Phospholipase C-gamma1 in tumor progression

The vast majority of cancer morbidity and mortality arises from tumor progression beyond the primary tumor site. Unfortunately, most therapies are not effective for advanced stage disease with regional extension or distant metastases. Thus, new treatments are needed to target rate limiting steps in tumor progression. The ability of cancers to invade and metastasize requires the acquisition of specific cell behaviors that enable the cell to escape from the localized site, breach the defined boundaries, reach a hospitable ectopic site and grow in this new locale. Recently, dysregulation of cell motility as stimulated by various extracellular factors has gained credence as a rate-limiting alteration in tumor progression in carcinomas and some other solid tumors. This has focused attention on initiators of signaling cascades that regulate tumor migration. In this effort, one molecule, phospholipase C-γ1 (PLCγ), has been shown to function as a key molecular switch. This revised version was published online in July 2006 with corrections to the Cover Date.     

Biologic therapy of inflammatory bowel disease

Advancing knowledge regarding the biology of chronic inflammation has led to the development of specific biologic therapies that mechanistically target individual inflammatory pathways. Many biologic therapies are being evaluated for the treatment of the chronic inflammatory bowel diseases, Crohn's disease and ulcerative colitis. Biologic compounds proven to be effective for Crohn's disease include monoclonal antibodies to tumor necrosis factor (infliximab and CDP571) and to the leukocyte adhesion molecule alpha4 integrin (natalizumab). Other biologic compounds for which there is insufficient evidence to judge efficacy for inflammatory bowel disease include: p55 tumor necrosis factor binding protein (onercept); interferon alpha; interferon beta-1a; anti-interferon gamma antibody; anti-interleukin 12 antibody; p65 anti-sense oligonucleotide (blocks NF-kappaB); granulocyte colony stimulating factor, and granulocyte macrophage colony stimulating factor; anti-interleukin 2 receptor antibody; epidermal growth factor; keratinocyte growth factor 2 (repifermin); human growth hormone; anti-CD4 antibody; and anti-alpha4beta7 antibody. Biologic therapies that have been proven ineffective for inflammatory bowel disease include: interleukin 10; interleukin 11; anti-sense intercellular adhesion molecule-1; and the tumor necrosis factor receptor fusion protein etanercept. Based on the early successes of infliximab, CDP571 and natalizumab, it seems certain that biologic therapy will play an important role in the future treatment of inflammatory bowel disease.     

ErbB1 and prostate cancer: ErbB1 activity is essential for androgen-induced proliferation and protection from the apoptotic effects of LY294002

BACKGROUND: Androgens play a critical role in proliferation and survival of prostate cancer cells, but the mechanisms leading to these effects are poorly understood. ErbB receptor tyrosine kinases have been implicated in the development of prostate cancer. METHODS: We examined the interaction between the ErbB receptors and androgens using the LNCaP androgen-sensitive prostate tumor model. RESULTS: In the absence of androgens, the cells have low levels of ErbB1 and relatively high levels of ErbB2. Addition of androgens to the medium reversed the ratio; ErbB1 levels rose and ErbB2 levels dropped in response to treatment with the synthetic hormone, R1881. Expression of ErbB activating ligands was found to be constitutive and androgen-independent. The androgen-induced proliferation of LNCaP cells was completely inhibited by the addition of the small molecule ErbB1 tyrosine kinase inhibitors CGP59326 and the bispecific inhibitor (PKI166) for ErbB1 and ErbB2 to the culture medium. Furthermore, in the absence of androgens the relatively low proliferative level was further significantly reduced in the presence of CGP59326. Inhibition of PI3K activity by LY294002 led to induction of apoptosis in androgen-deprived LNCaP cells. Androgen-mediated rescue from LY294002-induced apoptosis was inhibited by addition of CGP59326 to the cells. CONCLUSIONS: These results suggest a model whereby androgens promote an increase in the activity of the epidermal growth factor (EGF)-network by increasing ErbB1 levels, and this activity of is essential for androgen-induced proliferation and survival of the prostate cancer LNCaP cell line.     

Transactivation of ErbB1 and ErbB2 receptors by angiotensin II in normal human prostate stromal cells

BACKGROUND: Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is synthesized primarily in the stromal compartment of the human prostate and may regulate stromal as well as epithelial cell growth and survival. The primary cognate HB-EGF receptor, ErbB1, has been shown recently to be transactivated by G-protein coupled receptors (GPCRs) through regulated proteolytic cleavage of the membrane-bound, precursor form of HB-EGF. Previous studies have demonstrated that human prostate tissue, especially tissue from benign prostatic hyperplasia (BPH), has high angiotensin converting enzyme activity, and a high density of angiotensin (Ang) receptors in periurethral stromal cells. Because the pressor peptide Ang II signals through GPCRs, we tested the possibility that Ang II could transactivate ErbB1/ErbB2 in human prostate stromal (hPS) cells. METHODS: Primary and early passage hPS cells were used as an in vitro model. Cells were stimulated by recombinant HB-EGF or Ang II and total cell lysates were harvested for immunoprecipitation and Western blot. Cell growth was measured by [(3)H]thymidine incorporation assay and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazoliumbromide (MTT) assay. RESULTS: Ang II receptors AT1 and AT2 were expressed in hPS cells. ErbB1 and ErbB2 receptors were activated by HB-EGF. Furthermore, Ang II was able to transactivate both ErbB1 and ErbB2, and this transactivation activity could be abolished by pretreatment with [Glu-52]-diphtheria toxin/CRM197, a specific inhibitor of HB-EGF bioactivity. Consistent with its transactivation activity, Ang II modestly promoted hPS cell growth and this effect was abolished by pretreatment with the ErbB1 antagonist AG1478. CONCLUSION: These experiments suggest a regulatory role for Ang II in the prostate stroma and implicate the endogenous stromal growth factor HB-EGF as a mediator of Ang II signaling in the prostate.     

N-Terminal Site-Specific Mono-PEGylation of Epidermal Growth Factor

Purpose. N-terminal site-specific mono-PEGylation of recombinant human epidermal growth factor (EGF) was accomplished using polyethyleneglycol (PEG) derivatives (Mw = 2000 and 5000) through a reactive terminal aldehyde group. Methods. The site-specific PEG conjugation was conducted at a slightly acidic pH condition (pH 5.5). The mono-PEGylation was targeted to an -amine group at the N-terminal end of EGF to minimize reduction of biologic activity. Tryptic digestion mapping and MALDI-TOF MS techniques were applied to show the occurrence of mono-PEGylation at the N-terminus of EGF. Results. The site-specific mono-PEGylated EGF, when compared with native EGF, fully retained its in vitro biologic activities such as cell proliferation and intracellular signal transduction. This revealed that although a synthetic polymer of a PEG was covalently conjugated to EGF, the internalized complex of PEGylated EGF-receptor within cells did not hamper the intracellular signal transduction events. The PEGylated EGF also exhibited a prolonged circulation in blood stream in vivo and markedly enhanced physical stability when incubated with tissue homogenate. Conclusion. N-terminally mono-PEGylated EGF shows increased physical stability while retaining its biologic activity.     

Relevance of mitogen activated protein kinase (MAPK) and phosphotidylinositol-3-kinase/protein kinase B (PI3K/PKB) pathways to induction of apoptosis by curcumin in breast cells

Following observations that curcumin inhibited proliferation (IC(50)=1-5 microM), invasiveness and progression through S/G2/M phases of the cell cycle in the non-tumourigenic HBL100 and tumourigenic MDA-MB-468 human breast cell lines, it was noted that apoptosis was much more pronounced in the tumour line. Therefore, the ability of curcumin to modulate signalling pathways which might contribute to cell survival was investigated. After pre-treatment of cells for 20 min, curcumin (40 microM) inhibited EGF-stimulated phosphorylation of the EGFR in MDA-MB-468 cells and phosphorylation of extracellular signal regulated kinases (ERKs) 1 and 2, as well as ERK activity and levels of nuclear c-fos in both cell lines. At a lower dose (10 microM), it also inhibited the ability of anisomycin to activate JNK, resulting in decreased c-jun phosphorylation, although it did not inhibit JNK activity directly. In contrast, the activation of p38 mitogen activated protein kinase (MAPK) by anisomycin was not inhibited. Curcumin inhibited basal phosphorylation of Akt/protein kinase B (PKB) in both cell lines, but more consistently and to a greater extent in the MDA-MB-468 cells. The MAPK kinase (MKK) inhibitor U0126 (10 microM), while preventing ERK phosphorylation in MDA-MB-468 cells, did not induce apoptosis. The PI3K inhibitor LY294002 (50 microM) inhibited PKB phosphorylation in both cells lines, but only induced apoptosis in the MDA-MB-468 line. These results suggest that while curcumin has several different molecular targets within the MAPK and PI3K/PKB signalling pathways that could contribute to inhibition of proliferation and induction of apoptosis, inhibition of basal activity of Akt/PKB, but not ERK, may facilitate apoptosis in the tumour cell line.     

Induction by staurosporine of hepatocyte growth factor production in human skin fibroblasts independent of protein kinase inhibition

Staurosporine is one of the most potent and well known inhibitors of protein kinases, and it is often used to study the involvement of protein kinases in signal transduction pathways. We now report that staurosporine can induce the production of hepatocyte growth factor (HGF) independently of protein kinase inhibition. Staurosporine markedly stimulated the production of HGF in various cell types, including human skin fibroblasts. Its effect was accompanied by up-regulation of HGF gene expression. The inhibition of protein kinases appears not to be involved in staurosporine-induced HGF production, because other protein kinase inhibitors, K-252a, H-7, GF 109203X and genistein, had no HGF-inducing activity. UCN-01, 7-hydroxystaurosporine, which differs from staurosporine only in its aglycone moiety, also showed HGF-inducing activity, and inactive K-252a differs from staurosporine only in its sugar moiety. These results indicate that the sugar moiety, a six-atom ring structure, is important in the HGF-inducing activity of staurosporine. Experiments were then carried out to determine whether the characteristics of staurosporine-induced HGF production have similarities to those of HGF production stimulated by other HGF inducers. The effect of staurosporine like that of 8-bromo-cAMP and that of cholera toxin was marked in human skin fibroblasts from all four different sources, whereas the effects of epidermal growth factor and phorbol 12-myristate 13-acetate were variable depending on cells. The net increase in HGF production induced by staurosporine was not reduced in protein kinase C-depleted human skin fibroblasts. Moreover, synergistic induction of HGF was detected between staurosporine and interferon-gamma as well as between 8-bromo-cAMP and interferon-gamma. Staurosporine, however, did not increase intracellular cAMP levels in human skin fibroblasts. These results indicate that staurosporine induced HGF in different cell types via a signaling pathway similar to the cAMP-mediated pathway without increasing cAMP levels.     

Inhibited growth of colon cancer carcinomatosis by antibodies to vascular endothelial and epidermal growth factor receptors

Vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) regulate colon cancer growth and metastasis. Previous studies utilizing antibodies against the VEGF receptor (DC101) or EGF receptor (C225) have demonstrated independently that these agents can inhibit tumour growth and induce apoptosis in colon cancer in in vivo and in vitro systems. We hypothesized that simultaneous blockade of the VEGF and EGF receptors would enhance the therapy of colon cancer in a mouse model of peritoneal carcinomatosis. Nude mice were given intraperitoneal injection of KM12L4 human colon cancer cells to generate peritoneal metastases. Mice were then randomized into one of four treatment groups: control, anti-VEGFR (DC101), anti-EGFR (C225), or DC101 and C225. Relative to the control group, treatment with DC101 or with DC101+C225 decreased tumour vascularity, growth, proliferation, formation of ascites and increased apoptosis of both tumour cells and endothelial cells. Although C225 therapy did not change any of the above parameters, C225 combined with DC101 led to a significant decrease in tumour vascularity and increases in tumour cell and endothelial cell apoptosis (vs the DC101 group). These findings suggest that DC101 inhibits angiogenesis, endothelial cell survival, and VEGF-mediated ascites formation in a murine model of colon cancer carcinomatosis. The addition of C225 to DC101 appears to lead to a further decrease in angiogenesis and ascites formation. Combination anti-VEGF and anti-EGFR therapy may represent a novel therapeutic strategy for the management of colon peritoneal carcinomatosis.     

中药杏香兔耳风对大鼠宫颈粘膜EGF表达的影响

目的:探讨中药杏香兔耳风治疗宫颈炎的作用机理。方法:建立大鼠宫颈炎动物模型,分别于给药4天、10天后观察杏香兔耳风不同剂量组对大鼠宫颈粘膜表皮生长因子(EGF)表达的影响,并进行病理组织学检查。结果杏香兔耳风高、中剂量组对苯酚胶浆造成的实验性大鼠宫颈炎模型,给药4天后,可明显升高宫颈粘膜EGF表达,与模型对照组比较具有显著性差异(其中高剂量组P<0.01,中剂量组P<0.05);给药10天后,各组宫颈粘膜EGF表达无明显差异;病理组织学检查结果也表明,杏香兔耳风高、中剂量组对大鼠宫颈炎有一定的治疗作用,以高剂量组最为明显。结论:杏香兔耳风对大鼠宫颈炎治疗作用与其促进大鼠宫颈粘膜EGF的早期表达有一定关系。     

食管鳞状细胞癌组织中EGF蛋白和mRNA的表达

目的:观察食管鳞状细胞癌组织中EGF的表达情况及其与食管鳞状细胞癌浸润、转移及发生发展的关系.方法:采用免疫组化SP法和原位杂交法分别检测62例正常食管黏膜组织、31例癌旁不典型增生组织及62例食管鳞状细胞癌组织中EGF蛋白和mRNA的表达.结果:正常食管黏膜组织、癌旁不典型增生组织及癌组织中EGF蛋白的阳性表达率依次增高,分别为27.4%、45.2%、69.4%,3组间比较差异有统计学意义(x2=21.954,P＜0.01);正常食管黏膜组织、癌旁不典型增生组织及癌组织中EGF mRNA的阳性表达率依次升高,分别为40.3%,48.3%,77.4%,3组间比较差异有统计学意义(x2=18.494,P＜0.05);食管鳞状细胞癌组织中EGF蛋白和mRNA的表达与癌组织的分化程度、浸润深度及有无淋巴结转移有关(P均＜0.05).结论:EGF过表达与食管鳞状细胞癌的浸润、转移及发生发展有关.EGF有望成为食管鳞状细胞癌早期诊断和判断预后的辅助指标.