Study On Anti-breast Cancer Effect Of Maduramicin In Vitro And In Vivo

Breast cancer is a malignant tumor arising from mammary epithelial tissue.The incidence of breast cancer is increasing year by year and has tended to be younger.In recent years,polyether ionophore antibiotics have been found to have excellent anticancer activity.Among which,several compounds can selectively kill cancer stem cells and multidrug resistant cancer cells.Therefore,polyether ionophore antibiotics are considered as novel potential anticancer drugs.To understand the effect of polyether ionophore antibiotics on breast cancer cells,in this study,breast cancer cells and BALB/c mude mice were used as in vitro and in vivo models,repectively.We focus on the the anti-breast cancer effect and mechanism of maduramicin in the present study,the main contents are as follows:1 Study on the toxic effects of four polyether ionophore antibiotics on breast cancer cellsTo investigate the toxic effects of four polyether ionophore antibiotics on breast cancer cells,in this study,MCF-10 A cells and Ep H4-Ev cells were used as normal breast epithelial cell models to screen the safe concentration of drugs.Furthermore,the drugs that inhibit proliferation of human breast cancer MDA-MB-231 cells and mouse breast cancer 4T1 cells were screened out within the safe concentration range.The breast cancer cells and normal breast epithelial cells were treated with different concentrations(0 μM、0.0125 μM、0.25 μM、0.5 μM、1 μM、2 μM、4 μM、8 μM、16 μM)of maduramicin,salinomycin,monensin and hainanmycin for 24 h,and the cell activity was determined by CCK-8method.In addition,the breast cancer cells were treated with different concentrations(0μM、0.0625 μM、0.125 μM、0.25 μM、0.5 μM、1 μM)of maduramicin for 24 h,48 h and72 h,respectively,followed by morphological changes observation by microscope and the cell activity assessment by CCK-8 method.The results showed that salinomycin and maduramicin had no obvious toxicity on normal breast epithelial cells in the low concentration range(0-1 μM),and could significantly inhibit the proliferation of breast cancer cells,when the concentration was higher than 2 μM,salinomycin and maduramicin could significantly inhibit the proliferation of breast epithelial cells.Monensin showed significant toxicity to two kinds of normal mammary epithelial cells at 0.5 μM.However,hainanmycin significantly decreased the activity of normal mammary epithelial cells at 0.25μM.The activity of MDA-MB-231 and 4T1 cells was significant decreased by maduramicin(0.0625 μM ～ 1 μM)treatment,and cellular morphology was damaged severely.These findings suggest that maduramicin showed time-and concentration-dependent manner anti-breast cancer efficacy,which is higher than salinomycin,monensin and hainanmycin within the safe concentration.2 Study on the growth activity of maduramicin on breast cancer cells in vitroTo explore the effect of maduramicin on anti-breast cancer cells in vitro,MDA-MB-231 cells and 4T1 cells were used as in vitro model.Breast cancer cells were treated with different concentrations of maduramicin(0 μM、0.0625 μM、0.25 μM、1 μM)for 48 h,followed by Annexin V-FITC/PI staining to analyze the apoptosis rate of the cells by performing flow cytometry,PI single staining to determine cell cycle distribution,colony formation was observed by cell cloning test,cell migration ability was detected by scratching test,transwell chamber was used to detect cell migration and invasion ability,the expressions of MMP-2 、 MMP-9 、 E-cadherin and Vimentin were analyzed by performing western blotting.The results indicated that the apoptosis rate of breast cancer cells induced by maduramicin was significantly increased,and the cell cycle distribution was also affected,it blocks breast cancer cells in the S phase,the clone formation rate of breast cancer cells treated with different concentrations of maduramicin was significantly decreased,maduramicin significantly inhibited the "scratch" healing of breast cancer cells,and significantly reduced the migration and invasion ability of breast cancer cells in vitro.Maduramicin significantly inhibited the expression of MMP-2 、 MMP-9 and Vimentin proteins,and significantly up-regulated the expression of E-cadherin protein of breast cancer cells.The above fingdings suggest that maduramicin induces apoptosis and decreases the ability of metastasis and invasion of breast cancer cells in vitro.3 Study on the effect of anti-breast cancer of maduramicin in vivoTo further varify the antitumor effect of maduramicin on mice with breast cancer in situ and investigate anti-breast cancer mechanism,BALB/c nude mouse in situ breast cancer model was established.After successfully establishment of in situ breast cancer model,mice in the control group were given PBS intragastrically,and mice of the drug treatment group was given maduramicin with different concentrations(0.35 mg/kg,0.7mg/kg,3.5 mg/kg)every other day for 14 days according to the body weight of mice.During the whole period,the state of mice was observed,body weight was recorded and tumor volume was measured.After 14 days,the mice were sacrificed and their blood was collected for routine blood analysis and blood biochemical analysis,the tumor tissues followed by dissection,weighing and photograph.In addition,the heart,liver,spleen,lung,kidney,duodenal and tumor of mice were collected for histopathological examination.Tumor tissues in each group were stained by TUNEL,immunohistochemical staining of MMP-2,MMP-9,Vimentin and E-cadherin was performed of the tumor tissues.The results showed that all BALB/c nude mice were subcutaneous tumorigenesis after inoculation with4T1 cells,and the tumorigenesis rate was 100%.During the administration of maduramicin,there was no significant difference in body weight between the maduramicin group and the control group,the mean volume growth rate and size of tumor in maduramicin group were significantly lower than that in the control group,and the difference between different drug concentration groups was extremely significant,the tumor weight in maduramicin treatment group was significantly lower than that in control group.AST in the control group was significantly increased,and leukocytes and neutrophils in the control group,low-dose and medium-dose drug groups were significantly increased compared with the high-dose drug groups,which could slightly improve the degree of inflammation in mice.Compared with the control group,there was no obvious damage of the observed organs in the low,medium and high dose of maduramicin groups.Breast cancer cells in the control group were arranged and scattered,and the number of tumor cells was large and the atypia was large,while the number of tumor cells in mice of maduramicin treatment group was small and there were necrotic tumor cells.The number of TUNEL positive cells in the treatment group was significantly higher than that in the control group,suggesting that the maduramicin-induced apoptosis in breast cancer cells was obvious.Tumor metastasis occurred in the control group during the treatment process,but it was significantly improved in the treatment group.Furthermore,the immunohistochemical results exhibited that the expression of MMP-2,MMP-9,Vimentin and E-cadherin in the tumor tissues were positive.Compared with the control group,maduramicin decreased the expression of MMP-2 and MMP-9,and MMP-2 and MMP-9 protein expression quantity decreased significantly with the increasing of maduramicin dose.The expression of Vimentin reduced significantly with the increasing of maduramicin dose.On the contrary,the expression of E-cadherin was significant reduced with the dcreeasing dose maduramicin,which was consistent with the results of in vitro experiments.These above results indicated that maduramicin inhibits the growth of breast cancer via inducing apoptosis,and reduces the metastasis and invasion ability of breast caner by regulating the expression of some relevant proteins.