Gene Transfer of Pro-opiomelanocortin Prohormone Suppressed the Growth and Metastasis of Melanoma: Involvement of α-Melanocyte-Stimulating Hormone-Mediated Inhibition of the Nuclear Factor κB/Cyclooxygenase-2 Pathway

Abstract

Pro-opiomelanocortin (POMC) is a prohormone of various neuropeptides, including corticotropin, ␣-melanocyte-stimulating hormone (␣-MSH), and ␤-endorphin (␤-EP). POMC neuropeptides are potent inflammation inhibitors and immunosuppressants and may exert opposite influences during tumorigenesis. However, the role of POMC expression in carcinogenesis remains elusive. We evaluated the antineoplastic potential of POMC gene delivery in a syngenic B16-F10 melanoma model. Adenovirus-mediated POMC gene delivery in B16-F10 cells increased the release of POMC neuropeptides in cultured media, which differentially regulated the secretion of pro-and anti-inflammatory cytokines in lymphocytes. POMC gene transfer significantly reduced the anchorage-independent growth of melanoma cells. Moreover, pre-or post-treatment with POMC gene delivery effectively retarded the melanoma growth in mice. Intravenous injection of POMC-transduced B16-F10 cells resulted in reduced foci formation in lung by 60 to 70% of control. The reduced metastasis of POMC-transduced B16-F10 cells could be attributed to their attenuated migratory and adhesive capabilities. POMC gene delivery reduced the cyclooxygenase-2 (COX-2) expression and prostaglandin (PG) E 2 synthesis in melanoma cells and tumor tissues. In addition, application of NS-398, a selective COX-2 inhibitor, mimicked the antineoplastic functions of POMC gene transfer in melanoma. The POMC-mediated COX-2 down-regulation was correlated with its inhibition of nuclear factor B (NFB) activities. Exogenous supply of ␣-MSH inhibited NFB activities, whereas application of the ␣-MSH antagonist growth hormone-releasing peptide-6 (GHRP-6) abolished the POMC-induced inhibition of NFB activities and melanoma growth in mice. In summary, POMC gene delivery suppresses melanoma via ␣-MSH-induced inhibition of NFB/COX-2 pathway, thereby constituting a novel therapy for melanoma.POMC is a multifunctional polycistronic gene located on human chromosome 2p23.3. POMC is a 31 kDa prohormone that is processed into various neuropeptides, including corticotropin, melanotropins (␣-, ␤-, and ␥-MSH), lipotropins, and ␤-endorphin (␤-EP) (Solomon, 1999;Catania et al., 2004b). POMC peptides possess pleiotropic functions including pigmentation, adrenocortical function, regulation of energy stores, the immune system, and the central and peripheral

Downregulation of Hepatoma-Derived Growth Factor Contributes to Retarded Lung Metastasis via Inhibition of Epithelial–Mesenchymal Transition by Systemic POMC Gene Delivery in Melanoma

“…All the cell lines were used within 6 months after resuscitation, and no further cell line authentication was conducted. The GFP-and luciferaseexpressing B16-F10 melanoma cells were generated as previously described (20).…”

“…The recombinant adenovirus vectors containing GFP (Ad-GFP) and POMC (Ad-POMC) were generated as previously described (20,28).…”

“…Immunohistochemistry was conducted as described previously (20). The primary antibodies are CD31 (1:100; Novocastra), a-SMA (1:200), E-cadherin (1:250), and vimentin (1:200).…”

“…Whole-cell protein extracts were prepared as described previously (20). Proteins were separated on 8% to 16% Amersham ECL gels and western blotted with antia-SMA (1:1,000), E-cadherin, vimentin (1:500), and b-actin (1:5,000).…”

“…Recent studies indicate that the anti-inflammatory POMC therapy effectively suppresses the growth of murine primary tumors, including B16-F10 (20,21) and Lewis lung carcinoma (22). Furthermore, POMC-derived peptide, a-MSH, has been reported to trigger melanoma/melanocyte differentiation (23,24) and potently inhibits both the in vitro and in vivo invasion of highly invasive B16-BL6 melanoma cells (25,26).…”