Vasoactive intestinal polypeptide (VIP) exhibits effects on cell proliferation. Here, VIP, as well as the related peptide, pituitary adenylate cyclase activating peptide (PACAP), promoted human keratinocyte division. Stearyl-Nle 17 -VIP (SNV) was identified as a superior mitogen for the keratinocytic cell line, HaCaT, both in potency (fM^nM concentrations) and efficacy. Reverse transcription-polymerase chain reaction detected in keratinocytes only PACAP mRNA and the relevant type 1 (VPAC 1 R) and type 2 (VPAC 2 R) receptors, while VIP and the third receptor (PAC 1 ) transcripts were absent. Upon serum deprivation of HaCaT, the VPAC 1 R mRNA was apparently increased, while the VPAC 2 R transcript remained constant. Incubation of HaCaT with VIP or SNV increased nitric oxide and cGMP formation. In contrast to VIP, SNV did not augment cAMP. Thus, the paracrine VIP, and autocrine PACAP, related pathways leading to keratinocyte proliferation may involve VPAC 1 R/VPAC 2 R and nitric oxide/cGMP production. ß

BACKGROUND Vasoactive intestinal peptide (VIP) is one of several small neuropeptides that affect cancer growth. A lipophilic VIP analog, stearyl‐Nle17‐neuroten‐ sin6‐11VIP7‐28 (SNH) that inhibited lung carcinoma growth has been described previously. The experiments performed were clonogenic assays in vitro and tumor xenografts in nude mice in vivo. These studies were now extended to colon carcinoma and to combination therapy with chemotherapeutic agents. METHODS Assays were performed with cell lines, and tumor proliferation was assessed using the (3‐[4,5‐dimethylthiazol‐2‐yl‐5]‐[3‐carboxymethoxyphenyl]‐2‐[4‐sulfophenyl]‐2H tetrazolium) (MTS) colorimetric assay for mitochondrial function of living cells. RESULTS The lipophilic analog (SNH) enhanced the antiproliferative activity of diverse chemotherapeutic agents: doxorubicine (antibiotic); vinorelbine (vinca alkaloid, antimicrotubule formation); paclitaxel (antimicrotubule agent); gemcitabine (antimetabolite); irinotecan (topoisomerase I inhibitor); and cisplatin (platinum compound acting as an alkylating agent). In all cases, the antiproliferative effect of SNH and the chemotheraputic agent was at least additive and for some combinations and concentrations even synergistic. For example, 2 μM of the antagonist that produced a 15–20% growth inhibition in the nonsmall cell lung carcinoma cell line reduced the IC50 by 2–4‐fold for most of the chemotherapeutic agents tested. Higher analog concentrations were even more efficacious. Similar results were obtained with colon carcinoma cell lines. CONCLUSIONS Chemotherapeutic treatment of advanced solid tumors, such as nonsmall cell lung carcinoma, colon carcinoma, or prostate carcinoma, achieves a response rate of between 10% and 30% with significant toxicity. Combination therapy with the lipophilic VIP analog SNH and the preferred chemotherapeutic agent may greatly enhance the response rate, and by permitting a dose reduction, should significantly reduce side effects. Cancer 2001;92:2172–80. © 2001 American Cancer Society.

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Four novel 2,4-methano amino acids (MAAs, 1-aminocyclobutane-1-carboxylic acids) were synthesized. These include the basic MAA analogs of lysine (16), ornithine (5), and arginine (6) and the neutral methanovaline (22), related to proline. The above MAAs, as well as the MAA analog of homothreonine (7), were incorporated into the peptide chain of the immunomodulatory peptide tuftsin, Thr-Lys-Pro-Arg, known to enhance several biological activities mediated by phagocytic cells. The synthetic methano tuftsin analogs were assayed for their ability to stimulate interleukin-6 (IL-6) secretion by mouse peritoneal macrophages and for their stability in human serum toward enzymatic degradation. It was found that, at 2 x 10(-7) M, [MThr1]tuftsin (24) and an isomer of [MVal3]tuftsin (27a) were considerably more active than the parent peptide in augmentation of cytokine release. [MOrn2]Tuftsin (25) was equally potent. The analogs [MThr1]tuftsin (24) and [MOrn2]tuftsin (25), both pertaining to the proteolytically sensitive Thr-Lys bond of tuftsin, exhibited high resistance to enzymatic hydrolysis as compared to tuftsin. Using specific rabbit anti-tuftsin antibodies in a competitive enzyme-linked immunosorbent assay (ELISA) revealed that none of the MAA analogs can cross-react with tuftsin. It may indicate that the peptides assume global structures different than that of tuftsin.

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Vasoactive intestinal peptide (VIP) is a recognized growth factor affecting many cell types. We have previously developed a series of lipophilic VIP analogues containing an N-terminal covalently attached stearyl moiety. The current studies identified stearyl-Nle 17 -VIP and stearyl-Nle 17 -neurotensin 6À11 VIP 7À28 , acting at W WM concentrations, as cytotoxic to human keratinocytes. The core C-terminal active VIP-derived peptide, stearyl-Lys-Lys-Tyr-Leu-NH 2 (St-KKYL-NH 2 ), was identified as being responsible for the observed cytotoxicity. Cytotoxicity coincided with marked reduction in intracellular cyclic GMP and was abolished by co-treatment with the endonuclease inhibitor, aurine-tricarboxylic acid, suggesting apoptotic mechanisms. Stearyl-VIP derivatives thus offer lead compounds for future drug development against hyperproliferative skin conditions. ß

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