Epidermal growth factor-induced stimulation of epidermal growth factor-receptor synthesis in human cytotrophoblasts and A431 carcinoma cells

Epidermal growth factor (EGF)-receptor is a transmembrane glycoprotein whose intracellular degradation is known to be enhanced by EGF. We tested whether the receptor is replenished during this process by an enhanced rate of synthesis. Human A431 epidermoid carcinoma cells and primary cultures of human placental cytotrophoblasts were used in these studies. Cells were labeled with [35S]methionine, and EGF-receptor biosynthesis was quantitated by immunoprecipitation using a monoclonal anti-EGF-receptor antibody. EGF stimulated receptor biosynthesis at concentrations of 0.1 to 1 nM. The effect was seen within 2 h of EGF addition. At high EGF concentrations the stimulatory effect was diminished. In contrast, the effect of EGF on receptor degradation in these cells was negligible at low nanomolar concentrations and was pronounced only at saturating concentrations (greater than or equal to 10 nM). These results show that occupation of the cell surface EGF-receptor by its ligand can lead to the production of more receptor protein, thus counterbalancing the negative effect on receptor degradation. At low nanomolar concentrations of EGF the stimulatory effect on receptor synthesis predominates over degradation, indicating a positive regulatory role of EGF in receptor action.     

水通道蛋白3在表皮生长因子诱导的乳腺癌细胞迁移中的作用机制研究

目的研究水通道蛋白3（aquapofin3,AQm）与表皮生长因子（EGF）诱导的乳腺癌细胞迁移之间的关系,并对其可能的机制进行初步探索。方法采用Westernblot法检测4种乳腺癌细胞MDA—MB-231、T47D、MCF-7、ER—ZR-70中的AQP3与EGFR的表达情况,选取高表达的细胞株进行EGF诱导,观察细胞迁移（划痕愈合实验）及AQP3表达的变化。施加CuSO。（AQm抑制剂）后观察细胞迁移及AQP3表达的变化。为进一步证明EGF通过哪条通路来调节AQF3的表达,采用LY294002（P13K／AKT通路抑制剂）及U1026（MAPK／ERK1／2通路抑制剂）分别进行阻断,观察AQP3的表达变化。结果Western blot检测发现迁移性强的MDA—MB-231细胞具有EGFR和AQP3的高表达。针对此细胞用不同浓度的EGF进行诱导,发现AQ1Y3的反应性表达与伤口愈合程度呈现一致的趋势（r=0．885,P〈0．01）,采用不同浓度的CuSO。对AQP3的表达进行抑制,MDA—MB-231细胞的伤口愈合程度明显下降（r=0．959,P〈0．01）。采用LY294002阻断P13K／AKT通路后,AQm表达下降,而U1026作用后AQP3表达无明显变化。结论在乳腺癌细胞中存在AQP3表达;在EGF诱导的乳腺癌细胞迁移中,AQP3的反应性表达起到重要的作用,而EGF可能通过P13K／AKT通路来调节AQP3的表达。     

Inhibition of epidermal growth factor-induced RhoA translocation and invasion of human pancreatic cancer cells by 3-hydroxy-3-methylglutaryl-coenzyme a reductase inhibitors.

3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors prevent the conversion of HMG-CoA to mevalonate and thereby inhibit the synthesis of other products derived from this metabolite. This includes a number of small prenylated GTPases involved in cell growth, motility, and invasion. We studied the effect of HMG-CoA reductase inhibitors (fluvastatin and lovastatin) on in vitro invasion of human pancreatic cancer PANC-1 cells. Epidermal growth factor (EGF) induced a dose-dependent increase of PANC-1 cell invasion in a modified Boyden chamber assay. Stimulation of cancer cells with EGF induced translocation of RhoA from the cytosol to the membrane fraction and actin stress fiber assembly. Furthermore, Clostridium botulinum C3 transferase, a specific inhibitor of Rho, inhibited the ability of EGF to promote invasion, indicating that EGF-induced cancer cell invasion is regulated by Rho signaling. Treatment of PANC-1 cells with fluvastatin markedly attenuated EGF-induced translocation of RhoA from the cytosol to the membrane fraction and actin stress fiber assembly, whereas it did not inhibit the tyrosine phosphorylation of EGF receptor and c-erbB-2. The induction of cancer cell invasion by EGF was inhibited by the addition of fluvastatin or lovastatin in a dose-dependent manner. The effects of fluvastatin or lovastatin on cell morphology and invasion were reversed by the addition of all-trans-geranylgeraniol but not by the addition of all-trans-farnesol. These results suggest that HMG-CoA reductase inhibitors affect RhoA activation by preventing geranylgeranylation, which results in inhibition of EGF-induced invasiveness of human pancreatic cancer cells.     

Effects of the liver tumor promoter ethinyl estradiol on epidermal growth factor-induced DNA synthesis and epidermal growth factor receptor levels in cultured rat hepatocytes

The objective of this study was to determine whether DNA synthesis induced in the livers of female rats treated with ethinyl estradiol (EE) was due to direct effects of this synthetic estrogen on hepatocytes. Hepatocytes, obtained by collagenase perfusion from female Lewis rats, were cultured in serum-free medium containing low or no phenol red and supplemented with insulin, transferrin, and selenium. When present at 10-15 microM for the initial 30 h of culture, EE caused a subsequent 2-2.7-fold increase in hepatocyte DNA synthesis. Pretreatment of the hepatocytes with EE during the first 30 h of culture caused an EE concentration-dependent enhancement of their subsequent DNA synthetic response to epidermal growth factor (EGF). Pretreatment with EE shifted the EGF dose-response curve, causing a dramatic enhancement of the response to EGF beginning at 2 ng EGF/ml. The response to a saturating (25 ng/ml) dose of EGF was also greatly enhanced. Determination of the effect of EE on hepatocyte surface EGF receptors revealed that the increased responsiveness of DNA synthesis to EGF was accompanied by a twofold increase in EGF receptor number per cell. These results indicate that EE has direct, growth-related effects on hepatocytes which may contribute to liver growth induced in vivo by this tumor promoter.     

Sulindac sulfide inhibits epidermal growth factor-induced phosphorylation of extracellular-regulated kinase 1/2 and Bad in human colon cancer cells

Colorectal cancer is the second leading cause of cancer death in the United States. Nonsteroidal anti-inflammatory drugs including sulindac are promising chemopreventive agents for colorectal cancer. Sulindac and selective cyclooxygenase (COX)-2 inhibitors cause regression of colonic polyps in familial polyposis patients. Sulindac induces apoptotic cell death in cancer cells in vitro and in vivo. In tumor cells, activation of extracellular-regulated kinase (ERK) 1/2 results in phosphorylation of several ERK1/2 effectors, including the proapoptotic protein Bad. Phosphorylation of Ser112 by ERK1/2 inactivates Bad and protects the tumor cell from apoptosis. Sulindac metabolites and other nonsteroidal anti-inflammatory drugs selectively inhibit ERK1/2 phosphorylation in human colon cancer cells. In this study we show that epidermal growth factor (EGF) strongly induces phosphorylation of ERK1/2 and Bad in HT29 colon cancer cells. EGF-stimulated phosphorylation of ERK and Bad is blocked by pretreatment with U0126, a selective MAP kinase kinase (MKK)1/2 inhibitor. Similarly, pretreatment with sulindac sulfide blocks the ability of EGF to induce ERK1/2 and Bad phosphorylation, but also down-regulates total Bad but not ERK1/2 protein levels. The ability of sulindac to block ERK1/2 signaling by the EGF receptor may account for at least part of its potent growth-inhibitory effects against cancer cells.     

Dactylone inhibits epidermal growth factor-induced transformation and phenotype expression of human cancer cells and induces G1-S arrest and apoptosis

The marine natural chamigrane-type sesquiterpenoid, dactylone, is closely related to secondary metabolites of some edible species of red algae. In the present study, the effect of dactylone was tested on the mouse skin epidermal JB6 P+ Cl41 cell line and its stable transfectants as well as on several human tumor cell lines, including lung (H460), colon (HCT-116), and skin melanomas (SK-MEL-5 and SK-MEL-28). This natural product was effective at nontoxic doses as a cancer-preventive agent, which exerted its actions, at least in part, through the inhibition of cyclin D3 and Cdk4 expression and retinoblastoma tumor suppressor protein (Rb) phosphorylation. The inhibition of these cell cycle components was followed by cell cycle arrest at the G1-S transition with subsequent p53-independent apoptosis. Therefore, these data showed that application of dactylone and related compounds may lead to decreased malignant cell transformation and/or decreased tumor cell proliferation.     

Modulation of growth and apoptosis response in PC-3 and LNCAP prostate-cancer cell lines by FAS

Fas/APO-I is a cell-surface protein, a member of the TNF-receptor family, and it potentially induces apoptosis. In presence of an apoptosis-inducible anti-human Fas MAb, Fas-negative control PC-3 human prostate-cancer cells did not undergo morphological changes, while PC-3 human Fas transfectants showed apoptotic changes in vitro. However, LNCaP human Fas transfectants, as well as Fas-negative control LNCaP human prostate-cancer cells, were Fas-resistant. The growth of Fas-transfected PC-3 tumor was retarded compared with that of control PC-3 tumor in vivo without stimulation of anti-human Fas MAb. Anti-human Fas MAb administration in vivo caused macroscopic Fas-transfected PC-3 tumors formed in BALB/c nude mice to undergo apoptosis. © 1996 Wiley-Liss, Inc.     

Establishment and characterization of TSGH9201, a human gastric carcinoma cell line that is growth inhibited by epidermal growth factor

A human signet ring gastric carcinoma cell line TSGH9201 was established in vitro. The cells grew in vitro as a monolayer with polygonal morphology and had a population doubling time of 34 hours. The cells secreted tumor markers CEA and CA 125. They were, however, not tumorigenic in athymic nude mice. Karyotypic analysis demonstrated a near tetraploidy with a modal chromosome number of 98. Northern blotting and immunocytochemical analysis revealed the expression of both transforming growth factor α and high levels of epidermal growth factor receptor. Cell growth was inhibited by the epidermal growth factor in vitro. The cell line may be a useful tool to study autocrine growth regulation through the epidermal growth factor receptor. © 1995 Wiley-Liss, Inc.     

Clinicoprognostic implications of increased serum levels of vascular endothelial growth factor and basic fibroblastic growth factor in early B-cell chronic lymphocytic leukaemia

To assess the relative merit of increased serum levels of vascular endothelial growth factor and basic fibroblastic growth factor in predicting the risk of disease progression of patients with early B-cell chronic lymphocytic leukaemia we analyzed 81 Binet stage A patients whose sera were taken at the time of diagnosis and evaluated for the presence of vascular endothelial growth factor and basic fibroblast growth factor using an enzyme-linked immunosorbent assay. Serum levels of vascular endothelial growth factor positively correlated with Rai sub-stages (P=0.03), peripheral blood lymphocytosis (P=0.03), bone marrow histology (P=0.04) and beta2-microglobulin (beta2-m) (P=0.006). When dealing with basic fibroblast growth factor only a correlation with Rai sub-stages (P=0.02) could be found. Different cut-offs set on the basis of a stratification in quartiles, failed to demonstrate any correlation between serum levels of basic fibroblast growth factor and disease progression. In contrast, patients with increased serum levels of vascular endothelial growth factor (above median value, 203 pg ml(-1)) had a three times increased risk of disease progression, although, in multivariate analysis only Rai sub-stages (P=0.0001) and lymphocyte doubling time (P=0.002) retained their prognostic significance. Low levels of vascular endothelial growth factor were indicative of good clinical outcome in the subgroup of patients with either low (P=0.02) or high (P=0.03) beta2-m concentration. Finally, the highest prognostic power was obtained when serum vascular endothelial growth factor and beta2-m were examined in combination. Median of progression-free survival of patients who had both serum vascular endothelial growth factor and beta2-m higher than median value was only 13 months, in contrast median progression-free survival of patients with one marker increased (i.e. above the 50th percentile) was 40 months. Patients with both markers below the median experienced the best clinical outcome (median progression-free survival not reached at 40 months). In conclusion, serum levels of either vascular endothelial growth factor or basic fibroblast growth factor are high in patients with early chronic lymphocytic leukaemia, however, only vascular endothelial growth factor predicts behaviour of disease and helps to refine the prognosis of stage A patients.     

Characterization of the epidermal growth factor receptor in human meningioma

We have characterized the epidermal growth factor (EGF) receptor in human meningioma (biopsy) microsomes, cellularly derived microsomes, and intact meningioma cells in culture. Scatchard analysis of competition studies reveals both high and low affinity EGF binding sites in the meningiomas tested [dissociation constant (Kd) = 0.9 nM, maximum number of binding sites (Bmax) = 280 fmol/mg protein; Kd = 5.0 nM, Bmax = 660 fmol/mg protein, respectively]. The binding of 125I-EGF is specific since it is abolished by excess unlabeled EGF but not by excess unlabeled platelet-derived growth factor or insulin. Meningioma cultures preincubated with platelet-derived growth factor (10 ng/ml) at 37 degrees C shifted the 125I-EGF competition curve to the right but did not affect receptor number (100,000 sites/cell) when compared to cultures preincubated at 4 degrees C. Cross-linking studies performed with ethyleneglycol bis(succinimidyl succinate) followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography reveal a major band of specifically bound EGF (Mr approximately 150,000), although the normal (Mr approximately 170,000) and another putative proteolytic form (Mr approximately 125,000) can also be seen. These results indicate that human meningiomas contain a mixed population of EGF binding sites and exhibit properties of previously described EGF receptors.     

Biological effect of epidermal growth factor on the in vitro growth of human tumors

The effect of epidermal growth factor (EGF) on the in vitro growth of 186 malignant human tumor specimens (45 melanomas, 32 sarcomas, and 56 lung, 16 gynecological, 14 breast, 12 genitourinary, and 11 gastrointestinal carcinomas) was evaluated in the cellular adhesive matrix human tumor culture system supplemented with transferrin, insulin, hydrocortisone, and estradiol. EGF increased tumor growth by at least 50% in 81% of the 186 tumors and by over 100% in 54%. The enhanced growth induced by EGF was related to an accelerated cellular division independent of tumor type and not to an increase in the actual number of clonogenic units. The drug concentrations of cell cycle-independent Adriamycin and cisplatin needed to achieve a 90% tumor cell kill were not altered by the responsiveness of the tumor to EGF.     

Therapeutic targeting of the epidermal growth factor receptor in human cancer

The epidermal growth factor receptor (EGFR; also referred to as HER1 or ERBB1), is a member of the type 1 receptor tyrosine kinase family known as the ERBB family. Comprising 4 members-ERBB1, ERBB2 (also known as HER2), ERBB3 (HER3), and ERBB4 (HER4)-these receptors play a principal role in allowing cells to integrate and respond correctly to diverse external stimuli, ranging from soluble endocrine and paracrine factors to signaling molecules on neighboring cells. The cell must interpret these extracellular signals to produce an appropriate developmental or proliferative response, and aberrant activation of the kinase activity of these receptors, particularly EGFR and ERBB2, is important in the development and progression of human cancer. Given its roles in signal transduction and development of the malignant phenotype, EGFR has emerged as a critical target for therapeutic development against various forms of cancer. This review focuses on the current therapeutic approaches directed against EGFR, the emerging challenges of EGFR therapy resistance, and how our increasing knowledge of EGFR biology is driving more targeted or alternative approaches to cancer therapies.