Abstract B26: Enkephalin-encoding HSV-1 vectors demonstrate proinflammatory blunting and increased transgene met- and leu-enkephalin expression in cultured human pancreatic carcinoma cells - PANC-1.

Purpose: Previous studies demonstrated decreased nociceptive and tissue inflammatory scores in animal models of inflammatory arthritis and pancreatitis when treated with enkephalin (ENK) encoding Herpes simplex virus type 1 (HSV-1) recombinant vectors vs. vector controls. We hypothesized that HSV-Enk treated cell cultures would: 1) demonstrate increased expression of transgene products, and 2) blunt proinflammatory cytokine levels in cultured supernatants, in the absence of neuronal input. Methods: Cultures of PANC-1 human pancreatic cancer cells (ATCC, Bethesda, MD) were treated with replication deficient recombinant vectors (HSV-1 vector with inserted human preproenkephalin transgene, HSV-Enk) and control vectors, (HSV-1 replication deficient recombinant vector, HSV-beta-galactosidase, or HSV-beta-gal). Protein expression of cellular met-and leu-enkephalins, beta-galactosidase, RANTES and mu opioid receptor (MOR) was assessed by cell staining using specific antibodies and matched nonspecific antibody controls. Amplification of mRNA, by RT-PCR, Real Time PCR and cDNA nucleotide sequencing were performed. TNF-alpha, RANTES and interleukin 6 (IL-6) were measured in culture supernatants by immunoassays. Statistical analyses: Student's t-tests and Mann-Whitney U tests. P values <0.05 was considered significant. Results: Cell Staining: Increased staining of beta-gal, met-ENK and RANTES was noted in HSV-beta-gal treated cells vs. untreated cultures. Met-and leu-ENK and MOR staining were greatly increased in the HSV-Enk treated cells, whereas RANTES staining intensity was much less in HSV-Enk compared to HSV-beta-gal treated cells. Staining reflected the predicted 4:1 transcription ratio of met- to leu-enkephalin from the human preproenkephalin gene. Real Time PCR demonstrated a 5.5-fold increase in met-enkephalin mRNA from HSV-Enk compared to HSV-beta-gal treated cells. Sequencing: cDNA nucleotide sequences were obtained from untreated, LPS, HSV-beta-gal or HSV-Enk treated cells. Sequencing confirmed that the vector encoded transgene was actively transcribed, because the nucleotide sequence differed 1.2% from that of endogenous PANC-1 proenkephalin. Proinflammatory cytokines: Significantly decreased levels of TNF-alpha, RANTES and IL-6 were obtained in the supernatants of HSV-Enk-treated PANC-1 cells, compared to HSV-beta-gal-treated cells. Conclusion: These studies provide further evidence that endogenous and transgene enkephalins can be generated in peripheral non-neuronal cells, independent of neuronal input. This supports earlier studies by showing that enkephalins are immunomodulatory and physiologically relevant in peripheral tissue inflammation. HSV Enk-encoding vectors have demonstrated analgesia in the treatment of cancer pain in clinical trials and our studies point to their additional potential in tissue sparing, or tissue repair. Citation Format: Terry A. McNearney, Huaizhi Yin, Steven P. Wilson, Karin N. Westlund. Enkephalin-encoding HSV-1 vectors demonstrate proinflammatory blunting and increased transgene met- and leu-enkephalin expression in cultured human pancreatic carcinoma cells - PANC-1. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr B26.