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Influence of Naproxen Sodium on ERK Expression in the Breast Cancer Cells

Author: LiChunYang
Tutor: SongMin
School: China Medical University
Course: Pathology and Pathophysiology
Keywords: Extracellular signal-regulated kinase Phosphorylated extracellular signal-regulated kinase Naproxen sodium Breast Cancer
CLC: R737.9
Type: Master's thesis
Year: 2010
Downloads: 67
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Abstract

Preface activation of the ERK signaling pathway in tumorigenesis are closely related, ERK is a cell proliferation and apoptosis signaling protein, is also a key point to control the progression of breast cancer. ERK phosphorylation of anti-apoptotic molecules, while activation of transcription factors to stimulate the expression of survival gene and produce anti-apoptotic effects, ERK through phosphorylation reaction can adjust the activity of certain transcription factors, these transcription factors to further regulate their respective target genes transcription of, causing change of expression or activity of a specific protein, and ultimately regulate cell metabolism and function, affect cell to produce a specific biological effect. NSAIDs are widely used in clinical antipyretic analgesics, and its mechanism of action is inhibition of cyclooxygenase, blocking arachidonic acid (arachidonic acid, AA) prostaglandin (prostaglandins, PGs) biosynthesis, to play its anti-inflammatory effects. In this study, by observing the different concentrations of naproxen sodium apoptosis of breast cancer cells in vitro to clarify the role of the pro-apoptotic drugs on breast cancer cells, ERK and P-ERK expression was detected by Western blot clear the ways and means of drug-induced apoptosis. Materials and Methods cryopreserved material cell line MDA-MB-231 and MCF-7 human breast cancer cell lines for the Chinese Department of Pathology. Second, the main reagent concentrated rabbit anti-human ERK1 / 2 monoclonal antibody (L352) was purchased from American Bioworld and concentrated rabbit anti-human P-ERK1 / 2 monoclonal antibody (Y204) purchased in the United States Bioworld. Concentrated horseradish peroxidase-conjugated goat anti-rabbit antibody (BS10350) purchased in the United States Bioworld Company. Flow cytometry kit was purchased from Xinbo Sheng. Cell culture and grouping of the three experimental method (a) were cultured breast cancer cells MCF-7 and MDA-MB-231, two breast cancer cells were divided into four groups, respectively, for the control group, a low concentration of the drug-treated group, moderate concentration drug-treated group, and a high concentration drug-treated group. Control group, low concentration of drug-treated group, moderate concentration of drug-treated group, and a high concentration drug-treated group, respectively corresponding to the concentration of the drug Ommol / L, 1mmol / L, 5 mmol / L, and 10mmol / L,. (B) flow cytometry apoptosis using Annexin V / PI double staining, flow cytometry (BDFACScalibur) analysis results Cellquest software analysis and processing. (C) Western blot routinely collect different concentrations of naproxen sodium 24 h after the MDA-MB-231 and MCF-7 breast cancer cells, centrifuged supernatant packaging, the Bradford method determination of the protein concentration of the cell extract. Anti-total ERK1 / 2 antibody detection of total ERK1 / 2 the amount of an internal control to specifically recognize dual phosphorylation ERKl / 2 antibody detection of phosphorylated ERK1 / 2 amount; After the end of the SDS-PAGE electrophoresis, the PVDF membrane was placed in blocking buffer, and the room temperature and closed 1 h, rabbit anti-human of ERK-1/2 antibody, and rabbit anti-human P-ERK antibody overnight, adding HRP labeled goat anti- rabbit IgG, Western blotting reagents evenly on a PVDF membrane, luminescent reagent is decanted, cover with plastic wrap and the x-ray film is placed in cling film in a dark room PVDF membrane, rinse the x-ray film, camera and computer image analysis, application BandScar5.0 software results gray value detection. (D) statistical analysis application SPSS 12.0 statistical software for statistical analysis using t-test analysis of the image gray value of each group Western blot and flow cytometry apoptosis rate, P lt; 0.05 with a significant difference. 1, flow cytometry results MCF-7 and MDA-MB-231 between the two groups P gt; 0.05, no significant difference; different concentrations of the drug treatment groups P lt; 0.05, with a significant statistical difference ; concentration of the drug at the same time the rate of apoptosis has a significant correlation. 2, Western blot results of different drug concentrations between groups ERK expression p gt; 0.05, and no significant difference, P-ERK expression is P lt; 0.05, with a significant statistical difference, while the concentration of the drug with P-ERK The expression has a significant negative correlation. Discussion of ERK1 / 2 is equal to the early 1990s by Boulton has isolated and identified a protein kinase, ERK by the phosphorylation reaction can be adjusted some of the activity of transcription factors, these transcription factors further adjust their respective transcription of target genes, causing a lot of protein expression or activity of change, and ultimately regulate cell metabolism and function, affect cell to produce a specific biological effect. Closely related to the occurrence of the ERK signaling pathway activation and tumor, ERK is a cell proliferation and apoptosis signaling protein, is also a key point to control the progression of breast cancer. MAPK signal transduction pathway is involved in the regulation of a variety of cell functions, in particular, plays a key role in cell proliferation, differentiation, and apoptosis. This study demonstrated that apoptosis of breast cancer has a significant correlation with the expression of P-ERK and ERK expression no significant correlation, indicating that ERK phosphorylation pathway is one of the key anti-apoptotic pathways in breast cancer cells. NSAIDs are widely used in clinical antipyretic analgesics, in recent years, has become the people's research focus on the anti-tumor effect of NSAIDs,. This study breast cancer cells treated by different drug concentrations observed and found that naproxen sodium has obvious inducing apoptosis of breast cancer cells, and this effect is enhanced with the increase of drug concentration. Estrogen receptor-positive MCF-7 and MDA-MB-231, estrogen receptor-negative breast cancer cells, ERK and P-ERK expression detection, naproxen sodium had no significant effect on the expression of two cell ERK has a significant inhibitory effect on the expression of P-ERK, while the inhibition enhanced with the increase of the concentration of the drug, with a clear correlation, no correlation with the estrogen receptor, thus indicating that naproxen sodium by inhibition of ERK phosphorylation pathway to reduce P-ERK expression to promote breast cancer cell apoptosis. Conclusion 1, naproxen sodium no obvious effect on the expression of the ERK, while the expression of P-ERK was significantly inhibited, while the inhibitory effect is enhanced with increasing concentration of the drug, with a significant correlation. 2, naproxen sodium by inhibiting ERK phosphorylation pathway to reduce P-ERK expression to promote breast cancer cell apoptosis.

Influence of Naproxen Sodium on ERK Expression in the Breast Cancer Cells

Abstract

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