنین نتایج تست انکسین نیز تاییدکننده افزایش آپوپتوز در سلول­ها است. در نتیجه می­توان بیان کرد که هارمین به دلیل داشتن سیتوتوکسیسته موثر در مهار تکثیر و القای آپوپتوز، می­تواند در درمان سلول­های سرطانی کولون مورد استفاده قرار گیرد.

کلیدواژه‌ها


عنوان مقاله [English]

Apoptosis induction of Harmine on Human Colon Cancer Cell Line HT29 and Alteration in Apoptotic Genes Expression P53, Bax and Bcl-2

نویسندگان [English]

  • Reyhaneh Chitbandi 1
  • Javad Baharara 2
  • Maryam Tehranipour 1
1 Department of Biology, Islamic Azad University, Mashhad Branch, Mashhad, Iran
2 Department of Biology & Research Center for Animal Development Applied Biology, Islamic Azad University, Mashhad Branch, Mashhad, Iran
چکیده [English]

Alkaloids have anti-proliferative and apoptotic stimulatory effects on cancer cells. Harmine is also one of the important alkaloids that have been reported to have anticancer effects. The aim of this study was to investigate the effect of Harmine on apoptosis induction in human HT29 colon cancer cell line and expression alteration of apoptotic genes P53, Bax and Bcl-2. For this purpose, HT29 colon cancer cells were treated with different concentrations of harmine (0, 35, 45, 55, 65 μg/ml) for 24 hours. Then the cell viability was assessed using the MTT assay. Nuclear changes and chromatin condensation were investigated by DAPI staining. Also, apoptosis induction was determined by Annexin V-FITC testing and changes in P53, Bax and Bcl-2 genes expression was assessed by using Real Time PCR. The results showed that the concentration of Harmin reduced the viability of HT29 colon cancer cells. Also, the treatment of HT29 colon cancer cells with Harmin caused morphological changes in the cell nucleus, including chromatin condensation of the nucleus, the formation of apoptotic bodies, and the wrinkling of the cell membrane. All of these morphological features indicate apoptosis. The results of the Annexin test also confirmed this finding; with about 57% of the cells undergo apoptosis. On the other hand, the treatment of HT29 cancer cells with Harmin reduced the expression of Bcl-2 and at the same time increased the expression of P53 and Bax and, in general, reduced the ratio of Bcl-2 to Bax. As a result, Harmin can be used to treat colon cancer cells because of its cytotoxicity, which is effective in inhibiting cell prolifration and inducing apoptosis.

کلیدواژه‌ها [English]

  • Harmine
  • Colon cancer
  • Cytotoxicity
  • Apoptosis
  1. Abdalan S., Baghbani-Arani F., Sadat Shandiz S.A. 2018. Evaluation of Anticancer Effect of Aqueous and Hydroalcoholic Extracts of Quercusin Fectoria Leaf against Colon Cancer HT29 Cell Line. Journal of Arak University of Medical Sciences, 21(4) :48-57.
  2. Ahamed A., Panneerselvam A., Alaklabi A., Arif I.A., Ambikapathy V., Thajuddin N. 2020. Molecular perspective and anticancer activity of medicinal plants. Saudi Journal of Biological Sciences, 27(2): 666-675.
  3. Chen Q., Chao R., Chen H., Hou X., Yan H., Zhou S., Peng W., Xu A. 2013. Antitumor and neurotoxic effects of novel harmine derivatives and structure‐activity relationship analysis. International Journal of Cancer, 114(5):675-82.
  4. Ding Y., He J., Huang J., Yu T., Shi X., Zhang T., Yan G., Chen S., Peng C. 2019. Harmine induces anticancer activity in breast cancer cells via targeting TAZ. International journal of oncology, 54(6): 1995-2004.
  5. Gianchecchi E., Fierabracci A. 2020 Feb. Insights on the Effects of Resveratrol and Some of Its Derivatives in Cancer and Autoimmunity: A Molecule with a Dual Activity. Antioxidants, 9(2): 91.
  6. Hamsa T.P., Kuttan G. 2011. Harmine activates intrinsic and extrinsic pathways of apoptosis in B16F-10 melanoma. Chinese Medicine, 6(1):11.
  7. Hamsa T.P., Kuttan G. 2010. Harmine inhibits tumour specific neo-vessel formation by regulating VEGF, MMP, TIMP and Pro-Inflammatory Mediators Both in Vivo and in Vitro, 649(1-3):64-73.
  8. Hassan M., Watari H., AbuAlmaaty A., Ohba Y., Sakuragi N. 2014. Apoptosis and molecular targeting therapy in cancer. BioMed research international, 2014:150845.
  9. Ibraheam I.A., Hussein H.M., Hameed I.H. 2018. Potential uses and Analysis of Bioactive Natural Compounds of Peganum harmala. International Journal of Pharmaceutical Quality Assurance, 9(02):153-157.
  10. Ismail M.M., Farrag A.M., Harras M.F., Ibrahim MH, Mehany AB. 2020 Jan. Apoptosis: A target for anticancer therapy with novel cyanopyridines. Bioorganic Chemistry, 94:103481.
  11. Jimenez J., Riveron-Negrete L., Abdullaev F., Espinosa-Aguirre J., Rodríguez-Arnaiz R. 2008. Cytotoxicity of the β-carboline alkaloids harmine and harmaline in human cell assays in vitro. Experimental and Toxicologic pathology, 60(4-5): 381-389.
  12. Kola P., Metowogo K., Kantati Y.T., Lawson-Evi P., Kpemissi M., El-Hallouty SM, Mouzou AP, Eklu-Gadegbeku K., Aklikokou K.A. 2020. Ethnopharmacological Survey on Medicinal Plants Used by Traditional Healers in Central and Kara Regions of Togo for Antitumor and Chronic Wound Healing Effects. Evidence-Based Complementary and Alternative Medicine, 6940132:1-12.
  13. Liu J., Li Q., Liu Z., Lin L., Cao M., Jiang J. 2016. (Harmine induces cell cycle arrest and mitochondrial pathway-mediated cellular apoptosis in SW620 cells via inhibition of the Akt and ERK signaling pathways. Oncology Reports, 35(6): 3363-3370.
  14. Omara T., Kiprop A.K., Ramkat R.C., Cherutoi J., Kagoya S., Moraa Nyangena D., Azeze Tebo T., Nteziyaremye P., Nyambura Karanja L., Jepchirchir A., Maiyo A. 2020. Medicinal plants used in traditional management of cancer in Uganda: a review of ethnobotanical surveys, phytochemistry, and anticancer studies. Evidence-Based Complementary and Alternative Medicine, 2020:3529081.
  15. Pfeffer C.M., Singh A.T. 2018. Apoptosis: a target for anticancer therapy. International journal of molecular sciences, 19(2):448.
  16. Roostaee Z. 2018. Effect of alkaloids belong to β-carbolines family in Peganum harmala on cancer cells. Sarem Journal of Reproductive Medicine, 2(2):73-78.
  17. Roshankhah S., Arji Rodsari B., Jalili C., Salahshoor M.R. 2020. The Role of Harmine in Up-regulating p53 Gene Expression and Inducing Apoptosis in MCF-7 Cell Line. Middle East Journal of Cancer, 11(1):34-41.
  18. Ruan S., Jia F., Li J. 2017. Potential antitumor effect of harmine in the treatment of thyroid cancer. Evidence-Based Complementary and Alternative Medicine, 2017:9402615.
  19. Siegel R., DeSantis C., Jemal A. Colorectal cancer statistics. CA: a cancer journal for clinicians, 64(2):104-17.
  20. Siegel R.L., Miller K.D., Goding Sauer A., Fedewa S.A., Butterly L.F., Anderson J.C., Jemal A. 2020. Colorectal cancer statistics. CA: a Cancer Journal for Clinicians, 70(3):145-164.
  21. Wang C., Zhang Z., Wang Y., He X. 2015. Cytotoxic indole alkaloids against human leukemia cell lines from the toxic plant Peganum harmala. Toxins, 7(11): 4507-4718.
  22. Wijesinghe W.P., Mantilaka M.M., Weerasinghe A.M., de Silva K.N., Gamagedara T.P., Rajapakse R.M. 2016. Colloidal hydroxyapatite/poly (acrylic acid) hybrids using calcium sucrate and ammoniumdihydrogen orthophosphate. Journal of Applied Solution Chemistry and Modelling, 5:21-29.
  23. Wu L.W., Zhang J.K., Rao M., Zhang Z.Y., Zhu H.J., Zhang C. 2019. Harmine suppresses the proliferation of pancreatic cancer cells and sensitizes pancreatic cancer to gemcitabine treatment. OncoTargets and therapy, 12:4585.
  24. Zhang H., Jiao Y., Shi C., Song X., Chang Y., Ren Y., Shi X. 2018. Berbamine suppresses cell proliferation and promotes apoptosis in ovarian cancer partially via the inhibition of Wnt/β-catenin signaling. Acta Biochimica et Biophysica Sinica, 50(6): 532-539.
  25. Zou N., Wei Y., Li F., Yang Y., Cheng X., Wang C. 2017. The inhibitory effects of compound Muniziqi granule against B16 cells and harmine induced autophagy and apoptosis by inhibiting Akt/mTOR pathway. BMC Complement Alternative Medicine, 17:517.