Aberrant Elevation of Tyrosine-specific Phosphorylation in Human Gastric Cancer Cells

Phosphotyrosine-containing proteins in various human cancer cell lines were studied by immunoblotting with anti-phosphotyrosine antibody. Of 29 cell lines derived from oral epidermoid cancer, esophageal cancer, gastric cancer, colon cancer, pancreatic cancer, hepatocellular carcinoma and malignant melanoma, 3 of the 6 gastric cancer cells showed aberrant elevation of tyrosine-speciflc phosphorylation. On the other hand, both esophageal cancer cells and colon cancer cells, which were reported to have amplified epidermal growth factor receptor and activated p60^v-src kinase, respectively, showed no apparent elevation of tyrosine-specific phosphorylation, and their profiles of phosphorylation were similar to that of normal human fibroblasts. Two gastric cancer cells, NUGC-4 and MKN-45, showed similar profiles of phosphorylation but their responses to growth factors differed from each other. Tyrosine phosphorylation in NUGC-4 was strongly activated by treatment with epidermal growth factor and quickly reduced by the acid treatment which is effective in removing growth factors from cellular surface receptors. On the contrary, phosphorylation in MKN-45 did not respond to either growth factor or acid treatment. These results suggest that NUGC-4 and MKN-45 have tyrosine kinases which are activated by different mechanisms but share similar substrates.     

Aberrant elevation of tyrosine-specific phosphorylation in human gastric cancer cells.

Phosphotyrosine-containing proteins in various human cancer cell lines were studied by immunoblotting with anti-phosphotyrosine antibody. Of 29 cell lines derived from oral epidermoid cancer, esophageal cancer, gastric cancer, colon cancer, pancreatic cancer, hepatocellular carcinoma and malignant melanoma, 3 of the 6 gastric cancer cells showed aberrant elevation of tyrosine-specific phosphorylation. On the other hand, both esophageal cancer cells and colon cancer cells, which were reported to have amplified epidermal growth factor receptor and activated p60v-src kinase, respectively, showed no apparent elevation of tyrosine-specific phosphorylation, and their profiles of phosphorylation were similar to that of normal human fibroblasts. Two gastric cancer cells, NUGC-4 and MKN-45, showed similar profiles of phosphorylation but their responses to growth factors differed from each other. Tyrosine phosphorylation in NUGC-4 was strongly activated by treatment with epidermal growth factor and quickly reduced by the acid treatment which is effective in removing growth factors from cellular surface receptors. On the contrary, phosphorylation in MKN-45 did not respond to either growth factor or acid treatment. These results suggest that NUGC-4 and MKN-45 have tyrosine kinases which are activated by different mechanisms but share similar substrates.     

Krüppel-like factor 4 negatively regulates β-catenin expression and inhibits the proliferation, invasion and metastasis of gastric cancer

Krüppel-like factor 4 (KLF4) is a zinc-finger protein that plays an important role in the progression of gastric carcinoma. The abnormal activation of β-catenin frequently occurs in gastric cancer and has been associated with the promotion of tumor growth, invasion and metastasis. However, the potential interaction between KLF4 and β-catenin during gastric cancer development is unknown. In this study, a lentiviral KLF4 expression vector was constructed and utilized to transfect the human gastric cancer cell lines, SGC-7901, BGC-823, MKN-28 and MKN-45. KLF4 and β-catenin expression levels were measured by quantitative real-time RT-PCR and western blot analysis. Cell proliferation, colony formation and invasive potential were determined in the KLF4-transfected gastric cancer cells. The expression of E-cadherin and matrix metallopeptidase 2 (MMP2) was determined by western blot analysis. The overexpression of KLF4 significantly inhibited the expression of β-catenin in the MKN-45 gastric cancer cells. The restored expression of KLF4 suppressed proliferation, colony formation and inhibited the invasion and metastatic properties of MKN-45 gastric cancer cells. Furthermore, the forced expression of KLF4 in gastric tumor cell lines restored E-cadherin expression and inhibited MMP2 expression. Consistent with the in vitro findings, the enforced expression of the KLF4 gene in MKN-45 gastric carcinoma cells by lentiviral vector-mediated gene transfer effectively suppressed tumor growth in vivo. Our results show that KLF4 inhibits β-catenin expression and regulates the β-catenin-mediated biological behaviors of gastric cancer cells. The modulation of KLF4 expression may represent a novel therapeutic approach for β-catenin-driven malignancies.     

Downregulation of erbB3 abrogates erbB2-mediated tamoxifen resistance in breast cancer cells

Receptor tyrosine kinase activity is essential for erbB2 (HER2/neu) promotion of breast carcinogenesis, metastasis and therapeutic resistance. erbB2 kinase can be activated by dimerization with another erbB receptor, most of which bind ligands. Of these, the erbB2/erbB3 heterodimer is the most potent oncogenic complex. erbB2 reportedly requires erbB3 to promote cellular proliferation, although this may occur without changes in erbB2 tyrosine kinase activity in some model systems. Our investigations focus on the role(s) of erbB3 in erbB2-associated kinase activity and tamoxifen resistance. Using tumor-derived cell lines from wild type rat c-neu transgenic mice and human breast cancers, we demonstrate that erbB3 plays a critical role in the activation of erbB2 tyrosine kinase activity and erbB2-associated tumorigenesis. Mechanistically, downregulation of erbB3 by specific siRNA reduces erbB2 tyrosine phosphorylation, decreases the PI-3K/Akt signaling, and inhibits mammary/breast cancer cell proliferation and colony formation. Specific erbB3 siRNA sensitizes erbB2 transfected MCF-7 cells (MCF-7/erbB2) to tamoxifen-associated inhibition of both cell growth and colony formation and enhances tamoxifen-induced apoptosis, in contrast to control siRNA transfected MCF-7/erbB2 cells which are tamoxifen-resistant. Our data indicates that erbB2/erbB3 heterodimerization is a prerequisite for erbB2 tyrosine kinase activation in mammary/breast cancer cells and that downregulation of erbB3 inhibits erbB2-associated procarcinogenic activity via inactivation of the PI-3K/Akt pathway. Furthermore, erbB3 also contributes to erbB2-mediated tamoxifen resistance and therefore may be a clinically relevant therapeutic target in addition to erbB2.     

Troglitazone induces G1 arrest by p27(Kip1) induction that is mediated by inhibition of proteasome in human gastric cancer cells.

We examined in the present study whether human gastric cancer cells express peroxisome proliferator-activated receptor gamma (PPARgamma), the effect of PPARgamma activation by troglitazone, a selective ligand, on cellular growth, and the mechanism of the growth arrest by troglitazone in gastric cancer cells. RT-PCR, northern blot and western blot analysis demonstrated that all four tested human gastric cancer cell lines, MKN-28, MKN-45, MKN-74 and KATO-III, expressed PPARgamma mRNA and protein. WST-1 assay and flow cytometric analysis revealed that troglitazone inhibited the growth and induced G1 arrest in all four gastric cancer cell lines. To examine the role of p27(Kip1), a cyclin-dependent kinase inhibitor, in the G1 arrest by troglitazone, we determined p27(Kip1) protein expression by western blot analysis in gastric cancer cells that had been treated with troglitazone. Troglitazone increased p27(Kip1) in all four gastric cancer cell lines. Since it has been reported that the ubiquitin-proteasome system plays a vital role in the degradation of p27(Kip1) protein, we evaluated the hypothesis that inhibition of proteasome mediates the troglitazone-induced p27(Kip1) accumulation. Lactacystin, a proteasome inhibitor, inhibited cell growth and increased p27(Kip1) expression in MKN-74 cells. It was further demonstrated that troglitazone inhibited proteasome activity in a dose-dependent manner in MKN-74 cells. All these results suggest that troglitazone inhibited proteasome activity, followed by induction of p27(Kip1), which arrests cells at the G1 phase of the cell cycle in gastric cancer cells. The troglitazone-mediated inhibition of the proteasome suggests a novel mechanism for the anti-proliferative effect of this agent in cancer cells.     

Human cytomegalovirus induces cellular tyrosine kinase signaling and promotes glioma cell invasiveness

Given our previous findings that human cytomegalovirus (HCMV) nucleic acids and proteins are expressed in human malignant glioma in vivo, we investigated cellular signaling events associated with HCMV infection of human glioma and astroglial cells. HCMV infection caused rapid activation of the phosphatidylinositol-3 kinase (PI-3K) effector AKT kinase in human astro-glial and fibroblast cells, and induced tyrosine phosphorylation of phospholipase Cγ (PLCγ). Co-immunoprecipitation experiments revealed association of the p85 regulatory subunit of PI-3K with a high-molecular weight protein phosphorylated on tyrosine, following short-term exposure to HCMV. In contrast to a previous report, we were unable to confirm the identity of this high-molecular weight protein as being the epidermal growth factor receptor (EGFR). Stimulation of glioma and fibroblast cell lines over-expressing EGFR with HCMV (whole virus) or soluble glycoprotein B did not induce tyrosine phosphorylation of the receptor, as did the genuine ligand, EGF. Furthermore, we found that expression levels of the human ErbB1-4 receptors were not rate-limiting for HCMV infection. Dispensability of EGFR function during early HCMV infection was substantiated by demonstration of viral immediate early gene expression in cells lacking the EGFR gene, indicating that HCMV may promote oncogenic signaling pathways independently of EGFR activation. Among non-receptor cellular kinases, HCMV infection induced phosphorylation of focal adhesion kinase (FAK) Tyr397, which is indispensable for integrin-mediated cell migration and invasion. HCMV-induced FAK activation was paralleled by increased extracellular matrix-dependent migration of human malignant glioma but not normal astro-glial cells, suggesting that HCMV can selectively augment glioma cell invasiveness. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Suppression of tumor growth in human gastric cancer with HER2 overexpression by an anti-HER2 antibody in a murine model

New modalities are necessary for the treatment of patients with unresectable gastric cancer. The aim of this study was to investigate whether or not anti-HER2 antibody could suppress the growth of human gastric cancer cells with HER2 overexpression in vitro and in vivo. Four human gastric cancer cell lines, NCI-N87, MKN-45P, Kato-III, and MKN-1, were used in this study. The suppression of cell proliferation in vitro and of subcutaneous tumor growth in a nude mouse model after treatment with trastuzumab was examined. The expression of HER2 protein was investigated by Western blot analysis. The effect of trastuzumab on the survival rate of nude mice with peritoneal dissemination was examined. Trastuzumab significantly reduced proliferative activity in NCI-N87, a HER2-overexpressing human gastric cancer cell line, in vitro. In the nude mouse model with transplanted subcutaneous tumor, trastuzumab significantly suppressed the tumor growth of NCI-N87 cells, and then HER2 expression was reduced. Trastuzumab improved the survival rate of mice with peritoneal dissemination of MKN-45P cells. Trastuzumab therapy is a potential candidate for a novel treatment of HER2-overexpressing gastric cancer.     

Growth inhibition of gastric cancer cell lines by the tyrphostin RG13022 and its effects on intracellular signalling

Overexpression of EGFr and c-erbB-2 is related to poor prognosis in a variety of cancers including gastric cancer. Thus: the ability to modulate the functional activity of these receptors is an attractive target for diagnostic intervention. In this study we examined the effect of a well characterised tyrosine kinase inhibitor (RG13022) on the cellular proliferation and EGF activated tyrosine kinase signalling pathway of two primary gastric cell lines: MKN45 and N87. RG13022 has a dose dependent, antiproliferative effect on both gastric cell lines when grown either in serum-free conditions or in the presence of FCS. Western blotting revealed RG13022 caused an inhibition of EGF stimulated tyrosine phosphorylation of EGFr in A431 cells and both EGFr and c-erbB-2 in MKN45 cells. No clear modulation of EGFr or c-erbB-2 phosphorylation was observed in N87 cells. In both A431 cells and N87 cells (which overexpress EGFr and c-erbB-2 respectively) exposed to EGF, MAP2 kinase immunoblot analysis resulted in the detection of a second protein band with reduced migration in SDS-PAGE. In N87 cells, this protein appeared to co-mi,orate with a strongly tyrosine phosphorylated protein, which suggests that it is a hyper-phosphorylated form of MAP2 kinase. However, treatment with RG13022, whilst inhibiting phosphorylation of this protein, did not prevent a shift in gel mobility (suggestive of activation) of MAP2 kinase in response to EGF. These findings demonstrate that the tyrphostin RG13022 inhibits cell proliferation of two primary gastric cancer cell lines. Investigation of intracellular signalling pathways suggests that alterations in intracellular signalling are responsible for the actions of RG 13022 in these cells. The biochemical analysis revealed that in N87 and A431, cells which overexpress c-erbB-2 and EGFr respectively, the tyrphostin affects the MAP2 kinase immunoreactivity and migration on SDS gels but fails to affect this protein in the MKN45 cell line. This data questions the usefulness of MAP2 kinase gel shift assays as markers of activation but supports the further development of tyrosine kinase inhibitors as potential inhibitors of gastric tumour proliferation.     

KRC-408, a novel c-Met inhibitor,suppresses cell proliferation and angiogenesis of gastric cancer

Among many cancer therapeutic targets, c-Met receptor tyrosine kinase has recently given particular attention. This kinase and its ligand, hepatocyte growth factor (HGF), play a central role in cell proliferation and the survival of several human cancers. Thus, we developed KRC-408 as a novel c-Met inhibitor and investigated its anti-cancer effects on human gastric cancer. KRC-408 inhibited the phosphorylation of c-Met and its constitutive downstream effectors such as phosphatidylinositol 3-kinase (PI3K), Akt, Mek, and Erk. This compound was found to exert anti-cancer effects stronger than those of 5-fluorouracil (5-FU) on gastric cancer cells, especially cell lines that overexpressed c-Met. Interestingly, cytotoxicity of KRC-408 was lower than that of 5-FU in normal gastric cells. Apoptosis induced by KRC-408 was accompanied by increased levels of cleaved caspase-3 and PARP as well as DNA condensation and fragmentation. Flow cytometry analysis showed an accumulation of gastric cancer cells in the G2/M phase with concomitant loss of cells in the S phase following treatment with this drug. In the angiogenesis studies, KRC-408 inhibited tube formation and migration of human umbilical vein endothelial cells (HUVECs), and suppressed microvessel sprouting from rat aortic rings ex vivo along with blood vessel formation in a Matrigel plug assay in mice. Results of an in vivo mouse xenograft experiment showed that the administration of KRC-408 significantly delayed tumor growth in a dose-dependent manner, and suppressed Akt and Erk phosphorylation as well CD34 expression in tumor tissues. These findings indicate that KCR-408 may exert anti-tumor effects by directly affecting tumor cell growth or survival via the c-Met receptor tyrosine kinase pathway. We therefore suggest that KRC-408 is a novel therapeutic candidate effective against gastric cancers that overexpress c-Met.     

The tyrosine kinase Abl is required for Src-transforming activity in mouse fibroblasts and human breast cancer cells

The cytoplasmic tyrosine kinase Src has been implicated in signal transduction induced by growth factors and integrins. Src also shows oncogenic activity when deregulated. Accumulating evidence indicates that the tyrosine kinase Abl is an important substrate for Src signalling in normal cells. Here we show that Abl is also required for Src-induced transformation of mouse fibroblasts. Abl does not mediate tyrosine phosphorylation of Stat3 and Shc, two important regulators of Src oncogenic activity. In contrast, Abl controls the activation of the small GTPase Rac for oncogenic signalling and active Rac partly rescued Src transformation in cells with inactive Abl. Moreover, Abl mediates Src-induced extracellular regulated kinase 5 (ERK5) activation to drive cell transformation. Finally, we find that Abl/Rac and Abl/ERK5 pathways also operate in human MCF7 and BT549 breast cancer cells, where neoplastic transformation depends on Src-like activities. Therefore, Abl is an important regulator of Src oncogenic activity both in mouse fibroblasts and in human cancer cells. Targeting these Abl-dependent signalling cascades may be of therapeutic value in breast cancers where Src-like function is important.     

α-Lipoic acid-induced inhibition of proliferation and met phosphorylation in human non-small cell lung cancer cells

α-Lipoic acid (α-LA), a naturally occurring anti-oxidant and co-factor for metabolic enzymes, suppresses the growth of different types of tumor cells. The mechanisms that are responsible for these results, however, remain to be elucidated. In the present study, we investigated the effects of α-LA on the proliferation and activation status of definitive receptor tyrosine kinases, epidermal growth factor receptor (EGFR) and Met/hepatocyte growth factor (HGF) receptor, in gefitinib-sensitive human non-small cell lung cancer cells harboring EGFRs with an activating mutation. The enantiomers R-α-LA and S-α-LA suppressed cell proliferation and increased the level of reactive oxygen species in HCC-827 and PC-9 human non-small cell lung cancer cells in an indistinguishable dose-dependent fashion. A phospho-receptor tyrosine kinase array and cell cycle analysis indicated that α-LA decreased tyrosine phosphorylation levels of EGFR, ErbB2, and Met, and this was associated with an inhibition in the cell-cycle transition from the G1 phase to the S phase without inducing apoptosis. Gefitinib, an inhibitor for EGFR tyrosine kinase, inhibited EGFR tyrosine phosphorylation/activation and proliferation of the cells. Instead, the addition of HGF induced Met tyrosine phosphorylation, and this was associated with a resistance to gefitinib-induced growth inhibition, which meant a gain in proliferative ability. In the presence of gefitinib and HGF, the addition of α-LA suppressed Met tyrosine phosphorylation, and this was associated with an inhibition in cell growth. These results suggest that the suppression of tyrosine phosphorylation/activation of growth factor receptors that is critical for the proliferation of human non-small cell lung cancer cells is a mechanism by which α-LA exerts growth inhibition for cancer cells.     

维甲酸α受体介导全反式维甲酸抑制胃癌细胞生长的作用机理

目的 探讨维甲酸α受体（ＲＡＲａ）介导全反式维甲酸（ＡＴＲＡ）抑制胃癌细胞生长的作用机理。方法 应用Ｎｏｔｈｅｒｎ ｂｌｏｔ分析维甲酸受体在胃癌细胞中的表达水平,通过脂质体转染方法将ｓｅｎｓｅ ＲＡＲａ和ａｎｔｉｓｅｎｓｅ ＲＡＲａ分别转染到细胞中,通过ＭＴＴ方法和软琼脂集落形成实验分析ＡＴＲＡ对转染细胞生长和恶性程度的影响,瞬时转染和测定氯霉素乙酰转移酶（ＣＡＴ）活性,分析维甲酸应答元件（ＲＡＲ     

(-)-Epigallocatechin-3-gallate induces apoptosis in gastric cancer cell lines by down-regulating survivin expression

The polyphenol (-)-epigallocatechin-3-gallate (EGCG) is a green tea constituent, which has been shown to inhibit cancer cell growth in vitro, in vivo and in epidemiological studies. In this study, we investigated its effects in gastric cancer cell lines. Five gastric cancer cell lines, the MKN-1, MKN-28, MKN-45, NUGC-3 and TMK-1, were found to be sensitive to EGCG treatment. Of all the cell lines tested, NUGC-3 was the most sensitive. EGCG treatment of NUGC-3 cells induced apoptosis, which was confirmed by sub-G1 analysis, caspase-Glo assay and Western blotting against cleaved PARP and cleaved caspase-3. EGCG treatment lowered survivin and increased Bax and TRAIL expression. Furthermore, EGCG induced p73 activation in NUGC-3 cells. Small interfering RNA against p73 diminished EGCG effects on survivin expression and cell viability. These results show that EGCG induces cell death in gastric cancer cells by apoptosis via inhibition of survivin expression downstream of p73. This study provides a novel mechanism whereby EGCG potentially inhibits cancer cell growth, concluding that EGCG may be a potential candidate in anti-survivin cancer therapy.     

TGFα-PE40对人低分化胃癌细胞生长的抑制作用

 目的　探讨基因重组转化生长因子α-绿脓杆菌外毒素融合蛋白(TGFα-PE40,简称TP40)对人低分化胃癌细胞生长的导向抑制作用。方法　用免疫组化LSAB法检测人低分化胃癌MKN-45细胞表皮生长因子受体(EGFR)的表达,用结晶柴染色法和3H-亮氨酸掺入法检测TP40对MNK-45细胞生长及蛋白质合成的抑制,用过量EGF竞争拮抗TP40的细胞毒作用。结果　MKN-45细胞免疫组化显示出大量棕黄色的EGFR阳性染色。TP40浓度为1～100ng/ml时,对MKN-45细胞长生及蛋白质合成呈剂量依赖性抑制。过量EGF能完全拮抗TP40的细胞毒作用。结论　人低分化胃癌MKN-45细胞能高水平地表达EGFR,重组TP40对MKN-45细胞生长具有较强的抑制作用,作用部位为细胞的EGFR。     

4-Methyl-3-nitro-benzoic acid, a migration inhibitor, prevents breast cancer metastasis in SCID mice

The aim of this study was to determine the anticancer effects of seven licorice compounds in MKN-28, AGS, and MKN-45 gastric cancer cells and human gastric epithelium immortalized cells. We also explored the mechanism of action of licochalcone A (LCA), the most cytotoxic licorice compound, by analyzing its influence on cell cycle progression and apoptosis. The results indicated that LCA was the most cytotoxic licorice compound of those tested, and it inhibited gastric cancer cells growth in a dose-dependent manner, with an IC50 value of approximately 40μM. LCA affected gastric cancer cell viability by blocking cell cycle progression at the G2/M transition and inducing apoptosis. LCA treatment increased the expression of Rb and decreased the expression of cyclin A, cyclin B and MDM2 in MKN-28, AGS and MKN-45 cell lines. In addition, LCA-induced apoptosis by its effects on the expression of PARP, caspase-3, Bcl-2 and Bax. These data provide evidence that LCA has the potential to be used in the treatment of gastric cancer.