Densitometry of immunoblots was performed using Adobe Photoshop and normalized to controls. Immunoprecipitation. maximal phosphorylation was observed 5 to 10 min after addition of 30 nM TCDD. Both TCDD-induced ERK1/2 phosphorylation and cell proliferation were abolished by AhR small interfering RNA, AhR-specific inhibitor “type”:”entrez-nucleotide”,”attrs”:”text”:”CH223191″,”term_id”:”44935898″,”term_text”:”CH223191″CH223191, Src kinase inhibitor PP2, neutralizing antibodies against matrix metalloproteinase 7, heparin-binding-EGF-like growth factor and EGFR, EGFR inhibitors (AG1478 and PD168393), and MEK1 inhibitor PD98059. Coimmunoprecipitation experiments revealed that AhR forms a protein complex with Src and regulates Src activity by phosphorylating Src (Tyr416) and dephosphorylating Src (Tyr527). These data support novel observations that, in human colon cancer cells, Src-mediated cross talk between aryl hydrocarbon and EGFR results in ERK1/2 activation, thereby stimulating cell proliferation. mice, Kawajiri et al. (21) showed that activation of AhR with natural ligands suppresses intestinal tumor formation. Using AhR-deficient mice, Fan et al. (13) showed that AhR Rabbit Polyclonal to Granzyme B functioned as a tumor suppressor for liver carcinogenesis. Previously, we identified cross talk between the M3 muscarinic receptor, a G-protein-coupled receptor, and EGFR, a receptor tyrosine kinase (7, 9, 44). We demonstrated that M3 muscarinic receptor interacted with EGFR through activation of matrix metalloproteinase 7 (MMP7) and release of an EGFR ligand, heparin binding EGF-like growth factor. As a consequence of this interaction, muscarinic agonists stimulate colon cancer cell proliferation. These observations suggested to us that, in human intestinal epithelial cells, there might exist other examples of cross talk between EGFR and signaling pathways besides those with G-protein-coupled Dynasore receptors. The objective of the present Dynasore study was to seek evidence for AhR cross talk with EGFR and elucidate effects of this interaction. In particular, we asked whether activation of AhR results in pro-proliferative effects in human colon epithelial cells and what were the molecular mechanisms underlying these actions. Our results include the novel observation that TCDD-AhR interaction induces robust proliferation of human colon cancer cells through Src-mediated EGFR activation. MATERIALS AND METHODS Reagents. TCDD, MC, indirubin, indole-3-carbinol (I3C), Dynasore DIM, PP2, “type”:”entrez-nucleotide”,”attrs”:”text”:”CH223191″,”term_id”:”44935898″,”term_text”:”CH223191″CH223191, and Tiron were purchased from Sigma-Aldrich; PD168393, AG1478, PD98059, U0126, LY294002, PP3, GM6001, and GM6001 negative control (NC) from Calbiochem; CellTiter 96 AQueous one solution cell proliferation assay (MTS) kit from Promega; RPMI 1640, DMEM, and McCoy’s 5A growth media from Mediatech. TCDD was supplied in toluene solution. All other chemical inhibitors were dissolved in DMSO unless specified. Cell culture. H508 and SNU-C4 human colon cancer cells (ATCC) were grown in RPMI 1640 growth press Dynasore supplemented with 10% FBS. Adherent cells were passaged weekly at subconfluence after trypsinization. Ethnicities were managed in incubators at 37C in an atmosphere of 5% CO2-95% air flow. Cell proliferation assay. Cells were seeded in 96-well plates at 10% confluence and allowed to attach for 24 h. After 18 h of serum starvation, 200 l new serum-free growth press containing test agents were added. When chemical inhibitors and antibodies were used, they were added 30 min and 2 h, respectively, before test agents. All samples contained the same concentrations of solvents, i.e., either 0.1% toluene (TCDD and MC), 0.1% DMSO (“type”:”entrez-nucleotide”,”attrs”:”text”:”CH223191″,”term_id”:”44935898″,”term_text”:”CH223191″CH223191, PP2, PP3, PD168393, AG1478, PD98059, U0126, LY294002, GM6001, GM6001NC, and indirubin), 0.1% ethanol (I3C and DIM), or mixtures. Cells were incubated for 5 days at 37C in an atmosphere of 5% CO2-95% air flow without further improvements of test providers. After a 5-day time incubation, cell proliferation was determined by addition 20 l CellTiter 96 AQueous One remedy (Promega) to each well. After a 1- to 2-h incubation at 37C, absorbance was measured at 490 nm using a 96-well microtiter plate reader (SpectraMax384). Antibodies and immunoblotting. Rabbit polyclonal anti-ERK1/2 (p44/42) MAPK, mouse monoclonal anti-phospho-ERK1/2 MAPK, rabbit monoclonal anti-EGFR, rabbit polyclonal anti-phospho-EGFR (Tyr845), rabbit polyclonal anti-phospho-EGFR (Tyr992), rabbit polyclonal anti-phospho-EGFR (Tyr1173), rabbit polyclonal anti-phospho-Src (Tyr416), rabbit polyclonal anti-phospho-Src (Tyr527), and rabbit polyclonal anti-Src antibodies were from Cell Signaling Technology. Goat polyclonal anti-AhR and mouse monoclonal anti-AhR antibodies were from Santa Cruz Biotechnology and Sigma, respectively. Neutralizing anti-EGFR antibody (LA1) was from Millipore. Neutralizing anti-MMP7 and anti-heparin-binding-EGF-like growth element (HBEGF) antibodies were from R&D Systems. Immunoblotting was performed as explained previously (7). For chemical inhibitors (“type”:”entrez-nucleotide”,”attrs”:”text”:”CH223191″,”term_id”:”44935898″,”term_text”:”CH223191″CH223191, PP2, PP3, AG1478, PD168393, PD98059, U0126, LY294002, wortmannin, GM6001, and GM6001NC) that are soluble only in DMSO, all samples.
- The authors wish to acknowledge The Childrens Memorial Health Institute, Warsaw, Poland for the access to Q-TOF LC/MS; mass spectrometer purchase was supported by European POIG
- Notably, we found that the presence of sodium cholate in the assay buffer is definitely imperative to achieve separation and prevent aggregation of lipids
- The reaction was stirred at 23 C for 30 min
- The total email address details are representative of three separate experiments
- A) PCR was performed using primers for the wild-type Crry gene as well as for the Neo put utilized to disrupt the gene