Determination of Cytotoxic effect and Expression analyses of Apoptotic and Autophagic related genes in Thymoquinone-treated Colon Cancer Cells

Year 2020, Volume: 24 Issue: 1, 189 - 196, 01.02.2020

Esra Tokay

https://doi.org/10.16984/saufenbilder.585012

Cited By: 1

Abstract

Thymoquinone
(Tq; 2-isopropyl-5-methyl-1,4 benzoquinone) is the main ingredient present in
the volatile oil of Nigella sativa
(black seed). Recently, it has been reported that, Tq was inhibited cell
proliferation of many cancer cell lines including ovarian, lung, breast, osteosarcoma,
pancreatic, fibrosarcoma, lung cancer, squamous cell carcinoma and myeloblastic
leukemias. In this study, the anticancer effect
of Tq was investigated in human Colon Cancer Cells (HT-29) with MTT assay.
Positive control was achieved with 5-fluorouracil (5-FU). Also, the invasion
capability of HT-29 cellls was determined in presence of Tq and 5-FU with
scratch analyses. In addition, the autophagic and apoptotic effect of Tq were
investigated by qRT-PCR method to find out the pathway of anti-proliferative
effect in Tq-treated HT-29 cells. As a result, Tq was inhibited the cell
proliferation of HT-29 cells in time dependent manner, with 118  µM for 24 h and 84 µM for 48 h, respectively. A
concentration of 150 µM of Tq was able to significantly reduced the
invasiveness of HT-29 cells about  ̴8
fold compared with not treated cells at 6h. ATG-12, ATG-7 and LC3-II were
significantly downregulated in presence of Tq.  Conversaly, the expression of all of the
autophagy genes were downregulated in presence of 5-FU. Moreover, pro-apoptotic
gene Bax was significiantly upregulated nearly 15 fold whereas the expressions
of Bcl-2 and Bcl-XL (pro-survival genes) were decreased in presence of Tq. On
the other hand, in 5-FU treated HT-29 cells, the expression of  Bax, Bcl-2 and Bcl-XL genes and
autophagy related genes were significantly downregulated. 

Keywords

Tq, autophagy, apoptosis, HT-29

References

• [1] A. Bhandari, M. Woodhouse, and S. Gupta, “Colorectal cancer is a leading cause of cancer incidence and mortality among adults younger than 50 years in the USA: a SEER-based analysis with comparison to other young”, J Investig Med. vol.65, no.2:, pp. 311–315, 2017.
• [2] T.J Syed Ahmad Tajudin., H. Fatimah, I. Wan Iryani and A. Abdul Manaf, “Combinatorial Cytotoxic Effects of Gelam Honey and 5-Fluorouracil against Human Adenocarcinoma Colon Cancer HT-29 Cells In Vitro’. International Journal of Cell Biology”, vol. 2019, pp.10, 2019.
• [3] A.E. Ashour, A.R. Abd-Allah, H.M. Korashy, S.M. Attia , Alzahrani A.Z., Saquib Q., Bakheet S.A., Abdel-Hamied H.E., Jamal S., Rishi A.K., “Thymoquinone suppression of the human hepatocellular carcinoma cell growth involves inhibition of IL-8 expression, elevated levels of TRAIL receptors, oxidative stress and apoptosis”. Mol Cell Biochem. vol. 389 no.1-2,pp. 85-98, 2014
• [4] Raghunandhakumar S., Paramasivam A., Senthilraja S., Naveenkumar C., Asokkumar S., Binuclara J., Jagan S., Anandakumar P., Devaki T.,”Thymoquinone inhibits cell proliferation through regulation of G1/S phase cell cycle transition in N-nitrosodiethylamine-induced experimental rat hepatocellular carcinoma”. Toxicol Lett., vol.223, no.1,pp.60-72,2013.
• [5] Attoub S., Sperandio O., Raza H., Arafat K., Al-Salam S., Al Sultan M.A., Al Safi M., Takahashi T., Adem A., “Thymoquinone as an anticancer agent: evidence from inhibition of cancer cells viability and invasion in vitro and tumor growth in vivo”. Fundam Clin Pharmacol., vol. 27, no. 5, pp. 557-69,2013.
• [6] Shahin Y.R., Elguindy N.M., Abdel Bary A., Balbaa M., “The protective mechanism of Nigella sativa against diethylnitrosamine-induced hepatocellular carcinoma through its antioxidant effect and EGFR/ERK1/2 signaling”. Environ Toxicol. doi: 10.1002/tox.22574. [Epub ahead of print],2018.
• [7] Mohamed M S.A., Abdulaziz M.A., Salim S A.R., Abdulaziz A. Al-Y., Othman A. Al-S., Mohamed M.H., Mahmoud N.N., “Thymoquinone attenuates diethylnitrosamine induction of hepatic carcinogenesis through antioxidant signaling”. Oxid Med Cell Longev. Vol. 3, no. 2 , pp. 254–261., 2010.
• [8] El-Baba C., Mahadevan V., Fahlbusch F.B., Mohan S.S., Rau T.T., Gali-Muhtasib H., Schneider-Stock R., “Thymoquinone-induced conformational changes of PAK1 interrupt prosurvival MEK-ERK signaling in colorectal cancer”. Mol Cancer., vol. 13, pp. 201, 2014.
• [9] Fischer T.D., Wang J.H., Vlada A., Kim J.S., Behrns K.E.,”Role of autophagy in differential sensitivity of hepatocarcinoma cells to sorafenib”. World J Hepatol. Vol. 6, no. 10, pp. 752-8,2014.
• [10] Juan W., Qi W., Juan-Juan L., Chuang C., Si S., Chang-Hua W., Sheng-Rong S., “Autophagy mediates free fatty acid effects on MDA-MB-231 cell proliferation, migration and invasion”. Oncol Lett. Vol. 14, no. 4, pp. 4715–4721, 2017.
• [11] Liu J., Chen Z., Guo J., Wang L., Liu X., “Ambra1 induces autophagy and desensitizes human prostate cancer cells to cisplatin”. Biosci Rep. doi: 10.1042/BSR20170770. [Epub ahead of print], 2017.
• [12] Wan B., Dai L., Wang L., Zhang Y., Huang H., Qian G., Yu T., “Knockdown of BRCA2 enhances cisplatin and cisplatin-induced autophagy in ovarian cancer cells”. Endocr Relat Cancer. Vol. 25, no. 1, pp. 69-82, 2018.
• [13] Tokay E. and Kockar F. “Identification of intracellular pathways through which TGF-β1 upregulates URG-4/URGCP gene expression in hepatoma cells”, Life Sci., vol. 144, pp. 121-8, 2016.
• [14] Schneider C. A.; Rasband W. S. and Eliceiri, K. W., “NIH Image to ImageJ: 25 years of image analysis”, Nature methods, vol. 9, no. 7, pp. 671-675, 2012.
• [15] El-Najjar N., Chatila M., Moukadem H., Vuorela H., Ocker M., Gandesiri M., Schneider-Stock R., Gali-Muhtasib H., “Reactive oxygen species mediate thymoquinone-induced apoptosis and activate ERK and JNK signaling”, Apoptosis, vol. 15, no. 2, pp. 183-95, (2010).
• [16] Chen C., Lu L., Yan S., Yi H., Yao H., Wu D., He G., Tao X., Deng X. Autophagy and doxorubicin resistance in cancer. Anticancer Drugs, vol. 29, no. 1, pp. 1-9, 2018.