Journal of Mazandaran University of Medical Sciences (Feb 2024)
Evaluation of Antioxidant Properties and Cytotoxic Effect of Scutellaria Pinnatifida Extract on Survival and Expression of Genes Involved in Cell Cycle (P53 and CDK2) in Kidney Cancer
Abstract
Background and purpose: To treat kidney cancer, methods such as surgery, chemotherapy, immunotherapy, etc. are used, which cause many side effects, thus, we try to use methods that have fewer side effects, such as the use of plant compounds. Considering the importance of changes in the function of genes, especially vital genes during cancer, in this study, the genes involved in the cell cycle, including P53 and CDK2, were investigated. The purpose of this study was to investigate the antioxidant properties and cytotoxic effect of Scutellaria pinnatifida extract on the survival and expression level of genes involved in the cell cycle (P53 and CDK2) in kidney cancer. Materials and methods: In this experimental-laboratory study, Scutellaria pinnatifida extract was prepared by soaking method and the presence of phytochemical compounds in the extract was investigated qualitatively. Kidney cancer ACHN cells were cultured. The cytotoxic effect of an extract with concentrations of 1000, 500, 250, 125, 100, and 62.5 µg/ml was measured by MTT assay (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-Diphenyltetrazolium Bromide) which is a color method. The assay is to check the proliferation and survival of cells, it was measured at 24, 48 and 72 hours. Then, RNA extraction was performed from the treated cells with concentrations of 500 and 250 µg/ml, and the concentration and quantity of the extracted RNAs were measured using nano drops. Then cDNA was made from the extracted RNAs and the expression levels of P53 and CDK2 genes were analyzed using Real-Time PCR. DPPH (Diphenyl picrylhydrazyl) test was used to evaluate the antioxidant properties of the extract which is based on the measurement of antioxidant inhibition capacity. Results: According to the results obtained from the phytochemical tests, the spike plate plant extract contains flavonoids, triterpenoids, asteroids, sterols, and glycosides, but it does not contain alkaloids, tannins, and saponins. MTT assay showed that Scutellaria pinnatifida extract has a lethal effect on ACHN cells and this effect is dose and time-dependent. By decreasing the concentration of spike plate plant extract, the survival rate of cells increased significantly, and by increasing the duration of treatment from 24 to 72 hours, the survival rate in kidney cancer cells decreased significantly. Real-Time PCR also showed increased expression of the P53 gene and decreased expression of the CDK2 gene in ACHN cells treated with Scutellaria pinnatifida extract. Changes in expression are also dependent on dose and time. The antioxidant test confirmed the antioxidant properties of the extract and it showed that the antioxidant properties of this plant depend on the concentration. Value in this study is less than 0.05 (P<0.05). Conclusion: The findings show that Scutellaria pinnatifida extract can have an anti-cancer effect and destroy ACHN cancer cells. Changes in P53 and CDK2 gene expression in ACHN cells indicate that Scutellaria pinnatifida extract probably disturbs the cell cycle in kidney cancer cells and thereby reduces the survival of these cells. Therefore, it can be considered a compound with anti-cancer properties
