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Volume 3, Issue 2-2, March 2015, Page: 7-14
Cytotoxic, Cytostatic and Anti-Estrogenic Effect of Thymoquinone on Estrogen Receptor-Positive Breast Cancer MCF7 Cell Line
Marjaneh Motaghed, Oncology Science Cluster, Advanced Medical and Dental Institute, University Sains Malaysia, Penang, Malaysia
Received: Dec. 10, 2014;       Accepted: Dec. 13, 2014;       Published: Dec. 27, 2014
DOI: 10.11648/j.ajls.s.2015030202.12      View  3701      Downloads  223
About 80% of breast cancers are estrogen-receptor positive. The research carried out herein focused on the effect of Thymoquinone which is an active compound of Nigella sativa seed on estrogen-receptor positive breast cancer MCF7 cell line. The percentage of apoptotic cells was found using Annexin V-FITC apoptosis detection kit. CycleTEST PLUS DNA Reagent was used to distinguish distribution of treated cells between different cell cycle phases. DNA microarray identified the regulated genes, level of expressed genes, gene ontology and pathway networks. Significant arrest of treated cells at G1 phase suggested cytostatic effect of Thymoquinone 100 µM after 24 hours at p-value < 0.05 which was similar to anti-estrogenic compounds such as Tamoxifen. Cytotoxic effect of Thymoquinone 100 µM was found through highly significant accumulation of cells at sub-G1 phase after 72 hours at p-value < 0.0001. CYP1A1, CYP1B1, NQO1 and UGT1A8 genes were down regulated after 24 hours treatment with Thymoquinone 50 µM concentration which suggested reduction of catechol estrogens and rising in metoxy forms of estradiol and estrone. Reduction of ER would be predictable due to the down-regulation of CYP1B1 and UGT1A8 genes which reduced affinity of trans-tamoxifen-o-glucuronide to ER. The study proposed the benefits of using Thymoquinone to accelerate Tamoxifen effects in treating breast cancer and reducing its side effects.
Thymoquinone, Tamoxifen, Anti-Estrogenic Effect, Cell Cycle, Apoptosis Assay, cDNA Microarray
To cite this article
Marjaneh Motaghed, Cytotoxic, Cytostatic and Anti-Estrogenic Effect of Thymoquinone on Estrogen Receptor-Positive Breast Cancer MCF7 Cell Line, American Journal of Life Sciences. Special Issue: The Most Effective Medicinal Plants in Cancer Treatment. Vol. 3, No. 2-2, 2015, pp. 7-14. doi: 10.11648/j.ajls.s.2015030202.12
National Cancer Institute, Global Cancer Research Programs., 2013. [Online]. Available at:
Krishnan, A.V., Swami. S., Feldman, D., 2012. The potential therapeutic benefits of vitamin D in the treatment of estrogen receptor positive breast cancer. Steroids 77, 1107-1112.
Steigerová, J., Oklest’ková., Levková, M., Rárová, L., koláˇr, Z., Strnad, M., 2010. Brassinosteroids cause cell cycle arrest and apoptosis of human breast cancer cells. Journal of Chemico-Biological Interactions 188, 487-496.
Song, L., Dong, W., Gao, M., Li, J., Hu, M., Guo, N, Huang, C., 2010. A novel role of IKKα in the mediation of UVB-induced G0/G1 cell cycle arrest response by suppressing Cyclin D1 expression. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research 1803, 323-332.
Watts, C.K.V., Brady, A., Sarcevic, B., Defazio, A., Musgrove, E.A., Sutherland, R.L., 1995 Antiestrogen inhibition of cell cycle progression in breast cancer cells is associated with inhibition of cyclin-dependent kinase activity and decreased retinoblastoma protein phosphorylation. Molecular Endocrinology 9, 1804-1813.
Musgrove, E.A., Hamilton, J.A., Lee, C.S., Sweeney, K.J., Watts, C.K., Sutherland, R.L., 1993. Growth factor, steroid, and steroid antagonist regulation of cyclin gene expression associated with changes in T-47D human breast cancer cell cycle progression. Molecular. Cell. Biology 13, 3577-3587.
Yang, J.J., Park, S.K., CHO, L.Y., Han, W., Park, B., Kim, H., Lee, K.S., Haahn, S.K., Cho, S.I., Ahn, S.H., Noh, D.Y., korean breast cancer society., 2010. Cost-Effectiveness Analysis of 5 Years of Postoperative Adjuvant Tamoxifen Therapy for Korean Women with Breast Cancer. Clinical Therapeutics 32, 1122-1138.
Clemons, M., Danson, S., Howell, A., 2002. Tamoxifen ("Nolvadex"): a review. Cancer Treatment Review 28, 165-180.
Sutherland, R.L., Hall, R.E., Taylor, I.W., 1983. Cell Proliferation Kinetics of MCF-7 Human Mammary Carcinoma Cells in Culture and Effects of Tamoxifen on Exponentially Growing and Plateau-Phase Cells. Cancer Research 43, 3998-4006.
Gurib-fakim, A., 2006. Medicinal plants: Traditions of yesterday and drugs of tomorrow. Molecular Aspects of Medicine 27, 1-93.
Rates, S.M.K., 2001. Plants as source of drugs. Toxicon 39, 603–613.
Graham, J.G., Quinn, M.L., Fabricant., D.S., Farnsworth, N.R., 2000. Plants used against cancer - an extension of the work of Jonathan Hartwell. Journal Ethnopharmacology 73:347–377.
Da Silva, C.P., De Oliveira, C.R., DA Conceicao, P., De Lima, M., 1996. Apoptosis as a Mechanism of Cell Death Induced by Different Chemotherapeutic. Bio-chemical Pharmacology 51, 1331-1340.
Taylor, I.W., Hodson, P.J., Gren, M.D., Sutherland, R.L., 1983. Efects of Tamoxifen on Cell Cycle Progresion of Synchronous MCF-7 Human Mammary Carcinoma Cells. Cancer Research 43, 4007-4010.
Kara, M.I., Ereiyas, K., Altan, A.B., Ozkut, M., AY, S., Inan, S., 2012. Thymoquinone accelerates new bone formation in the rapid maxillary expansion procedure archives of oral biology 57, 357-363.
Gullett, N.P., Ruhul Amin, A.R., Bayraktar, S., Pezzuto, J.M., Shin, D.M., Khuri, F.R., Aggarwal, B.B., Surh, Y.J., Kucuk, O., 2010. Cancer Prevention with Natural Compounds. Seminars in Oncology 37, 258-281.
Shoieb, A.M., Elgayyar, M., Dudrick, P.S., Bell, J.L., Tithof, P.k., 2003. In vitro inhibition of growth and induction of apoptosis in cancer cell lines by Thymoquinone. International Journal of Oncology 22, 107-113.
Gali-Muhtasib, H., Diab-Assaf, M., Boltze, C., Al-Hmaira, J., Hartig, R., Roessner, A., Schneider-Stock, R., 2004a. Thymoquinone extracted from black seed triggers apoptotic cell death in human colorectal cancer cells via a p53-dependent mechanism. International Journal Oncology 25, 857-66.
Worthen, D.R., Ghosheh, O.A,, Crooks, P.A., 1998. The in vitro anti-tumor activity of some crude and purified components of blackseed, Nigella sativa L. Anticancer Research 18, 1527–1532.
Torres, M.P., Ponnusamy, M.P., Chakraborty, S., Smith, L.M., Das, S., Arafat, H.A., Batra, S.K., 2010. Effects of Thymoquinone in the Expression of Mucin 4 in Pancreatic Cancer Cells: Implications for the Development of Novel Cancer Therapies. Molecular Cancer Therapy 9, 1419-1431.
Kaseb, A., Chinnakannu, K., Chen, D., Sivanandam, A., Tejwani, S., Menon, M., Dou, Q., Reddy, G., 2007. Androgen receptor and E2F-1 targeted Thymoquinone therapy for hormone-refractory prostate cancer. Cancer Research 67, 7782-8.
Gali-Muhtasib, H., Abou Kheir, W., Khei, L., Darwiche, N. & Crooks, P., 2004b. Molecular pathway for Thymoqui-none-induced cell-cycle arrest and apoptosis in neoplastic keratinocytes. Anti-Cancer Drugs 15, 389-399.
Roepke, M., Diestel, A., Bajbouj, K., Walluscheck, D., Schonfeld, P., Roessner, A., Schneider-Stock, R., Gali-Muhtasib, H., 2007. Lack of p53 augments Thymoquinone-induced apoptosis and caspase activation in human osteosarcoma cells. Cancer Biology and Therapy 6, 160-169.
Ragheb, A., Attia, A., Eldin, W.S., Elbarbry, F., Gazarin, S., Shoker, A., 2009. The protective effect of Thymoquinone, an anti-oxidant and anti-inflammatory agent, against renal injury. Saudi Journal of Kidney Diseases and Transplantation 20, 741-752.
Okoh, V., Deoraj, A., Roy, D., 2011. Estrogen-induced reactive oxygen species-mediated signallings contribute to breast cancer. Biochimica et Biophysica Acta 1815, 115-133.
Mansur, A.D.P., Silva, T.C.B.F., Takada, J.Y., Avakian, S.D., Strunz, C.M.C., Cesar, L.A.M., Aldrighi, J.M., Ramires, J.A.F., 2012. Long-Term Prospective Study of the Influence of Estrone Levels on Events in Postmenopausal Women with or at High Risk for Coronary Artery Disease. The Scientific World Journal 38, 149-156.
Zhou, J., Seidel, E., 2010. Estrogens induce visfatin expression in 3T3-L1 cells. Peptides 31, 271-274.
Arahamsson, A., Morad, V., Saarinen, N., Dabrosin, C., 2012. Estradiol, Tamoxifen, and Flaxseed Alter IL-1β and IL-1Ra Levels in Normal Human Breast Tissue in Vivo. Journal Clinical Endocrinology Metababolism 97, 2044-54.
Marucci, C., Fishman, J., 1993. P450 enzymes of estrogen metabolism. Pharmacology and Therapeutic 57, 237-57.
Mikstack, R., Rimando, A.M., Dutkiewicz, Z., Stefanski, T., Sobiak, S., 2012. Design, synthesis and evaluation of the inhibitory selectivity of novel transresveratrol analogues on human recombinant CYP1A1, CYP1A2 and CYP1B1. Bioorganic & Medicinal Chemistry 20, 5117-26.
Gao, H., Chakraborty, G., Lee-Lim, A.P., Mo, Q., Decker, M., Vonica, A., Shen, R., Brogi, E., Brivanlou, A.H., Giancotti, F.G., 2012. The BMP inhibitor coco reactivates breast cancer cells at lung metastatic sites. Cell 150, 764-779.
Gajjar, K., Martin-Hirsch, P.L., Martin, F.L., 2012. CYP1B1 and hormone-induced cancer. Cancer Letters 324, 13-30.
Mcfadyen, M.C.E., Murray, G.I., 2005. Cytochrome P450 1B1: a novel anticancer therapeutic target. Future Oncology 1, 259-263.
Kawajiri, K., 1999. CYP1A1. IARC Science Publication 148, 159-172.
Furukawa, M., Nishimura, M., Ogino, D., Chiba, R., Ikai, I., Ueda, N., Naito, S., Kuribayashi, S., Moustafa, M. A., Uchida, T., Sawada, H., Kamataki, T., Funae, Y., Fukumoto, M. 2004. Cytochrome p450 gene expression levels in peripheral blood mononuclear cells in comparison with the liver. Cancer Science 95, 520-529.
Lin, Y., Yao, Y., Liu, S., Wang, L., Moorthy, B., Xiong, D., Cheng, T., Ding, X., Gu, J., 2012. Role of mammary epithelial and stromal P450 enzymes in the clearance and metabolic activation of 7,12-dimethylbenz(a)anthracene in mice. Toxicology Letters 212, 97–105.
Williams-Brown, M.Y., Salih, S.M., Xu, X., Veenstra, T.D., Saeed, M., Theiler, S.K., Diaz-Arrastia, C.R., Salma, S.A., 2012. The effect of Tamoxifen and raloxifene on estrogen metabolism and endometrial cancer risk. Journal of Steroid Biochemical Molecular Biology 126, 78–86.
Ashley-Martin, J., Vanleeuwen, J., Cribb, A., Andreou, P., Guernsey, J. R., 2012. Breast Cancer Risk, Fungicide Exposure and CYP1A1*2A Gene-Environment Interactions in a Province-Wide Case Control Study in Prince Edward Island, Canada. International Journal of Environmental Research and Public Health 9, 1846-1858.
Fu, J., Weise, A., Falany, J., Falany, C., Thibodeau, B., Miller, F., Kocarek, T., Runge-Morris, M. 2010. Expression of es-trogenicity genes in a lineage cell culture model of human breast cancer progression. Breast Cancer Research Treatment 120, 35-45.
Zhao, Y.N., Zhang, W., Chen, Y.C., Fang, F., Xiao-Quan, L., 2012. Relative imbalances in the expression of catechol-O-methyltransferase and cytochrome P450 in breast cancer tissue and their association with breast carcinoma. Maturitas 72, 139-145.
Guengerich, P.F., Chun, Y., Kim, D., Gillam, E., Shimada, T., 2003. Cytochrome P450 1B1: a target for inhibition in anti-carcinogenesis strategies. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 523-524, 173-182.
Scornaienchi, M.L., Thornton, C., Willett, K.L., & Wilson, J.Y., 2010. Cytochrome P450-mediated 17b-estradiol metabolism in zebrafish (Danio rerio). Journal of Endocrinology 206, 317–325.
Han, E.H., Kim, H.G., Hwang, Y.P., Song, G.Y., Jeong, H.G., 2010. Prostaglandin E2 Induces CYP1B1 Expression via Ligand-Independent Activation of the ERa Pathway in Human Breast Cancer Cells. Toxicological Sciences 114, 204–216.
Zahid, M., Kohli, E., Saeed, M., Rogan, E., Cavalieri, E., 2006. The Greater Reactivity of Estradiol-3,4-quinone vs Estradiol-2,3-quinone with DNA in the Formation of Depuri-nating Adducts: Implications for Tumour-Initiating Activity. Chemistry Research Toxicology 19, 164-172.
Kemp, D.C., Fan, P.W., Stevens, J.C., 2002. Characterization of Raloxifen Glucuronidation in Vitro: Contribution of Intestinal Metabolism to Presystemic Clearance. Drug Metabolism and Disposition 30, 694-700.
Lakhani, N.J., Sarkar, M.A., Venitz, J., Figg, W.D., 2003. 2-Methoxyestradiol, a Promising Anticancer Agent. Pharmacotherapy: The Journal of Human Pharmacology and Drug Therapy 23, 165-172.
Ogura, K., Ishikawa, Y., Kaku, T., Nishiyama, T., Ohnuma, T., Muro, K., Hiratsuka, A., 2006. Quaternary ammonium-linked glucuronidation of trans-4-hydroxyTamoxifen, an active metabolite of Tamoxifen, by human liver microsomes and UDP-glucuronosyltransferase 1A4. Biochemical Pharmacology 71, 1358-1369.
Berno, V., Amazit, L., Hinojos, C., Zhong, J., Mancini, M.G., Sharp, Z.D., Mancini, M.A., 2008. Activation of Estrogen Receptor-α by E2 or EGF Induces Temporally Distinct Patterns of Large-Scale Chromatin Modification and mRNA Transcrip-tion. PlosOne 3, e2286.
Broker, L.E., Kruyt, F.A.E., Giaccone, G., 2005. Cell Death Independent of Caspases: A Review. Clinical Cancer Research 11 3155-3162.
Rajput, S., Kumar, B.N.P., Sarkar, S., Das, S., Azab, B., Santhekadur, P.K., Das, S.K., Emdad, L., Sarkar, D., Fisher, P.B., Mandal, M., 2013. Targeted Apoptotic Effects of Thymoquinone and Tamoxifen on XIAP Mediated Akt Regulation in Breast Cancer. PlosOne 8, e61342.
Jaiswal, A., Mcbride, O., Adesnik, M., Nebert, D., 1988. Human dioxin-inducible cytosolic NAD(P)H:menadione oxi-doreductase. cDNA sequence and localization of gene to chromosome 16. Journal Biology Chemestry 263, 13572-13578.
Liao, K., Niu, F., Hao, H.P., Wang, G.J., 2012. Advances on structure-activity relationship of NQO1-targeting antitumour quinones. Chinese Journal of Natural Medicines 10, 0170-0176.
Yuan, W., Xu, L., Chen, W., Wang, L., Fu, Z., Pang, D., Li, D., 2011. Evidence on the association between NQO1 Pro187Ser polymorphism and breast cancer risk in the current studies: a meta-analysis. Breast Cancer Research and Treatment 125, 467-472.
Dawling, S., Roodi, N., Parl, F.F., 2003. Methoxyestrogens Exert Feedback Inhibition on Cytochrome P450 1A1 and 1B1. Cancer Research 63, 3127-3132.