Reactive oxygen species (ROS) are a crucial factor in the anticancer activity of Oliveria decumbens extract against the A431 human skin cell line

Abstract

Globally, skin cancer is a main public health challenge whose incidence is continuously increasing. Given the limitations of conventional t herapies, new research and novel therapies may be promising for reducing skin cancer morbidity and mortality. Phytochemicals are attractive resources for new therapy design in cancer research due to their cost-effectiveness and lower side effects. In the present study, the anti-cancer activity of Oliveria decumbens (O. decumbens) extract was investigated on the human skin cancer A431 cell line A431. The aqueous extract of the O. decumbens plant was prepared using the traditional method. Then IC50 was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay under different concentrations of O. decumbens. Cell apoptosis was investigated by Annexin V-FITC/Propidium Iodide (PI) and flow cytometry. Cell cycle was investigated by PI staining and flow cytometry. Reactive oxygen species (ROS) production was analyzed by DCFH-DA (2', 7' -dichlorofluorescein-diacetate) staining and flowcytometry.IC50 for cell viability was determined 475g/ml. Cell cycle analyses showed G1 arrest in treated cells compared to control cell. Results also confirmed significant increase of apoptotic cells (8.2%1, P<0.05) under IC50 concentration of the extract in comparison to the control group (2.50.99%). A significant increase in ROS level was observed in O.ecumbens treated cells compared to control cells (738 170 vs 31655 in the control group, P<0.05.).Overall, the present results indicate that O. decumbens extract can inhibit skin cancer cell proliferation via inhibition of cell cycle and apoptosis. It seems that ROS production plays a critical role in the anticancer effect of O. decumbens extract. Therefore, its potential option for future treatment of skin cancer should be considered.

Keywords: A431cell line; Aqueous extract; Oliveriadecumbens; ROS; Skin cancer.

Figure 1

.A) Cell viability was assessed by PI staining and flow cytometry after 48 hours of treatment with 1000g/ml. B), MTT assay was performed to determine IC50 after 48 hours of extract treatment.

Figure 2

A,B).Apoptotic cells were assessed by Annexin V-FITC/Propidium iodide (PI) staining using flow cytometry. Dot plots represent early apoptotic cells in the lower right quadrant, late apoptotic cells in the upper right quadrant, and viable cells in the lower left quadrant (LL). The Percentage of apoptotic cells from three biological replicates is expressed as the mean in the bar graph. Data are presented as mean±SD. *p<0.05.

Figure 3

A,B)After 48 hours of treatment, cells were labeled with PI and analyzed by DNA flow cytometry. The data show the percentage of cells in each phase of the cell cycle. G2/S and G1.The Percentage of apoptotic cells of three biological replicates is expressed as the mean in the bar graph. All experiments were performed in triplicate. Data are presented as mean±SD; **p<0.01,*p<0.05

Figure 4

Figure4: A, B) Detection of ROS in treated and control cells by flow cytometry using a DCFH-DA assay. A) Plots of the fluorescence intensities of the DCF dye. Cells are exposed to 900 μM H2O2 for 60 minutes before their incubation with the DCFH-DA probe for flow cytometry measurements. B) Quantitative histograms of the mean fluorescence intensity of the DCFH-DA probe with regard to the background level of untreated cells. Data are expressed as mean ± SD of three independent experiments (*p < 0.05)