Effect Of Recombinant Human TWEAK Induced The Expression Of TRAF1 In Breast Cancer Cell Lines

ObjectiveTumor necrosis factor(TNF)-like weak inducer of apoptosis(TWEAK,TNFSF12) is a member of the TNF superfamily,and a typeⅡtransmember protein.TWEAK activates the Fn14 receptor,and may regulate apoptosis,proliferation,and inflammation,processes that play a significant role in pathological conditions.As for a type of important intracellular connection proteins,TRAFs(Tumor necrosis factor receptor-associated factors)can participate in many signal transducers pathways to active genes such as NF-κB and JNK.Seven TRAFs members have already been found. TRAF1 is the most special,which doesn't have the TRAF-N structural domain.Several model systems have been used to examine TRAF1 function and the results obtained have led to a variety of often contradictory conclusions about whether TRAF1 is a positive or negative regulator of TNFR signaling pathways and in which pathway it is important.This study investigated the expression of Fn14 in the breast cancer cell lines through immunofluorescence staining.Co-immunoprecipitation and western blot is used to study the expression of TRAF1 in the condition of stimulated by rhTWEAK.We also explored whether rhTWEAK can activate the NF-κB signaling pathway.Materials and Methods1.MaterialsThe normal breast cell line MCF-10A,the low metastasis breast cancer cell line MDA-MB-231 and the high metastasis breast cancer cell line MDA-MB-435s. 2,ReagentsGoat anti-Fn14 polyclonal antibody,Mouse anti-TRAF1 monoclonal antibody and Mouse anti-TRAF2 monoclonal antibody were purchased from Santa Cruz Company. Mouse anti-Phospho-IκBαmonoclonal antibody was purchased from Cell signaling Company.Recombinant human TWEAK(rhTWEAK)was obtained from Peprotech Company.The DAB agent kit and HRP-banding second antibody were bought from Beijing Zhongshan Jinqiao Biological Company.μColumns andμMACS Protein G MicroBeads were purchased from Miltenyi Biotec Company in Germany.Cell lysis Buffer was bought from Beyotime Company.3.Methods(1)The expressions of Fn14 in the above-mentioned cultured cell lines were examined through immunofluorescence staining.(2)The expressions of TRAF1 in the condition of stimulated by rhTWEAK in the above-mentioned cultured cell lines were examined with Western blot.(3)The relationships between TRAF1 and TRAF2 with co-immunoprecipitation in the condition of stimulated by rhTWEAK in the above-mentioned cultured cell lines were also examined.(4)The activation of NF-κB were examined with Western blot after the induction of rhTWEAK in MDA-MB-231 and MDA-MB-435s cell lines.(5)A statistical software Statistical Product and Services Solutions(SPSS) (version 12.0)was utilized for data analysis.Analysis of variance was done to compare the expressions of TRAF1 in different cell lines.A p-value less than 0.05 was considered valid in terms of statictics and Values less than 0.01 were considered predominantly significant.Results1.Expressions of Fn14 through immunofluorescence staining in normal breast and breast cancer cell lines (1)The expression of Fn14 in MDA-MB-231 cell line is positive.(2)Fn14 is negatively expressed in MCF-10A and MDA-MB-435s cell lines.2.Expression of TRAF1 through western blot in the condition of stimulated by rhTWEAK in normal breast and breast cancer cell lines(1)The effect of rhTWEAK on TRAF1 protein expression is dose- dependent up-regulating in Fn14-positive cell line MDA-MB-231(p＜0.01)and down- regulating in Fn14-negative cell line MCF-10A and MDA-MB-435s.(p＜0.01)(2)The effect of rhTWEAK on TRAF1 protein expression is time- dependent up-regulating in Fn14-positive cell line MDA-MB-231(p＜0.01)and down-regulating in Fn14-negative cell line MCF-10A and MDA-MB-435s.(p＜0.01)3.Relationships between TRAF1 and TRAF2 in the condition of stimulated by rhTWEAK in normal breast and breast cancer cell lines(1)It is found through co-immunoprecipitation that TRAF1 and TRAF2 combined in normal human breast cell line MCF-10A,human breast cancer cell line MDA-MB-231 and MDA-MB-435s.(2)The quantity of TRAF1 in combination with TRAF2 is significant up-regulated by rhTWEAK(100ng/ml,24h)in Fn14-positive cell line MDA-MB-231(p＜0.01)and down- regulated in Fn14-negative cell line MCF-10A and MDA-MB-435s.(p＜0.05)4.The activation of NF-κB stimulated by rhTWEAK in MDA-MB-231 and MDA-MB-435s cell linesThe phosphorylation level of IκBαwas transient up-regulated(15-30min)by rhTWEAK in Fn14-positive cell line MDA-MB-231 but not in Fn14-negative cell line MDA-MB-435s.Conclusions1.The expression of Fn14 in MDA-MB-231 cell line is positive while negative in MCF-10A and MDA-MB-435s cell lines. 2.The effect of rhTWEAK on TRAF1 protein expression is dose and time dependent up-regulating in Fn14-positive cell line MDA-MB-231 and down- regulating in Fn14-negative cell line MCF-10A and MDA-MB-435s.3.RhTWEAK can stimulate the activition of NF-κB signaling pathway in Fn14-positive cell line MDA-MB-231.The induction of rhTWEAK to TRAF1 is potentialy Mediated by Fn14,and the mechanism of action of TRAF1 may be interact with TRAF2,which may play an important role in the activition of NF-κB signaling pathway induced by rhTWEAK in human breast cancer cell lines.