Targeted Inhibition of Inducible Nitric Oxide Synthase Inhibits Growth of Human Melanoma In vivo and Synergizes with Chemotherapy

Abstract

Purpose: Aberrant expression of inflammatory molecules, such as inducible nitric oxide (NO) synthase (iNOS), has been linked to cancer, suggesting that their inhibition is a rational therapeutic approach. Whereas iNOS expression in melanoma and other cancers is associated with poor clinical prognosis, in vitro and in vivo studies suggest that iNOS and NO can have both protumor and antitumor effects. We tested the hypothesis that targeted iNOS inhibition would interfere with human melanoma growth and survival in vivo in a preclinical model.Experimental Design: We used an immunodeficient non-obese diabetic/severe combined immunodeficient xenograft model to test the susceptibility of two different human melanoma lines to the orallygiven iNOS-selective small molecule antagonistand without cytotoxic cisplatin chemotherapy.Results: L-nil significantly inhibited melanoma growth and extended the survival of tumor-bearing mice. L-nil treatment decreased the density of CD31+ microvessels and increased the number of apoptotic cells in tumor xenografts. Proteomic analysis of melanoma xenografts with reverse-phase protein array identified alterations in the expression of multiple cell signaling and survival genes after L-nil treatment. The canonical antiapoptotic protein Bcl-2 was downregulated in vivo and in vitro after L-nil treatment, which was associated with increased susceptibility to cisplatin-mediated tumor death. Consistent with this observation, combination therapy with L-nil plus cisplatin in vivo was more effective than either drug alone, without increased toxicity.Conclusions: These data support the hypothesis that iNOS and iNOS-derived NO support tumor growth in vivo and provide convincing preclinical validation of targeted iNOS inhibition as therapy for solid tumors. Clin Cancer Res; 16(6); 1834-44. ©2010 AACR.Upregulation of proinflammatory molecules by tumor cells is a poorly understood phenomenon, which can play a role in both the induction and maintenance of certain human cancers (1). One such molecule, inducible nitric oxide (NO) synthase (iNOS) is constitutively overexpressed in many cancers, including melanoma and gastric (2), breast (3), colon (4), and head and neck (5, 6) carcinomas. iNOS and its product NO have wide-ranging and varied effects on cellular physiology, signal transduction, and cell survival. At high levels, such as produced by activated macrophages during inflammatory responses to pathogens, NO alone or in combination with reactive oxygen species can have a direct cytotoxic effect on pathogens or tumor cells (7). At lower levels, NO can affect signal transduction pathways by interacting with metal ligands of proteins (8) or covalently modifying proteins through nitration and nitrosylation (9, 10). These protein Authors' Affiliations:

Pharmacological Inhibition of NOS Activates ASK1/JNK Pathway Augmenting Docetaxel-Mediated Apoptosis in Triple-Negative Breast Cancer

“…In the present study, we demonstrate that the pan-NOS inhibitor L-NMMA interacts with docetaxel-based chemotherapy to overcome treatment resistance through a mechanism that redirects cell fate toward apoptosis (as opposed to survival) through activation of procell death ASK1/JNK pathway. Our results are similar to other in vitro and in vivo studies showing the feasibility of pharmacologic NOS inhibition combined with cytotoxic chemotherapy as a treatment for cancer (43,49,50). In this study, we describe the cross-talk between treatment resistance and EnR stress, and targeting NOS signaling may overcome this resistance.…”

“…iNOS is an inflammatory mediator (42) capable of promoting the survival and proliferation of different cancer types such as melanoma (43)(44)(45), liver (46), colon, head and neck (44,47), and glioblastoma (48). In TNBC and metaplastic breast cancer, iNOS expression levels are correlated with aggressiveness, poor survival, and treatment resistance (8,14,16,18,30).…”

Unique antineoplastic profile of Saquinavir-NO, a novel NO-derivative of the protease inhibitor Saquinavir, on the in vitro and in vivo tumor formation of A375 human melanoma cells

“…The presently observed inhibitory activity of L-NAME in the growth of melanoma is consistent with recent data from Sikora et al (27) who found that oral treatment with the specific iNOS-selective small molecule antagonist N(6)-(1-iminoethyl)-l-lysine-dihydrochloride (L-nil) significantly inhibited the growth of two human melanoma cell lines mel624 and mel528 xenotransplanted into severe combined immunodeficient (SCID) mice and extended the survival of tumor-bearing animals. In their experiments, iNOS inhibition was associated with tumor growth suppression, decrease in tumor microvessels, down-regulation of the anti-apoptotic gene Bcl-2, increased number of intratumoral apoptotic cells and enhanced efficacy when L-nil treatment was combined with cisplatin in vivo.…”

“…This synergism closely resembles that observed in our study between L-NAME and both Saq and Saq-NO. Although dismantling the molecular and pharmacological mechanisms underlying this synergism was outside the phenomenological scope of our work and has not been studied, it is possible that in a similar manner to that described by Sikora et al (27) for N(6)-(1-iminoethyl)-l-lysine-dihydrochloride (L-nil) and cisplatin it may be due to combined and higher effective influence on key oncogenic pathways of A375 cells. Taken together these data support the hypothesis that targeting NOS may represent a valuable novel target of melanoma therapies that may be combined with other chemotherapeutic agents including soft NO donor such as Saq-NO.…”

Biochemistry of Nitric Oxide

“…The importance of these isoenzyme specific inhibitors is that they inhibit selective NO biosynthesis rather than suppressing whole NO biosynthesis (Table 1). L-NIL is an exception with 30 folds more specificity towards iNOS [147]. Among these inhibitors Galaxo Wellcome compound 1400 w (N-(3-aminomethyl) benzyl) acetamide) is most selective iNOS inhibitor and Astra Arcus compound ARL 17477 is most selective for nNOS (Fig.…”