TGF-β1Promotes Prostate Cancer Epithelial-Mesenchymal Transition And Metastasis By Regulating Expression Of CD44

In the world, the incidence of prostate cancer ranks the second out of all the male malignant tumors, and mortality ranks the sixth out of the male malignant tumors. The incidence of prostate cancer has obviously geographical and ethnic differences, The incidence in developed countries is higher than it in developing countries. Although the incidence of prostate cancer in Asia is much lower than it in the European and American countries, it is on the rise in recent years,and the growth is more quick than it in European and American developed countries. As early as1941, androgen deprivation therapy (ADT) become the main way of prostate cancer treatment, Which block the combination of androgen and androgen receptor,so as to achieve therapeutic purposes.However, there is a problem in this treatment that a part of androgen dependent prostate cancer (ADPC) by androgen deprivation therapy (ADT), done the standard treatment for1to2years, translate into castration resistant prostate cancer (CRPC). In the process, some studies found that the chemotherapy resistance,metastasis, invasion and recurrence of tumor cells enhances. This may be closely related to the increase of cancer stem cells (CSCs) and the occurrence of epithelial mesenchymal transition (EMT). However, the evidence of this aspect is still little.Part1ADT activates TGF-β1signaling pathwaysObjective:To explore relationship between ADT and TGF-β1signaling pathwaysMethods:1. The study population was divided into three groups,which are ADPC group,ADT group and CRPC group.2. We detected TGF-β1expression in the above three groups by Immunohistochemical method.Results: We compared the results of three groups, in tumor specimens of CRPC group TGF-β1expression increased.Conclusions:In the process that ADPC converts into CRPC after the treatment with ADT TGF-β1signaling pathways are activated.Part2TGF-β1induces EMT and increases the expression of CD44 Objective: To research the role which TGF-β1has in EMT and CD44Methods: We cultured androgen dependent prostate cancer cells(LNCaP) and androgen independent prostate cancer cells (CWR22RV1) in vitro.Respectively, we used TGF-(31(5ng/ml) stimulating the cells,and then detected CD44、p-Smad2and Smad2expression level when0、3、6、9hr,and then detected E-cadherin and vimentin expression level when0、12、24、36、48hr by Western blot.Results:1.TGF-β1increased the expression of CD44.2.TGF-β1promoted the occurrence of EMT.Conclusions:TGF-β1induces EMT and increases the expression of CD44. Part3TGF-β1adjusts the CD44which leads to EMTObjective:To research the mechanism that TGF-β1induces EMTMethods:1. We cultured LNCaP cells and CWR22RV1cells in vitro. Respectively, the cells was divided into four groups.First,we added SB431542(TGF-β1receptor inhibitor) into the third group and the fourth group.Second, we added TGF-β1(5ng/ml) into the second group and the fourth group after2hours.Then,we extracted the protein after3hours. We detected CD44、p-Smad2and Smad2expression level by Western blot.2. We cultured LNCaP cells and CWR22RV1cells in vitro. Respectively, the cells was divided into three groups which are the control group, experimental group1, experimental group2. We put the sicontrol plasmid into the control group,and put the siCD44plasmid into experimental group1for24hours,and put the siCD44plasmid into experimental group2for48hours. Respectively, we extracted the protein and detected Expression of CD44level by Western blot.3. We cultured LNCaP cells and CWR22RV1cells in vitro. The cells was divided into four groups, respectively. First, we put the sicontrol plasmid into the first group and the second group. Second, we put the siCD44plasmid into the third group and the fourth group for48hours.Then we added TGF-β1(5ng/ml) into the second group and the fourth group.After24hours we extracted the protein,and detected CD44、 E-cadherin and Vimentin expression level by Western blot,respectively. Results:1.In LNCaP and CWR22RV1cells,compared with the control group, Expression of CD44level significantly increased in the groups which we added TGF-β1alone into. Compared with the control group respectively, Expression of CD44level had no obvious change in the groups which we added SB431542alone into and we added SB431542and TGF-β1into.2. After puting the plasmids into LNCaP and CWR22RV1cells, Expression of CD44level (from high to low)in turn showed that the group put sicontrol plasmid into、the group put siCD44plasmid into for24hours and the group put siCD44plasmid into for48hours.3.In LNCaP and CWR22RV1cells, compared with the control group, CD44and Vimentin expression level increased and E-cadherin expression level decreased in the groups which we added TGF-β1alone into. Compared with the control group respectively, CD44and Vimentin expression level decreased and E-cadherin expression level increased in the groups which we put siCD44plasmid alone into and we put siCD44plasmid and TGF-β1into.Conclusions:TGF-β1upregulates the CD44which leads to EMT.Part4Targeted inhibition of CD44in vivo impedes prostate cancer metastasis.Objective:To generate prostate cancer mouse model and study the relationship between targeted inhibition of CD44in vivo and prostate cancer metastasis.Methods:1.We cultured LNCaP cells and CWR22RV1cells in vitro. Respectively,with salinomycin processing for0,24,48hours, we extracted the protein and detected Expression of CD44level by Western blot.2.We generated prostate cancer mouse model,and the mice were divided into tow groups at random which are experimental group(Intraperitoneal injections of salinomycin) and control group(Intraperitoneal injection of corn oil).we took out tumor in situ and metastases,and compared the situation of metastases between groups.3. We detected CD44、E-cadherin and Vimentin expression level in the above groups by immunohistochemical method. Results:1. Salinomycin inhibited Expression of CD44.2. The number of metastases in the experimental group were significantly less than the control group.3. CD44and Vimentin expression level in the experimental group is less than the control group. E-cadherin expression level in the experimental group is more than the control group.Conclusions: Targeted inhibition of CD44in vivo effectively impedes prostate cancer metastasis and the occurrence of EMT.