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

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.