Enteroaggregative Escherichia coli induced activation of epidermal growth factor receptor contributes to IL-8 secretion by cultured human intestinal epithelial cells.

Enteroaggregative Escherichia coli (EAEC) has been identified as a new enteropathogen that causes acute and chronic diarrhea in children and travelers. One defining aspect of EAEC-pathogenesis is the induction of an inflammatory response in intestinal epithelium. In this study, we have found that EAEC-induced EGFR activation in human small intestinal and colonic epithelial was attenuated in the presence of a specific inhibitor of EGFR (Tyrphostin AG1478). Further, the aggregative stacked-brick type of adherence of this organism to both the cell lines and this pathogen-induced cytoskeletal rearrangement of these cells was also reduced in the presence of Tyrphostin AG1478. Moreover, EAEC-induced activation of downstream effectors (ERK-1/2, PI3K and Akt) of EGFR mediated cell signaling pathways were found to be suppressed in the presence of EGFR inhibitor. A decrease in IL-8 response in EAEC infected both the cell types were also noted in the presence of specific inhibitors of these downstream effectors, transcription factors and Tyrphostin AG1478. We propose that EAEC-induced activation of EGFR is quintessential for stacked-brick adherence of EAEC to human intestinal epithelial cells, their cytoskeletal rearrangements and stimulation of ERK-1/2 and PI3K/Akt mediated signal transduction pathways, resulting in the activation of NF-κB, AP-1, STAT-3 and finally IL-8 secretion by these cells.

Investigation of Adsorption Tyrphostin AG528 Anticancer Drug Upon the CNT(6,6-6) Nanotube: A DFT Study.

OBJECTIVE: In the present study, the interaction between drug Tyrphostin AG528 and CNT(6,6-6) nanotube by Density Functional Theory (DFT) calculations in solvent water has been investigated for the first time. METHODS AND RESULTS: According to the calculations, intermolecular hydrogen bonds take place between an active position of the molecule Tyrphostin AG528 and hydrogen atoms of the nanotube which play an important role in the stability of complex CNT(6,6- 6)/Tyrphostin AG528. The non-bonded interaction effects of the molecule Tyrphostin AG528 with CNT(6,6-6) nanotube on the electronic properties, chemical shift tensors and natural charge have also been detected. The natural bond orbital (NBO) analysis suggested that the molecule Tyrphostin AG528 as an electron donor and the CNT(6,6-6) nanotube play the role of an electron acceptor at the complex CNT(6,6-6)/Tyrphostin AG528. CONCLUSION: The electronic spectra of the Tyrphostin AG528 drug and complex CNT(6,6-6)/Tyrphostin AG528 in solvent water were calculated by Time-Dependent Density Functional Theory (TD-DFT) for the investigation of adsorption effect of the Tyrphostin AG528 drug over nanotube on maximum wavelength. Then, the possibility of the use of CNT(6,6-6) nanotube for Tyrphostin AG528 delivery to the diseased cells has been established.

Regulation of Tight-Junction Integrity by Insulin in an In Vitro Model of Human Blood-Brain Barrier.

Although insulin receptor is expressed at the human blood-brain barrier (BBB), the physiological and pathologic roles of insulin signaling in biologic responses at the BBB remain unclear. Here, we investigate insulin signaling at the human BBB using human cerebral microvascular endothelial cell line (hCMEC/D3) as a well-established in vitro model. Western blot analysis showed that insulin induced phosphorylation of extracellular signal-regulated kinase and insulin receptor substrate-1 in hCMEC/D3 cells. Short-term insulin stimulation increased cell proliferation via the canonical phosphoinositide-3 kinase/protein kinase B and mitogen-activated protein kinase signaling pathways, suggesting that insulin signaling is involved in the regulation of biologic responses in the human BBB. We also found that insulin rapidly increased tight-junction integrity of hCMEC/D3 cells via the phosphoinositide-3 kinase/protein kinase B/glycogen synthase kinase-3 ß signaling pathway. Inhibition of insulin/insulin-like growth factor-1 receptor kinase by AG1024 blocked the increase of tight-junction integrity. In addition, high-insulin/high-glucose treatment (as a model of hyperglycemia and hyperinsulinemia) synergistically reduced the tight-junction integrity in hCMEC/D3 cells, although either condition alone had little or no effect. Our findings suggest that, in addition to the established role of interactions of astrocytes and pericytes with brain capillary endothelial cells, insulin signaling from the blood side of the BBB contributes to maintenance of homeostasis by regulating cell proliferation and tight-junction integrity.

ADAM17 and EGFR regulate IL-6 receptor and amphiregulin mRNA expression and release in cigarette smoke-exposed primary bronchial epithelial cells from patients with chronic obstructive pulmonary disease (COPD).

Aberrant activity of a disintegrin and metalloprotease 17 (ADAM17), also known as TACE, and epidermal growth factor receptor (EGFR) has been suggested to contribute to chronic obstructive pulmonary disease (COPD) development and progression. The aim of this study was to investigate the role of these proteins in activation of primary bronchial epithelial cells differentiated at the air-liquid interface (ALI-PBEC) by whole cigarette smoke (CS), comparing cells from COPD patients with non-COPD CS exposure of ALI-PBEC enhanced ADAM17-mediated shedding of the IL-6 receptor (IL6R) and the EGFR agonist amphiregulin (AREG) toward the basolateral compartment, which was more pronounced in cells from COPD patients than in non-COPD controls. CS transiently increased IL6R and AREG mRNA in ALI-PBEC to a similar extent in cultures from both groups, suggesting that posttranslational events determine differential shedding between COPD and non-COPD cultures. We show for the first time by in situ proximity ligation (PLA) that CS strongly enhances interactions of phosphorylated ADAM17 with AREG and IL-6R in an intracellular compartment, suggesting that CS-induced intracellular trafficking events precede shedding to the extracellular compartment. Both EGFR and ADAM17 activity contribute to CS-induced IL-6R and AREG protein shedding and to mRNA expression, as demonstrated using selective inhibitors (AG1478 and TMI-2). Our data are consistent with an autocrine-positive feedback mechanism in which CS triggers shedding of EGFR agonists evoking EGFR activation, in ADAM17-dependent manner, and subsequently transduce paracrine signaling toward myeloid cells and connective tissue. Reducing ADAM17 and EGFR activity could therefore be a therapeutic approach for the tissue remodeling and inflammation observed in COPD.

Suppression of Mcl-1 induces apoptosis in mouse peritoneal macrophages infected with Mycobacterium tuberculosis.

The effect of myeloid cell leukemia-1 (Mcl-1) inhibition on apoptosis of peritoneal macrophages in mice infected with Mycobacterium tuberculosis was investigated and the primary signaling pathway associated with the transcriptional regulation of Mcl-1 was identified. Real-time PCR and western blotting indicated that Mcl-1 transcript and protein expression are upregulated during infection with virulent M. tuberculosis H37Rv and Xinjiang strains but not with attenuated M. tuberculosis strain H37Ra or Bacillus Calmette-Guérin. Mcl-1 transcript and protein expression were downregulated by specific inhibitors of the Janus kinase/signal transducer and activator of transcription (JAK/STAT), mitogen-activated protein kinase (MAPK) and phosphoinositol 3-kinase (PI3K) pathways (AG490, PD98059 and LY294002, respectively). The strongest inhibitor of Mcl-1 expression was PD98059, the MAPK inhibitor. Flow cytometry demonstrated that the rate of apoptosis in peritoneal macrophages is significantly higher in mice infected with M. tuberculosis and the rate of apoptosis is correlated with the virulence of the strain of M. tuberculosis. Apoptosis was found to be upregulated by AG490, PD98059 and LY294002, whereas inhibition of the MAPK pathway sensitized the infected macrophages to apoptosis. Taken together, these results suggest that specific downregulation of Mcl-1 significantly increases apoptosis of peritoneal macrophages and that the MAPK signaling pathway is the primary mediator of Mcl-1 expression.

Molecular mechanism of constitutive endocytosis of Acid-sensing ion channel 1a and its protective function in acidosis-induced neuronal death.

Acid-sensing ion channels (ASICs) are proton-gated cation channels widely expressed in the peripheral and CNSs, which critically contribute to a variety of pathophysiological conditions that involve tissue acidosis, such as ischemic stroke and epileptic seizures. However, the trafficking mechanisms of ASICs and the related proteins remain largely unknown. Here, we demonstrate that ASIC1a, the main ASIC subunit in the brain, undergoes constitutive endocytosis in a clathrin- and dynamin-dependent manner in both mouse cortical neurons and heterologous cell cultures. The endocytosis of ASIC1a was inhibited by either the small molecular inhibitor tyrphostin A23 or knockdown of the core subunit of adaptor protein 2 (AP2) µ2 using RNA interference, supporting a clathrin-dependent endocytosis of ASIC1a. In addition, the internalization of ASIC1a was blocked by dominant-negative dynamin1 mutation K44A and the small molecular inhibitor dynasore, suggesting that it is also dynamin-dependent. We show that the membrane-proximal residues (465)LCRRG(469) at the cytoplasmic C terminus of ASIC1a are critical for interaction with the endogenous adaptor protein complex and inhibition of ASIC1a internalization strongly exacerbated acidosis-induced death of cortical neurons from wild-type but not ASIC1a knock-out mice. Together, these results reveal the molecular mechanism of ASIC1a internalization and suggest the importance of endocytic pathway in functional regulation of ASIC1a channels as well as neuronal damages mediated by these channels during neurodegeneration.

Inhibition of protein kinase CK2 by flavonoids and tyrphostins. A structural insight.

Sixteen flavonoids and related compounds have been tested for their ability to inhibit three acidophilic Ser/Thr protein kinases: the Golgi apparatus casein kinase (G-CK) recently identified with protein FAM20C, protein kinase CK1, and protein kinase CK2. While G-CK is entirely insensitive to all compounds up to 40 µM concentration, consistent with the view that it is not a member of the kinome, and CK1 is variably inhibited in an isoform-dependent manner by fisetin and luteolin, and to a lesser extent by myricetin and quercetin, CK2 is susceptible to drastic inhibition by many flavonoids, displaying with six of them IC(50) values < 1 µM. A common denominator of these compounds (myricetin, quercetin, fisetin, kaempferol, luteolin, and apigenin) is a flavone scaffold with at least two hydroxyl groups at positions 7 and 4'. Inhibition is competitive with respect to the phospho-donor substrate ATP. The crystal structure of apigenin and luteolin in complex with the catalytic subunit of Zea mays CK2 has been solved, revealing their ability to interact with both the hinge region (Val116) and the positive area near Lys68 and the conserved water W1, the two main polar ligand anchoring points in the CK2 active site. Modeling experiments account for the observation that luteolin but not apigenin inhibits also CK1. The observation that luteolin shares its pyrocatechol moiety with tyrphostin AG99 prompted us to solve also the structure of this compound in complex with CK2. AG99 was found inside the ATP pocket, consistent with its mode of inhibition competitive with respect to ATP. As in the case of luteolin, the pyrocatechol group of AG99 is critical for binding, interacting with the positive area in the deepest part of the CK2 active site.

Human ether-à-go-go gene potassium channels are regulated by EGFR tyrosine kinase.

Human ether á-go-go gene potassium channels (hEAG1 or Kv10.1) are expressed in brain and various human cancers and play a role in neuronal excitement and tumor progression. However, the functional regulation of hEAG channels by signal transduction is not fully understood. The present study was therefore designed to investigate whether hEAG1 channels are regulated by protein tyrosine kinases (PTKs) in HEK 293 cells stably expressing hEAG1 gene using whole-cell patch voltage-clamp, immunoprecipitation, Western blot, and mutagenesis approaches. We found that the selective epidermal growth factor receptor (EGFR) kinase inhibitor AG556 (10 µM), but not the platelet growth factor receptor (PDGFR) kinase inhibitor AG1295 (10 µM) or the Src-family inhibitor PP2 (10 µM), can inhibit hEAG1 current, and the inhibitory effect can be reversed by the protein tyrosine phosphatase (PTP) inhibitor orthovanadate. Immunoprecipitation and Western blot analysis revealed that tyrosine phosphorylation level of hEAG1 channels was reduced by AG556, and the reduction was significantly countered by orthovanadate. The hEAG1 mutants Y90A, Y344A and Y485A, but not Y376A and Y479A, exhibited reduced response to AG556. Interestingly, the inhibition effect of AG556 was lost in triple mutant hEAG1 channels at Y90, Y344, and Y485 with alanine. These results demonstrate for the first time that hEAG1 channel activity is regulated by EGFR kinase at the tyrosine residues Tyr90, Try344, and Try485. This effect is likely involved in regulating neuronal activity and/or tumor growth.

High expression of ErbB2 contributes to cholangiocarcinoma cell invasion and proliferation through AKT/p70S6K.

AIM: To compare the impact of ErbB2 on cell invasion and proliferation in cholangiocarcinoma (CCA) cell lines. METHODS: Level of endogenous ErbB2 expression in three CCA cell lines, namely HuCCA-1, KKU-100 and KKU-M213, was determined by real-time reverse-transcriptase polymerase chain reaction. Two ErbB2 inhibitory methods, a small molecule ErbB2 kinase inhibitor (AG825) and siRNA, were used to disrupt ErbB2 function in the cell lines. CCA cell invasion, motility and proliferation under ErbB2-disrupted conditions were detected using Transwell and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. In addition, ErbB2 downstream effectors were investigated by Western blotting analysis. RESULTS: Suppression of ErbB2 activity, using a specific kinase inhibitor (AG825), reduced invasion, motility and proliferation of all three CCA cell lines. The ability of this drug to inhibit neoplastic properties (invasion, motility and proliferation) increased concomitantly with the level of ErbB2 expression. Similarly, knockdown of ErbB2 level by siRNA inhibited cell invasion and proliferation of KKU-M213, a high-ErbB2-expressing cell, better than those of the lower-ErbB2-expressing cells, HuCCA-1 and KKU-100. Thus, both inhibitory methods indicated that there is more ErbB2-dependency for malignancy of the high-ErbB2-expressing cell, KKU-M213, than for that of low-ErbB2-expressing ones. In addition, interrupting ErbB2 activity decreased phosphorylation of AKT and p70S6K, but not extracellular signal-regulated kinase 1/2, in the high-ErbB2-expressing CCA cell line. CONCLUSION: Our data indicated that high ErbB2 expression enhances CCA invasion, motility and proliferation via the AKT/p70S6K pathway, which suggests the possibility of targeting these molecules for CCA therapy.

P2Y2 nucleotide receptors mediate metalloprotease-dependent phosphorylation of epidermal growth factor receptor and ErbB3 in human salivary gland cells.

The G protein-coupled receptor P2Y(2) nucleotide receptor (P2Y(2)R) has been shown to be up-regulated in a variety of tissues in response to stress or injury. Recent studies have suggested that P2Y(2)Rs may play a role in immune responses, wound healing, and tissue regeneration via their ability to activate multiple signaling pathways, including activation of growth factor receptors. Here, we demonstrate that in human salivary gland (HSG) cells, activation of the P2Y(2)R by its agonist induces phosphorylation of ERK1/2 via two distinct mechanisms, a rapid, protein kinase C-dependent pathway and a slower and prolonged, epidermal growth factor receptor (EGFR)-dependent pathway. The EGFR-dependent stimulation of UTP-induced ERK1/2 phosphorylation in HSG cells is inhibited by the adamalysin inhibitor tumor necrosis factor-alpha protease inhibitor or by small interfering RNA that selectively silences ADAM10 and ADAM17 expression, suggesting that ADAM metalloproteases are required for P2Y(2)R-mediated activation of the EGFR. G protein-coupled receptors have been shown to promote proteolytic release of EGFR ligands; however, neutralizing antibodies to known ligands of the EGFR did not inhibit UTP-induced EGFR phosphorylation. Immunoprecipitation experiments indicated that UTP causes association of the EGFR with another member of the EGF receptor family, ErbB3. Furthermore, stimulation of HSG cells with UTP induced phosphorylation of ErbB3, and silencing of ErbB3 expression inhibited UTP-induced phosphorylation of both ErbB3 and EGFR. UTP-induced phosphorylation of ErbB3 and EGFR was also inhibited by silencing the expression of the ErbB3 ligand neuregulin 1 (NRG1). These results suggest that P2Y(2)R activation in salivary gland cells promotes the formation of EGFR/ErbB3 heterodimers and metalloprotease-dependent neuregulin 1 release, resulting in the activation of both EGFR and ErbB3.

Regulation of human cardiac KCNQ1/KCNE1 channel by epidermal growth factor receptor kinase.

The aim of the present study was to investigate whether/how the recombinant human cardiac I(Ks) could be regulated by epidermal growth factor receptor kinase in HEK 293 cells stably expressing hKCNQ1/hKCNE1 genes using the approaches of perforated patch clamp technique, immunoprecipitation and Western blot analysis. It was found that the broad spectrum isoflavone tyrosine kinase inhibitor genistein and the selective epidermal growth factor receptor kinase inhibitor tyrphostin AG556 suppressed the recombinant I(Ks), and their inhibition was countered by the protein tyrosine phosphatase inhibitor orthovanadate. The Src-family kinase inhibitor PP2 reduced the current, but the effect was not antagonized by orthovanadate. Immunoprecipitation and Western blot analysis revealed that tyrosine phosphorylation level of hKCNQ1 protein was decreased by genistein or AG556, but not by PP2. These results provide the novel information that epidermal growth factor receptor kinase, but not Src-family kinases, regulates the recombinant cardiac I(Ks) stably expressed in HEK 293 cells via phosphorylating KCNQ1 protein of the channel.

[Effects of theaflavins on the signal transduction pathway of epidermal growth factor receptor in airway mucous hypersecretion].

OBJECTIVE: To investigate the effects of theaflavins (TFs) on airway mucous hypersecretion and on the signal transduction pathway of epidermal growth factor receptor (EGFR). METHODS: The cell model of mucous hypersecretion was made by human lung A549 cell stimulated by human neutrophil elastase (HNE), and treated with TFs and AG1478, a blocking agent of EGFR. The expression of mucin (MUC) 5AC, EGFR, P-EGFR, phosphorylation extracellular signal regulated kinase 1/2 (P-ERK1/2), P-p38, phosphorylation c-Jun N-terminal kinase (P-JNK) were detected. The cell activity after TFs treatment was assessed by methyl thiazolyl tetrazolium method. The cells were divided into 5 groups: a negative control group, an HNE treatment group, a TFs pre-treatment group, an AG1478 pre-treatment group and a TFs + AG1478 group. The changes of MUC5AC mRNA and EGFR mRNA were examined by the use of reverse transcriptase-polymerase chain reaction (RT-PCR). The protein expression changes of EGFR, P-EGFR, P-ERK1/2, P-p38 and P-JNK were measured by Western blot. The protein expression changes of MUC5AC were detected by enzyme-linked immunosorbent assay, while the protein morphological changes of MUC5AC were observed by immunofluorescence and confocal laser technology. The data were analyzed with SPSS 12.0 software. Differences between groups were assessed for significance by t test. RESULTS: The expression levels of MUC5AC mRNA and its protein in the HNE group were (0.99 +/- 0.03) and (169 +/- 6) microg/mg, and those of EGFR were (0.98 +/- 0.02) and (0.89 +/- 0.03), both of them increased significantly as compared to those in the control group [(0.53 +/- 0.02), (105 +/- 4) microg/mg and (0.61 +/- 0.11), (0.21 +/- 0.05)]. The protein expressions of P-EGFR, P-ERK1/2, P-p38 were increased significantly as compared with the control group. But the increase of P-p38 was not significant as compared to P-ERK1/2. The protein expression of P-JNK did not change markedly. After the cells were pre-treated with TFs and AG1478 respectively, the above measurements were decreased significantly as compared with the HNE group. The decrease was more significant in the TFs + AG1478 group (t = 3.02, P < 0.05). CONCLUSIONS: Theaflavins decreased the level of EGFR and inhibited the activation of EGFR, and attenuated airway mucous hypersecretion via the ERK pathway.

KGF and EGF signalling block hair follicle induction and promote interfollicular epidermal fate in developing mouse skin.

A key initial event in hair follicle morphogenesis is the localised thickening of the skin epithelium to form a placode, partitioning future hair follicle epithelium from interfollicular epidermis. Although many developmental signalling pathways are implicated in follicle morphogenesis, the role of epidermal growth factor (EGF) and keratinocyte growth factor (KGF, also known as FGF7) receptors are not defined. EGF receptor (EGFR) ligands have previously been shown to inhibit developing hair follicles; however, the underlying mechanisms have not been characterised. Here we show that receptors for EGF and KGF undergo marked downregulation in hair follicle placodes from multiple body sites, whereas the expression of endogenous ligands persist throughout hair follicle initiation. Using embryonic skin organ culture, we show that when skin from the sites of primary pelage and whisker follicle development is exposed to increased levels of two ectopic EGFR ligands (HBEGF and amphiregulin) and the FGFR2(IIIb) receptor ligand KGF, follicle formation is inhibited in a time- and dose-dependent manner. We then used downstream molecular markers and microarray profiling to provide evidence that, in response to KGF and EGF signalling, epidermal differentiation is promoted at the expense of hair follicle fate. We propose that hair follicle initiation in placodes requires downregulation of the two pathways in question, both of which are crucial for the ongoing development of the interfollicular epidermis. We have also uncovered a previously unrecognised role for KGF signalling in the formation of hair follicles in the mouse.

Heparin-binding epidermal growth factor and Src family kinases in proliferation of renal epithelial cells.

Our recent studies have shown that proliferation of renal proximal tubular cells (RPTC) in the absence of growth factors requires activation of the epidermal growth factor (EGF) receptor. We sought to identify the endogenous EGF receptor ligand and investigate the mechanism(s) by which RPTC proliferate in different models. RPTC expressed both pro- and cleaved forms of heparin-binding epidermal growth factor (HB-EGF) and several metalloproteinases (MMP-2, -3, -9, and ADAM10, ADAM17) that have been reported to cleave HB-EGF. Treatment of RPTC with CRM 197, an inhibitor of HB-EGF binding to the EGF receptor, or downregulation of HB-EGF with small interfering RNA inhibited RPTC proliferation following plating. Furthermore, GM6001 (pan-MMP inhibitor), tumor-necrosis factor protease inhibitor-1 (TAPI-1; MMP and ADAM17 inhibitor), and GW280264X (ADAM10 and -17 inhibitor), but not GI254023X (ADAM10 inhibitor), attenuated the proliferation after plating. Although EGF receptor activation is required for RPTC proliferation after oxidant injury, CRM197, GM6001, and TAPI-1 did not block this response. In contrast, inhibition of Src with PP1 blocked EGF receptor activation and RPTC proliferation after oxidant injury. In addition, PP1 treatment attenuated HB-EGF-enhanced RPTC proliferation. We suggest that RPTC proliferation after plating is mediated by HB-EGF produced through an autocrine/paracrine mechanism and RPTC proliferation following oxidant injury is mediated by Src without involvement of HB-EGF.

S-adenosylmethionine-dependent protein methylation in mammalian cytosol via tyrphostin modification by catechol-O-methyltransferase.

It has previously been shown that incubation of mammalian cell cytosolic extracts with the protein kinase inhibitor tyrphostin A25 results in enhanced transfer of methyl groups from S-adenosyl-[methyl-3H]methionine to proteins. These findings were interpreted as demonstrating tyrphostin stimulation of a novel type of protein carboxyl methyltransferase. We find here, however, that tyrphostin A25 addition to mouse heart cytosol incubated with S-adenosyl-[methyl-3H]methionine or S-adenosyl-[methyl-14C]methionine stimulates the labeling of small molecules in addition to proteins. Base treatment of both protein and small molecule fractions releases volatile radioactivity, suggesting labile ester-like linkages of the labeled methyl group. Production of both the base-volatile product and labeled protein occurs with tyrphostins A25, A47, and A51, but not with thirteen other tyrphostin family members. These active tyrphostins all contain a catechol moiety and are good substrates for recombinant and endogenous catechol-O-methyltransferase. Inhibition of catechol-O-methyltransferase activity with tyrphostin AG1288 prevents both base-volatile product formation and protein labeling from methyl-labeled S-adenosylmethionine in heart, kidney, and liver, but not in testes or brain extracts. These results suggest that the incorporation of methyl groups into protein follows a complex pathway initiated by the methylation of select tyrphostins by endogenous catechol-O-methyltransferase. We suggest that the methylated tyrphostins are further modified in the cell extract and covalently attached to cellular proteins. The presence of endogenous catechols in cells suggests that similar reactions can also occur in vivo.

Treatment of HNSCC cell lines with the EGFR-specific inhibitor cetuximab (Erbitux) results in paradox phosphorylation of tyrosine 1173 in the receptor.

Overexpression of the epidermal growth factor receptor (EGFR, ErbB1, HER1) is frequent in head and neck squamous cell carcinomas (HNSCCs) and correlates with disease progression. Inhibition of EGFR with the kinase inhibitor AG1478 abolished receptor phosphorylation and reduced cell proliferation. However, treatment of HNSCC cells with cetuximab (Erbitux), a monoclonal antibody designed to block the EGFR ligand binding site, led to paradox EGFR activation due to hyperphosphorylation of tyrosine 1173, however, with a concomitant reduction in Erk1/2 phosphorylation levels. No pronounced influence on cell proliferation levels could be observed after treatment with this antibody. Since cetuximab appears able to activate EGFR in HNSCC cell lines, it is necessary to rethink the exact mechanisms by which cetuximab that recently was approved for the treatment of advanced head and neck cancer, inhibits tumor growth.

Tyrphostins and other tyrosine kinase inhibitors.

The development of tyrosine phosphorylation inhibitors has transformed the approach to cancer therapy and is likely to affect other fields of medicine. In spite of the conservation among protein tyrosine kinases (PTKs), one can develop small molecules that block the activity of a narrow spectrum of PTKs and that exhibit much less toxicity than the currently used chemotherapeutic agents. In this review, we discuss principles for inhibiting specific PTKs. We discuss (a) the birth of the concept of generating targeted, nontoxic signal transduction inhibitors, (b) the potential of substrate-competitive versus the more common ATP-competitive PTK inhibitors, (c) the combination of PTK inhibitors with other signal transduction inhibitors to induce apoptosis-the best way to induce the demise of the cancer cell, and (d) the potential to utilize PTK inhibitors/tyrphostins to attenuate nonmalignant pathological conditions, such as immune disorders, tissue rejection, and restenosis.

Effect of surface-adsorbed proteins and phosphorylation inhibitor AG18 on intracellular protein expression in adherent macrophages.

Macrophages are believed to play an important role in the host inflammatory response to implanted biomaterials. However, the mechanism of macrophage adhesion to protein-adsorbed substrates and the subsequent activation and inflammation is unresolved. Previously the effect of various surface-adsorbed proteins and increasing concentrations of phosphorylation inhibitor AG18 on intracellular protein expression levels in adherent human monocytic cell line U937 was identified using SDS-PAGE and densitometry. The protein ligands and AG18 concentrations up or down regulated the expression of a set of proteins ranging from approximately 200 to approximately 23 kDa. In the present work, HPLC coupled tandem mass spectroscopy (LC/MS) was used to identify proteins in these bands. We hypothesized that key proteins in macrophage adhesion and activation could be identified by observing protein expression resulting from various surface-adsorbed ligands and AG18 concentrations. Increasing concentrations of AG18 down or up regulate protein expression in adherent U937 on PBS-adsorbed TCPS at approximately 52, approximately 42 and approximately 23 kDa. AG18 concentrations had no effect on cells on albumin (Alb)-adsorbed surfaces but regulated different protein expression in adherent U937 on fibronectin (FN)-adsorbed TCPS at 40 and 80 microm AG18. Both Alb and FN regulate distinct sets of proteins in adherent cells as surface-adsorbed ligands. Based on the data from LC/MS, both surface associated ligand and increasing concentrations of AG18 modulate shifts in intracellular signaling.

Selective induction of cyclooxygenase-2 plays a role in lysophosphatidic acid regulated Fas ligand cell surface presentation.

Previous studies found that lysophosphatidic acid (LPA) upregulated Fas ligand (FasL) presentation on the ovarian cancer cell surface and lead to apoptosis of activated lymphocytes. In this report, we investigated the role of selective induction of cyclooxygenase-2 (Cox-2) in FasL cell surface presentation stimulated by LPA. Ovarian cancer cells pretreated with general aspirin derivative acetylsalicylic acid and specific Cox-2 inhibitor (NS-398) before stimulation with LPA, FasL cell surface presentation was significantly blocked, so was the apoptosis of activated lymphocytes mediated by increasing FasL on the ovarian cancer cell surface. Using the specific inhibitors PD98059, AG1478 or dominant-negative epidermal-growth-factor receptor (EGFR-DN) plasmid, we found that the activation of ERK1/2 played a role in Cox-2 induction, and the transactivation of EGFR worked as an upstream signaling pathway in ERK1/2 phosphorylation. This study first revealed the selective induction of Cox-2 by LPA led to FasL presentation on ovarian cancer cell surface and provide cancer cell immune privilege, and might provide important information of Cox-2 in cancer progression and Cox-2 inhibitors' application in cancer chemoprevention.

[Construction of a three-dimensional human angiogenesis model in vitro for antiangiogenic drug selection].

OBJECTIVE: To develop a simple human angiogenesis model that simulates all the critical steps of the angiogenic process for screening antiangiogenic substances. METHODS: Human umbilical vein endothelial cells (HUVECs) were sandwiched into three-dimensional collagen gels for culturing to produce the angiogenesis model in vitro, on which the antiangiogenic effects of genistein, tyrphostin A23 and lavendustin C were assessed. RESULTS: HUVECs had the capacity to repidly form highly organized capillary-like structures in this model with uniformity of the entire culture, and was easily reproducible. Tyrosine kinase inhibitors, genistein, tyrphostin A23 and lavendustin C were found to inhibit the capillary tube formation. CONCLUSION: This new human angiogenesis model has proved to be simple, responsive, and reproducible for testing potential angiogenic inhibitors, and is suitable for screening anti-angiogenic drugs.