Celastrol Upregulates Protein TMEM100 To Surpress Metastasis Of Breast Cancer Cells

Breast cancer is one of the three most common cancers worldwide(the others are lung and colon cancer)and the highest morbidity of malignant tumors in women worldwide.It severely affects women's health,and is the highest incidence of malignant tumors for the female.Metastasis is the major cause of mortality for the patients with breast cancer.Therefore,metastasis of breast cancer is the problem to be solved.Celastrol is a natural compound purified from the traditional Chinese medicine,having efficiently low toxicity and comprehensive antitumor activity,inducing tumor cell apoptosis and inhibiting tumor metastasis.However,it lacks enough understanding in metastasis of breast cancer.Tumour necrosis factor-alpha(TNF-?)is a member of the TNF family and a multi-functional proinflammatory cytokine produced by macrophages and tumor cells.It is reported that TNF-? participates in metastasis process in a variety of tumor,promoting tumor metastasis.In this study,we researched that the effct of celastrol on metastasis of breast cancer cells and pro-metastasis of TNF-?.The main works are as follow:The cell viability of MDA-MB-231 cells was measured by MMT assay after treatment of celastrol and/or TNF-? for some times.The cell adhesion ability was measured by cell adhesion assay and flow cytometry.The cell migration ability was measured by wound healing assay.The cell invasive ability was measured by Transwell assay.The angiogenesis ability was measured by tube formation assay.The expression level of TMEM100 and SMAD1 and the phosphorylation of SMAD1/5 were measured by western blot.The transcription expression of gene ATF2,ACVR1(ALK1),SMAD4 and TGF? was measured by qPCR.The results are as follows:1.The results of anti-metastasis of celastrol in breast cancer cells were as follows:(1)By MMT assay,it showed that cell activity of MDA-MB-231 cells were weaken with increasing concentrations of celastrol,celastrol obviously surpressed cell activity in a dose-and time-dependent manner.(2)The concentrations of celastrol were bigger,the adherence rates were lower.Celastrol inhibited adhesion of MDA-MB-231 cells.(3)The number of MDA-MB-231 cells that adhered to P-selectin was decreased with increasing concentration of celastrol.Further this suggested that celastrol inhibited adhesion of MDA-MB-231 cells.(4)With increasing concentration of celastrol,distances of cell migration were decreased.So celastrol inhibited migration of MDA-MB-231 cells.(5)The number of cells that traversed the small room was gradually reduced with increasing concentration of celastrol.This suggested that celastrol inhibited invasion of MDA-MB-231 cells.(6)With increasing concentration of celastrol,the number of small tubes was gradually reduced,and the lumens were not complete.Celastrol inhibited angiogenesis of MDA-MB-231 cells in vitro.(7)Celastrol promoted the expression of protein TMEM100 and SMAD1,and enhanced the phosphorylation of protein SMAD1/5.(8)Celastrol promoted the expression of ACVR1 gene,but inhibited the expression of TGF? signaling pathways related genes.2.The results that celastrol surpresses pro-metastasis of TNF-? were as follows:(1)TNF-? promoted cell activity of MDA-MB-231 cells,but celastrol obviously surpressed the activity induced by TNF-?.(2)TNF-? increased the adherence rates of MDA-MB-231 cells,so it enhanced the adhesion ability of MDA-MB-231 cells.While celastrol decreased the adhesion ability increased by TNF-?.This suggested that celastrol inhibited adhesion induced by TNF-?.(3)TNF-? promoted the adhesion of P-selectin/MDA-MB-231 cells,so it promoted cell adhesion.But celastrol decreased the adhesion increased by TNF-?.Further this suggested that celastrol inhibited adhesion promoted by TNF-?.(4)Under the treatment of TNF-?,the distance of cell migration was increased.This suggested TNF-? enhanced the migration ability of MDA-MB-231 cells.Howere,celastrol reduced the distance by induced TNF-?.So celastrol inhibited migration induced by TNF-?.(5)Under the treatment of TNF-?,the number of cells that traversed the smallroom was increased,while celastrol decreased the increasion induced by TNF-?.Celastrol inhibited invasion induced by TNF-?.(6)TNF-? promoted angiogenesis;but with the treatment of celastrol,the number of small tubes was gradually reduced,and the lumens were not complete.So celastrol inhibited angiogenesis induced by TNF-?.(7)TNF-? upregulated the expression of TGF? signaling pathways related genes,while celastrol inhibited this upregulation.In conclusion,we demonstrated that celastrol inhibited invasion and metastasis of breast cancer cells,and surpressed pro-metastasis of TNF-?.Celastrol up-regulated the expression of protein TMEM100 to inhibite metastasis of breast cancer by enhancing the phosphorylation of SMAD1/5 and promoting ACVR1/SMAD1/5 signaling.Besides Celastrol inhibited the transcription of TGF? signaling pathway to promote the expression of protein TMEM100.Celastrol suppressed metastasis induced TNF-? in breast cancer cells by downregulating TGF? signaling.Therefore,the research of the molecular mechanisms on celastrol provieded a scientific basis for its application in the area of anticancer.