Inhibition Effects of Scorpion Venom Extracts (Buthus matensii karsch) on the Growth of Human Breast Cancer MCF-7 cells
African Journal of Traditional, Complementary and Alternative Medicines
To observe the inhibition effects of the Buthus matensii Karsch (BmK) scorpion venom extracts on the growth of human breast cancer MCF-7 cells, and to explore its mechanisms. Methods: Two common tumor cells (SMMC7721, MCF-7) were examined for the one which wasmore sensitivity to scorpion venom by MTT method. Cell cycle was determined by flow cytometry. Immunocytochemistry was applied to detect apoptosis-related protein Caspase-3 and Bcl-2 levels, while the expression of cell cycle-related
... cycle-related protein Cyclin D1 was shown by Western blotting. Results: Our data indicated that MCF-7 was the more sensitive cell line to scorpion venom. The extracts of scorpion venom could inhibit the growth and proliferation of MCF-7 cells. Furthermore, the extract of scorpion venom induced apoptosis through Caspase-3 up-regulation while Bcl-2 down-regulation in MCF-7 cells. In addition, the extracts of scorpion venom blocked the cells from G 0 /G 1 phase to S phase and decreased cell cycle-related protein Cyclin D1 level after drug intervention compared with the negative control group. Conclusions: These results showed that the BmK scorpion venom extracts could inhibit the growth of MCF-7 cells by inducing apoptosis and blocking cell cycle in G 0 /G 1 phase. The BmK scorpion venom extracts will be very valuable for the treatment of breast cancer. Cyclin-dependent kinases the more sensitive cell line human breast cancer cells (MCF-7). These results will provide an experimental basis for further purification of the anti-cancer composition in BmK scorpion venom and development and clinical application of this anti-cancer drug. Materials and Methods Cell culture (Fracchiolla et al., 1997) Two cell lines used in this project were human hepatoma cell line (SMMC 7721), human breast cancer cell line (MCF-7). All cell lines were stored in our laboratory and subcultured RPMI 1640 medium (GIBCO, USA) supplemented with 10% fetus bovine serum (FBS) (GIBCO, USA) at 37℃ with 5% CO 2 . Cells were treated with serial concentrations of the BmK scorpion venom extracts (SVE) and the untreated cells were used as negative controls. Cell survival experiments (Btieler H, 2010) Cell survival was examined with tetrazolium salt (MTT) assay (Amerco, USA). Two types of tumor cells (MCF-7 and SMMC7721) were seeded at 3×10 3 cells /well in 96-well plates. When the cells were completely adhere to the wells after 16 h, 20μl different concentration of experimental BmK scorpion venom protein was added to each well. Six wells were used for each concentration. After being incubated for 24 h, MTT (5 mg/mL) was added to each well and then after 4h incubation, each well was detected. (Hirsch T et al., 1998) Climbing cell slices were treated with the BmK scorpion venom (600μg/mL) for 24 h and fixed in 4% paraformaldehyde for 15 min at room temperature. After being washed twice with PBS for 3 min each, the cells were incubated in 3% H 2 O 2 deionized water for 15 min, preincubated with 2% blocking serum for 15 min at room temperature. After incubation with the primary antibody for 1 h at room temperature, the slides were washed twice with PBS for 3 min each again. Then the cells were added with the secondary antibody and incubated at 37℃ for 20 min. After washing twice with PBS for 3 min each, the cells were visualized with dimethylbenzene solution. The following antibodies were used in immunocytochemistry staining: caspase-3 (1:1000, Santa Cruz, USA) and Bcl-2 (1:1000, Santa Cruz, USA). (Jamil Z et al., 2001) Changes of the cell cycle were examined with PI labeling by flow cytometry (FCM). The cells were inoculated to a 24-well culture plate (1x10 5 cells /well) and cultured for 16 h, they were added with 600μg/mL of BmK scorpion venom and cultured for 24 hours before being harvested. The cells were washed twice with PBS, and added with 50 μl RNase (10μg/mL) and 200 μl PI (5 μg/mL) (Sigma, USA). After being incubated for 30 min at 4℃ in dark, the cells were detected by flow cytometry. Li et al., Afr J Tradit Complement Altern Med. (2014) 11(5):105-110 http://dx.doi.org/10.4314/ajtcam.v11i5.17 109 Figure 4: Effects of experimental BmK scorpion venom on the cell cycle of MCF-7 cells. Percentage of cells in different cell cycle phases after incubated with 600 μg/mL BmK scorpion venom extracts (SVE) for 24 h in MCF-7 cells by flow cytometry. Data shown are mean levels (+ SEM) from three independent experiments. T test was applied for statistical analysis. *, P < 0.05. Effects of the BmK scorpion venom on the expression of cell cycle related protein Cyclin D1 Compared with the control cells without BmK scorpion venom, the expression of cell cycle-related protein CyclinD1 decreased in MCF-7 cells treated with 600 μg/mL of BmKs corpion venom after 24 h. The results were shown in Fig. 5 Detection of apoptosis proteins by immunocytochemistry Changes of cell cycle measured by flow cytometry Figure 5: Effects of the BmK scorpion venom on the expression of cell cycle related protein Cyclin D1. Effects of scorpion venom on Cyclin D1 protein. MCF-7 cells were treated with 600 μg/mL of BmK scorpion venom extracts (SVE) for 24 h. Protein from the total cell lysate was subjected to Western blot analysis for cyclin D1 protein. β-actin was used as an internal control. Representative results are shown from three independent experiments. Discussion Scorpion venoms have been used in Chinese folk medicine to cure cancer patients. The scorpion venoms extracts were mixtures of different toxins and peptides which showed different functions (Tiago EH, 2011). Our study demonstrated the anti-tumor effects of scorpion venoms extracts on breast cancer cells and liver cancer cells. It has been shown that cell proliferation and apoptosis is to adjust a balanced state. Once the balance is broken by external factors, it will be followed with excessive cell proliferation or a large number of cells undergoing apoptosis. There are mainly two apoptotic pathways: activation of caspase-8 initiates protease cascade to induce apoptosis; the release of cytochrome C-mediated activation of Caspase-3 to induce apoptosis (Zhang X, 2002). Bcl-2 is another protein related to apoptosis through regulating mitochondrial PTP opening and closing. Bcl-2 and Bcl-xL can inhibit PTP opening thereby inhibit the release of apoptotic factors and prevent the occurrence of apoptosis (Tsujimoto Y et al., 1985). Our work indicated that scorpion venoms inhibited proliferation of breast cancer and liver cancer cells in a dose and time dependent manner. More important, our results showed that the expression of pro-apoptotic protein caspase-3 increased while that of apoptosis suppression protein Bcl-2 decreased after the MCF-7 cells treated with scorpion venom. It could be preliminarily considered that the inhibition to the proliferation of MCF-7 cells by experimental BmK scorpion venom could be achieved by inducing apoptosis.