Inhibition of interferon-gamma-stimulated melanoma progression by targeting neuronal nitric oxide synthase (nNOS)

Abstract

Interferon-gamma (IFN-γ) is shown to stimulate melanoma development and progression. However, the underlying mechanism has not been completely defined. Our study aimed to determine the role of neuronal nitric oxide synthase (nNOS)-mediated signaling in IFN-γ-stimulated melanoma progression and the anti-melanoma effects of novel nNOS inhibitors. Our study shows that IFN-γ markedly induced the expression levels of nNOS in melanoma cells associated with increased intracellular nitric oxide (NO) levels. Co-treatment with novel nNOS inhibitors effectively alleviated IFN-γ-activated STAT1/3. Further, reverse phase protein array (RPPA) analysis demonstrated that IFN-γ induced the expression of HIF1α, c-Myc, and programmed death-ligand 1 (PD-L1), in contrast to IFN-α. Blocking the nNOS-mediated signaling pathway using nNOS-selective inhibitors was shown to effectively diminish IFN-γ-induced PD-L1 expression in melanoma cells. Using a human melanoma xenograft mouse model, the in vivo studies revealed that IFN-γ increased tumor growth compared to control, which was inhibited by the co-administration of nNOS inhibitor MAC-3-190. Another nNOS inhibitor, HH044, was shown to effectively inhibit in vivo tumor growth and was associated with reduced PD-L1 expression levels in melanoma xenografts. Our study demonstrates the important role of nNOS-mediated NO signaling in IFN-γ-stimulated melanoma progression. Targeting nNOS using highly selective small molecular inhibitors is a unique and effective strategy to improve melanoma treatment.

A Small Peptide Increases Drug Delivery in Human Melanoma Cells

“…In vitro, we did not observe any significant difference in cytotoxicity between MAC-3-190 and co-administration of KK-11 and MAC-3-190 ( Figure 3 D). In addition, our previous studies and preliminary observations demonstrated that MAC-3-190 (5 mg/kg/day) does not have any significant antitumor activity in vivo [ 8 ]. Therefore, we evaluated if co-administration with KK-11 peptide would affect the antitumor activity of MAC-3-190 in vivo in mice carrying melanoma xenografts.…”

“…The tumor size was measured with a caliper, and body weight was recorded once a week. When the tumor size reached approximately 800–1000 mm 3 , as calculated using the following formula: [Length × (Width 2 )]/2 (in mm) [ 8 ], mice were processed for in vivo or ex vivo imaging [ 26 ].…”

“…The growth of the tumors was monitored three times a week and measured using digital Vernier calipers. The tumor volume (mm 3 ) was calculated as [Length × (Width 2 )]/2 (in mm) [ 8 ]. The mice were sacrificed after 21 days per IACUC policy when the control group’s average tumor size reached 2000 mm 3 .…”

“…Similar resistance was also reported with cisplatin treatment in melanoma [ 7 ]. An enzyme, neuronal nitric oxide synthase (nNOS), was found to be overexpressed in CM, and has been identified as a key player in melanomagenesis by enhancing tumor growth and interferon-gamma-stimulated melanoma progression [ 8 , 9 ]. Our previous studies demonstrated that novel nNOS inhibitors, such as MAC-3-190 and HH044, exhibit promising anti-melanoma activities by inhibiting nNOS-mediated nitric oxide signaling [ 8 , 9 , 10 , 11 ].…”

“…Two types of anti-cancer drugs were studied, cytotoxic DOX and nNOS inhibitor MAC-3-190. The latter is a targeted therapy for melanoma [ 8 , 9 ]. Co-administration of KK-11 significantly enhanced the cytotoxicity of DOX in vitro, and the in vivo antitumor activity of MAC-3-190 in a melanoma xenograft mouse model [ 11 ].…”