Herpes simplex virus-1 (HSV-1) enkephalin-encoding vector increases enkephalin and decreases inflammatory cytokine expression in cultured PANC-1 pancreatic cells

The current in vitro study was designed to assess opioid peptides expression and inflammatory blunting in two rat experimental pancreatitis models, using human cultured cells, including PANC-1 cells derived from pancreatic cancer. Replication-defective HSV-1 vectors with human transgene proenkephalin cDNA (HSV-Enk), beta-galactosidase cDNA (control vector, HSV-beta-gal) or vehicle control were applied to cell cultures at Multiplicity of Infection ranges of 5.0-0.005. Enkephalin expression and aspects of inflammatory blunting were measured by immunocytochemistry, RT-PCR, cDNA microarrays, and immunoassays. All HSV vector treated PANC-1 cells expressed met-enkephaIin, leu-enkephalin, the mu opioid receptor, and beta-galactosidase. However, HSV-Enk treated cells expressed more cellular met-enkephalin and leu-enkephalin, products of the proenkephalin gene. In the c-DNA microarray analysis from PANC-1 cells, the HSV-Enk treated cells induced fewer IL-6 and apoptosis-related signaling pathway genes compared to the HSV-beta-gal(control)treated cells. RT-PCR analysis demonstrated an average 5-fold increase of met-enkephalin cDNA transcripts in HSV-Enk infected cells, compared to HSV-beta-gal treated controls. In nucleotide sequence analyses, met-enkephalin cDNA from HSV-Enk treated cells demonstrated 98% sequence homogeneity compared to vector untreated but LPS-stimulated PANC-1 cellular controls or HSV-beta-gal treated cells. Immunoassays demonstrated that HSV-Enk treated PANC-1 cells expressed significantly less IL-6 and TNF-alpha in the culture supernatants compared to the HSV-beta-gal infected cells. Thus, there is svidence that met-enkephalin transcripts are dervied from both cellular and transgene sources in the HSV-Enk treated cells. Furthermore, the inflammatory blunting observed in the animal models of HSV vector treated pancreatitis and arthritis may utilize central and peripheral mechanisms.

(Supported by NIH RO1 NS 39041, the Keck Foundation and an anonymous gift)