| Objectives To investigate the effects and mechanism of extracts from Phellinus igniarius(EPI)on cell apoptosis from tumor of MDA-MB-231 triple negative breast cancer subcutaneous transplantation nude mice.Methods MDA-MB-231 cells were routinely cultured in DMEM culture medium,and stimulated with vehicle or different concentrations(20,40,80,160,320?g/ml)of EPI for48 hours,CCK-8 was used to detect the inhibition of proliferation of MDA-MB-231 cells by different concentrations of EPI,and the expression levels of caspase-3 and VEGF were detected by Western Blot.Culture human breast cancer MDA-MB-231 cells.Prepare 1×107/ml cell suspension,and inject 200?l cell suspension in the right subcutaneous groin of each nude mouse.After the tumors grow,five mice were randomly selected,HE staining and immunohistochemistry of three markers estrogen receptor(ER),progesterone receptor(PR)and human epidermal growth factor receptor 2(HER2),proved that the triple negative breast cancer model was successfully established.Thirty nude mice were then selected for follow-up experiments.All the mice were randomly divided into 5 groups with 6 mice in each: Control group(Each nude mouse was injected 0.2ml 0.9% sodium chloride injection daily),the EPI lowdose group(Each nude mouse was injected 30mg/kg EPI every day),the EPI medium-dose group(Each nude mouse was injected 60mg/kg EPI every day),the EPI high-dose group(Each nude mouse was injected 100mg/kg EPI every day),paclitaxel group(As a positive control group,each nude mouse was injected 50mg/kg paclitaxel every two days).During the intervention period,the maximum diameter and minimum diameter of tumor were measured with vernier calipers every 2 days.After 3 weeks,nude mice in each group were sacrificed by cervical dissection.Tumor tissues of nude mice in each group were harvested,and the tumor average weight and tumor inhibition rate were assessed and calculated to observe the effect of EPI on the growth of transplanted tumor.Microscopic changes of tumor sections were observed under the microscope after HE staining.TUNEL staining was used to detect the apoptosis of tumor cells.Immunohistochemistry staining was used to compare the expression of cysteine aspartic protease 3(caspase-3)and cysteine proliferation-related nuclear antigen(ki-67)in each group to evaluate the effect of mulberry extracts on apoptosis and proliferation of transplanted tumor cells.Results CCK-8 results showed: Compared with the control group,the cell proliferation rate of each concentration group was reduced,and inhibition effect is concentrationdependent.In all groups,the EPI groups with the concentration of 160 and 320?g/ml changed the most significantly.Western Blot results showed that the expression level of the caspase-3 in the triple negative breast cancer cells treated with mulberry extract of 160 and 320?g/ml was significantly higher than that in the 20,40 and 80?g/ml group and the control group.The expression level of VEGF in triple negative breast cancer cells treated with EPI at concentrations of 160 and 320?g/ml was significantly lower than that in the 20,40 and 80?g/ml group and the control group.MDA-MB-231 cells were inoculated under the skin of nude mice for about 2 days.Small rice-like nodules were observed 4 days after inoculation,then the subcutaneous nodules gradually increased with partially irregular boundary.Four weeks later,the maximum diameter of tumor was greater than or equal to 0.5cm,which reached the standards of tumor formation.Thirty nude mice were randomly divided into 5 groups.In addition to thecontrol group,each group was given corresponding treatment for 3 weeks.After administration,the tumor size of the medium and high dose EPI(60,100mg/kg)and paclitaxel groups were significantly smaller than the low dose group and the control group.The results of HE staining,TUNEL assay and immunohistochemical staining of tumor tissues in each group showed that the medium and high dose groups(60,100mg/kg)and paclitaxel groups all had significant differences compared with the control group and the low dose group(30mg/kg).Conclusions EPI could significantly inhibit the proliferation of breast cancer cells in the model,in the range of 20,40,80,160 and 320?g/ml,as the concentration increases,the inhibitory effect on cells is enhanced;EPI can induce apoptosis of triple negative breast cancer xenograft cells,the mechanism of action is that it can up-regulate the expression of caspase-3 in triple-negative breast cancer cells,and simultaneously down-regulate the expression of VEGF and ki-67 to induce cells apoptosis.Figure 12;Table 1;Reference 93... |
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