Have been made in understanding the function and regulation of PKM2 as a pyruvate kinase and protein kinase in cancer cells [5]. A recent study confirmed that the PKM2 inducedby epidermal growth factor (EGF) translocates into the nucleus of glioblastoma cells, interacts with b-catenin and leads to cyclinD1 expression, which promotes cell proliferation and Control cells. As expected, TG significantly increased apoptosis in both control tumorigenesis [6]. These findings reveal a novel role for PKM2 as a transcriptional coactivator. However, there are some controversies regarding the specificity and potential of PKM2 as an anti-cancer target in cancer therapy. A recent finding revealed that PKM2 expression is strongly correlated with gastric cancer differentiation. Differentiated types of cancers express more PKM2 protein than do the undifferentiated ones. PKM2 was an adverse prognostic factor in signet ring cell gastric cancer [7]. The biological role of PKM2 in different differentiation phases and in the development of gastric cancer needs to be further elucidated. Previous studies regarding PKM2 have focused on tumor metabolism and tumor growth. There have been only a fewPkM2 Regulates the EGF/EGFR Signalreports on tumor metastasis. E-Cadherin plays a critical role in maintaining epithelial integrity, and the loss of E-cadherin affects the adhesive repertoire of a cell [8]. Previous studies [9] in vitro have shown that the loss of E-cadherin in human carcinoma cell lines is associated with poor differentiation and a fibroblastoid morphology. The EGF-dependent activation of the EGFR has been reported to be inhibited in an E-cadherin adhesiondependent manner, which inhibits the ligand-dependent activation of diverse receptor tyrosine kinases [10]. Our research demonstrated that the knockdown of PKM2 decreased the activity of Ecadherin and enhanced the EGF/EGFR signaling pathway in the cell lines BGC823 and SGC7901 that were positive for E-cadherin expression. However, in the undifferentiated gastric carcinoma cell line AGS, which lacks E-cadherin expression, PKM2 promoted cell migration and invasion. The aim of this study was to elucidate the function and mechanism of PKM2 with regard to cell motility in differently differentiated cell lines.Protein Extraction and Western Blot AnalysisCells were re-suspended in lysis buffer containing a protease inhibitor cocktail, and the extracted proteins were separated using 8-10 SDS AGE gels. b-Tubulin was used as a loading control. Antibodies against E-cadherin and p-E-cadherin were obtained from Epitomics. The phospho-EGFR (Tyr1068), phospho-PLCc1 (Tyr783), phospho-AKT (Ser473), phospho-Gab1 (Tyr627), phospho-c-cbl (Tyr700), and phospho-ERK1/2 (Thr202/Tyr204) antibodies were obtained from Cell Signaling Technology.RNA Extraction, Reverse Transcription and Real-time PCRTotal RNA was extracted using the TRIzol reagent (Invitrogen, CA, USA). The samples were then treated with DNase for 15 min at room temperature, and the RNA was further purified using an RNA cleanup kit (Qiagen, CA, USA). The reverse transcription (RT) reaction for the first-strand cDNA synthesis was performed using reverse transcriptase (Bio-Rad) with 2 mg of total RNA. Quantitative RT-PCR analysis was performed with the ABI 7500 (Applied Biosystems), and the gene expression levels for each individual sample were normalized to GAPDH. The mean relative gene expression was determined and differences were calculated using the 2-DDCt method of agarose gel electrophoresis. The RTPCR Ncentration.Histological AnalysisDuring the experiment no crab died and no remarkable primer sequences were as follows:.Have been made in understanding the function and regulation of PKM2 as a pyruvate kinase and protein kinase in cancer cells [5]. A recent study confirmed that the PKM2 inducedby epidermal growth factor (EGF) translocates into the nucleus of glioblastoma cells, interacts with b-catenin and leads to cyclinD1 expression, which promotes cell proliferation and tumorigenesis [6]. These findings reveal a novel role for PKM2 as a transcriptional coactivator. However, there are some controversies regarding the specificity and potential of PKM2 as an anti-cancer target in cancer therapy. A recent finding revealed that PKM2 expression is strongly correlated with gastric cancer differentiation. Differentiated types of cancers express more PKM2 protein than do the undifferentiated ones. PKM2 was an adverse prognostic factor in signet ring cell gastric cancer [7]. The biological role of PKM2 in different differentiation phases and in the development of gastric cancer needs to be further elucidated. Previous studies regarding PKM2 have focused on tumor metabolism and tumor growth. There have been only a fewPkM2 Regulates the EGF/EGFR Signalreports on tumor metastasis. E-Cadherin plays a critical role in maintaining epithelial integrity, and the loss of E-cadherin affects the adhesive repertoire of a cell [8]. Previous studies [9] in vitro have shown that the loss of E-cadherin in human carcinoma cell lines is associated with poor differentiation and a fibroblastoid morphology. The EGF-dependent activation of the EGFR has been reported to be inhibited in an E-cadherin adhesiondependent manner, which inhibits the ligand-dependent activation of diverse receptor tyrosine kinases [10]. Our research demonstrated that the knockdown of PKM2 decreased the activity of Ecadherin and enhanced the EGF/EGFR signaling pathway in the cell lines BGC823 and SGC7901 that were positive for E-cadherin expression. However, in the undifferentiated gastric carcinoma cell line AGS, which lacks E-cadherin expression, PKM2 promoted cell migration and invasion. The aim of this study was to elucidate the function and mechanism of PKM2 with regard to cell motility in differently differentiated cell lines.Protein Extraction and Western Blot AnalysisCells were re-suspended in lysis buffer containing a protease inhibitor cocktail, and the extracted proteins were separated using 8-10 SDS AGE gels. b-Tubulin was used as a loading control. Antibodies against E-cadherin and p-E-cadherin were obtained from Epitomics. The phospho-EGFR (Tyr1068), phospho-PLCc1 (Tyr783), phospho-AKT (Ser473), phospho-Gab1 (Tyr627), phospho-c-cbl (Tyr700), and phospho-ERK1/2 (Thr202/Tyr204) antibodies were obtained from Cell Signaling Technology.RNA Extraction, Reverse Transcription and Real-time PCRTotal RNA was extracted using the TRIzol reagent (Invitrogen, CA, USA). The samples were then treated with DNase for 15 min at room temperature, and the RNA was further purified using an RNA cleanup kit (Qiagen, CA, USA). The reverse transcription (RT) reaction for the first-strand cDNA synthesis was performed using reverse transcriptase (Bio-Rad) with 2 mg of total RNA. Quantitative RT-PCR analysis was performed with the ABI 7500 (Applied Biosystems), and the gene expression levels for each individual sample were normalized to GAPDH. The mean relative gene expression was determined and differences were calculated using the 2-DDCt method of agarose gel electrophoresis. The RTPCR primer sequences were as follows:.
