Experimental Study On Doxycycline As An Inhibitor Of The Epithelial-to-mesenchymal Transition And Vasculogenic Mimicry In Hepatocellular Carcinoma

Background and Objective:Hepatocellular carcinoma(HCC)is one of the most common malignant tumors.Although recently the diagnosis and the therapy of HCC has made a big progress,the morbidity and mortality didn’t improve notably.Epithelial-to-mesenchymal transition(EMT)and vasculogenic mimicry(VM)are hot topics in recent years,they are closely related tumor Invasion,metastasis and prognosis of tumor patients.As the study of EMT and VM is deepening constantly,the researches of drugs that inhibit EMT and VM is getting more and more popular.In this study we studied how doxycycline inhibited VM and EMT in HCC,preliminarily clarified the mechanism of the inhibition,and provided experimental basis for new therapy pathway.Methods:1.Model of transplanted tumor on nude mouse were used to study the effects of doxycycline on HCC microcirculation,tumor proliferation and the survival time of animals.2.Use Immunohistochemistry and endomucin/periodic acid Schiff(PAS)double staining methods to get the microvessel density(MVD)and the number of VM,and test the expression of EMT related proteins(E-cadherin,vimentin,Twistl,Snail)and VM related protein(VE-cadherin),and discuss the relationship between doxycycline EMT,VM.3.Observe the inhibition of doxycycline on HCC through in vitro studies such as cell proliferation experiment,adhesion experiment,migration experiment,invasion experiment and tunnel formation experiment.4.Test the effects of doxycycline on E-cadherin,vimentin,Twist,Snail,VE-cadherin expression by using Western blotting,RT-PCR,real-time PCR,immunofluorescence,study the mechanism of the inhibition on molecular level.5.Screen out low expression E-cadherin HCC cell lines using transfection,the add doxycycline,observe the expression of E-cadherin,vimentin,Twist,Snail and VE-cadherin,furtherly illuminate the effect of doxycycline on EMT and VM.6.Use an EpiQuikTM DNA Methyltransferase Activity/Inhibition Assay Ultra Kit and Gelatin-Zymography to detect the activities of DNMT and MMPs,additionally,DNA methylation assay and methylation-specific polymerase chain reaction(MSP)were used to detect the methylation and ummethylation of CDH1,further to detect the mechanisms of inhibition of EMT and VM by doxycycline.Results:1.The engrafted MHCC97H tumors in the doxycycline treatment group developed slower than those in the control group,and the tumor sizes in the treatment group were smaller than those in the control group.The tumor sizes of the drug group and control group were 1.083 cm3 and 1.991 cm3(P<0.05)respectively.The anti-tumor rate was 43.39%.Kaplan-Meier survival analysis results showed that the mice in the control group exhibited shorter survival time than those in the doxycycline treatment group.2.In vivo,the number of microcirculation patterns in the tumors indicated that fewer VM channels and endothelium-dependent vessels were in the treatment group than in the control group.The tumors in the treatment group expressed less VE-cadherin protein than the control group(P<0.05).The tumors in the treatment group expressed E-cadherin protein at a higher extent than the control group,while vimentin,Twist,Snail in the treatment group were less expressed than those in the control group(P<0.05).3.In vitro,experiment results showed doxycycline could suppress the cloning and the growth of tumor cells,promote the adherence and inhibit the migration,invasion and the ability of VM formation of tumor cells.4.The results of western blotting,RT-PCR,real-time PCR and IF all showed that doxycycline could promote the expression of E-cadherin and suppress the expression of vimentin,Twist,Snail,VE-cadherin.5.E-cadherin was upregulated in the doxycycline-treated E-cadherin shRNA group compared with the E-cadherin shRNA group,while vimentin,Twist,Snail,VE-cadherin were down regulated.6.Gelatin zymography results showed that the activated MMP-2(72 kDa)and MMP-9(92 kDa)were downregulated in the doxycycline-pre-treated group.DNMT activity assay showed that a high concentration of doxycycline significantly decreased DNMT activity.The MSP of E-cadherin also showed that a high concentration of doxycycline could partially inhibit CpG methylation in the CDH1 promoter region;doxycycline could then inhibit EMT progression.Conclusions:1.Doxycycline could inhibit tumor growth in vivo and extend the duration of animals’ life.2.Doxycycline has the ability to inhibit EMT progression and VM formation.3.Doxycycline has the ability to inhibit the activities of DNMT and MMPs,and can inhibit the DNA methylation of CDH1.