Molecular Studying the effect of simultaneous treatment of Thymoquinone and Cobalt (II) chloride on the expression of genes involved in self-renewal, proliferation, migration and DNA methylation in breast cancer line MCF7 and normal fibroblastic cell line HDF

Nowadays, much attention is paid to the effects of natural factors on physiological and pathological processes in human body. In this regard, lack of oxygen or hypoxia is one of the crucial biological factors involved in various physiological processes such as wound healing and pathological processes such as cancer. Moreover, it is very important to find natural compounds affecting the characteristics and functions of cells. Thymoquinone (TQ) is a natural compound derived from certain plants such as Nigella Sativa. It has many biopharmacological effects, including anti-bacterial, anti-oxidant, anti-inflammatory, anti-diabetic, anti-aging, anti-cancer, etc. Given the biopharmacological properties of TQ and the importance of hypoxia as an important factor affecting physiological and pathological processes, this study was designed to investigate the effect of TQ under cobalt (II) chloride-mediated hypoxia on breast cancer and wound healing by evaluating the expression of SOX2, CDK4, c-MET, and DNMT1 genes in a breast cancer cell line (MCF7) and a normal fibroblastic cell line (HDF) that treated with these compounds.

In the present study, after the cultivation of MCF7 and HDF cell lines, each of the cells were divided into two groups. The treatment group was treated simultaneously with 500 ng/ml of TQ and 100 μM of cobalt (II) chloride for 24 h and the control group was only treated with cobalt (II) chloride. After incubation time, total RNA extraction, DNase I treatment, and cDNA synthesis were carried out and finally, the expression of target genes was examined by real-time PCR assay. In this study, relative threshold method was used to determine the amount of gene expression changes, and SPSS software and Student's t-test statistical method were used to find the significance of gene expression changes in the treated groups compared to the controls.

The results showed that simultaneous treatment of MCF7 cells with TQ and cobalt (II) chloride significantly (P < 0.05) reduced the expression of CDK4, c-MET, and DNMT1 genes at about 4.35-, 1.89-, and 2.08-fold, respectively, compared to the control group. However, the treatment of MCF7 cells caused a limited increase in the expression of SOX2 at about 1.14-fold, which was not significant according to the significance level of ≥ 1.5. Moreover, simultaneous treatment of HDF cells with TQ and cobalt (II) chloride significantly increased c-MET gene expression by about 1.86-fold. In addition, the treatment of HDF cells caused a slight increase in the expression of CDK4 at about 1.26-fold, which was not significant according to the significance level of ≥ 1.5. Also, the expression of SOX2 and DNMT1 genes has decreased at about 1.28- and 1.32-fold in the treatment group compared to the control group, which were as not significant according to the significance level of  ≥ 1.5.

Overall, it can be concluded that TQ under cobalt (II) chloride-mediated hypoxia may inhibit breast cancer by inhibiting the expression of genes involved in proliferation and migration. In addition, due to the important role of fibroblasts in the wound healing process, TQ may help wound healing under hypoxic conditions by increasing the migration potential of fibroblast cells.