ABSTRACT
OBJECTIVE Pancreatic ductal adenocarcinoma (PDAC), a lethal cancer in need of new, effective therapies, has a unique tumor microenvironment characterized by a dense fibrotic stroma (desmoplasia) that is generated by pancreatic cancer- associated fibroblasts (PCAFs) derived from pancreatic stellate cells (PSCs) and pancreatic fibroblasts (PFs). METHEDS and RESULTS Hypothe?sizing that G protein-coupled receptors (GPCRs) may regulate PCAFs, we used an unbiased GPCRomic array approach to compare GPCR expression in PCAFs, PFs and PSCs and identified 82 GPCRs commonly expressed by PCAFs derived from primary tumors of five PDAC patients. We discovered that PCAFs have increased expression of numerous GPCRs, in particular aGPCR with much higher expression in PCAFs compared to both PFs and PSCs. Immunohistochemistry revealed increased expression of this GPCR in PDAC tumors. Co- culture of PSCs with PDAC cells or incubation with TNFα induced its expression. Activation of the GPCR in PCAF sincreased expression of interleukin-6 (IL-6) via a cAMP/PKA/CREB signaling pathway. GPCR knockdown with siRNA diminished IL-6 production and secretionby PCAFs and ability of PCAF conditioned media to enhance proliferation of PDAC cells. CONCLUSION We conclude that PDAC cells induce expression by PCAFs of a novel GPCR, resulting in increased IL-6 production by PCAFs and promotion of PDAC cell proliferation. This PCAF-expressed GPCR thus contributes to PDAC cell-PCAF interaction and as such, may be a novel therapeutic target for PDAC tumors.
ABSTRACT
Background The sea cucumber lysozyme belongs to the family of invertebrate lysozymes and is thought to be a key defense factor in protecting aquaculture animals against bacterial infection. Recently, evidence was found that the sea cucumber lysozyme exerts broad spectrum antimicrobial action in vitro against Gram-negative and Gram-positive bacteria, and it also has more potent antimicrobial activity independent of its enzymatic activity. To explore the antimicrobial role of this non-enzymatic lysozyme and model its structure to novel antimicrobial peptides, the peptide from the C-terminal amino acid residues 70-146 of the sea cucumber lysozyme in Stichopus japonicus (SjLys-C) was heterologously expressed in Escherichia coli Rosetta(DE3)pLysS. Results The fusion protein system led to over-expression of the soluble and highly stable product, an approximate 26 kDa recombinant SjLys-C protein (rSjLys-C). The present study showed that rSjLys-C displayed strong antimicrobial activity against the tested Gram-positive and Gram-negative bacteria. In particular, the heat-treated rSjLys-C exhibited more inhibitive activity than the native rSjLys-C. The structural analysis of SjLys-C showed that it is a typical hydrophilic peptide and contains a helix-loop-helix motif. The modeling of SjLys-C molecular structures at different temperatures revealed that the tertiary structure of SjLys-C at 100°C underwent a conformational change which is favorable for enhancing antimicrobial activity. Conclusion These results indicate that the expressed rSjLys-C is a highly soluble product and has a strong antimicrobial activity. Therefore, gaining a large quantity of biologically active rSjLys-C will be used for further biochemical and structural studies and provide a potential use in aquaculture and medicine.