| Choriocarcinoma is a highly malignant trophoblastic tumor which originatedfrom the trophoblast. It may occur after abortion or in the process ofpregnancy. It grows quickly and can also widely metastasize to other organs ortissues through both the venous and lymphatic systems. Early venous andlymphatic metastasis could cause patients’ rapid death. The transforminggrowth factor-beta1(TGF-β1) belongs to a growth factor super-family becamehotspot of oncology field in recent years and has been suggested to play acritical role in regulating genesis and development of choriocarcinoma. TGFβis a kind of biological multifunctional cytokine that consist of5isoforms（TGF-β1～5）. TGF-β1is the main isoform that can be secreted by almost allkinds of cell. it regulates a variety of cellular processes such as celldifferentiation, proliferation, extracellular matrix production and embryonicdevelopment, and it is also closely related with tumor development. TGF-β1initiates signaling via a cell surface transmembrane serine/threonine kinasereceptor complex which then activates its downstream Smads protein. Afterthat, receptor binding induces phosphorylation of its downstream proteinSmad2and Smad3. Smad4works as a mediator, carries phosphorylatedSmad2and Smad3into the nuclear where target gene is processed. P38mitogen-activated protein kinase (p38MAPK) became a focus of oncologyfield in investigating the invasion and metastasis of tumor. Previous studiesindicated that TGFβ can activate the p38MAPK pathway, and there is acrosstalk between TGFβ and p38MAPK pathway in gastric tumor and ratcardiac fibroblasts, but the mechanism of the interaction between TGF-β andp38MAPK pathway still remain unclear. Therefore in this study, weinvestigated the mechanism of the interaction between Smad and p38MAPKsignaling in JEG-3choriocarcinoma cells by using p38MAPK inhibitor and TGF-β receptor inhibitor and detect the proteins in Smad and p38MAPKpathways.Objective:Cross-talk between Smad3and p38MAPK through TGF-β1in JEG-3choriocarcinoma cellsMethod:1. JEG-3cells were cultured in incubator with5%CO2at37C in RPMI-1640supplemented with10%fetal bovine serum (FBS, Hangzhou SijiqingBiological Engineering Materials Co., Ltd, Hangzhou, China),200mMglutamine,100mM pyruvic acid Na,100μg/ml streptomycin and100U/mlpenicillin. When the cells reached approximately70-80%, they weresubcultured with0.25%trypsin and0.02%EDTA.2. JEG-3cells were seeded and cultured in24-well plate with an initialconcentration of5×104cells/ml in incubator with5%CO2at37C. The nextday culture medium was exchanged to new RPMI-1640without fetal bovineserum and continued to culture for48h. Cells were divided into8groups, andgroups were as follows: control group,2h TGF-β1group,6h TGF-β1group.TGF-β1of5ng/ml was added to each well and cultured for2h and6hrespectively except control group. P-Smad3and nuclear translocation weredetected by immunofluorescence assay. Each group has3duplicate wells.3. Cells were incubated in6-well plate with an initial concentration of5×104cells/ml in incubator with5%CO2at37C. The next day culture medium wasexchanged to new RPMI-1640without fetal bovine serum. The next dayculture medium was exchanged to new RPMI-1640without fetal bovineserum and continued to culture for48h. Cells were divided into6groups, andgroups are as follows: control group, TGF-β1group,1μM SB203580,3μMSB203580,1μM LY364947,3μM LY364947. Cells in appropriate wellswere pretreated with different concentrations of TGF-β1receptorinhibitor(LY36494) and p38MAPK inhibitor (SB203580) and cultured for2h,then5ng/ml TGF-β1was added into each well, except control group,continuing incubated for2h. Protein expression levels of Smad3 and P-Smad3were detected by western blotting assay.4. Cells were incubated in6-well plate with an initial concentration of5×104cells/ml in incubator with5%CO2at37C. The next day culture medium wasexchanged to new RPMI-1640without fetal bovine serum. The next dayculture medium was exchanged to new RPMI-1640without fetal bovineserum and continued to culture for48h. the groups have been mentionedabove. Cells in appropriate wells were pretreated with different concentrationsof TGF-β1receptor inhibitor(LY36494) and p38MAPK inhibitor (SB203580)and cultured for2h, then5ng/ml TGF-β1was added into each well, exceptcontrol group, continuing incubated for2h. Transcription levels ofSmad3were detected by Real-time quantitative PCR.Result:1. We observed that phospho-Smad3showed a diffuse cyto-plasmic stainingpattern in control group and few staining in nucleus For the group of2hTGF-β1treatment, phospho-Smad3staining appeared in both cytoplasmand nucleus. whereas in group of6h TGF-β1treatment, most ofphospho-Smad3staining was in nucleus and P-Smad3almost finishednuclear translocation.2. The protein expression of Smad3and phospho-Smad3significantlyincreased in TGF-β1group, comparing with the control group. With theincreasing concentrations of inhibitors, the Smad3and phospho-Smad3protein levels in LY364947group gradually reduced compared with thecontrol and TGF-β1groups(P<0.05) With the increasing concentrations ofinhibitors, the Smad3SB203580groups gradually reduced compared withthe other two groups (P<0.05).3. Comparing with the control group, the mRNA expression levels of Smad3significantly elevated in TGF-β1group. With the increasing concentrationsof inhibitors, the Smad3transcriptional levels in LY364947and SB203580groups gradually reduced compared with the other two groups(P<0.05)Conclusion:1. TGF-β1induced activation and nuclear translocation of Smad3in JEG-3 cells.2. Inhibition of p38MAPK decreased protein expression level of Smad3and TGF-β1-induced activation of Smad3in a dose-dependent manner.3. Inhibition of p38MAPK decreased the transcriptional level of Smad3in adose-dependent manner. |
