Expression Of GSK-3β In Breast Cancer Cells And The Relationship With P38MAPK Pathway

IntroductionGSK3P is one of the subtypes of glycogen synthesis kinase(GSK3), which is a multi-kinase express extensively in normal and cancer tissues. GSK3βis not only involved in glycogen metabolism, but also play a key role in protein synthesis, cell proliferation, cell differentiation, cell movement, and other aspects of tumor. It is reported recently that GSK3βinvolved in breast cancer cell growth and apoptosis signal pathway. However, whether GSK3βis involved in the invasion and metastasis of breast cancer cells remains to be studied. p38MAPK is one important member of mitogen-activated protein kinases (MAPKs). Recently, more and more attention has been paid that p38MAPK signaling pathway is involved in the process of tumor invasion and metastasis by positively regulating mechanism. Therefore, we investigated the expression of p-p38,p-GSK3βser9 and GSK3βprotein in breast cancer cells to study the effect of GSK3βon the invasion of breast cancer and the effect of p38MAPK signaling pathway on the GSK3P activity. Our studies suggested that GSK3βand p38MAPK pathway might be a potential therapeutic target for anti-breast cancer treatment.Material and Methods1. Samples(1)MCF-7, MDA-MB-231 and MDA-MB-435s were purchased from the Cell Center of Peking Union Medical College.(2)Reagents The murine anti-human p-p38 monoclonal antibody was purchased from Cell signaling company. The rabbit anti-human GSK3βmonoclonal antibody was purchased from Bioworld company. The rabbit anti-human p-GSK3βser9 monoclonal antibody was purchased from Santa company. SB203580, the specifical inhibitor of p38MAPK was provided by Calbiochem company. SB216763, the specifical inhibitor of GSK3βwas provided by Cayman company, and the transwell chamber was provided by Corning company.2. Methods(1) Western blot:Protein concentration was measured by Bradford method. Tissue lysates were electrophoresed in polyacrylamide SDS gel and transferred onto polyvinylidene difluride membranes, Protein bands were blocked and incubated with the first and second antibody and visualized with ECL. Protein contents were calculated by densitometry.(2)Cell immunoflorescence:MDA-MB-435s cells blocked were incubated with primary antibody (p-p38 1:100, p-GSK3βser91;100) overnight at 4℃, then incubated with FITC-conjugated secondary antibody(1:50)and TRITC-conjugated Phalloidin(1:50) away from light,30min, they were incubated with Hoechest for nuclear counterstaining.(3)Matrigel Invasion Assay:Inoculated MDA-MB-435s cells in up-cave, then added SB216763, incubated at 37℃,24h. Wiped out the cells in up-cave, fixed, stained and counted the cells in down-cave.3. Statistical analysisAll the data are analyzed with SPSS for Windows 13.0 software. The result of Western Blot and Matrigel Invasion Assay is analyzed by t-test. The statiscal significance is defined as P<0.05.Results1. Expression of p-p38, p-GSK3βser9 and GSK3βprotein in breast cancer cell lines(1)Cell Western Blot shows:The expression of p-p38 and p-GSK3βser9 presents progressively increase in MCF-7, MDA-MB-231 and MDA-MB-435s as well as the potential malignancy (P<0.05). The expression of GSK3βhas no obvious difference(P>0.05).(2) MDA-MB-435s cells Immunoflorescence shows:p-p38 mainly localized in nucleus and manipulus in cytoplasm. p-GSK3βser9 mainly localized in cytoplasm and manipulus in nucleus. 2. The changes of MDA-MB-435s dealed with LiCL and SB216763, the inhibitor of GSK3β.(1)The MDA-MB-435s cells which were incubated with LiCL and SB203580 increased the expression of p-GSK3βser9 (P<0.05). But the expression of GSK3βhad no change (P>0.05).(2)Cells Immunoflorescence shows:the cytoplasm p-GSK3βser9 decreased as well as an increase of nucleus p-GSK3pser9.(3)The SB216763 promoted the potential of invasion and migratory of MDA-MB-435s with a dose-dependent manner (P<0.05).3. Influence of SB203580, the specifical inhibitor of p38MAPK to the activity of GSK3P.Western Blot shows:SB203580 decreased the expression of p-p38 and p-GSK3βser9 with a dose-dependent manner in MDA-MB-435s cells(P<0.05). The expression of GSK3βhad also no change (P>0.05).DiscussionGSK3 is a serine/threonine kinase that regulates multiple signaling pathways. There are two highly homologous isoforms, GSK3a and GSK3β. GSK3βis a constitutively active kinase that is regulated by phosphorylation, localization, and GSK3P-binding proteins. GSK3βis activated by phosphorylation of tyrosine 216 and negatively regulated by phosphorylation of serine 9. GSK3P is a negative regulator of several signaling networks in cells. p38MAPK is one important member of mitogen-activated protein kinase(MAPK) family. The p38MAPK undergoes phosphorylation at both tyrosine and threonine sites and can be activated by a wide spectrum of stimuli, including inflammatory cytokines, growth factors and cellular stress. p38MAPK signaling pathway has been implicated in cell growth, apoptosis, motion and invasive phaenotype and mediate cell migration, tumor invasion and metastasis.Our studies found that the change of p-GSK3βser9 expression assumes an increasing tendency in MCF-7, MDA-MB-231 and MDA-MB-435s as well as the potential malignancy and the Matrigel Invasion Assay showed that the SB216763, which is a specifical inhibitor of GSK3βpromoted the potential of invasion and migratory of MDA-MB-435s with a dose-dependent manner which suggested that the change of GSK3βactivity might concerned with invasiveness of the breast cancer cell lines. Our Cell immunoflorescence showed that p-GSK3pser9 mainly locates in the cytoplasma. But after 24h SB216763 affects MDA-MB-435s cell line, the nucleus expression of p-GSK3pser9 increases. Showing the movement of p-GSK3βser9 can changes GSK30 activeness, and may adjusts the invasion and metastasis of breast cancer cell.Many research had showed GSK3βand P38MAPK have certain relationship. We found that the P38MAPK inhibitor SB203580 decreased the expression of p-p38 and p-GSK3βser9 with a dose-dependent manner. In other words, suppresses the P38MAPK pathway possibly direct or indirect decline the expression of p-GSK3βser9 level, thus increases GSK3βactiveness, then plays certain role in the invasion and metastasis of breast cancer, but its exact mechanism remains to be further studied.Our studies showed that inhibiting GSK3P activity can promote the invasive ability of breast cancer cell lines and in breast cancer cell lines. GSK3βhas a negative correlation between P38MAPK. At present, GSK3βand p38MAPK signaling pathway inhibitors become cancer treatment hot spots. Our studies provide a new theory for the treatment of breast cancer.Conclusion1. GSK3βactivity inhibits could increase the invasive ability of breast cancer cell lines.2. GSK3βactivity was negatively correlated with the p38MAPK in breast cancer cell lines.