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.     

Expression and tyrosine phosphorylation of phosphatidyl-inositol-3 kinase in human gastric-cancer cells - its correlation with cell-growth

To reveal the signaling pathway leading to oncogenecity of human cancer cells, we examined the expression and tyrosine-phosphorylation of phosphatidylinositol (PI)-3 kinase in cancer cell lines. Of the 14 cell lines examined, two poorly differentiated human gastric cancer cell lines, NUGC-4 and MKN-45, which were previously found to have aberrant elevation of tyrosine phosphorylation showed elevated levels of PI-3 kinase 85-kDa subunit expression. In these cells, tyrosine-phosphorylation and overall activity of PI-3 kinase were apparently elevated, compared with normal human fibroblasts and another well differentiated gastric cancer cell line, MKN-28. Treatment of these cells with tyrosine kinase inhibitor, genistein, strongly suppressed the PI-3 kinase activity. Furthermore, wortmannin, a potent inhibitor of PI-3 kinase, strongly suppressed the growth of these gastric cancer cells. These results suggest that the growth signaling via tyrosine phosphorylation is required for the activation of PI-3 kinase in NUGC4 and MKN-45, and that this activation plays an important role in oncogenic growth of these cells. However, these two cell lines showed different responses of PI-3 kinase to acid-treatment and tyrosine kinase inhibitors. In MKN-45, activation of PI-3 kinase appeared to be constitutive, and could be relevant to the oncogenic nature of the cell line.     

Differential expression and function of caveolin-1 in human gastric cancer progression

Caveolin-1 is a scaffold protein of caveolae that acts as a tumor modulator by interacting with cell adhesion molecules and signaling receptors. The role of caveolin-1 in the pathogenesis of gastric cancer (GC) is currently unknown. We show by confocal immunofluorescence microscopy and immunohistochemistry of biopsies from GC patients (n = 41) that the nonneoplastic mucosa expressed caveolin-1 in foveolar epithelial cells and adjacent connective tissue. GC cells of only 3 of 41 (7%) patients expressed caveolin-1 and were all of the intestinal type. Quantitative PCR and Western blotting confirmed that, compared with nonneoplastic tissue, the overall caveolin-1 mRNA was decreased in 14 of 19 (74%) GC patients and protein in 7 of 13 (54%), respectively. Strong caveolin-1 reactivity was found in the nonepithelial compartment (myocytes, fibroblasts, perineural, and endothelial cells) in both tumor-free and GC samples. In a series of human GC cell lines, caveolin-1 expression was low in cells derived from a primary tumor (AGS and SNU-1) but was increased in cell lines originating from distant metastases (MKN-7, MKN-45, NCI-N87, KATO-III, and SNU-5). Ectopic expression of caveolin-1 in AGS cells decreased proliferation but promoted anchorage-independent growth and survival. RNAi-mediated knockdown of endogenous caveolin-1 in MKN-45 cells accelerated cell growth. These data indicate that caveolin-1 exhibits a stage-dependent differential expression and function in GC and may thereby contribute to its pathogenesis.     

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.     

MicroRNA-15a-5p suppresses cancer proliferation and division in human hepatocellular carcinoma by targeting BDNF

We examined the expression pattern and functional roles of microRNA 15a-5p (miR-15a-5p) in human hepatocellular carcinoma (HCC). Possible miR-15a-5p aberrant expression in HCC cell lines or clinical HCC specimens was examined by quantitative real-time PCR (qRT-PCR). In HCC HepG2 and SNU-182 cells, miR-15a-5p was ectopically overexpressed by lentiviral transduction. Its effect on HCC proliferation, cancer division, and in vivo tumor growth were examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, cell cycle assay, and tumorigenicity assay, respectively. The targeting of miR-15a-5p on its downstream gene, brain-derived neurotrophic factor (BDNF), was examined by dual-luciferase assay, qRT-PCR, and Western blot, respectively. BDNF was then overexpressed in HepG2 and SNU-182 cells to evaluate its selective effect on miR-15a-5p in HCC modulation. MiR-15a-5p is aberrantly downregulated in in vitro HCC cell lines and in vivo HCC clinical specimens. Ectopic overexpression of miR-15a-5p suppressed cancer proliferation, induced cell cycle arrest in HepG2 or SNU-182 cells in vitro, and inhibited HCC tumor growth in vivo. MiR-15a-5p selectively and negatively regulated BDNF at both gene and protein levels in HCC cells. Forced overexpression of BDNF effectively reversed the tumor suppressive functions of miR-15a-5p on HCC proliferation and cell division in vitro. Our study demonstrated that miR-15a-5p is a tumor suppressor in HCC and its regulation is through BDNF in HCC.     

An orally available small-molecule inhibitor of c-Met, PF-2341066, exhibits cytoreductive antitumor efficacy through antiproliferative and antiangiogenic mechanisms

The c-Met receptor tyrosine kinase and its ligand, hepatocyte growth factor (HGF), have been implicated in the progression of several human cancers and are attractive therapeutic targets. PF-2341066 was identified as a potent, orally bioavailable, ATP-competitive small-molecule inhibitor of the catalytic activity of c-Met kinase. PF-2341066 was selective for c-Met (and anaplastic lymphoma kinase) compared with a panel of >120 diverse tyrosine and serine-threonine kinases. PF-2341066 potently inhibited c-Met phosphorylation and c-Met-dependent proliferation, migration, or invasion of human tumor cells in vitro (IC(50) values, 5-20 nmol/L). In addition, PF-2341066 potently inhibited HGF-stimulated endothelial cell survival or invasion and serum-stimulated tubulogenesis in vitro, suggesting that this agent also exhibits antiangiogenic properties. PF-2341066 showed efficacy at well-tolerated doses, including marked cytoreductive antitumor activity, in several tumor models that expressed activated c-Met. The antitumor efficacy of PF-2341066 was dose dependent and showed a strong correlation to inhibition of c-Met phosphorylation in vivo. Near-maximal inhibition of c-Met activity for the full dosing interval was necessary to maximize the efficacy of PF-2341066. Additional mechanism-of-action studies showed dose-dependent inhibition of c-Met-dependent signal transduction, tumor cell proliferation (Ki67), induction of apoptosis (caspase-3), and reduction of microvessel density (CD31). These results indicated that the antitumor activity of PF-2341066 may be mediated by direct effects on tumor cell growth or survival as well as antiangiogenic mechanisms. Collectively, these results show the therapeutic potential of targeting c-Met with selective small-molecule inhibitors for the treatment of human cancers.     

COX-2 correlates with F-box protein, Skp2 expression and prognosis in human gastric carcinoma

The expression of cyclooxygenase (COX)-2 is induced by growth factors, tumor promoters and cytokines, and is correlated with carcinogenesis, tumor progression and inhibition of apoptosis. To clarify the pathological significance of COX-2, we examined the effect of a selective COX-2 inhibitor, NS398, on two human gastric carcinoma cell lines, MKN-45 and KATO-III, and the expression of Skp2, P27/Kip1 and COX-2 protein in human gastric carcinomas. NS398 inhibited cell growth in a time- and dose-dependent manner and exerted cell cycle arrest in the G0/G1 phase without induction of apoptosis in MKN-45, but had no effect in KATO-III. In MKN-45, NS398 induced up-regulation of P27/Kip1 and down-regulation of COX-2, cyclin D1 and Skp2. Immunohistochemistry using 63 surgically resected gastric carcinomas disclosed that COX-2 expression was correlated with Skp2 expression and that P27/Kip1 expression was inversely correlated with COX-2 and Skp2 expression. High levels of COX-2 or Skp2 were significantly correlated with poor survival (P=0.02 and P=0.004). Our results suggested that: a) NS398 induced inhibition of cell proliferation through cell cycle arrest and suppressed the expression of Skp2 in COX-2-expressing gastric carcinoma cells, and b) COX-2 contributes to the expression of Skp2 and poor survival in human gastric carcinomas.     

Mitogenic synergy through multilevel convergence of hepatocyte growth factor and interleukin-4 signaling pathways

Hepatocyte growth factor (HGF) regulates various physiological and developmental processes in concert with other growth factors, cytokines and hormones. We examined interactions between cell signaling events elicited by HGF and the cytokine interleukin (IL)-4, in the IL-3-dependent murine myeloid cell line 32D transfected with the human HGF receptor, c-Met. HGF was a potent mitogen in these cells, and prevented apoptosis in response to IL-3 withdrawal. IL-4 showed modest anti-apoptotic activity, but no significant mitogenic activity. IL-4 synergistically enhanced HGF-stimulated DNA synthesis, whereas only additive prevention of apoptosis was observed. IL-4 did not enhance HGF-dependent tyrosine phosphorylation of c-Met or Shc. In contrast, HGF-stimulated activation of MAP kinases was enhanced by IL-4, suggesting that the IL-4 and HGF signaling pathways converge upstream of these events. Although phosphatidylinositol 3-kinase (PI3K) inhibitors diminished HGF-induced mitogenesis, anti-apoptosis, and MAP kinase activation, IL-4 enhanced HGF signaling persisted even in the presence of these inhibitors. IL-4 enhancement of HGF signaling was partially blocked in 32D/c-Met cells treated with inhibitors of MEK1 or c-Src kinases, completely blocked by expression of a catalytically inactive mutant of Janus kinase 3 (Jak3), and increased in 32D/c-Met cells overexpressing STAT6. Our results suggest that the IL-4 and HGF pathways converge at multiple levels, and that IL-4-dependent Jak3 and STAT6 activities modulate signaling events independent of PI3K to enhance HGF-dependent mitogenesis in myeloid cells, and possibly other common cellular targets.     

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.     

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.     

组蛋白去乙酰化转移酶抑制剂MS-275对胃癌细胞增殖的影响

目的：探讨组蛋白去乙酰化转移酶抑制剂MS-275对胃癌细胞（MKN-45）和人正常胃黏膜细胞（GES-1）及张氏肝细胞（changliver）生长及凋亡的影响。方法：用不同浓度的MS-275分别处理体外培养的胃癌细胞和人正常胃黏膜细胞及张氏肝细胞,用水溶性四唑盐（WST-1）法检测对细胞的增殖抑制作用;流式细胞仪和TUNEL法检测细胞凋亡情况。结果：MS-275可明显抑制胃癌细胞的生长且呈时间和浓度依赖性,细胞凋亡率明显增加,但对正常细胞没有促凋亡作用。低浓度的MS-275能诱导MKN-45细胞发生G0/G1期阻滞,随着药物处理时间的延长,可以明显检测到G0/G1期比例增大。结论：去乙酰化转移酶抑制剂MS-275对胃癌细胞MKN-45具有选择性细胞毒作用,为其临床应用提供了有价值的实验依据,有望成为新的抗胃癌药物。     

PPAR gamma ligand-induced apoptosis through a p53-dependent mechanism in human gastric cancer cells

We have recently demonstrated that the PPARγ ligand troglitazone induced cell growth arrest and evoked apoptosis in a gastric cancer cell line, MKN–45. Since in general, p53 plays an important role in the induction of apoptosis and growth inhibition, we tried to clarify whether or not p53 mediates troglitazone-induced apoptosis and growth arrest in gastric cancer cells. Troglitazone increased the number of apoptoic cells in MKN-28, MKN-45 and MKN-74, but not in KATO-III cells. The troglitazone-induced apo-ptotic change was significantly reduced by coincubation with bisphenol A digycidyl ether (BADGE), a synthetic PPARy antagonist, in MKN-74 cells, suggesting that PPARγ mediates the apo-ptotic effect of troglitazone. Since KATO-III lacks the p53 gene, we speculated that p53 might be implicated in the PPARγ ligand-induced apoptosis. Western blot analysis revealed that p53 expression was increased by troglitazone in a time-dependent manner in MKN-74 cells, further suggesting that p53 may mediate the ap-optotic process induced by troglitazone. We next established a dominant-negative p53 mutant by stable transfection of p53 mutant into MKN-74 cells. In the dominant-negative p53 mutant cells, troglitazone failed to induce apoptosis, strongly supporting the hypothesis that p53 indeed mediates the process of the troglitazone-induced apoptosis. In the dominant-negative p53 mutant cells, troglitazone significantly induced cell growth arrest and increased expression of p27^Kip1 protein, which is thought to be the key molecule to evoke growth arrest, suggesting that p53 is not involved in the growth inhibition by troglitazone. All these results suggest that p53 mediates the PPARy ligand-induced apoptosis, but not the cell growth inhibition. (Cancer Sci 2003; 94: 338–343)     

RNA干扰乙醛脱氢酶1A1表达对胃癌细胞生物学行为的影响

目的 研究小RNA干扰乙醛脱氢酶1A1基因（ALDH1A1）表达对胃癌细胞生物学行为包括增殖、克隆形成和侵袭能力的影响。方法 构建表达ALDH1A1的siRNA的PGPU6／GFP／Neo-ALDH1A1真核细胞表达载体,转染人胃癌细胞系MKN-45细胞为实验组,同时设阴性对照组和空白对照组,Western blotting检测干扰ALDH1A1基因表达的效果。MTT法、克隆形成实验、Transwell 小室侵袭实验检测ALDH1A1表达下降后MKN-45细胞增殖、克隆形成和侵袭能力的情况。结果 Western blotting检测显示实验组MKN-45细胞ALDH1A1蛋白表达低于阴性对照组（0.36±0.04 vs. 0.72±0.08,P＜0.05）;MTT法显示实验组MKN-45细胞的增殖抑制率高于阴性对照组［（52.56±1.81）％ vs.（30.32±2.23）％,P＜0.05］;克隆形成实验显示实验组MKN-45细胞的克隆形成能力低于阴性对照组（21.67±2.00 vs. 36.78±3.53,P＜0.05）;Transwell 小室侵袭实验显示实验组胃癌细胞的侵袭能力低于阴性对照组（55.11±7.98 vs. 84.78±6.00,P＜0.05）。结论 通过RNA干扰技术可明显下调ALDH1A1蛋白在MKN-45细胞中表达,并抑制肿瘤细胞的增殖、克隆形成和侵袭能力。ALDH1A1有望成为胃癌治疗的一个新的靶点。     

Blocking of FGFR signaling inhibits breast cancer cell proliferation through downregulation of D-type cyclins

Overexpression of fibroblast growth factor receptor (FGFR) tyrosine kinases has been found in many human breast cancers and has been associated with poor patient prognosis. In order to understand the mechanism by which FGFR mediates breast cancer cell proliferation, we used a low molecular weight compound, PD173074, that selectively inhibits FGFR tyrosine kinase activity and autophosphorylation. This potential anticancer agent caused a G1 growth arrest of MDA-MB-415, MDA-MB-453 and SUM 52 breast cancer cells. Our analyses revealed that FGFR signaling links to the cell cycle machinery via D-type cyclins. PD173074-mediated inhibition of FGFR activity caused downregulation of cyclin D1 and cyclin D2 expression, inhibition of cyclin D/cdk4 activity and, as a consequence, reduction of pRB phosphorylation. Retroviral-mediated ectopic expression of cyclin D1 prevented pRB hypophosphorylation and the cell cycle G1 block in PD173074-treated cells, suggesting a central role for D cyclins in proliferation of FGFR-driven breast cancer cells. The repression of FGFR activity caused downregulation of MAPK in MDA-MB-415 and MDA-MB-453 cells. In SUM 52 cells, both MAPK and PI3K signaling pathways were suppressed. In conclusion, results shown here describe a mechanism by which FGFR promotes proliferation of breast cancer cells.     

miR-26a/b调控p53/MDM2通路对胃癌细胞凋亡的影响

目的 探讨miR-26a/b调控p53/MDM2通路对胃癌细胞凋亡的影响。方法 实时荧光定量PCR检测胃癌细胞系MGC803、MKN-45和MKN-28中miR-26a/b的表达水平,荧光素酶报告系统分析miR-26a/b与MDM2 3’非翻译区（3’UTR）的结合情况;将化学合成miR-26a/b前体分子mimics和无关序列转染胃癌细胞MKN-45,化学合成miR-26a/b inhibitor转染胃上皮细胞GES-1后,Western blotting检测MDM2、p53及其下游分子p21和Bcl-2的表达,MTT法检测转染24、48、72 和96 h的细胞增殖情况;通过Annexin Ⅴ/PI双染检测miR-26a/b对MKN-45细胞凋亡情况。结果 与永生化胃上皮细胞GES-1相比,miR-26a/b在肿瘤细胞系MGC803、MKN-45和MKN-28中的表达均下调,在MKN-45中表达水平最低;荧光素酶活性检测显示,miR-26a/b过表达抑制MDM2 3’UTR报告载体（Wild）的荧光素酶活性,而对3’UTR突变型报告载体（Mutation）荧光素酶活性无明显影响。miR-26a/b抑制MKN-45细胞中MDM2表达并增强p53及下游分子表达,而在GES-1细胞中抑制miR-26a/b可以增强MDM2表达,降低p53及下游分子表达。MTT结果显示,miR-26a/b抑制MKN-45细胞的增殖,miR-26a/b inhibitor则明显促进GES-1细胞的增殖。通过AnnexinⅤ/PI检测细胞凋亡发现,miR-26a/b过表达的MKN-45细胞的凋亡率高于对照细胞（P＜0.01）。结论 miR-26a/b 能与MDM2 3’UTR 特异结合,调控p53/MDM2通路影响胃癌细胞的增殖及凋亡。     

白藜芦醇靶向EGFR和c-Met信号通路抑制非小细胞肺癌细胞增殖的机制

 目的 探讨白藜芦醇抑制非小细胞肺癌细胞增殖的分子机制。方法 非小细胞肺癌细胞用白藜芦醇处理24、48和72 h,MTS和软琼脂集落形成实验检测白藜芦醇对非小细胞肺癌的抑制作用,免疫印迹检测白藜芦醇对表皮生长因子受体（epidermal growth factor receptor, EGFR）和肝细胞生长因子受体（c-Met）表达水平及下游信号通路的影响,流式细胞术检测白藜芦醇对细胞周期的影响。结果 白藜芦醇剂量依赖性抑制非小细胞肺癌细胞的增殖。白藜芦醇抑制EGFR和c-Met信号通路磷酸化活化的同时下调了EGFR总蛋白及EGFR在胞膜和胞核的表达,同时降低了c-Met蛋白在胞膜的表达及c-Met蛋白60 kD大小剪切体在胞核的表达。白藜芦醇促进了A549细胞G₀/G₁期阻滞。结论 白藜芦醇通过靶向EGFR和c-Met信号通路抑制非小细胞肺癌细胞的增殖。     

Anti-tumor activity of oridonin on SNU-5 subcutaneous xenograft model via regulation of c-Met pathway

Gastric cancer is the leading cause of cancer death worldwide. Oridonin, a diterpenoid isolated from Rabdosia rubescens, has attracted considerable attention as a potential treatment for gastric cancer based on its anti-tumor effects in many tumor cell lines. However, detailed anti-tumor mechanisms of oridonin remain a matter of speculation. In the present study, a gastric carcinoma cell line harboring c-Met gene amplification SNU-5 was used to investigate the underlying mechanisms. The results showed that in vitro, oridonin potently inhibited c-Met phosphorylation and c-Met-dependent cell proliferation (IC50 value, 36.8 μM), meanwhile down-regulated the expression of the downstream signaling molecules including phospho-c-Raf, phospho-Erk, and phospho-Akt. In vivo, oridonin showed efficacy at well-tolerated doses, including marked cytoreductive anti-tumor activity in SNU-5 subcutaneous xenograft model. The anti-tumor efficacy of oridonin was dose-dependent and showed strong inhibition of c-Met phosphorylation. Additional mechanism of action studies showed dose-dependent inhibition of c-Met-dependent signal transduction, tumor cell proliferation (Ki67), and reduction of microvessel density (CD31). These results suggested that the anti-tumor activity of oridonin may be mediated by direct effects on tumor cell growth or survival as well as anti-angiogenic mechanisms. In summary, the results indicated that oridonin exerted anti-tumor growth on human gastric cancer SNU-5 in vitro and in vivo by direct regulation of c-Met signaling pathway and the anti-tumor effects was mainly based on its anti-proliferation and anti-angiogenesis.     

An Orally Available Small-Molecule Inhibitor of c-Met,PF-2341066, Reduces Tumor Burden and Metastasis in a Preclinical Model of Ovarian Cancer Metastasis

Deregulated expression of the hepatocyte growth factor (HGF) receptor, c-Met, in cancer contributes to tumor progression and metastasis. The objective of this study was to determine whether blocking c-Met with an orally available c-Met inhibitor, PF-2341066, reduces tumor burden and increases survival in a xenograft model of ovarian cancer metastasis. Treatment of mice injected interperitoneally with SKOV3ip1 cells showed reduced overall tumor burden. Tumor weight and the number of metastases were reduced by 55% (P < .0005) and 62% (P < .0001), respectively. Treatment also increased median survival from 45 to 62 days (P = .0003). In vitro, PF-2341066 reduced HGF-stimulated phosphorylation of c-Met in the tyrosine kinase domain as well as phosphorylation of the downstream signaling effectors, Akt and Erk. It was apparent that inhibition of the pathways was functionally important because HGF-induced branching morphogenesis was also inhibited. In addition, proliferation and adhesion to various extracellular matrices were inhibited by treatment with PF-2341066, and the activity of matrix metalloproteinases was decreased in tumor tissue from treated mice compared with those receiving vehicle. Overall, these data indicate that PF-2341066 effectively reduces tumor burden in an in vivo model of ovarian cancer metastasis and may be a good therapeutic candidate in the treatment of patients with ovarian cancer.