Prostaglandins, acting through different receptors of the GPCR family, regulate many cellular functions [27]. In epithelial cells, prostaglandins often enhance proliferation and survival, and several lines of evidence implicate them in oncogenesis [28]. In many tumours, cyclooxygenases (COX-1 and COX-2), which catalyze the rate-limiting step in prostaglandin synthesis, are overexpressed, and the levels of prostaglandins, notably prostaglandin E2 (PGE2), are elevated [28–31]. In hepatocytes,

PGE2 and other prostaglandins enhance learn more DNA synthesis [15, 32–34], and COX-2 is overexpressed in many hepatocarcinomas [35, 36]. In the study presented here we examined the Morris hepatocarcinoma cell line MH1C1, which was chosen due to its Eltanexor research buy responsiveness to both EGF and the prostaglandins PGE2 and PGF2α, and investigated the interaction between the pathways mediated by prostaglandin receptors and EGFR. We previously observed that while there was no evidence of transactivation of EGFR induced by prostaglandins or other GPCR agonists in hepatocytes, PGE2 induced phosphorylation of the EGFR in the MH1C1 cells [37, 38]. We have now investigated further the signalling mechanisms involved in this effect. Methods Chemicals Dulbecco’s Modified Eagle’s Medium, Dulbecco’s phosphate-buffered saline, William’s Medium E, glutamine, and Pen-Strep (10.000 U/ml) were from Lonza(Verviers,

Belgium). HEPES was from Gibco (Grand Island, NY). Dexamethasone, insulin, bovine serum albumin, collagen (type I, rat tail), prostaglandin F2α (Tris salt) and epidermal growth factor

(EGF) were obtained from Sigma-Aldrich selleckchem (St.Louis, MO). GF109203X ([2-[1-(3-dimetylaminopropyl)-1 H-indol-3-yl]-male-imide]) and GM6001/Galardin (N-[(2R)-2 (hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide) were from Calbiochem (San Diego, CA). Gefitinib was a gift from AstraZeneca (Cheshire, UK). [6-3 H]thymidine (20–30 Ci/mmol) and myo-[2-3 H]inositol (15.0 Ci/mmol) were from PerkinElmer (Boston, MA). AL8810 (9α,15R-dihydroxy-11β-fluoro-15-(2,3-dihydro-1 H-inden-2-yl)-16,17,18,19,20-pentanor-prosta-5Z,13E-dien-1-oic acid),L161982 (N-[[4'-[[3-butyl-1,5-dihydro-5-oxo-1-[2-(trifluoromethyl)phenyl]-4 H-1,2,4-triazol-4-yl]methyl][1,1'-biphenyl]-2-yl]sulfonyl]-3-methyl-2-thiophenecarboxamide), (+)fluprostenol, Oxymatrine and prostaglandin E2 (PGE2) were from Cayman Chemical (Ann Arbor, MI). SC51322 (8-chloro-2-[3-[(2-furanylmethyl)thio]-1-oxopropyl]hydrazide, dibenz[b,f][1,4]oxazepine-10(11 H)-carboxylic acid) was obtained from BIOMOL Research Laboratories (Plymouth Meeting, PA). The Src inhibitor CGP77675 was a gift from Novartis Pharma AG (Basel, Switzerland). All other chemicals were of analytical quality. Antibodies against phosphorylated AktSer473, total Akt, dually phosphorylated ERKThr202/Tyr204, GAPDH and phospho-ShcTyr239/240 were obtained from Cell Signaling Technology (Boston, MA).