Effect Of Dihydroartemisinin On Canine Mammary Tumor Cell Invasion And EMT Mechanism Related Factors

Canine breast tumor is a kind of tumor disease which is seriously harmful to health.At present,most of the treatment for breast cancer is surgery,radiotherapy and chemotherapy,but the results are more prone to relapse or side effects.As a traditional Chinese medicine extract,dihydroartemisinin(DHA)has been widely recognized for its antimalarial effect.With the deepening of the research,its anti-tumor effect has been gradually revealed.This study was conducted to investigate the effects of Dihydroartemisinin on the invasion and EMT relatedfactors of canine breast tumor cells.Take the logarithmic growth phase of the same CHMm cells,using low(5 M),medium(10 M),high(20M)concentration of DHA treatment and were cultured for 24,48,72 hours.Cells were collected using CCK-8 staining;cell scratch test for cell migration;invasion detection of transwell cells;ELISA matrix metalloproteinase(MMP-9)detection of cell content and matrix metalloproteinase inhibitor 1(TIMP-1)content;Real-time fluorescence quantitative PCR method for detection of cell E-cadherin(CDH1),zinc finger binding protein(ZEB)and the zinc finger transcription factor(SLUG)cells of epithelial mesenchymal transition(EMT)expression related factors.1.The cytotoxicity of CHMm cells was detected by CCK-8.Compared with the blank control group,the low,medium and high test group had significant inhibition(p<0.01).The same concentration of DHA,72h and 48h culture compared with 24h culture on the cell inhibitory effects were significant(p<0.01).The inhibition rate of DHA on CHMm cells was obviously drug time and concentration dependent.2.The cell migration rate of cell scratch test showed that,in the same incubation time,compared with the blank control group,the low,medium and high test group had significant inhibitory effect on the migration of CHMm cells(p<0.01).The same concentration of DHA,72h and 48h culture compared with 24h culture on the cell migration rate effects were significant(p<0.01).The migration rate of DHA on CHMm cells was obviously drug time and concentration dependent.3.Cell invasion test cell invasion.The results show that the culture of the same time,compared with the control group,the low,medium and high in the experimental group through the basement membrane staining cell number increased significantly,indicating that DHA on CHMm cells invasion inhibition effect significantly(p<0.01).The same concentration of DHA,72h and 48h culture compared with 24h culture on the cell invasion effects were significant(p<0.01).The invasion rate of DHA on CHMm cells was obviously drug time and concentration dependent.4.The results showed that the content of MMP-9 in each concentration group was significantly lower than that of the control group at the same time(p<0.01).With the same concentration,the content of MMP-9 was not significantly changed with the increase of culture time(p>0.05).DHA significantly inhibited the secretion of MMP-9 in CHMm cells,which was significantly dependent on drug concentration.5.The results showed that the content of TIMP1 in each concentration group did not change significantly compared with the blank control group at the same incubation time(p>0.05).with the same concentration of DHA,the TIMP1 content in each experimental group did not change significantly(p>0.05).6.The expression of EMT related factors in mRNA expression,compared with the blank control group,the expression of CDH1 in different concentrations of mRNA at the same time was significantly higher than that of the control group DHA(p<0.01).The mRNA expression of SLUG,ZEB1,ZEB2,Twist,HMGA2,MMP-9 and VEGF was significantly lower(p<0.01).Dihydroartemisinin can inhibit the proliferation of canine breast tumor cells,and inhibit the invasion of EMT cells through the regulation of the expression of related factors.