Decrease In Epidermal Growth Factor Receptor Research Articles

Hydrogen peroxide-induced oxidative damage and protective role of peroxiredoxin 6 protein via EGFR/ERK signaling pathway in RPE cells.

Damage to retinal pigment epithelium (RPE) cells caused by oxidative stress is closely related to the pathogenesis of several blinding retinal diseases, such as age-related macular degeneration (AMD), retinitis pigmentosa, and other inherited retinal degenerative conditions. However, the mechanisms of this process are poorly understood. Hence, the goal of this study was to investigate hydrogen peroxide (H2O2)-induced oxidative damage and protective role of peroxiredoxin 6 (PRDX6) protein via EGFR/ERK signaling pathway in RPE cells. Cells from a human RPE cell line (ARPE-19 cells) were treated with H2O2, and then cell viability was assessed using the methyl thiazolyl tetrazolium assay. Cell death and reactive oxygen species (ROS) were detected by flow cytometry. The levels of PRDX6, epidermal growth factor receptor (EGFR), P38 mitogen-activated protein kinase (P38MAPK), c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) were detected by Western blot assay. PRDX6 and EGFR were also detected via immunofluorescence staining. Our results show that H2O2 inhibited cell viability, induced cell death, and increased ROS levels in ARPE-19 cells. It was also found that H2O2 decreased the levels of PRDX6, EGFR, and phosphorylated ERK but increased the levels of phosphorylated P38MAPK and JNK. PRDX6 overexpression was found to attenuate H2O2-induced inhibition of cell viability and increased cell death and ROS production in ARPE-19 cells. PRDX6 overexpression also increased the expression of EGFR and alleviated the H2O2-induced decrease in EGFR and phosphorylated ERK. Moreover, inhibition of epidermal growth factor-induced EGFR and ERK signaling in oxidative stress was partially blocked by PRDX6 overexpression. Our findings indicate that PRDX6 overexpression protects RPE cells from oxidative stress damage caused by decreasing ROS production and partially blocking the inhibition of the EGFR/ERK signaling pathway induced by oxidative stress. Therefore, PRDX6 shows promise as a therapeutic target for the prevention of RPE cell damage caused by oxidative stress associated with retinal diseases.

Forkhead Box q1 promotes invasion and metastasis in colorectal cancer by activating the epidermal growth factor receptor pathway

BACKGROUNDColorectal cancer (CRC) is an extremely malignant tumor with a high mortality rate. Little is known about the mechanism by which forkhead Box q1 (FOXQ1) causes CRC invasion and metastasis through the epidermal growth factor receptor (EGFR) pathway.AIMTo illuminate the mechanism by which FOXQ1 promotes the invasion and metastasis of CRC by activating the heparin binding epidermal growth factor (HB-EGF)/EGFR pathway.METHODSWe investigated the differential expression and prognosis of FOXQ1 and HB-EGF in CRC using the Gene Expression Profiling Interactive Analysis (GEPIA) website (http://gepia.cancer-pku.cn/index.html). Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were used to detect the expression of FOXQ1 and HB-EGF in cell lines and tissues, and we constructed a stable low-expressing FOXQ1 cell line and verified it with the above method. The expression changes of membrane-bound HB-EGF (proHB-EGF) and soluble HB-EGF (sHB-EGF) in the low-expressing FOXQ1 cell line were detected by flow cytometry and ELISA. Western blotting was used to detect changes in the expression levels of HB-EGF and EGFR pathway-related downstream genes when exogenous recombinant human HB-EGF was added to FOXQ1 knockdown cells. Proliferation experiments, transwell migration experiments, and scratch experiments were carried out to determine the mechanism by which FOXQ1 activates the EGFR signaling pathway through HB-EGF, and then to evaluate the clinical relevance of FOXQ1 and HB-EGF.RESULTSGEPIA showed that the expression of FOXQ1 in CRC tissues was relatively high and was related to a lower overall survival rate. PCR array results showed that FOXQ1 is related to the HB-EGF and EGFR pathways. Knockdown of FOXQ1 suppressed the expression of HB-EGF, and led to a decrease in EGFR and its downstream genes AKT, RAF, KRAS expression levels. After knockdown of FOXQ1 in CRC cell lines, cell proliferation, migration and invasion were attenuated. Adding HB-EGF restored the migration and invasion ability of CRC, but not the cell proliferation ability. Kaplan–Meier survival analysis results showed that the combination of FOXQ1 and HB-EGF may serve to predict CRC survival.CONCLUSIONBased on these collective data, we propose that FOXQ1 promotes the invasion and metastasis of CRC via the HB-EGF/EGFR pathway.

A novel cyclic NP1 reveals obstructionof EGFR kinase activity and attenuation of EGFR-driven cell lines.

Aberrations of the epidermal growth factor receptor (EGFR), for example, mutations and overexpression, play pivotal roles in various cellular functions, such as proliferation, migration, and cell differentiation. Approved small molecule-based inhibitors, including gefitinib and erlotinib, are used clinically to target the tyrosine kinase domain of EGFR (TK-EGFR). However, the severity of the side effects, off-target effects, and drug resistance is a concern. Cyclic peptides are a well-known peptide format with high stability and are promising molecules for drug development. Herein, the Ph.D.™-C7C phage display library was used to screen cyclic peptides against TK-EGFR. Biopanning, both with and without propagation methods, was performed to assess the highest capacity peptides using the enzymatic activity of TK-EGFR. Interestingly, NP1, a peptide selected during biopanning without propagation demonstrated an inhibitory effect against TK-EGFR at IC50 within the nanomolar range; this effect was better than that of P1 obtained using biopanning with propagation. Moreover, NP1 elicited EGFR with an affinity binding (KD ) value of 18.40 ± 5.50 µM by surface plasmon resonance (SPR). Introducing cell-penetrating peptides or Arginine-9 (Arg9) at the N-terminus of NP1 thus improves cell-penetrability and can lead to the inhibition of EGFR-driven cancer cell lines; however, it exhibits no hepatotoxicity. Furthermore, NP1 caused a decrease in phosphorylated EGFR after activation within cells. A docking model shows that NP1 interacted primarily with TK-EGFR via hydrogen bonding. Together, this suggests that NP1 is a novel EGFR peptide inhibitor candidate with specificity and selectivity toward TK-EGFR, and may be applied to targeted therapy.

Maternal birth weight is associated with milk epidermal growth factor in Filipino women.

Lactational programming, through which milk-borne bioactives influence both neonatal and long-term biological development, is well established. However, almost no research has investigated how developmental stimuli during a mother's early life may influence her milk bioactives in adulthood. Here, we investigated the association between maternal birth weight and milk epidermal growth factor (EGF) and epidermal growth factor receptor (EGF-R) in later life. We predicted there would be a decrease in both milk EGF and EGF-R in the milk produced by mothers who were themselves born low birth weight. Study participants are from the Cebu Longitudinal Health and Nutrition Survey. Mothers (n = 69) were followed longitudinally since birth with prospective data collection. Anthropometrics, health, and dietary recalls were collected with early morning milk samples when mothers were 24 to 25 years of age. Milk samples were analyzed for EGF and its receptor (EGF-R). Analysis of variance was used to test for differences in milk EGF and EGF-R between low and average birthweight mothers after adjustment for parity, age, and maternal dietary energy intake. Mothers who were low birth weight produced milk with significantly less EGF and more EGF-R which resulted in a lower ratio of EGF to EGF-R. These associations persisted after adjustment for infant age, maternal adiposity, and dietary energy. While this is a small sample size, these preliminary findings suggest that maternal early life characteristics, such as birth weight, may be important contributors to variation in milk bioactives. Future work is necessary to understand how variation in maternal early life may influence milk composition in adulthood.

Upregulation of ADAM10 in oral squamous cell carcinoma and its correlation with EGFR, neoangiogenesis and clinicopathologic factors

ADAM10 (a disintegrin and metalloproteinase-10) is a known sheddase for EGFR (epidermal growth factor receptor) ligands and has been suggested to modulate angiogenesis. We aimed to evaluate the expression of ADAM10 in patients with oral squamous cell carcinoma (OSCC) and to determine its correlation with EGFR, CD105 and clinicopathologic parameters. Fifty primary OSCCs with clinical data were graded according to the histologic risk assessment (HRA) model and subjected to immunohistochemical staining using antibodies against ADAM10, EGFR1 and CD105. ADAM10 was assessed in both epithelial and stromal components. The associations among all three proteins and clinicopathologic factors including tumor size, lymph node status and distant metastasis (TNM) were statistically analyzed (P < 0.05). Epithelial-ADAM10, stromal-ADAM10 and EGFR were overexpressed in 92%, 40% and 56% of the OSCCs, respectively. EGFR expression occurred in peripheral and diffuse patterns, which were also separately considered in our analyses. A significant correlation was found between ADAM10 and CD105 (r = −0.455; P < 0.001). Lymphocytic infiltration scores (P = 0.04) and tumor size (P = 0.001) showed significant differences between EGFR+ and EGFR− tumors, but none of the other variables had any relationships with either clinicopathologic factors or each other (P > 0.05). ADAM10 was upregulated in OSCC but had no correlation with survival-associated factors such as TNM or the HRA model. At the protein level, epithelial ADAM10 negatively regulated neoangiogenesis, but its interaction with EGFR was minimal. Reduction in host immunologic responses was associated with a decrease in EGFR. These findings, if corroborated, could be interesting in combination therapies used for cancer treatment.

Functional cooperation between HIF-1α and c-Jun in mediating primary and acquired resistance to gefitinib in NSCLC cells with activating mutation of EGFR

ObjectiveHypoxia-inducible factor 1 (HIF-1) and activator protein 1 (AP-1) are important transcription factors regulating expression of genes involved in cell survival. HIF-1α and c-Jun are key components of HIF-1 and AP-1, respectively, and are regulated by epidermal growth factor receptor (EGFR)-mediated cell signaling and tumor microenvironmental cues. The roles of HIF-1α and c-Jun in development of resistance to EGFR tyrosine kinase inhibitor (TKI) in non-small cell lung cancer (NSCLC) with activating mutation of EGFR have not been explored. In this study, we investigated the roles of HIF-1α and c-Jun in mediating primary and acquired resistance to gefitinib in NSCLC cells with activating mutation of EGFR. Materials and methodsChanges in HIF-1α protein and in total and phosphorylated c-Jun levels in relation to changes in total and phosphorylated EGFR levels before and after gefitinib treatment were measured using Western blot analysis in NSCLC cells sensitive or resistant to gefitinib. The impact of overexpression of a constitutively expressed HIF-1α (HIF-1α/ΔODD) or a constitutively active c-Jun upstream regulator (SEK1 S220E/T224D mutant) on cell response to gefitinib was also examined. The effect of pharmacological inhibition of SEK1-JNK-c-Jun pathway on cell response to gefitinib was evaluated. ResultsDownregulation of HIF-1α and total and phosphorylated c-Jun levels correlated with cell inhibitory response to gefitinib better than decrease in phosphorylated EGFR did in NSCLC cells with intrinsic or acquired resistance to gefitinib. Overexpression of HIF-1α/ΔODD or SEK1 S220E/T224D mutant conferred resistance to gefitinib. There exists a positive feed-forward regulation loop between HIF-1 and c-Jun. The JNK inhibitor SP600125 sensitized gefitinib-resistant NSCLC cells to gefitinib. ConclusionsHIF-1α and c-Jun functionally cooperate in development of resistance to gefitinib in NSCLC cells. The translational value of inhibiting HIF-1α/c-Jun cooperation in overcoming resistance to EGFR TKI treatment of NSCLC cells with activating mutation of EGFR deserves further investigation.

Cetuximab sensitivity of head and neck squamous cell carcinoma xenografts is associated with treatment-induced reduction in EGFR, pEGFR, and pSrc.

The aims of this study were to validate in vitro drug sensitivity testing of head and neck squamous cell carcinoma (HNSCC) cell lines in an in vivo xenograft model and to identify treatment-induced changes in the epidermal growth factor receptor (EGFR) signaling pathway that could be used as markers for cetuximab treatment response. The in vitro and in vivo cetuximab sensitivity of two HNSCC cell lines, UT-SCC-14 and UT-SCC-45, was assessed using a crystal violet assay and xenografts in nude mice, respectively. The expression of EGFR, phosphorylated EGFR (pEGFR), phosphorylated Src (pSrc), and Ki-67 was investigated by immunohistochemistry. To verify these results, the in vitro expression of EGFR and pEGFR was analyzed with ELISA in a panel of 10 HNSCC cell lines. A close correlation was found between in vitro and in vivo cetuximab sensitivity data in the two investigated HNSCC cell lines. In treatment sensitive UT-SCC-14 xenografts, there was a decrease in EGFR, pEGFR, and pSrc upon cetuximab treatment. Interestingly, in insensitive UT-SCC-45 xenografts, an increased expression of these three proteins was found. The change in EGFR and pEGFR expression in vivo was confirmed in cetuximab-sensitive and cetuximab-insensitive HNSCC cell lines using ELISA. High sensitivity to cetuximab was strongly associated with a treatment-induced reduction in pEGFR both in vivo and in vitro in a panel of HNSCC cell lines, suggesting that EGFR and pEGFR dynamics could be used as a predictive biomarker for cetuximab treatment response.

High mannose-binding Pseudomonas fluorescens lectin (PFL) downregulates cell surface integrin/EGFR and induces autophagy in gastric cancer cells.

BackgroundPseudomonas fluorescens lectin (PFL) belongs to a recently discovered anti-HIV lectin family and induces anoikis-like cell death of MKN28 gastric cancer cells by causing α2 integrin internalization through recognition of high mannose glycans; however, the detailed anti-cancer mechanism is not fully elucidated.MethodsCell adherence potency of MKN28 upon PFL treatment was assessed using a colorimetric assay. Cell surface molecules to which PFL bound were identified by peptide mass finger printing with Matrix Assisted Laser Desorption/Ionization-time of flight mass spectrometry and their cellular localization determined by immunofluorescence microscopy. Gene and protein expression in PFL-treated MKN28 cells were evaluated by microarray analysis and western blot, and the function of these genes was evaluated by siRNA knock-down. A proliferation assay measured the sensitivity of PFL-treated cancer cells to anti-cancer drugs. The effect of PFL on subcutaneous MKN28 tumor growth and hepatic tumor formation in BALB/c nude mice was evaluated.ResultsThe strength of MKN28 cell adherence in vitro to the extracellular matrix was impaired by PFL treatment, consistent with the observation that PFL induces rapid downregulation of surface integrins. PFL also was found to bind to cell surface epidermal growth factor receptor (EGFR). Surface EGFR molecules were endocytosed following PFL binding, and were degraded in a time-dependent fashion. This degradation process was largely the result of autophagy, as revealed by the increased expression of autophagic proteins. PFL-induced EGFR degradation was partly inhibited by RAB7 siRNA as well as LC3 siRNA, and internalized EGFR colocalized with ATG9 at 48 h post-PFL treatment, suggesting that these proteins contribute to dynamic degradation induced by PFL. PFL-induced decrease in surface EGFR rendered MKN28 cells susceptible to gefitinib, a selective inhibitor of EGFR tyrosine kinase. In vivo experiments showed that PFL-treated MKN28-EGFP cells injected in the portal vein of BALB/c nude mice failed to form tumor colonies on the liver, and intratumoral injection of PFL significantly inhibited tumor growth.ConclusionPFL-mediated downregulation of integrin and EGFR contributes to the inhibition of tumor growth in vitro and in vivo. This novel anti-cancer mechanism of PFL suggests that this lectin would be useful as an anti-cancer drug or an adjuvant for other drugs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2099-2) contains supplementary material, which is available to authorized users.

Dasatinib is synergistic with cetuximab and cisplatin in triple-negative breast cancer cells

BackgroundPatients presenting with triple-negative breast cancers (TNBCs) have a poorer prognosis compared with those with other subtypes of breast cancer. The majority of TNBCs overexpress epidermal growth factor receptor (EGFR). However, EGFR inhibition as a monotherapy, as with the monoclonal antibody cetuximab, is ineffective. Src family tyrosine kinases play a critical role in signal transduction downstream of growth factor receptors and are involved in the development of EGFR inhibitor resistance. We hypothesize that dasatinib, an Src family tyrosine kinase inhibitor, may help overcome EGFR resistance to cetuximab, and in combination with cisplatin may enhance growth inhibition and apoptosis and reduce metastatic potential. MethodsGrowth inhibition, apoptosis, cell migration and invasion, and effects on EGFR, Akt, and mitogen-activated protein kinase phosphorylation were examined in a panel of breast cancer cell lines, including seven TNBC cell lines. ResultsSix out of seven TNBC cell lines demonstrated a synergistic interaction using the triple-drug combination, compared with only two TNBC cell lines with the cisplatin and cetuximab combination. An induction of apoptosis and decrease in EGFR and mitogen-activated protein kinase phosphorylation, and thus resensitization to EGFR inhibition, was observed using the three-drug treatment regimen. A significant reduction (P < 0.001) in tumor cell migration and invasion was also found following dasatinib treatment alone or in combination. ConclusionsThese findings may have important clinical implications in treating TNBC patients whose tumors co-overexpress both EGFR and c-Src. Identification of this subset of patients may be beneficial in the design of a clinical trial using this treatment regimen.

Presenilin-1 Regulates Neural Progenitor Cell Differentiation in the Adult Brain

Presenilin-1 (PS1) is the catalytic core of the aspartyl protease γ-secretase. Previous genetic studies using germ-line deletion of PS1 and conditional knock-out mice demonstrated that PS1 plays an essential role in neuronal differentiation during neural development, but it remained unclear whether PS1 plays a similar role in neurogenesis in the adult brain. Here we show that neural progenitor cells infected with lentiviral vectors-expressing short interfering RNA (siRNA) for the exclusive knockdown of PS1 in the neurogenic microenvironments, exhibit a dramatic enhancement of cell differentiation. Infected cells differentiated into neurons, astrocytes and oligodendrocytes, suggesting that multipotentiality of neural progenitor cells is not affected by reduced levels of PS1. Neurosphere cultures treated with γ-secretase inhibitors exhibit a similar phenotype of enhanced cell differentiation, suggesting that PS1 function in neural progenitor cells is γ-secretase-dependent. Neurospheres infected with lentiviral vectors expressing siRNA for the targeting of PS1 differentiated even in the presence of the proliferation factors epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), suggesting that PS1 dominates EFG and bFGF signaling pathways. Reduction of PS1 expression in neural progenitor cells was accompanied by a decrease in EGF receptor and β-catenin expression level, suggesting that they are downstream essential transducers of PS1 signaling in adult neural progenitor cells. These findings suggest a physiological role for PS1 in adult neurogenesis, and a potential target for the manipulation of neural progenitor cell differentiation.

Epidermal Growth Factor Receptor as a Potential Modulator of Protection and Potentiation of Colon Carcinogenesis

Purpose: The epidermal growth factor receptor (EGFR) is overexpressed in ˜50% of colorectal cancer. Targeting EGFR has promise as a chemopreventive/chemotherapeutic strategy depending on the molecular context of the cell (Karapetis NEJM 2008). Furthermore, EGFR may have a kinase independent role such as altering intracellular glucose metabolism (Weihua et al., Cancer Cell 2008). This is particularly relevant given that diabetes/metabolic syndrome is an established colorectal cancer promotional factor. Nonsteroidal anti-inflammatory drugs (NSAIDs), on the other hand, are potent chemopreventive agents that may act through a myriad of molecular targets (COX-2, β-catenin, NF-κB etc). Little is known about the role of EGFR in NSAID prevention or hyperglycemic promotion. Methods: Two colon cancer cell lines, SW620 and HT-29, were used. SW620 cells do not express EGFR whereas HT-29 cells do. Cells were treated with sulindac sulfide or vehicle for 48-72h or with 10X the amount of glucose found in their appropriate media. MTS proliferation assays were done with several doses of glucose. Standard immunoblotting techniques were used; lysates were separated onto SDS-PAGE gels and transferred onto PVDF membranes. Immunoblots were probed with EGFR or PCNA as a marker for proliferation. Blots were probed with β-actin as a loading control. Results: Treatment with sulindac sulfide resulted in a marked expression of EGFR in HT-29 cells (53.7% of control, p<0.005) with a concomitant reduction in proliferation as assessed by PCNA (59.7% of control, p<0.005). HT-29 cells when treated with glucose caused a dose-dependent curve assessed by MTS assay. Glucose at very high doses (10X the normal level of glucose was added to the media) caused a marked suppression of EGFR protein expression (53.8% of control in HT-29 cells) implicating decreased glucose homeostasis in HT-29. Finally, to further evaluate this we used SW620 cells, which do not express EGFR. In the absence of EGFR there was a strong increase in cell number as assessed by MTS and proliferation (129.3% of control, p<0.05). Conclusion: We demonstrate that NSAIDs cause a marked suppression in EGFR. For glucose, the relationship is more complex. If cells express EGFR at baseline, then a decrease in EGFR is noted with resulting suppression. On the other hand, if the cells are EGFR null, the glucose has a marked promotional effect. This work underscores the need to assess the EGFR context in both early and late colon carcinogenesis in designing interventional strategies.

Inhibition of histone deacetylase suppresses EGF signaling pathways by destabilizing EGFR mRNA in ER-negative human breast cancer cells.

Estrogen receptor alpha (ER)-negative human breast cancer cells frequently overexpress epidermal growth factor receptor (EGFR) and respond poorly to endocrine therapies. Our previous studies demonstrate that histone deacetylation plays a key role in ER gene silencing, and ER expression can be restored with histone deacetylase (HDAC) inhibitors in ER-negative human breast cancer cells. Whether inhibition of HDAC also alters epidermal growth factor (EGF) signaling pathways is not defined. Here we present evidence that reexpression of ER protein by a clinically available HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA or vorinostat), is coupled with loss of EGFR in ER-negative human breast cancer cells. Consistent with this observation, MDA-MB-231 cells, which are ER-negative and overexpress EGFR, that are engineered to express ER show a decrease in EGFR protein expression. Down-regulation of EGFR by SAHA results from attenuation of its mRNA stability. We also confirm that new protein synthesis is required for maintaining EGFR mRNA stability. Further experiments indicate that a decrease in EGFR abolished EGF-initiated signaling pathways including phosphorylated PAK1, p38MAPK and AKT. Thus, SAHA may not only reactivate silenced ER, but also simultaneously deplete EGFR expression. These data suggest that inhibition of HDAC is a promising epigenetic therapy for ER-negative human breast cancer.

(-)-Epigallocatechin gallate causes internalization of the epidermal growth factor receptor in human colon cancer cells

We recently found that the inhibitory effect of (-)-epigallocatechin gallate (EGCG) on epidermal growth factor (EGF) binding to the epidermal growth factor receptor (EGFR) is associated with alterations in lipid organization in the plasma membrane of colon cancer cells. Since changes in lipid organizations are thought to play a role in the trafficking of several membrane proteins, in this study we examined the effects of EGCG on cellular localization of the EGFR in SW480 cells. Treatment of the cells for 30 min with as little as 1 microg/ml of EGCG caused a decrease in cell surface-associated EGFRs and this was associated with internalization of EGFRs into endosomal vesicles. Similar effects were seen with a green fluorescent protein (GFP)-EGFR fusion protein. As expected, the EGFR protein was phosphorylated at tyrosine residues, ubiquitinated and partially degraded when the cells were treated with EGF, but treatment with EGCG caused none of these effects. The loss of EGFRs from the cell surface induced by treating the cells with EGF for 30 min persisted for at least 2 h. However, the loss of EGFRs from the cell surface induced by temporary exposure to EGCG was partially restored within 1-2 h. These studies provide the first evidence that EGCG can induce internalization of EGFRs into endosomes, which can recycle back to the cell surface. This sequestrating of inactivated EGFRs into endosomes may explain, at least in part, the ability of EGCG to inhibit activation of the EGFR and thereby exert anticancer effects.

Exisulind in Combination with Celecoxib Modulates Epidermal Growth Factor Receptor, Cyclooxygenase-2, and Cyclin D1 against Prostate Carcinogenesis:In vivoEvidence

Nonsteroidal anti-inflammatory drugs mediate anticancer effects by modulating cyclooxygenase-2 (COX-2)-dependent and/or COX-2-independent mechanism(s); however, the toxicity issue is a concern with single agents at higher doses. In this study, we determined the combined effect of celecoxib, a COX-2 inhibitor, along with exisulind (sulindac sulfone/Aptosyn) at low doses in prostate cancer. We used a sequential regimen of N-methyl-N-nitrosourea + testosterone to induce prostate cancer in Wistar-Unilever rats. Following carcinogen treatment, celecoxib and exisulind individually and their combination at low doses were given in NIH-07 diet for 52 weeks. We determined the incidence of prostatic intraepithelial neoplasia, adenocarcinomas, rate of tumor cell proliferation, and apoptosis. Immunohistochemical and Western blot analysis were done to determine COX-2, epidermal growth factor receptor (EGFR), Akt, androgen receptor, and cyclin D1 expression. Serum prostaglandin E2 and tumor necrosis factor-alpha levels were determined using enzyme immunoassay/ELISA assays. The rats that received celecoxib in combination with exisulind at low doses showed a significant decrease in prostatic intraepithelial neoplasia and adenocarcinomas as well as an enhanced rate of apoptosis. An overall decrease in COX-2, EGFR, Akt, androgen receptor, and cyclin D1 expression was found associated with tumor growth inhibition. Reduced serum levels of COX-2 protein, prostaglandin E2, and tumor necrosis factor-alpha indicated anti-inflammatory effects. A strong inhibition of total and phosphorylated form of EGFR (Tyr(992) and Tyr(845)) and Akt (Ser(473)) was significant in rats given with these agents in combination. In this study, we show for the first time that the combination of celecoxib with exisulind at low doses could prevent prostate carcinogenesis by altering key molecular events.

In vivo Therapeutic Synergism of Anti–Epidermal Growth Factor Receptor and Anti-HER2 Monoclonal Antibodies against Pancreatic Carcinomas

Pancreatic carcinoma is highly resistant to therapy. Epidermal growth factor receptor (EGFR) and HER2 have been reported to be both dysregulated in this cancer. To evaluate the in vivo effect of binding both EGFR and HER2 with two therapeutic humanized monoclonal antibodies (mAb), we treated human pancreatic carcinoma xenografts, expressing high EGFR and low HER2 levels. Nude mice, bearing xenografts of BxPC-3 or MiaPaCa-2 human pancreatic carcinoma cell lines, were injected twice weekly for 4 weeks with different doses of anti-EGFR (matuzumab) and anti-HER2 (trastuzumab) mAbs either alone or in combination. The effect of the two mAbs, on HER receptor phosphorylation, was also studied in vitro by Western blot analysis. The combined mAb treatment significantly inhibited tumor progression of the BxPC-3 xenografts compared with single mAb injection (P = 0.006) or no treatment (P = 0.0004) and specifically induced some complete remissions. The two mAbs had more antitumor effect than 4-fold greater doses of each mAb. The significant synergistic effect of the two mAbs was confirmed on the MiaPaCa-2 xenograft and on another type of carcinoma, SK-OV-3 ovarian carcinoma xenografts. In vitro, the cooperative effect of the two mAbs was associated with a decrease in EGFR and HER2 receptor phosphorylation. Anti-HER2 mAb has a synergistic therapeutic effect when combined with an anti-EGFR mAb on pancreatic carcinomas with low HER2 expression. These observations may open the way to the use of these two mAbs in a large panel of carcinomas expressing different levels of the two HER receptors.

Effect of the anti-receptor ligand-blocking 225 monoclonal antibody on EGF receptor endocytosis and sorting

The anti-receptor antibody, 225 mAb, is known to block binding of ligand to the epidermal growth factor receptor (EGFR). However, the effect of this neutralizing antibody on EGFR endocytosis, trafficking and degradation remains unclear. Here, we demonstrate that endocytosis of 125I-225 mAb occurs, albeit with a slower rate than that of EGF. Using pulse chase assays, we show that internalized 125I-225 mAb is recycled to the surface much more efficiently than internalized 125I-EGF. Also, we found that internalization of 125I-225 mAb, in contrast to that of EGF, is independent of receptor tyrosine kinase activity, as evidenced by its insensitivity to AG1478, a specific EGFR tyrosine kinase inhibitor. Analysis of the levels of cell surface and total EGFR showed that treatment with 225 mAb results in a 30–40% decrease in surface EGFR and a relatively slow downregulation of total EGFR. Taken together, these data indicate that 225 mAb induces internalization and downregulation of EGFR via a mechanism distinct from that underlying EGF-induced EGFR internalization and downregulation.

Treatment of Human Tumor Xenografts with Monoclonal Antibody 806 in Combination with a Prototypical Epidermal Growth Factor Receptor–Specific Antibody Generates Enhanced Antitumor Activity

Monoclonal antibody (mAb) 806 is a novel epidermal growth factor receptor (EGFR) antibody with significant antitumor activity that recognizes a mutant EGFR commonly expressed in glioma known as delta2-7 EGFR (de2-7 EGFR or EGFRvIII) and a subset of the wild-type (wt) EGFR found in cells that overexpress the receptor. We have used two human xenograft mouse models to examine the efficacy of mAb 806 in combination with mAb 528, a prototypical anti-EGFR antibody with similar specificity to cetuximab. Treatment of nude mice, bearing s.c. or i.c. tumor human xenografts expressing the wt or de2-7 EGFR, with mAbs 806 and 528 in combination resulted in additive and in some cases synergistic, antitumor activity. Interestingly, mAb 528 was also effective against xenografts expressing the ligand independent de2-7 EGFR when used as a single agent, showing that its antitumor activity is not merely mediated through inhibition of ligand binding. When used as single agents, neither mAbs 806 or 528 induced down-regulation of the de2-7 EGFR either in vitro or in vivo. In contrast, the combination of antibodies produced a rapid and dramatic decrease in the total cell surface de2-7 EGFR both in vitro and in xenografts. Consistent with this decrease in total cell surface de2-7 EGFR, we observed up-regulation of the cell cycle inhibitor p27(KIP1) and a decrease in tumor cell proliferation as measured by Ki-67 immunostaining when the antibodies were used in combination in vivo. Thus, mAb 806 can synergize with other EGFR-specific antibodies thereby providing a rationale for its translation into the clinic.

Herpes simplex virus 1 infected cell protein 0 forms a complex with CIN85 and Cbl and mediates the degradation of EGF receptor from cell surfaces

Infected cell protein 0 (ICP0) is a 775-residue multifunctional herpes simplex virus protein associated with numerous functions related to transactivation of gene expression and repression of host defenses to infection. We report that an uncharted domain of ICP0 located between residues 245 and 510 contains multiple SH3 domain binding motifs similar to those required for binding to CIN85, the M(r) 85,000 protein that interacts with Cbl. CIN85 and Cbl are involved in endocytosis and negative regulation of numerous receptor tyrosine kinases. We report that ICP0 binds CIN85 in a reciprocal manner and that the complexes pulled down by ICP0 also contain Cbl. We tested the role of ICP0 in the down-regulation of receptor tyrosine kinases by using epidermal growth factor receptor (EGFR) as a prototypic receptor. In transfection assays, ICP0, in the absence of other viral genes, down-regulated EGF-dependent expression of a reporter gene (luciferase). ICP0 also down-regulated both total and cell surface levels of EGFR in EGF-independent manner. In wild-type virus-infected cells, the surface levels of EGFR were also decreased in the absence of EGF stimulation. Stimulation by EGF enhanced the decrease in surface EGFR. We conclude that ICP0 encodes SH3 domain binding sites that function to down-regulate signaling pathways associated with receptor tyrosine kinases. The results suggest that ICP0 precludes signaling to the infected cells through the receptor tyrosine kinases.

Imatinib mesylate induces apoptosis in human cholangiocarcinoma cells

Cholangiocarcinoma is a highly malignant, usually fatal cancer with limited therapeutic options. Receptor tyrosine kinases contribute to the development and progression of this cancer. The relatively selective tyrosine kinase inhibitor imatinib mesylate (STI-571 or Gleevec(R)) has recently been licensed. However, the ability of this drug to inhibit signal transduction and induce apoptosis in human cholangiocarcinoma cells is incompletely studied. Thus, our goal was to examine the ability of STI-571 to induce apoptosis in KMCH-1 cells, a human cholangiocarcinoma cell line. Apoptosis was assessed morphologically and also biochemically by measuring caspase activity and the mitochondrial membrane potential. STI-571 induced apoptosis and inhibited growth of KMCH-1 cells in a time- and concentration-dependent manner. The induction of apoptosis was accompanied by mitochondrial depolarization followed by a 4.5-fold increase in caspase activation and was abrogated by the pancaspase inhibitor z-VAD(OMe)-fmk. Interestingly, cholangiocarcinoma cells do not express detectable PDGFR, c-Abl or c-Kit, which are protein kinases known to be directly inhibited by STI-571. However, a significant decrease in epidermal growth factor receptor (EGFR) and focal adhesion kinase (FAK) phosphorylation was observed following treatment with STI-571. This decrease in EGFR and FAK phosphorylation was associated with a reduction in Akt activity resulting in loss of Mcl-1, a potent anti-apoptotic Bcl-2 family protein. These results indicate that STI-571 induces caspase-dependent apoptosis in a human cholangiocarcinoma cell line and suggest that STI-571 might warrant further investigation as a possible agent for treatment of human cholangiocarcinoma.

Oestrogen receptor-mediated modulation of the EGFR/MAPK pathway in tamoxifen-resistant MCF-7 cells.

Oestrogen receptor (ER) levels are usually maintained on acquisition of tamoxifen resistance in the clinic, however, tumour re-growth is associated with increased expression of epidermal growth factor receptor (EGFR) and activation of the mitogen activated protein kinase (MAPK) pathway. In the present study we have used the ER down-regulator fulvestrant ('Faslodex') to investigate the influence of the ER on growth of a tamoxifen-resistant (TAM-R) human breast cancer cell line. Expression levels of ER mRNA and protein were equivalent in parental wild-type MCF-7 (WT) and TAM-R cells. Fulvestrant eliminated ER protein expression and inhibited proliferation in both cell lines. The growth inhibitory effects of fulvestrant were associated with a decrease in basal EGFR, c-erbB2 and ERK1/2 activity in TAM-R but not WT cells. ER functionality as determined by oestrogen response element (ERE)-luciferase reporter activity and expression of PgR, pS2 and transforming growth factor alpha (TGFalpha) was significantly reduced in TAM-R compared to WT cells and was further decreased by fulvestrant treatment in both cell lines. Epidermal growth factor (EGF) and TGFalpha significantly increased EGFR/MAPK pathway activity in both cell lines. Ligand-induced EGFR/MAPK activation promoted TAM-R cell growth in both the absence and presence of fulvestrant, whereas no proliferative activity was observed under the same conditions in WT cells. These results suggest that the ER modulates EGFR/MAPK signalling efficiency in TAM-R cells possibly through the regulation of TGFalpha availability. This effect may be overcome by the action of exogenous EGFR ligands, which strengthen EGFR/MAPK signalling activity to generate endocrine-insensitive cell growth.