In vitro anti-tumor activity of the tanshinone IIA against SKOV3 cells

Abstract The aim of this study was to determine the anti-tumour activity of tanshinone IIA in SKOV3 cells. Results suggested that tanshinone IIA could significantly inhibit (IC50 value  =  19.6 μM) the proliferation and induce apoptosis of SKOV3 cells as demonstrated by flow cytometry analysis. In addition, tanshinone IIA treatment induced G2/M phase cell cycle arrest in SKOV3 cells. The results of Western blotting indicated that tanshinone IIA can suppress the expression of anti-apoptotic protein Bcl-2, increase (0.28 vs. 0.62) the expression of pro-apoptotic protein Bax (0.83 vs. 0.24) in SKOV3 cells. It can be concluded that the tanshinone IIA may be a possible therapeutic candidate having cytotoxic and anti-tumour potential. Graphical abstract


Introduction
Ovarian cancer is the leading cause of death from gynecological malignancies and one of the most common causes of death from cancer among women in the western world. Often the diagnosis of ovarian cancer occurs late and the treatment options are limited. Epithelial ovarian cancer arises primarily from neoplastic transformation of the ovarian surface epithelium, a layer of flat or cuboidal cells that cover the surface of the ovary (Jarboe et al. 2008).

ABSTRACT
To determine the anti-tumor activity of tanshinone IIA in SKOV3 cells. Results suggested that tanshinone IIA could significantly inhibit (IC 50 value = 19.6 μM) the proliferation of SKOV3 cells and induce apoptosis of SKOV3 cells demonstrated by flow cytometry analysis. In addition, tanshinone IIA treatment induced G2/M phase cell cycle arrest in SKOV3 cells. The results of western blotting indicated that tanshinone IIA can suppress the expression of anti-apoptotic protein Bcl-2, increase (0.28 vs 0.62) the expression of pro-apoptotic protein Bax (0.83 vs 0.24) in SKOV3 cells. It can be concluded that the tanshinone IIA may be a possible therapeutic candidate having cytotoxic and anti-tumor potential.
Tanshinone IIA is a diterpene naphthoquinone from the root of the Salvia miltiorrhiza Bunge, reported to possess anti-inflammatory, anti-oxidative and cytotoxic activities (Dong et al. 2009). It is also known to improve blood circulation and treat chronic hepatitis and hepatic fibrosis (Han et al. 2008;Liu et al. 2009). Traditional Chinese medicine considers Tanshinone IIA as potential drug for cancer treatment.
In the present study, we investigated the anti-tumor effect of tanshinone IIA on the proliferation, migration and invasion of SKOV3 cells. The expression of Bax and Bcl-2 was also examined.

Results and discussion
Anti-tumor activities of tanshinone IIA have been reported in previous studies (Su 2014;Li et al. 2015). In our study, as shown in Figure S1, tanshinone IIA showed a dose-dependent effect within the concentration ranges tested. The inhibitory activities of tanshinone IIA for SKOV3 cell proliferation increased significantly (p < 0.05) with increasing concentration. This indicated that tanshinone IIA can significantly inhibit SKOV3 cells growth.
To further confirm that programmed cell death was involved in the cytotoxic effect of tanshinone IIA on SKOV3 cells, tanshinone IIA -treated SKOV3 cells were subjected to apoptosis assays by DAPI staining and Annexin V-FITC and a PI double staining assay. In Figure  S2, the percentage of apoptotic cells reached ~ 26, ~ 58, and ~ 77% for cells treated with tanshinone IIA at 10, 20, 30 μg/μL for 48 h, respectively.
To investigate the effects of tanshinone IIA on cell cycle distribution, SKOV3 cells were treated with different concentrations of tanshinone IIA for 48 h ( Figure S3). In the untreated control cells, the highest percentage of cells were in the G0/G1 phase, followed by the S, and G2/M phases. In the cells treated with 2.5 or 5 μM tanshinone IIA, the G0/G1 phase was the most abundant, followed by the S, and G2/M phases. In the cells treated with 10 μM tanshinone IIA, the ratio of G0 to G1 cells declined while the percentage of G2-M cells increased considerably. These results indicated that tanshinone IIA could induce G2/M arrest to decelerate the cell cycle, preventing the cells from entering the mitotic period and proliferating (Knight et al. 2011).
Among the several apoptotic genes is Bax, a pro-apoptotic member of the Bcl-2 family that is capable of directly triggering apoptosis. Bcl-2, an anti-apoptotic gene is critical for regulation of apoptosis across diverse cell types and acts along intrinsic mitochondrial apoptosis pathway that is activated in response to a number of stress stimuli including oxidative stress (Gong et al. 2014). In this study, the data of Western blot analysis revealed that tanshinone IIA at concentration of 2.5 μM dose-dependently and substantially reduced Bcl-2 protein expression and increased Bax protein expression in SKOV3 cells ( Figure S5).

Conclusion
Tanshinone IIA treatment induced cell cycle arrest at the G2/M phase, decreased the anti-apoptotic factor Bcl-2 expression, increased the pro-apoptotic factor Bax expression, promoted human ovarian cancer cells SKOV3 cell apoptosis, and thus inhibited cell proliferation and viability.