Reduced expression of epidermal growth factor receptor related protein in gastric cancer

OBJECTIVES: The recently cloned epidermal growth factor receptor related protein (ERRP) has been proposed to be a negative regulator of the epidermal growth factor receptor (EGFR). Because of the causal involvement of EGFR and its ligands in gastric cancer growth, we investigated expression of ERRP and cell proliferation in human gastric cancer. METHODS: We examined ERRP expression and localisation in surgical specimens of gastric cancers from 47 patients versus non-malignant gastric mucosa and determined their relationship to cell proliferation and differentiation. We also examined expression of ERRP by western blotting in three different gastric cancer cell lines. To further determine the functional properties of ERRP, we examined the effect of ERRP on epidermal growth factor (EGF) induced EGFR phosphorylation essential for its activation in MKN-28 gastric cancer cells. RESULTS: ERRP expression was dramatically reduced in gastric cancers (34% of all specimens positive) compared with non-malignant gastric mucosa (66% of specimens positive). Expression of ERRP in cancer cells inversely correlated with cell proliferation and grade of malignancy. Cell lines derived from metastatic gastric cancers had reduced ERRP expression compared with cell lines derived from a non-metastatic cancer. Exogenous ERRP protein markedly inhibited EGF induced EGFR phosphorylation in gastric cancer cells providing a novel molecular mechanism of its action. CONCLUSIONS: Our data indicate that downregulation of ERRP could play an important role in gastric cancer differentiation and progression. ERRP is a negative regulator of tumour cell proliferation and may exert its inhibitory effect, in part, by attenuating EGFR activation.     

表皮生长因子及其受体对结肠癌干细胞的增殖调控

背景：肿瘤干细胞是肿瘤组织中一小部分具有自我更新、多向分化以及高度增殖能力的肿瘤细胞。研究发现表皮生长因子（EGF）可促进肿瘤干细胞增殖。目的：探讨EGF及其受体（EGFR）在结肠癌干细胞增殖调控中的作用。方法：结肠癌细胞株HT29、HCTll6培养于无血清培养基中,以EGF、碱性成纤维细胞生长因子（bFGF）、胰岛素样生长因子（IGF）分别干预细胞。MTT11法检测EGFR抑制剂吉非替尼对结肠癌细胞球细胞的增殖抑制作用,体外实验检测EGFR抑制剂吉非替尼、PDl53035对细胞球形成的抑制作用,流式细胞术检测细胞凋亡。体内实验检测结肠癌细胞球和细胞株的成瘤能力,real、timePCR检测两者干细胞标记LGR5、Musashi-1和分化标记CK20表达。结果：EGF组HCT116细胞形成的细胞球数量显著高于空白对照、bFGF、IGF组（P〈0．05）。吉非替尼能抑制HCT116细胞球细胞增殖和细胞球形成,并诱导细胞凋亡,作用呈浓度依赖性。HCT116细胞球成瘤时间较细胞株显著缩短,移植瘤体积显著增大（P〈0．05）。LGR5、Musashi-1在细胞球中的表达显著高于细胞株,而CK20在细胞株中的表达显著高于细胞球（P〈0．05）。结论：EGF对结肠癌细胞株HCTll6、HT29形成细胞球具有促进作用。EGFR抑制剂可抑制结肠痛细胞球增殖并诱导细胞凋亡,相关作用可能与调控LGR5、Musashi-1和CK20表i大有关。     

The secretory leukocyte protease inhibitor gene is a target of epidermal growth factor receptor action in endometrial epithelial cells

The over-expression of epidermal growth factor receptor (EGFR) and its ligands, epidermal growth factor (EGF) and transforming growth factor-alpha, is a common feature of epithelial carcinomas and correlates with neoplastic progression. Secretory leukocyte protease inhibitor (SLPI), a member of the Kazal superfamily of serine anti-proteases, induces proliferation and promotes malignancy of epithelial cells and is expressed at high levels in multiple tumor types. In the present study, we have demonstrated that EGF increases SLPI expression in the human endometrial epithelial cell line Ishikawa in a dose- and time-dependent manner. We have shown that this effect of EGF occurs, in part, at the level of the SLPI promoter and involves the MAP kinase signaling pathway. We have further shown that EGF promotion of cell proliferation, but not induction of cyclin D1 gene expression, involves SLPI. Our results suggest that the regulation of SLPI expression by EGFR ligand(s) may represent a 'feed-forward' mechanism by which the enhanced proliferative and migratory properties of EGFR over-expressing cancer cells are sustained. Increased SLPI expression is likely an important component of altered EGFR signaling in human tumors and may have significant therapeutic implications in cancer progression.     

表皮生长因子受体单克隆抗体对人肺腺癌细胞的生长抑制 …

观察表皮生长因子受体单克隆抗体对人肺癌细胞体外增殖的作用。探讨其在肺癌抗体生物治疗中的应用价值。方法 应用杂交瘤技术制备ＥＧＦＲ单在隆抗体并以噻唑蓝比色法检测ＥＧＦＲ单克隆抗体对人肺癌细胞体外增殖能力的影响。     

Cimetidine inhibits epidermal growth factor-induced cell signaling

BACKGROUND: Cimetidine, a histamine-2 (H2) receptor antagonist, has been demonstrated to have anticancer effects on colorectal cancer, melanoma and renal cell carcinoma. In the current study, we clarified that cimetidine inhibits both epidermal growth factor (EGF)-induced cell proliferation and migration in hepatocellular carcinoma (HCC) cell lines. METHOD: HCC cell lines (Hep3B, HLF, SK-Hep-1, JHH-2, PLC/PRF/5 and HLE) were used and cell proliferation was assessed by [3H]-thymidine incorporation assay. Cell migration was measured by in vitro cell migration assay. Biological effects of cimetidine were assessed with human EGF receptor (EGFR)-expressing mouse fibroblast cells (NR6-WT). The autophosphorylation of EGFR and the activation of other downstream effectors were analyzed by immunoprecipitation and immunoblotting. The concentration of intracellular cyclic AMP (cAMP) was measured by competitive enzyme immunoassay. RESULTS: Cimetidine inhibited both EGF-induced cell proliferation and migration in Hep3B, HLF, SK-Hep-1 and JHH-2, while cimetidine did not affect EGF-induced cell proliferation and migration in PLC/PRF/5 and HLE. Cimetidine was revealed to disrupt the EGF-induced autophosphorylation of EGFR and its downstream effectors, mitogen activated protein kinases and phospholipase C-gamma. To define the molecular basis of this negative regulation, we identified that cimetidine significantly decreased intracellular cAMP levels and that decrement of cAMP inhibited autophosphorylation of EGFR. The cell permeable cAMP analog, CPT-cAMPS reversed the cimetidine-induced inhibition of EGF-induced cell proliferation and cell migration by restoring autophosphorylation of EGFR. CONCLUSION: Cimetidine inhibited EGF-induced cell proliferation and migration in HCC cell lines by decreasing the concentration of intracellular cAMP levels. Cimetidine may be a candidate chemopreventive agent for HCC.     

Epidermal growth factor and 17beta-estradiol effects on proliferation of a human gastric cancer cell line (AGS)

BACKGROUND: Recent evidence suggests that both estrogens and growth factors play an important role in the growth of gastrointestinal tumors. The expression of estrogen receptors (ER) and epidermal growth factor receptors (EGFR) in the gastrointestinal tract might therefore result in functional cross-talk between estrogens and EGF. The aim of the present study was to evaluate in vitro the effects of 17beta-estradiol and EGF administration on cell proliferation of a human gastric adenocarcinoma cell line (AGS) and investigate whether any interaction of these compounds may play a role in regulating gastric cancer cell proliferation. METHODS: Estrogen and EGFRs were detected by enzyme immunoassay. Cell proliferation was assessed with the MTT test. RESULTS: Exposure of AGS cells to increasing concentrations of 17beta-estradiol showed an anti-proliferative action at concentrations of 2 microM or higher. The addition of increasing concentrations of EGF stimulated cell growth, with a maximal response at 50 ng/ml EGF. The effect of increasing 17beta-estradiol concentrations combined with 50 ng/ml EGF was to increase cell growth at the lower estradiol concentrations. At the highest estradiol concentration the EGF proliferative effect was suppressed, and a decrease in proliferation rates occurred. Moreover, a significant negative correlation was found between 17beta-estradiol concentrations and EGFR expression. CONCLUSIONS: These findings suggest that growth of cultured gastric cancer cells (AGS) might be modulated by sex steroid hormones through interaction with EGF.     

Prostate cancer cell proliferation is strongly reduced by the epidermal growth factor receptor tyrosine kinase inhibitor ZD1839 in vitro on human cell lines and primary cultures

PURPOSE: To investigate the effects of the epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) ZD1839 ('Iressa') on the cellular proliferation of androgen-sensitive and androgen-independent human prostatic cancer cell lines and primary cultures in vitro. EXPERIMENTAL DESIGN: In this study, we investigated the effects of the quinazoline ZD1839, a potent, selective EGFR-TKI, on the EGFR autophosphorylation and cellular proliferation of androgen-sensitive (ND1, LNCaP, and ALVA-31) and androgen-independent (PC3, DU145, and TSU-Pr1) human prostatic cancer cell lines and 20 primary cultures derived from human prostatic cancer tissue. RESULTS: EGFR was present and phosphorylated in all cell lines tested. ZD1839 reduced EGFR autophosphorylation in intact cell lines with IC(50)s of 0.46-0.97 microM, and inhibited cellular proliferation with IC(50)s of 0.37-1.03 microM. Constitutive EGFR autophosphorylation was low in primary cell cultures, but addition of EGF (50 ng/ml) caused marked EGFR autophosphorylation; cellular proliferation in the presence of EGF was inhibited by ZD1839 with a mean IC(50) of 0.45 microM. At doses >1 microM, ZD1839 induced apoptosis in both androgen-dependent and androgen-independent PCa cell lines. CONCLUSION. Our experiments suggest that EGFR-TKIs such as ZD1839 may have potential in blocking the growth and progression of human prostatic cancers even in early phases of the disease.     

Autonomous proliferation of colon cancer cells that coexpress transforming growth factor alpha and its receptor. Variable effects of receptor-blocking antibody.

Four human colon adenocarcinoma cell lines, SNU-C1, SNU-C4, SNU-C5, and NCI-H716, that are capable of proliferating autonomously in serum-free medium containing no added peptide growth factors were identified. All four cell lines show epidermal growth factor (EGF) receptors (EGFRs), express transforming growth factor alpha (TGF-alpha) messenger RNA, and release anti-TGF-alpha-immunoreactive molecules. The blocking anti-EGFR monoclonal antibody (mAb) 225 blocks autonomous proliferation of SNU-C1 and SNU-C4 cells. In both of these cell lines, the inhibitory effect of mAb 225 is reversible by the addition of EGF, TGF-alpha, or conditioned medium from any of the four cell lines. In contrast, autonomous proliferation of SNU-C5 and NCI-H716 cells is not inhibited by mAb 225 and is not affected by exogenous EGF, TGF-alpha, or conditioned medium. Together, these data confirm the previous finding that anti-EGFR antibodies can inhibit the proliferation of some carcinoma cell lines that coexpress TGF-alpha and EGFR. However, here it is shown that the mechanisms of autonomous proliferation of colon carcinoma cell lines are heterogeneous and not always sensitive to antibody disruption of TGF-alpha/EGFR autocrine interactions.     

Gefitinib (‘IRESSA’, ZD1839) inhibits EGF-induced invasion in prostate cancer cells by suppressing PI3 K/AKT activation

Purpose Androgen-independent prostate cancer (AI-PC) is characterized by a higher invasive potential compared to hormone-responsive prostate cancer. A therapeutic option for AI-PC should thus be targeted to suppress not only cell proliferation, but also the invasive ability of the cells. Here, we investigated the effect of the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor gefitinib (IRESSA, ZD1839) on EGF-stimulated invasion and proliferation in two androgen-independent prostate cancer cell lines PC3 and DU145. In addition, we determined the effect of the compound on EGF-stimulated PI3 K/AKT pathway activation, in view of the key role exerted by this pathway in carcinoma cell invasion.Methods Cell proliferation was determined by thymidine incorporation in the nuclei. Cell cycle analysis was performed by flow cytometry. Invasion through matrigel in vitro was measured by using Boyden chambers. PI3 K activity was measured by immunokinase assay and AKT phosphorylation was evaluated by Western blot analysis.Results Gefitinib inhibits invasion through matrigel and collagen in response to EGF in both cell lines. In addition, we confirm the inhibitory effect of the compound on basal and EGF-induced cell proliferation. Such an effect was accompanied by accumulation of the cells in the G0/G1 phase of the cell cycle. The effect of the compound is due, as expected, to suppression of EGF-induced autotransphosphorylation of EGFR. In addition, we demonstrate here that gefitinib inhibits EGF-induced activation of PI3 K/AKT pathway in both cell lines.Conclusion Overall, our results demonstrate that gefitinib is able to suppress invasion and proliferation of AI-PC cells by suppressing EGF-stimulated activation of the PI3 K/AKT pathway and support a possible use of the drug in the treatment of advanced PC to limit not only proliferation but also invasion to other districts.Abbreviations EGF Epidermal growth factor - EGFR Epidermal growth factor receptor - AI-PC Androgen-independent prostate cancer - PI3 K Phosphatidylinositol 3-kinase     

Genetic analysis of epidermal growth factor action: assignment of human epidermal growth factor receptor gene to chromosome 7.

Purified murine epidermal growth factor (EGF) binds to mouse and human cells. Two mouse transformed cell lines of different origins, PG19 and B82, were found to lack EGF receptors (EGFR). The defect in each of these two cell lines seems to be identical because they fail to complement each other. Somatic cell hybrids between these EGFR-deficient mouse cells and human cells expressing EGFR were produced. Several of these hybrids bound labeled EGF. Detailed cytogenetic analysis of these cell hybrids, followed by correlation of EGFR expression with human chromosomes revealed that EGFR presence correlated with human chromosome 7. The results suggest that the structural gene or a gene necessary for expression of the human EGF receptor is located on human chromosome 7.     

ShRNA靶向干扰EGFR抑制结直肠癌细胞增殖的机制

目的:从细胞周期的角度探讨RNA干扰抑制表皮生长因子受体(epidermal growth factor receptor,EGFR)表达对结直肠癌细胞增殖影响的机制.方法:以人结直肠癌细胞HCT-15为研究对象,构建EGFR-短发夹RNA(short hairpin RNA,shRNA)载体,采用脂质体转染的方法转染细胞,G418筛选稳定细胞株以获得稳定的转染细胞模型,分为4组:转染shRNA-NC,转染shRNA-1组,转染shRNA-2组,转染shRNA-3组;应用半定量RT-PCR和流式细胞术检测转染细胞模型的mRNA和蛋白表达水平;应用平板克隆实验检测转染后细胞增殖能力;应用流式细胞术检测转染后细胞周期的变化.结果:正确构建了shRNA质粒表达载体,成功转染;转染shRNA-1、2、3组较转染shRNA-NC组mRNA、蛋白表达水平均有下降,以转染shRNA-1和2组的抑制效应好(40.2%±3.2%,52.8%±11.3%);转染shRNA-1、2两组较对照组增殖能力下降,G0/G1期细胞增多(63.69±2.75%,60.10±2.00%vs51.08±3.42%)、S期细胞减少(28.20%±...     

Enhanced expression of epidermal growth factor receptor correlates with alterations of chromosome 7 in human pancreatic cancer.

Recently, the gene for the epidermal growth factor (EGF) receptor has been mapped to chromosome 7p, the short arm of chromosome 7 [Shimizu, N., Kondo, I., Gamou, M. A., Behzadian, A. & Shimizu, Y. (1984) Somatic Cell Mol. Genet. 10, 45-53]. Utilizing EGF binding in saturation studies, karyology, and cDNA hybridization experiments, we have sought to determine whether there is a correlation between dosage or alteration of chromosome 7 and enhanced expression of EGF receptor in cultured human pancreatic carcinoma cells. Saturation binding studies with 125I-labeled EGF were performed at 4 degrees C with four established human pancreatic cancer cell lines: T3M4, PANC-1, COLO 357, and UACC-462. Analysis of binding data revealed enhanced numbers of EGF receptors in all four cell lines. Chromosome banding analysis revealed clonal structural alterations of chromosome 7p in the cell lines T3M4, PANC-1, and COLO 357, whereas UACC-462 displayed multiple copies of chromosome 7. Hybridization studies using a radiolabeled EGF receptor cDNA probe failed to demonstrate DNA sequence amplification in any cell line but confirmed the presence of EGF receptor mRNA in these cells in approximate proportion to EGF receptor number. Our results suggest that enhanced expression of EGF receptor in human pancreatic cancer can be associated with either structural or numerical alterations of chromosome 7.     

RNA干扰技术抑制非小细胞肺癌表皮生长因子受体的表达

目的　观察RNA干扰技术是否能有效抑制非小细胞肺癌 (NSCLC)细胞株SPC A 1细胞表皮生长因子受体 (EGFR)的表达。方法　体外化学合成EGFR序列特异性双链RNA(dsRNA) ,与Lipofectamine 2 0 0 0结合后转染细胞 (分 4个组 ,A组 :加入无血清DMEM 5 0 0 μl;B组 :加入Lipofectamine2 0 0 0稀释液 2 5 0 μl及无血清DMEM 2 5 0 μl;C组 :加入非特异性dsRNA/Lipofectamine复合物 5 0 0 μl;D组 :加入dsRNA EGFR/Lipofectamine复合物 5 0 0 μl) ,用Westernblot和流式细胞仪检测EGFR表达。流式细胞仪测细胞周期 ,结合集落形成、化疗敏感性分析观察SPC A 1细胞生物学特性的改变。结果　与A组比较 ,D组EGFR数量减少了 71 31% (P <0 0 0 1) ,B组和C组分别降低了 9 0 %和 16 2 % (P >0 0 5 )。与A组比较 ,D组细胞生长抑制了 78 3% ,集落形成抑制了 6 6 8%。D组G0 G1期细胞百分数较A组增加了 17 4 8% ,D组进入S期的细胞百分数较A组减少了 19 2 0 %。据Origin 6 0计算的IC50 ,dsRNA EGFR可将细胞对顺铂的敏感性提高约 7倍。结论　dsRNA EGFR可有效抑制SPC A 1细胞EGFR表达 ,将更多的细胞阻滞在G0 G1期 ,抑制细胞增生 ,提高细胞对顺铂的敏感性 ,RNA干扰技术为NSCLC基因治疗提供了新策略。     

Growth-promoting effect of muscarinic acetylcholine receptors in colon cancer cells

PURPOSE: G-protein-coupled receptors are known to mediate cell growth via divergent signaling pathways. It has been reported that colon cancer cells express muscarinic acetylcholine receptor (mAChR) although their functional role is largely unknown. The aim of this study is to elucidate possible mechanisms responsible for the growth-promoting effect of mAChRs in colon cancer cells by using colon cancer cell line T84. METHODS: Carbachol, a stable mAChR agonist, dose-dependently induced cell growth with a maximal effect observed at 100 microM, equipotent with 1 nM EGF. 4-DAMP, a specific antagonist of subtype 3 mAChR, inhibited the stimulatory effect by carbachol, suggesting that the growth-promoting effect was receptor-mediated. Carbachol also dose-dependently stimulated extracellular signal-regulated protein kinase (ERK) activation. This effect was inhibited by PD98059, an inhibitor of extracellular signal-regulated protein kinase kinase, which also blocked carbachol activation of cell proliferation, indicating that the p21Ras-ERK pathway is an important signaling cascade in the mitogenic effect. To investigate how mAChR activated the p21Ras-ERK pathway, transactivation of epidermal growth factor receptor (EGFR) was examined. RESULTS: Carbachol induced tyrosine phosphorylation of EGFR, which was abolished by an EGFR tyrosine kinase inhibitor AG1478. Transactivation by carbachol was also abrogated by a metalloproteinases (MMPs) inhibitor GM6001 or an EGFR-blocking antibody (LA-1), suggesting that binding of EGFR ligand(s) produced by MMPs may initiate transactivation in a manner dependent on EGFR tyrosine kinase. The tyrosine-phosphorylated EGFR was immunoprecipitated together with GRB2 and tyrosine-phosphorylated Shc, indicating that transactivated EGFR is able to generate downstream signals. AG 1478 and LA-1 inhibited carbachol stimulation of cell growth. CONCLUSIONS: Taken together, our results indicate that the growth-promoting effect of subtype 3 mAChR in colon cancer cells may depend on transactivated EGFR-ERK pathways. EGFR not only receives external stimuli but also serves as a scaffold for downstream signaling molecules.