Activation of EGFR/ERBB2 via Pathways Involving ERK1/2, P38 MAPK, AKT and FOXO Enhances Recovery of Diabetic Hearts from Ischemia-Reperfusion Injury

This study characterized the effects of diabetes and/or ischemia on epidermal growth factor receptor, EGFR, and/or erbB2 signaling pathways on cardiac function. Isolated heart perfusion model of global ischemia was used to study the effect of chronic inhibition or acute activation of EGFR/erbB2 signaling on cardiac function in a rat model of type-1 diabetes. Induction of diabetes with streptozotocin impaired recovery of cardiac function (cardiac contractility and hemodynamics) following 40 minutes of global ischemia in isolated hearts. Chronic treatment with AG825 or AG1478, selective inhibitors of erbB2 and EGFR respectively, did not affect hyperglycemia but led to an exacerbation whereas acute administration of the EGFR ligand, epidermal growth factor (EGF), led to an improvement in cardiac recovery in diabetic hearts. Diabetes led to attenuated dimerization and phosphorylation of cardiac erbB2 and EGFR receptors that was associated with reduced signaling via extracellular-signal-regulated kinase 1/2 (ERK1/2), p38 mitogen activated protein (MAP) kinase and AKT (protein kinase B). Ischemia was also associated with reduced cardiac signaling via these molecules whereas EGF-treatment opposed diabetes and/or ischemia induced changes in ERK1/2, p38 MAP kinase, and AKT-FOXO signaling. Losartan treatment improved cardiac function in diabetes but also impaired EGFR phosphorylation in diabetic heart. Co-administration of EGF rescued Losartan-mediated reduction in EGFR phosphorylation and significantly improved cardiac recovery more than with either agent alone. EGFR/erbB2 signaling is an important cardiac survival pathway whose activation, particularly in diabetes, ischemia or following treatment with drugs that inhibit this cascade, significantly improves cardiac function. These findings may have clinical relevance particularly in the treatment of diabetes-induced cardiac dysfunction

Single-step synthesis of chemically cross-linked polysilastyrene and its conversion to β -silicon carbide

A new method for chemically cross-linking polysilastyrene using divinylbenzene as the cross-linking agent is reported. The procedure involves a single-step synthesis using the alkali-metal sodium to promote the polymerization of dimethyldichlorsilane in the presence of the comonomers phenylmethyldichlorosilane and divinylbenzene. The cross-linked polymer can be readily converted to β-SiC on pyrolysis at 1500° C. The β -SiC obtained by this procedure is nanocrystalline and has a grain-size distribution of 8-20 nm

Cationic Polyamidoamine Dendrimers As Modulators Of Egfr Signaling In Vitro And In Vivo

Cationic polyamidoamine (PAMAM) dendrimers are branch-like spherical polymers being investigated for a variety of applications in nanomedicine including nucleic acid drug delivery. Emerging evidence suggests they exhibit intrinsic biological and toxicological effects but little is known of their interactions with signal transduction pathways. We previously showed that the activated (fragmented) generation (G) 6 PAMAM dendrimer, Superfect (SF), stimulated epidermal growth factor receptor (EGFR) tyrosine kinase signaling - an important signaling cascade that regulates cell growth, survival and apoptosis-in cultured human embryonic kidney (HEK 293) cells. Here, we firstly studied the in vitro effects of Polyfect (PF), a non-activated (intact) G6 PAMAM dendrimer, on EGFR tyrosine kinase signaling via extracellular-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) in cultured HEK 293 cells and then compared the in vivo effects of a single administration (10mg/kg i.p) of PF or SF on EGFR signaling in the kidneys of normal and diabetic male Wistar rats. Polyfect exhibited a dose- and time-dependent inhibition of EGFR, ERK1/2 and p38 MAPK phosphorylation in HEK-293 cells similar to AG1478, a selective EGFR inhibitor. Administration of dendrimers to non-diabetic or diabetic animals for 24h showed that PF inhibited whereas SF stimulated EGFR phosphorylation in the kidneys of both sets of animals. PF-mediated inhibition of EGFR phosphorylation as well as SF or PF-mediated apoptosis in HEK 293 cells could be significantly reversed by co-treatment with antioxidants such as tempol implying that both these effects involved an oxidative stress-dependent mechanism. These results show for the first time that SF and PF PAMAM dendrimers can differentially modulate the important EGFR signal transduction pathway in vivo and may represent a novel class of EGFR modulators. These findings could have important clinical implications for the use of PAMAM dendrimers in nanomedicine

EVALUATION OF IN VITRO ANTICANCER AND ANTIOXIDANT ACTIVITIES FROM LEAF EXTRACTS OF MEDICINAL PLANT CLIDEMIA HIRTA

Objective: To evaluate the anticancer and antioxidant activity of medicinal plant Clidemia hirta extracted in different solvents.Methods: Crude extracts were prepared from the leaves of Clidemia hirta using ethanol, petroleum ether and chloroform solvents. Anticancer activities and antioxidant properties were assayed using standard yellow dye 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and 1, 1-diphenyl-2-picryl hydrazyl (DPPH) free radical scavenging assay respectively.Results: We found that the ethanol extract had higher inhibition activities against Dalton's lymphoma ascites (DLA) cancer cell line, 50% DLA cell line inhibition at 68µg/ml, while 50% inhibition by petroleum ether and chloroform extracts were at 160µg/ml and 172µg/ml, respectively. The antioxidant activity requires5µg/ml of ethanol extract to trap 50% of DPPH (IC50), whereas the positive control ascorbic acid trapped 50% of DPPH (IC50) at 3.5µg/ml.Conclusion: The prepared leaf extracts with different solvents of Clidemia hirta showed the antiproliferative and antioxidant activity in dose-dependent manner. Further works is required to identify the biologically active chemical constituents, responsible for cancer cell growth inhibition from this plant

Chronic administration of nano-sized PAMAM dendrimers in vivo inhibits EGFR-ERK1/2-ROCK signaling pathway and attenuates diabetes-induced vascular remodeling and dysfunction.

We investigated whether chronic administration of nano-sized polyamidoamine (PAMAM) dendrimers can have beneficial effects on diabetes-induced vascular dysfunction by inhibiting the epidermal growth factor receptor (EGFR)-ERK1/2-Rho kinase (ROCK)-a pathway known to be critical in the development of diabetic vascular complications. Daily administration of naked PAMAMs for up to 4 weeks to streptozotocin-induced diabetic male Wistar rats inhibited EGFR-ERK1/2-ROCK signaling and improved diabetes-induced vascular remodeling and dysfunction in a dose, generation (G6 > G5) and surface chemistry-dependent manner (cationic > anionic > neutral). PAMAMs, AG1478 (a selective EGFR inhibitor), or anti-EGFR siRNA also inhibited vascular EGFR-ERK1/2-ROCK signaling in vitro. These data showed that naked PAMAM dendrimers have the propensity to modulate key (e.g. EGFR) cell signaling cascades with associated pharmacological consequences in vivo that are dependent on their physicochemical properties. Thus, PAMAMs, alone or in combination with vasculoprotective agents, may have a beneficial role in the potential treatment of diabetes-induced vascular complications.This research is funded by a grant from the Research Sector at Kuwait University (MR01/13). We also acknowledge support from the OMICS Research Unit/RCF and the General Facility Grant (SRUL02/13)

Schematic model summarising our findings on the role of EGFR/erbB2 signaling in diabetes-induced cardiac dysfunction.

<p>Diabetes and/or hyperglycemia via attenuation of the EGFR/erbB2 signaling and through subsequent modulation of downstream effectors such as ERK1/2, p38 MAPK or AKT/FOXO can lead to cardiac dysfunction. The effects of diabetes on EGFR/erbB2 pathway are exacerbated by blockade of this pathway by AG1478 or AG825 which leads to worsening cardiac recovery from I/R. However, the inhibitory effects of diabetes on EGFR/ErbB2 pathway may be opposed by administering EGF that also leads to improved cardiac function. The Angiotensin II (Ang II)/AT₁ receptor pathway can also activate EGFR/erbB2 pathway that can be blocked by Losartan. Co-administration of EGF with Losartan attenuates losartan-mediated EGFR blockade and improves cardiac function in diabetes beyond that attained by either drug alone.</p

Diabetes is associated with reduced expression and phosphorylation of EGFR receptor at multiple tyrosine sites that can be further inhibited by chronic treatment with AG1478.

<p><b>a</b>) Representative Western blots showing levels of phosphorylated EGFR at Y992, Y1068, Y1086, and Y1148 as well as total EGFR (t-EGFR) and Actin as a control protein in non-diabetic control hearts (C), diabetic hearts (D) and diabetic hearts chronically treated with AG1478 (+AG1478). <b>b</b>) quantification of EGFR expression relative to actin and <b>c–f</b>) quantification of EGFR phosphorylation at the stated tyrosine site relative to total EGFR expression for all the groups studied by densitometry. N=4; * significantly different from control (p<0.05); ** significantly different from diabetes (p<0.05).</p

Acute EGF treatment in diabetic hearts opposes the diabetes and/or ischemia-induced changes in phosphorylation of EGFR/erbB2 signaling cascade.

<p><b>a</b>) A representative Western blot of phosphorylation changes in key molecules following acute administration of EGF before ischemia (DEP) or after ischemia (DER) is compared to diabetic hearts subjected to 40 mins ischemia (DI); b–g) densitometry plots quantifying the relative intensity of bands for the stated molecule relative to actin. N=4; * significantly different to DI.</p

Diabetes is associated with reduced expression and phosphorylation of erbB2 receptor at multiple tyrosine sites that can be further inhibited by chronic treatment with AG825.

<p><b>a</b>) Representative Western blots showing levels of phosphorylated erbB2 at Y877, Y1248, Y1248-a (which represents detection of Y1248 using an alternative antibody (p- erbB2-Antibody (Tyr1248)/EGFR (Tyr1173)) and Y12221/2 as well as total erbB2 (t-erbB2) and actin as a control protein in non-diabetic control hearts (C), diabetic hearts (D) and diabetic hearts chronically treated with AG825 (+AG825). <b>b</b>) quantification of erbB2 expression relative to actin and <b>c–f</b>) quantification of erbB2 phosphorylation at the stated tyrosine site relative to total erbB2 expression for all the groups studied by densitometry. N=4; * significantly different from control (p<0.05); ** significantly different from diabetes (p<0.05).</p