M

M. and EGFR phosphorylation proteins were detected. Results: 13f was confirmed as an inhibitor of EGFR with an IC50 value against the tyrosine kinase of EGFR of 22 nM and IC50 values for 48 h incubation period were 1.3 1.9, 1.5 0.4 and 1.7 1.1 M of KKU-100, KKU-452 and KKU-M156, respectively through dose- and time-dependent induction of early apoptosis of CCA cells. The compound also suppressed the clonogenic ability of KKU-100 and KKU-M156 cells stronger than Gefitinib, while potently inhibiting EGF-stimulated CCA cell migratory activity in KKU-452 Chrysophanic acid (Chrysophanol) cells. It was observed that under normal conditions EGFR was activated in CCA cells. EGF-stimulated basal expression of EGFR in KKU-452 cells was suppressed following 13f treatment, which was significantly greater than that of the marketed EGFR inhibitor Gefitinib. Conclusion: In summary, our study showed that 13f has potent anti-cancer activities including antiproliferation, clonogenic ability and migration through the modulation of EGFR signaling pathway in CCA for the first time. The compound represents an interesting starting point as a potential chemotherapeutic agent in ongoing efforts to improve response rate in CCA patients. and expression. Open in a separate window Physique 1 13f is an Inhibitor of the Tyrosine Kinase of EGFR. The tyrosine kinase of EGFR (TK-EGFR) assay was performed using the ADP-Glo? Kinase Assay kit. Data represent the percent of EGFR relative inhibition of by 13f with the IC50 value analysis of three impartial experiments. The structure of 13f was shown gene have been found in 15% and 5% of CCA these abnormalities correlate with increased phosphorylation of downstream MAPK or Akt (Gwak et al., 2005; Nakazawa et al., 2005; Leone et al., 2006). Overexpression of EGFR has been associated with invasive CCA and poor prognosis (Yoshikawa et al., 2008; Harder et al., 2009). In this study our data found all CCA cell lines, KKU-100, KKU-452 and KKU-M156 cells, were resistant to Cisplatin but high response to Gefitinib and 13f. As EGFR is usually a promising target of CCA treatment we investigated the effect of 13f as a new synthetic of 4-aryl-N-phenylpyrimidin-2-amine on anti-cancer activities of CCA compared with Gefitinib as a traditional EGFR inhibitor. Gefitinib is usually a small molecule of EGFR tyrosine kinase inhibitors (TKIs) used for second- or third-line treatment of advanced non-small cell lung cancer Chrysophanic acid (Chrysophanol) (de Marinis and Grossi, 2008; Tiseo et al., 2010). It binds to the adenosine triphosphate (ATP)-binding site of the intracellular tyrosine kinase and inhibits autophosphorylation consequent of blocking of EGFR signaling pathways (Giaccone, 2004). Gefitinib has been exerted anticancer activities including anti-proliferation, anti-apoptosis, and anti-angiogenesis in several human malignancy cell lines with expression (Ciardiello et al., 2000; Ciardiello and Tortora, 2001; Sirotnak, 2003). Gefitinib has been shown to inhibit the proliferation of the CCC cell lines at high concentration (Nakajima et al., 2012). The effect of Gefitinib combination with Gemcitabine (pyrimidine analog) suppressed the proliferation of HuCCT1- and RBE- CCA cell lines by induction of apoptosis. This combination also has shown the synergistic effect of HuCCT1 xenografts in vivo (Nakajima et al., 2012). Thus, we used a Gefitinib for comparing the efficacy of anti-cancer effects with 13f a new EGFR inhibitor. In the present study, we exhibited that 13f inhibited potential the tyrosine kinase of EGFR with the IC50 value is usually 22.74 nM. 13f is usually a more potent anti-cancer agent, which includes inhibition of proliferation by induced early apoptosis, inhibition of clonogenic ability and migration than Gefitinib by modulated EGFR signaling pathway in CCA. This data suggested that it is due to EGFR-TKI resistance. Previous report was found mutation of the gene mutations was correlation with clinical efficacy of EGFR inhibitors.LT and PrS contributed new reagents or analytical tools. performed. Levels of total expression of EGFR and EGFR phosphorylation proteins were Chrysophanic acid (Chrysophanol) detected. Results: 13f was confirmed as an inhibitor of EGFR with an IC50 value against the tyrosine kinase of EGFR of 22 nM and IC50 values for 48 h incubation period were 1.3 1.9, 1.5 0.4 and 1.7 1.1 M of KKU-100, KKU-452 and KKU-M156, respectively through dose- and time-dependent induction of early apoptosis of CCA cells. The compound also suppressed the clonogenic ability of KKU-100 and KKU-M156 cells stronger than Gefitinib, while potently inhibiting EGF-stimulated CCA cell migratory Chrysophanic acid (Chrysophanol) activity in KKU-452 cells. It was observed that under normal conditions EGFR was activated in CCA cells. EGF-stimulated basal expression of EGFR in KKU-452 cells was suppressed following 13f treatment, which was significantly greater than that of the marketed EGFR inhibitor Gefitinib. Conclusion: In summary, our study showed that 13f has potent anti-cancer activities including antiproliferation, clonogenic ability and migration through the modulation of EGFR signaling pathway in CCA for the first time. The compound represents an interesting starting point as a potential chemotherapeutic agent in ongoing efforts to improve response rate in CCA patients. and expression. Open in a separate window Physique 1 13f is an Inhibitor of the Tyrosine Kinase of EGFR. The tyrosine kinase of EGFR (TK-EGFR) assay was performed using the ADP-Glo? Kinase Assay kit. Data represent the percent of EGFR relative inhibition of by 13f with the IC50 value analysis of three impartial experiments. The structure of 13f was shown gene have been found in 15% and 5% of CCA these abnormalities correlate with increased phosphorylation of downstream MAPK or Akt (Gwak et al., 2005; Nakazawa et al., 2005; Leone et al., 2006). Overexpression of EGFR has been associated with invasive CCA and poor prognosis (Yoshikawa et al., 2008; Harder et al., 2009). In this study our data found all CCA cell lines, KKU-100, KKU-452 and KKU-M156 cells, were resistant to Cisplatin but high response to Gefitinib and 13f. As EGFR is usually a promising target of CCA treatment we investigated the effect of 13f as a new synthetic of 4-aryl-N-phenylpyrimidin-2-amine on anti-cancer activities of CCA compared with Gefitinib as a traditional EGFR inhibitor. Gefitinib is usually a small molecule of EGFR tyrosine kinase inhibitors (TKIs) used for second- or third-line treatment of advanced non-small cell lung cancer (de Marinis and Grossi, 2008; Tiseo et al., 2010). It binds to the adenosine triphosphate (ATP)-binding site of the intracellular tyrosine kinase and inhibits autophosphorylation consequent of blocking of EGFR signaling pathways (Giaccone, 2004). Gefitinib has been exerted anticancer activities including anti-proliferation, Rabbit polyclonal to PHF10 anti-apoptosis, and anti-angiogenesis in several human malignancy cell lines with expression (Ciardiello et al., 2000; Ciardiello and Tortora, 2001; Sirotnak, 2003). Gefitinib has been shown to inhibit the proliferation of the CCC cell lines at high concentration (Nakajima et al., 2012). The effect of Gefitinib combination with Gemcitabine (pyrimidine analog) suppressed the proliferation of HuCCT1- and RBE- CCA cell lines Chrysophanic acid (Chrysophanol) by induction of apoptosis. This combination also has shown the synergistic effect of HuCCT1 xenografts in vivo (Nakajima et al., 2012). Thus, we used a Gefitinib for comparing the efficacy of anti-cancer effects with 13f a new EGFR inhibitor. In the present study, we exhibited that 13f inhibited potential the tyrosine kinase of EGFR with the IC50 value is usually 22.74 nM. 13f is usually a more potent anti-cancer agent, which includes inhibition of proliferation by induced early apoptosis, inhibition of clonogenic ability and migration than Gefitinib by modulated EGFR signaling pathway in CCA. This data suggested that it is due to EGFR-TKI resistance. Previous report was found mutation of the gene mutations was correlation with clinical efficacy of EGFR inhibitors (Leone et al., 2006). Moreover, activation of EGFR increases resistance to erlotinib EGFR-TKI inhibitor of CCA cells (Jimeno et al., 2005). In addition, HuCCT-1 (CCA cell line) cells now are resistant to Gefitinib and increase the sensitivity by combination treatment with CI-1040 which is an extracellular signal-regulated kinase (ERK) kinase 1/2 blocker. This combination treatment with Gefitinib and CI-1040 could be inhibited EGFR activation and block ERK1/2 phosphorylation downstream of EGFR pathway (Hidalgo et al., 2006). Our goal is to therefore to identify EGFR inhibitors with greater efficiency and specificity for CCA treatment 4-aryl-N-phenylpyrimidin-2-amine derivatives have been confirmed as EGFR inhibitors using a variety of methods discussed herein. Related compounds have also previously reported.