Plantamajoside Inhibits Breast Cancer Growth And Pulmonary Metastasis By Decreasing The Activity Of Matrix Metalloproteinase-9

Background:Metastasis is the major cause of death in breast cancers.MMPs play a key role in tumor microenvironment that facilitates metastasis.The existing researches suggest that the high expression of gelatinase B(MMP-9)promote the metastasis of breast cancer.Other researchers argue that the high expression of MMP-9 protein also promote the development and growth of CMT.Therefore,theoretically,gelatinase inhibitor can effectively suppress tumor metastasis.The aim of this study was to select MMP-9 inhibitors from a variety of small molecule drugs,to investigate the inhibitory effect of the molecule on tumor cells in vitro,and the inhibition of formation and metastasis of tumor in vivo,also to analyze the mechanism of drug function.To provide the basis for clinical drug use,and partly reveal the mechanism of tumor metastasis.Methods:We investigated the expression matrix metalloproteinase(MMP)-9 in canine mammary gland tissue by immunohistochemistry assay.We screened gelatinase inhibitor among Chinese herbal medicine by molecular docking technology;investigated the proliferation,migration and invasion of MDA-MB-231 human breast cancer cell line and 4T1 mouse breast cancer cell line in response to the treatment with the screened inhibitor by growth inhibition assay,colony formation assay,invasion assay and gelatin zymography and western blotting,it was to explore PMS’s antitumor effect on cell level;then further examined the effects of inhibitor in vivo on allograft mammary tumors of mice bearing 4T1 cells,explained the mechanism of antitumor by detecting associated factors with immunohistochemistry.Results:Higher rates MMP-9(70.6%)expression were found in malignant CMT tissues than in normal tissues and benign tumor.And expression of this market was significantly positive associated with tumor nuclear grade,infiltrative growth and lymphatic invasion,in other words it has positive correlations with the malignance and metastasis of CMT.Thus,the results from this study suggested that high expression of MMP-9 may contribute to increased malignancy of canine mammary gland tumor.We successfully screened an Chinese herbal medicine-Plantamajoside(PMS)-which can reduce the gelatinase activity of MMP-9(P<0.01)and docking to MMP-9 molecule(AG=-10.39 kcal/mol).In vitro,250 μg/mL PMS significantly inhibit the proliferation of MDA-MB-231 human breast cancer cell line and 4T1 mouse breast cancer cell line,100 μg/mL PMS significantly inhibit the colony formation of these two cell lines.250 μg/mL PMS treated cell 36h,the inhibiton rates of wound healing were79.3(±8.2)%and 56.4(±4.2)%for MDA-MB-231and 4T1 respectively,the difference were extremely significant compared with control groups(P<0.01).Transwell assays showed that invasion inhibition effect of PMS on breast cancer cells were dose-dependent,the higher the dose was the lower the ivasion ability was.The invasion inhibition effect of PMS on MDA-MB-231 or 4T1 cells can be rescued by exogenous MMP-9.The results of gelatin zymography assay show the activity of MMP-9 was dramatically decreased by PMS both in MDA-MB-231 cells and 4T1 cells.However,PMS didn’t change the expression of MMP-9 protein according to the results of western blotting.PMS suppressed 4T1 allograft tumor in vivo,quantitative analysis displays that the weight and volume of tumors in treatment group were less than that in control group(P<0.05).Number of lung metastases was counted,result shows that the metastasis fociof the control group(92.8 ± 11.9)were significantly more than those of the PMS treatment group(29.5 ± 8.4),and there were statistically significant differences between the two groups.Immunohistochemistry staining of ki67 further conformed that tumor proliferation was synergistically inhibited by PMS with,and the difference between these two groups.The IHC staining of Ki67 of control group(24.3%± 2.1%)is significantly more than PMS treatment group(10.6%± 4.5%)(P<0.05).Biomarker of angiogenesis CD31and a-SMA in primary tumor tissues by 1HC staining shows there were significant differences between the two groups(P<0.01)Conclusions:MMP-9 is highly expressed in malignant CMT which can be used as a marker for the diagnosis of CMT.Our results indicate that PMS may inhibit migraton and metastasis abililty of breast cancer cells by inhibiting the activity of MMP-9 in vitro and display antitumor effect by inhibiting MMP-9 to decrease angiogenesis in vivo.