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Expression of GSK-3β in Breast Cancer Cells and the Relationship with P38MAPK Pathway

Author: XuMan
Tutor: SongMin
School: China Medical University
Course: Pathology and Pathophysiology
Keywords: p-p38 p-GSK3βser9 GSK3β Breast Cancer Shift
CLC: R737.9
Type: Master's thesis
Year: 2010
Downloads: 131
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Abstract

Introduction GSK3β glycogen synthase kinase-3 (glycogen synthesis kinase, GSK-3) one of the subtypes. In recent years, more and more studies confirm, GSK3β not only to participate the glycogen metabolism process, it is a multi-functional kinase, protein synthesis, cell proliferation, cell differentiation, cell motility, and tumor aspects play an important role. Recent studies have found that, GSK3β plays an important role in the incidence of breast cancer in the development process, and to participate in a breast cancer cell growth and apoptosis signaling pathway regulation. However, GSK3β is involved in the metastasis of breast cancer cells remains to be studied. p38MAPK is an important member of the mitogen-activated protein kinase (mitogen-activated protein kinase, MAPK) family. Recently, p38MAPK signaling pathways involved in tumor invasion and metastasis positively regulated manner much attention. To this end, this study examined the p-p38, p-GSK3βser9 and GSK3β expression in breast cancer cell lines, and further study of the role of GSK3β in breast cancer invasion and GSK3β and the p38MAPK pathway of relations for the treatment of breast cancer theoretical basis. Materials and methods, materials (1) cell lines of human breast cancer cell line MCF-7, MDA-MB-231, MDA-MB-435s, originally purchased from the Beijing Union Medical College of Basic Medical Cell Center, the experiment by the China Medical University has some cell recovery derived. (2) The the main reagent mouse anti-human p-p38 antibodies were purchased from Cell signaling. Rabbit anti-human GSK3β antibody was purchased from the the Bioworld company. Rabbit anti-human the p-GSK3pser9 antibodies were purchased from Santa. p38MAPK specific inhibitor SB203580 was purchased from Calbiochem. GSK3p specific inhibitor SB216763 were purchased from Cayman. Transwell chamber (aperture 8um) were purchased from Corning Incorporated. 2, the method (1) Western blotting was collected cells were added to RIPA buffer lysis buffer, sonication, high-speed centrifuging at a low temperature to extract total protein by the Bradford method for protein concentration determination. SDS-PAGE electrophoresis, transfer, after closure, plus a corresponding one of secondary antibodies. ECL luminescence, the observation camera, and the determination of the gray value. (2) immune staining taken logarithmically growing cells inoculated into the orifice. Fixed, TritonX-100 treatment, closed plus the corresponding primary antibody, secondary antibody, stained nucleus, fluorescence microscopy pictures. (3) Transwell experiment Transwell chambers inoculated cell culture, wipe on indoor cells fixed after staining observed taking pictures, cell count. 3, statistical analysis using SPSS13.0 statistical software line data processing: the t-test analysis, Western Blot image gray value, the Transwell penetrating cell number, LSD-t test several lines of each sample twenty-two compare P lt; 0.05 was considered statistically significant. 1, p-p38, p-GSK3βser9, of GSK3β in breast cancer cell lines in the expression (1) Western blotting in three breast cancer cell line MCF-7 and MDA-MB-231, MDA-MB-435s , p-GSK3βser9 and p-p38 expression showed an increasing trend, GSK3β expression was no significant difference. (2) select p-p38 the p-GSK3βser9 are highly expressed high aggressive and highly metastatic cell line MDA-MB-435s, Immunofluorescence localization: p-p38 expression in the nucleus, cytoplasm in the MDA-MB-435s only weak expression; the p-GSK3pser9 in MDA-MB-435s was mainly expressed in the cytoplasm, and only weakly expressed in the nucleus. Cell line MDA-MB-435s, of GSK3β the inhibitor LiCL and SB216763 role after 24h the p-GSK3βser9 and the GSK3β change (1) Western blotting: LiCL SB216763 can be raised the p-GSK3βser9 expression levels, GSK3P no significant change. (2) Immunofluorescence localization: 24h the p-GSK3βser9 after cytoplasmic expression reduce the the SB216763 role of MDA-MB-435s cell lines, increased expression. (3) Transwell experiments showed that: SB216763 energy concentration-dependent increase in the invasive and metastatic potential of breast cancer cells MDA-MB-435s. 3 of P38MAPK inhibitor SB203580 against GSK3β activity in Western blotting detection lowered p-p38, p-GSK3βser9 protein expression in the MDA-MB-435s, SB203580, concentration dependent manner, but does not affect the GSK3β protein. Discussion GSK3β is a multifunctional protein kinases, which play an important role in protein synthesis, cell proliferation, cell differentiation and cell movement. GSK3β activity mainly through 3 different ways to adjust: (1) the phosphorylation status of the different sites regulate GSK3β activity. Serine 9 (Ser9) at the N-terminal phosphorylation reduces GSK3β activity, phosphorylation at tyrosine 216 (Tyr216) at can enhance its activity; regulating GSK3β activity, (2) intracellular localization. GSK3β mostly present in the cytoplasm, but translocates into the nucleus of GSK3β activity to promote tumor cell cycle regulation, was able to play; (3) different regulation of substrate phosphorylation levels of GSK3β activity. Most GSK3β the substrate must first be other kinases phosphorylation (one phosphorylation), and can be recognized by a GSK3β and subject to phosphorylation (secondary phosphorylation). Accordingly, the phosphorylation level of the substrate to change the substrate can be identified, thereby regulating the GSK3β activity. GSK3β on tumor whether tumor suppressor or tumor promoters, have been inconclusive. p38MAPK signaling pathway through phosphorylation activates a variety of transcription factors involved in the regulation of cellular responses, including c-Jun, c-fos, and ATF2. Also activated p38MAPK activation of the transcription factor MEF2C ELK-1 MAX and CHOP, P38 pathway is also involved in the synthesis of cytoskeletal proteins, application p38MAPK inhibitor SB203580 inhibits cell movement. This article by observing the of GSK3β activity of breast cancer cell invasion ability, found with the breast cancer cell line MCF-7, MDA-MB-231, MDA-MB-435s invasion force increase, p-GSK3βser9 protein expression was increasing trend; Total GSK3β was no significant difference in negative correlation, suggesting that GSK3β activity and invasiveness of breast cancer. The Transwell experiments results show that the invasiveness of MDA-MB-435s cells is gradually increased with the increase in the concentration of the inhibitor GSK3β inhibitors SB216763. In other words, the reduction of GSK3β activity can increase the invasive ability of breast cancer cell line MDA-MB-435s. We further Immunofluorescence localization of breast cancer cell lines, the results show: p-GSK3βser9 mainly localized in the cytoplasm. The the SB216763 role of MDA-MB-435s cell lines after 24h the p-GSK3βser9 nuclear expression increased, the plasma decreased expression. Description the p-GSK3βser9 change GSK3β activity inside and outside the movement through nuclear, thereby regulating the invasive ability of breast cancer cells. Has been confirmed, GSK3β are present in the cytoplasm, nucleus and mitochondria, compared with the cytoplasm, nucleus and mitochondria parts of GSK3β activity higher. Nucleus of GSK3β in regulating many transcription factors in the nucleus, and its level is not constant, but dramatic changes according to the change of a number of factors in the intracellular. Therefore, we hypothesized that the nuclei GSK3β may play a major role in regulating breast cancer invasion gene. Many studies have found that the of GSK3β and P38MAPK exist certain contact, then the relationship between both breast how? We have adopted the the different concentration P38MAPK specific inhibitor SB203580 role in the cell line MDA-MB-435s, experimental results show that: SB203580 can concentration-dependent manner down the highly metastatic cell line MDA-MB-435s pGSK3βser9 protein expression levels in breast cancer. That inhibition P38MAPK pathway may be directly or indirectly to cut pGSK3βser9 expression level, thereby increasing GSK3β activity, and thus play a role in breast cancer invasion, but the exact mechanism needs further study. Our results show that inhibition of GSK3β activity can promote the invasion of breast cancer cell lines, and a negative correlation between GSK3β and P38MAPK in breast cancer cell lines. Currently, GSK3β and p38MAPK signaling pathway inhibitors become hot spots of cancer treatment, our experimental results provide a new theoretical basis for GSK3β and p38MAPK as a target for breast cancer treatment. Conclusion, inhibition of GSK3β activity can increase the capacity of the invasion of breast cancer cell lines. GSK3β activity in breast cancer cell lines with p38MAPK negatively correlated.

Expression of GSK-3β in Breast Cancer Cells and the Relationship with P38MAPK Pathway

Abstract

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