Knockdown of c-Fos suppresses the growth of human colon carcinoma cells in athymic mice

Here we have investigated whether inhibition of c-Fos expression in RKO human colon carcinoma cells (HCCCs) would result in reduced TGFβ1 expression and suppression of tumor growth in athymic mice. We stably transfected RKO cells with c-Fos small interfering RNA (siRNA) or with the corresponding control siRNA. Using these stable cell lines, we demonstrated that siRNA-c-Fos significantly suppressed both AP-1 binding, promoter reporter activity at the proximal AP-1 site in the TGFβ1 promoter, and TGFβ1 production. Further, we established colon cancer xenografts with each of RKO-siRNA-EV, RKO-siRNA-Ctrl and RKO-siRNA-c-Fos cells. By 24 days, the tumor size of RKO-siRNA-c-Fos xenografts was 40% that of either RKO-EV or RKO-siRNA-Ctrl. Immunohistochemistry (IHC) of tumor xenografts demonstrated that siRNA-c-Fos significantly blocked c-Fos expression, and consequently expression of TGFβ1. However, expression of TGFβ2 and TGFβ3 were unaffected. Overall, our results demonstrate that blockade of TGFβ1 production by siRNA-c-Fos effectively suppressed tumor growth in vivo.     

奥曲肽对胃癌细胞转录活化蛋白-1的抑制作用

背景与目的:生长抑素是一种多功能神经肽,其本身及其类似物能抑制多种内分泌肿瘤及某些胃肠肿瘤的生长,但是否能抑制胃癌的生长并不清楚。因此,本研究拟探讨生长抑素类似物奥曲肽对胃癌细胞生长的影响及其机制。方法:不同浓度的奥曲肽作用于人胃癌SGC-7901细胞24h后,用免疫印迹法检测奥曲肽对SGC-7901细胞中c-Fos、ERK蛋白表达的影响。建立人胃癌细胞裸鼠原位移植瘤模型,奥曲肽治疗8周,观察奥曲肽对胃癌生长的影响;免疫组化法检测裸鼠人胃癌组织c-Fos和ERK表达的变化。在此基础上,采用EMSA技术检测奥曲肽对胃癌细胞AP-1活化的影响。结果:1×10-5mol/L奥曲肽可明显降低胃癌细胞的3H-TdR的掺入;奥曲肽能抑制人胃癌细胞裸鼠原位移植瘤的生长,抑瘤率为62.3%。SGC-7901胃癌细胞经不同浓度奥曲肽作用后,其c-Fos和ERK的表达水平均有不同程度下降;组织标本检测亦有类似变化。EMSA分析显示,胃癌细胞有基础水平的AP-1活化,20%血清能刺激AP-1的结合力,奥曲肽能抑制这种刺激作用。结论:奥曲肽通过抑制胃癌c-Fos、ERK蛋白的表达而抑制AP-1的结合活性,从而抑制胃癌的生长。     

Mechanism of AP-1-mediated gene expression by select organochlorines through the p38 MAPK pathway

Organochlorine compounds have been demonstrated to have detrimental health effects in both wildlife and humans, an effect largely attributed to their ability to mimic the hormone estrogen. Our laboratory has studied cell signaling by environmental chemicals associated with the estrogen receptor (ER) and more recently via ER-independent mechanisms. Here, we show that the organochlorine pesticide dichlorodiphenyltrichloroethane (DDT) and its metabolites induce a stress mitogen-activated protein kinase (MAPK) that leads to AP-1 activation. Through the use of a dominant negative c-Fos mutant, we show that DDT exposure induces the collagenase promoter in an AP-1-dependent manner. DDT stimulates an AP-1 complex shift at the DNA to one favoring c-Jun/c-Fos dimers through both increasing c-Jun levels and by post-translational activation of c-Jun and c-Fos in HEK 293 and human endometrial Ishikawa cells. DDT treatment induces phosphorylation of ERK and p38, while JNK phosphorylation levels are slightly decreased. Using pharmacological and molecular inhibitors of the various MAPKs, we implicate the p38 signaling cascade, and to a lesser extent ERK, as necessary pathways for AP-1-mediated gene expression induction by organochlorines. Taken together, these results demonstrate that organochlorines induce the collagenase promoter via sequential activation of the p38 kinase cascade and AP-1.     

Silibinin suppresses human osteosarcoma MG-63 cell invasion by inhibiting the ERK-dependent c-Jun/AP-1 induction of MMP-2

Silibinin is a natural flavonoid antioxidant with anti-hepatotoxic properties and pleiotropic anticancer capabilities. We tested the hypothesis that silibinin inhibits cellular invasiveness by down-regulating the focal adhesion kinase (FAK) and extracellular signal-regulated protein kinase (ERK)-dependent c-Jun/activator protein-1 (AP-1) induction, which leads to inhibition of urokinase-type plasminogen activator (u-PA) and matrix metalloproteinase-2 (MMP-2) expressions in human osteosarcoma MG-63 cells. We found that silibinin decreased cell adhesion and invasiveness, as well as inhibited u-PA and MMP-2 expressions. Silibinin reduced ERK 1/2 phosphorylation, but had no effects on the phosphorylation of c-Jun N-terminal kinases (JNKs) 1/2, p38 and Akt. Silibinin suppressed AP-1-binding activity and c-Jun levels and its phosphorylation without changes of c-Fos and Ets-1 levels. Silibinin also inhibited interleukin-6-induced ERK 1/2 and c-Jun phosphorylation, and cell invasiveness. Thus, silibinin may possess an anti-metastatic activity in MG-63 cells.     

The proto-oncoprotein c-Fos negatively regulates hepatocellular tumorigenesis

Hepatocytes adopt an invasive and metastatic phenotype caused by the cooperation of transforming growth factor (TGF)-beta and oncogenic Ha-Ras. In the initial phase of this process, c-Fos is rapidly induced by TGF-beta, but then decreases to undetectable levels. Here, we investigated the functional implications of c-Fos activation and its contribution to hepatocellular tumorigenesis. By employing conditional c-Fos expression, we observed that continuous activation of c-Fos and consequently AP-1 activity leads to depolarization of differentiated murine epithelial hepatocytes. Most remarkably, this change in morphology was associated with inhibition of proliferation and induction of cell death. Coexpression of antiapoptotic Bcl-XL or scavenging of reactive oxygen species was sufficient to prevent the c-Fos-mediated phenotype. In contrast, the cooperation of c-Fos with oncogenic Ha-Ras or a Ras mutant selectively activating the MAPK pathway even enhanced c-Fos-induced effects. Showing the negative role in hepatocellular tumorigenesis, c-Fos repressed oncogenic Ras-driven anchorage-independent growth in vitro and strongly suppressed tumour formation in vivo. Taken together, we demonstrate that c-Fos modulates plasticity of epithelial hepatocytes and acts tumour suppressive in neoplastic hepatocytes by stimulating cell cycle inhibition and cell death.     

Celecoxib inhibits phorbol ester-induced expression of COX-2 and activation of AP-1 and p38 MAP kinase in mouse skin

Celecoxib, the first US FDA-approved selective cyclooxygenase-2 (COX-2) inhibitor initially developed for the treatment of adult rheumatoid arthritis and osteoarthritis, was reported to reduce the polyp burden in patients with familial adenomatous polyposis. This specific COX-2 inhibitor also protects against experimentally induced carcinogenesis, but molecular mechanisms underlying its chemopreventive activities remain largely unresolved. In the present work, we found that celecoxib inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced expression of COX-2 in female ICR mouse skin when applied topically 30 min prior to TPA as determined by both immunoblot and immunohistochemical analyses. In another study, celecoxib attenuated the DNA binding activity of activator protein 1 (AP-1) through suppression of c-Jun and c-Fos expression in TPA-treated mouse skin. In addition, celecoxib inhibited both the catalytic activity and phosphorylation of p38 mitogen-activated protein (MAP) kinase. In the same animal model, TPA treatment resulted in rapid activation via phosphorylation of extracellular signal-regulated protein kinase (ERK)1/2 and p38 MAP kinase, which are upstream of AP-1 in mouse skin. In order to clarify the roles of p38 and ERK in TPA-induced AP-1 activation, we utilized the pharmacologic inhibitors of these enzymes. The p38 inhibitor SB203580 blocked TPA-mediated AP-1 activation, while the MEK1/2 inhibitor U0126 was not inhibitory despite suppression of c-Fos expression in mouse skin. Furthermore, SB203580 markedly inhibited COX-2 expression induced by TPA. Taken together, these findings suggest that celecoxib down-regulates COX-2 by blocking activation of p38 MAP kinase and AP-1, which may represent molecular mechanisms underlying antitumor promoting effects of this drug on mouse skin tumorigenesis.