Relation Of ERK And Cell Proliferation And Apoptosis Of Breast Caner Cells

Objective:Breast cancer is the most common malignancy in women, and metastasis is the main cause of death of all breast cancer victims. However, the mechanism of breast cancer development and metastasis has been unclear up to now. The mitogen-activated protein kinases (MAPKs) are serine/threonine protein kinases that ,upon stimulation, phosphorylate their specific substrate at serine and/or theronine residues. MAPKs can be activated by various stimuli including cytokine, growth factor, neurotransmitter, hormone, a variety of cellular stress and adhesion. Activated MAPKs can phosphorylate numerous substrates including transcription factors and other protein kinases, modulate related gene expression, then participate in diverse physiological processes including proliferation, differentiation , mitosis, motility, metabolism and programmed death'apoptosis'. Aberrant activation of MAPK results in deprivation of differentiation and apoptosis, induces cell malignant transformation, aberrant proliferation, oncogenesis, increasing invasion, finally cause tumor metastasis.Conventional MAPKs consist of three family members: the extracellular signal-regulated kinase (ERK); the c-Jun NH2-terminal kinase (JNK); and the p38-MAPK. Additional MAPK, ERK5 is known as big MAPK 1 (BMK1) because of higher in molecular weight MAPK, including ERK7 and ERK8. However, the functions and activation pathways for ERK7 and ERK8 are not fully characterized.Each MAPK pathway contains a three-tiered kinase cascade comprising a MAPK kinase kinase (MAPKKK, MAP3K, MEKK or MKKK), a MAPK kinase (MAPKK, MAP2K, MEK or MKK) and the MAPK. This three-tier module mediates ultrasensitive switch-like responses to stimuli.ERKs, one of the earliest-identified and most important subfamily of MAPKs , are well-characterized MAPKs activated in response to growth stimuli. Phospho-ERK (p-ERK) is activation pattern of ERK. Activated ERK can enter into nucleus, regulate specific gene expression through phosphorylating and activating specified transcription factors.The level of activated ERK elevated in highly metastatic potential breast cancer cells and tissues. Activated ERK has a positive correlation with clinical-pathological parameters. Activated ERK ordinarily promoted breast cancer cells proliferation, anti-apoptosis and metastasis. However, their mechanism is not well understood. Professor Ye Li-Hong etc of Nankai University built sub-clone LM-MCF-7 from MCF-7 with high metastatic potential and obtained National invention patent(patent number:ZL 0310107316). The paired cell lines supply a cell model for studying molecular mechanisms of tumor metastasis. In this study the paired cell lines were used to research the relation and mechanisms of ERK pathways in proliferation and apoptosis of breast cancer cell.Materials and methods:1. cell lines and materialsLow metastatic potential breast cancer cell line MCF-7 and its subclone LM-MCF-7 with high metastatic potential , which was built by Ye Li-Hong's laboratory and obtained China national patent invention (patent number ZL 0310107316), were obtained from Pro. Ye Li-Hong of Nankai University (refer to Ye Li-hong, Wu Lian-Ying, Guo Wei etc. Screening of a sub-clone of human breast cancer cells with high metastasis potential. Zhonghua Yi Xue Za Zhi. 2006 Jan 3;86(1):61-5). Each cell line was maintained on plastic in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 1×penicillin-streptomycin, 2 mmol/L L-glutamine and 20mmol/L Hepes, at 37℃in a humidified atmosphere of 5% CO2-95% air. Cell passages were trypsinized with 0.25% trypsin.Mouse anti-human survivin monoclonal antibody, rabbit anti-human polyclonal ERK and Phospho-ERK antibodies were purchased from Cell Signaling Technology, Inc. ;rabbit anti-human polyclonal Bcl-2,cyclin D1 and caspase9 were from NeoMarker, Inc.; ERK inhibitor PD98059 was sigma production.2. Western Blot AnalysisExponentially growing MCF-7 and LM-MCF-7 cells were lysed and quantitated. Proteins were resolved by 12% SDS-polyacrylamide gel electrophoresis and transferred onto PVDF transfer membranes. The membranes were blocked overnight in PBS containing 5% skim milk and 0.1% Tween 20. ERK and p-ERK were detected in the cell lysate of the paired cell lines. So did LM-MCF-7 cell lines pretreated with various indicated concentrations of PD98059 to detect the change of p-ERK and the proteins involving in the functions of cell proliferation and apoptosis. The gray value was taken through gray scan software BandScan 4.5. The relative value represents objective band gray value divided by the sampleβ-actin orβ-tubulin gray value.3. statistical analysisAll data were expressed in mean±S.D( x±s) from at least three independent experiments. Asterisk (*) demonstrates significance at p<0.05(student's t-test). Asterisk (**) demonstrates significance at p<0.01(student's t-test).Results:1. Western blot showed activated ERK, ie p-ERK level of LM-MCF-7 cell line was remarkably higher than that of MCF-7 cell line (Fig.1). LM-MCF-7 cell line pretreated by different concentration of ERK specific inhibitor, PD98059 resulted in downregulation of p-ERK obviously and dose-dependently decreased p-ERK level (Fig.2).2. LM-MCF-7 cell line over-expressed cyclin D1 protein than MCF-7. LM-MCF-7 cell line pretreated by PD98059 downregulated cyclin D1 protein of LM-MCF-7 cell line, showing that ERK inhibitor blocked upregulation of cyclin D1(p<0.05, Fig.3)3. LM-MCF-7 cell line overexpressed anti-apoptotic protein survivin than MCF-7 cell line. Pretreated LM-MCF-7 with high expression of p-ERK by different concentration of ERK specific inhibitor, PD98059, it revealed that PD98059 inhibited dose-dependently upregulation of survivin protein (p<0.05, Fig.4)4. LM-MCF-7 cell line overexpressed anti-apoptotic protein Bcl-2 than MCF-7 cell line. Pretreated LM-MCF-7 with high expression of p-ERK by ERK specific inhibitor, PD98059, it demonstrated that PD98059 blocked upregulation of Bcl-2 protein (p<0.05, Fig.5). 5. LM-MCF-7 cell line under-expressed apoptotic protein enzyme caspase 9 than MCF-7 cell line. Pretreated LM-MCF-7 with high expression of p-ERK by ERK specific inhibitor, PD98059, it demonstrated that PD98059 blocked downregulation of caspase 9(Fig.6).Conclusions:1. p-ERK promoted LM-MCF-7 cell line proliferation through upregulating cyclin D1 expression.2. p-ERK promoted LM-MCF-7 cell line ani-apoptosis through upregulating anti-apoptotic protein survivin and Bcl-2, and downregulating caspase 9.