St louis pancreatic cancer treatment

Glucagon-like peptide 1 (GLP-1) (7-36) amide, a potent glucoincretin hormone (1,2), is secreted by the intestinal L-cells in response to fat meals and carbohydrates (3,4). It is a potentially important drug in the treatment of type 2 diabetes in light of its ability to improve insulin secretion in both subjects with impaired glucose tolerance and type 2 diabetes (5,6). GLP-1 is also an insulinotropic agent through its ability to stimulate insulin gene expression and proinsulin biosynthesis (7,8). GLP-1 increases the expression level of the [beta]-cell-specific transcription factor pancreatic and duodenal homeobox gene-1 (PDX-1) (9), which is implicated in the regulation of the expression of insulin, GLUT2, and glucokinase genes and in [beta]-cell differentiation (10-12). In addition, the glucoincretin increases in vitro [beta]-cell proliferation nonadditively with glucose via a phosphatidylinositol (PI) 3-kinase/protein kinase C [xi] signaling pathway in [beta](INS-I) cells (9,13), as well as the islet mass in mouse pancreas in vivo (14). Finally, GLP-1 induces several immediate early response genes and proto-oncogenes in INS cells that are implicated in cell growth/apoptosis control, such as c-fos, c-jun, junD, and nur77 (15,16).

GLP-1 signal transduction in the [beta]-cell is now being extensively studied. GLP-1 interaction with its specific high-affinity receptor (GLP-1R), a member of the G protein-coupled receptor (GPCR) superfamily, increases cAMP levels in several [beta]-cell models to activate the protein kinase A signal transduction system (4,17,18). There is also evidence that this rise in cAMP levels leads to an increase in cytosolic [Ca.sub.2+] (19,20), possibly via cAMP-regulated guanine nucleotide exchange factor II (Epac2) (21). However, the precise mechanism by which GLP-1R activates the PI 3-kinase signaling pathway to mediate the proliferative action of GLP-1 has not been elucidated.

Recent studies investigating the mitogenic effects of GPCR agonists have led to the concept that some GPCRs that lack intrinsic kinase activity transactivate the epidermal growth factor (EGF) receptor (EGFR) to promote cell proliferation and mitogenic signal transduction pathways (22-24). The EGFR has been recently identified as an essential element in the GPCR-mediated activation of mitogen-activated protein kinase and PI 3-kinase signaling pathways in cells treated with various GPCR agonists, such as lysophosphatidic acid (23,24), thrombin (24), and angiotensin II (22,25). In some instances, the EGFR transactivation by GPCRs has been documented to require the non-receptor-type tyrosine kinase c-Src (26,27). The mechanism involves the production of endogenous EGFR ligands from transmembrane precursors via GPCR-induced c-Src activation of an endoprotease (28).

Receptor molecules for EGF-like growth factors are encoded by the c-erbB gene and its relatives. Upon binding of their ligands, EGFRs undergo dimerization and autophosphorylation on tyrosine residues in order to recruit Src homology 2-containing proteins responsible for signal transduction (24). There are at least four known members for the EGFR family: c-erbB-1 (EGFR), c-erbB2, c-erbB-3, and c-erbB4. The c-erbB-1/EGFR is expressed throughout the human fetal pancreas and in adult [beta]-cells (29). Huotari et al. (29) showed that only the c-erbB-1/EGFR gene is expressed in INS-1 cells and that betacellulin (BTC), a member of the EGF family and activator of erbB-1/EGFR and erbB-4 (30), displayed mitogenic activity as it stimulated INS-1 cell replication at picomolar concentrations (29). BTC, a 9.5-kDa glycoprotein expressed in several mouse tissues, including kidney, liver, and pancreas (31), is synthesized from a 32-kDa membrane-anchored precursor that is thought to be proteolitically cleaved to generate an active and soluble secreted form of the molecule (32), as occurs with other members of the EGF family. Since BTC is expressed in all pancreatic [beta]-cell types examined thus far, such as various insulinomas (33), the [beta]-cell line BTC3 (31), and normal [beta]-cells (34), as well ms in pancreatic duct cells (34), it is reasonable to think that BTC could link the GLP-1R to the EGFR and be responsible for the mitogenic action of GLP-1 on the [beta]-cell.

In this study, our aim was to investigate the possible implication of EGFR transactivation in GLP-1-induced [beta]-cell proliferation and PI 3-kinase stimulation. Using both pharmacological and molecular biology approaches, we provide evidence supporting the concept that e-Src-dependent transactivation of the EGFR and proteolytic processing of membrane-anchored BTC or other EGF-like ligands link GLP-1R signaling to PI 3-kinase activation and [beta]-cell proliferation.

RESEARCH DESIGN AND METHODS

Reagents. Pertussis toxin was purchased from Calbiochem (La Jolla, CA). AG1478 and PP1 were purchased from Biomol (Plymouth Meeting, PA). Human GLP-1 fragment 7-36 amide and human recombinant BTC were obtained from Sigma (St. Louis, Me). The anti-EGFR antibody was purchased from Cell Signaling Technology (Mississauga, ON). The PY20 and 4G10 anti-phosphotyrosine antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA) and Upstate Biotechnology (Lake Placid, NY), respectively. Monoclonal anti-BTC primary antibody was from R&D System (Minneapolis, MN). GM6001, a pan-specific pharmacological metalloproteinase inhibitor, was from Chemicon International (Temecula CA). RPMI 1640 and the cell culture supplements, including FCS, were purchased from Gibco BRL (Burlington, Canada). Methyl-[[sup.3]H]thymidine was from ICN (Costa Mesa, CA). Protein concentrations were determined using the Bio-Rad protein assay (Bin Rad, Hercules, CA).

Cell culture and incubation. INS832/13 (35) cells (passages 36-70) were grown in monolayer cultures as described previously (36) in regular RPMI1640 medium supplemented with 10 mmol/l HEPES, 10% heat-inactivated FCS, 2 mmol/l L-glutamine, 1 mmol/l sodium pyruvate, 50 [micro]mol/l [beta]-mercaptoethanol, 100 IU/ml penicillin, and 500 [micro] g/ml streptomycin at 37[degrees]C in a humidified (5% C[O.sub.2]/95% air) atmosphere. This clone (832/13) of INS-1 cell was used because it shows better differentiation characteristics in terms of glucose-stimulated insulin secretion than the original INS 1 cell line (35). When cells reached 80% confluence (after ~7 days), they were washed with PBS and preincubated at 37[degrees]C for 90 rain in a Krebs-Ringer bicarbonate HEPES (KRBH) medium containing 1 mmol/l [CaCl.sub.2], 5 mmol/l [NaHCO.sub.3], 25 mmol/l HEPES (pH 7.4) supplemented with 3 mmol/l glucose, and 0.1% defatted BSA (Fraction V; Sigma). The pharmacological inhibitors were added to the medium during the last 30 rain of the preincubation period. Cells were then washed with PBS and incubated for the indicated times in the same supplemented KRBH medium containing the substances to be tested.

LNCaP cells were kindly given by Dr. Mes-Masson (University of Montreal) and cultured as described above for INS(832/13) cells.

[[sup.3]H]thymidine incorporation assay. A previously described procedure (9,37) was used for [3H]thymidine incorporation assay. In brief, cells were seeded 2 (lays before use in 96-well plates [8 X [10.sup.4] INS(832/13) cells or 1.5 x [10.sup.4] LNCaP cells per well] and cultured in regular complete RPMI medium ms described above. Cells were then washed with PBS and preincubated for a period of 24 h in minimal RPMI medium (i.e., without serum but with 3 mmol/l glucose and 0.1% BSA). They were then incubated for 24 h in minimal RPMI medium with various test substances. Proliferation was determined by incorporation of [[sup.3]H]thymidine (1 pCi/well) during the final 4 h of the 24-h incubation period. Cells were then harvested with a PHD cell harvester from Cambridge Technology (Watertown, MA), and the radioactivity retained on the dried glass fiber filters was measured by liquid scintillation.