He percentage of wound sealing was observed after 24 h. The invading cells in the transwell assay were quantified 24 h after EGF (100 ng/ml) was added to the lower chamber. To our surprise, we found that the treatment of AGS-sipk cells with EGF following the wound scratch and in the transwell significantly decreased the rate of wound sealing and invasion compared with that of the control cells (Fig. 3B, C). There were conspicuous differences between the BGC823/SGC7901 and AGS cells. To further illustrate the role of PKM2 in cell motility, we did the PKM2 rescuing experiments. We taked stably transfected method by using over-expression plasmid vector pcDNA6.0-mock and pcDNA6.0-PKM2 to deal with BGC823 and AGS cells which stable knockdown PKM2. The expression of p-EGFR, E-cadherin were shown in the PKM2 rescuing experiments (Fig. 3D). We observed that when the PKM2 expression recovered, the phosphorylation of EGFR has significantly reduced in BGC823 cells and increased in AGS cells. Moreover, cell motility of BGC823 cells was decreased and AGS cells were declined after PKM2 rescuing (Fig. 3E). To Title Loaded From File clarify the mechanism of these differences, we then analyzed the activity of the EGF/EGFR signaling pathway.lated with each other. In addition, we observed a high level of ERK1/2 phosphorylation in the nucleus of cancer cells without Ell 100 ml of TBST buffer and removing the liquid by applying Ecadherin expression. In areas of ERK1/2 phosphorylation, we also found higher levels of PKM2 expression. However, we did not find the phosphorylation of ERK1/2 in areas positive for E-cadherin expression (Fig. 4C). A correlation analysis among PKM2, Ecadherin and P-ERK1/2 was performed using Image-pro Plus software (Fig. 4D). The mean density (IOD/area) was recorded in different positive areas of 15 human gastric cancer specimens. We found a significant correlation between PKM2 and E-cadherin in E-cadherin-positive areas. Moreover, there was a significant correlation between PKM2 and p-ERK1/2 in E-cadherinnegative areas.DiscussionThe invasive and metastatic stage of cancer progression correlates with poor clinical prognosis and represents the most formidable barrier to successful treatment. Cell motility and invasiveness are the defining characteristics of malignant tumors, which enable tumor cells to migrate into adjacent tissues or through limiting basement membranes and extracellular matrices. Cell motility is required for the physiological processes of wound repair and organogenesis and for the pathological process of tumor invasion [13]. Invasive tumor cells are characterized by dysregulated cell motility in response to extracellular signals from growth factors and cytokines. Human tumors express high levels of growth factors and their receptors, and many types of malignant cells appear to exhibit autocrine- or paracrine-stimulated growth. Among the most well-studied growth factor receptor systems is the EGF receptor family [14]. Signals from the extracellular milieu dictate cell motility. Many growth factors, including the ligands that act through the epidermal growth factor receptor (EGFR), enhance cell motility [15]. At least two distinct intracellular signaling pathways are required for EGFR-mediated cell motility: the pathways utilizing PLC c and the MAP kinase pathway. PLC c activity has been proposed to enhance cell motility through the mobilization of actin-modifying proteins from an inactive membrane-associated localization to an active sub-membrane cytoskeletal locale [16]. The Erk MAP kinases transmi.He percentage of wound sealing was observed after 24 h. The invading cells in the transwell assay were quantified 24 h after EGF (100 ng/ml) was added to the lower chamber. To our surprise, we found that the treatment of AGS-sipk cells with EGF following the wound scratch and in the transwell significantly decreased the rate of wound sealing and invasion compared with that of the control cells (Fig. 3B, C). There were conspicuous differences between the BGC823/SGC7901 and AGS cells. To further illustrate the role of PKM2 in cell motility, we did the PKM2 rescuing experiments. We taked stably transfected method by using over-expression plasmid vector pcDNA6.0-mock and pcDNA6.0-PKM2 to deal with BGC823 and AGS cells which stable knockdown PKM2. The expression of p-EGFR, E-cadherin were shown in the PKM2 rescuing experiments (Fig. 3D). We observed that when the PKM2 expression recovered, the phosphorylation of EGFR has significantly reduced in BGC823 cells and increased in AGS cells. Moreover, cell motility of BGC823 cells was decreased and AGS cells were declined after PKM2 rescuing (Fig. 3E). To clarify the mechanism of these differences, we then analyzed the activity of the EGF/EGFR signaling pathway.lated with each other. In addition, we observed a high level of ERK1/2 phosphorylation in the nucleus of cancer cells without Ecadherin expression. In areas of ERK1/2 phosphorylation, we also found higher levels of PKM2 expression. However, we did not find the phosphorylation of ERK1/2 in areas positive for E-cadherin expression (Fig. 4C). A correlation analysis among PKM2, Ecadherin and P-ERK1/2 was performed using Image-pro Plus software (Fig. 4D). The mean density (IOD/area) was recorded in different positive areas of 15 human gastric cancer specimens. We found a significant correlation between PKM2 and E-cadherin in E-cadherin-positive areas. Moreover, there was a significant correlation between PKM2 and p-ERK1/2 in E-cadherinnegative areas.DiscussionThe invasive and metastatic stage of cancer progression correlates with poor clinical prognosis and represents the most formidable barrier to successful treatment. Cell motility and invasiveness are the defining characteristics of malignant tumors, which enable tumor cells to migrate into adjacent tissues or through limiting basement membranes and extracellular matrices. Cell motility is required for the physiological processes of wound repair and organogenesis and for the pathological process of tumor invasion [13]. Invasive tumor cells are characterized by dysregulated cell motility in response to extracellular signals from growth factors and cytokines. Human tumors express high levels of growth factors and their receptors, and many types of malignant cells appear to exhibit autocrine- or paracrine-stimulated growth. Among the most well-studied growth factor receptor systems is the EGF receptor family [14]. Signals from the extracellular milieu dictate cell motility. Many growth factors, including the ligands that act through the epidermal growth factor receptor (EGFR), enhance cell motility [15]. At least two distinct intracellular signaling pathways are required for EGFR-mediated cell motility: the pathways utilizing PLC c and the MAP kinase pathway. PLC c activity has been proposed to enhance cell motility through the mobilization of actin-modifying proteins from an inactive membrane-associated localization to an active sub-membrane cytoskeletal locale [16]. The Erk MAP kinases transmi.