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Inflammation plays a key role in tumor promotion and development. Indeed, cyclooxygenase-2 (COX-2) expression is strongly associated with different types of cancer. An emerging class of compounds with significant anti-inflammatory properties is the hydrogen sulfide-releasing non-steroidal anti-inflammatory drugs (H 2 S-NSAIDs). They consist of a traditional NSAID to which an H 2 S-releasing moiety is covalently attached. We have recently demonstrated that H 2 S donors inhibit melanoma cell proliferation. In the current study, we evaluated the potential beneficial effects of a new H 2 S-releasing derivative of naproxen, ATB-346 [2-(6-methoxynapthalen-2-yl)-propionic acid 4-thiocarbamoyl phenyl ester] which inhibits COX activity but also releases H 2 S. We used cell culture and a mouse melanoma model to evaluate the effect of ATB-346 on: i) in vitro growth of human melanoma cells; ii) in vivo melanoma development in mice. Cell culture studies demonstrated that ATB-346 reduced the in vitro proliferation of human melanoma cells and this effect was associated to induction of apoptosis and inhibition of NF-κB activation. Moreover, ATB-346 had novel Akt signaling inhibitory properties. Daily oral dosing of ATB-346 (43 μmol/kg) significantly reduced melanoma development in vivo. This study shows that ATB-346, a novel H 2 S-NSAID, inhibits human melanoma cell proliferation by inhibiting pro-survival pathways associated with NF-κB and Akt activation. Furthermore, oral treatment with ATB-346 inhibits melanoma growth in mice. In conclusion, the combination of inhibition of cyclooxygenase and delivery of H 2 S by ATB-346 may offer a promising alternative to existing therapies for melanoma. [ABSTRACT FROM AUTHOR]

Background: The need for new drugs in melanoma treatment is of great relevance. Indeed, current therapies for the treatment of metastatic melanoma offer a limited clinical benefit and only in recent years there has been an advancement due to the identification of new molecular targets . Hydrogen sulphide (H2S) is endogenously produced by the action of three enzymes CBS, CSE and the newly discovered 3-MST . While H2S is cytoprotective at physiological concentrations, it seems to have pro-apoptotic actions in cancer cells . However, to date there are not definitive reports on the role played by H2S in cancer development. Aim of this study was to determine the possible involvement of H2S in human melanoma. Methods: The study has been performed by using some relevant human melanoma cell lines such as A375, WM115 and SK-Mel-28. HaCat, a normal human fibroblast cell line, has been used as control. Cellular proliferation was evaluated by the MTT assay. Apoptosis was assayed by flow cytometry analysis by double staining with Annexin V and propidium iodide (PI). NF-kB/DNA-binding activity was evaluated by electrophoretic mobility shift assay. Expression of CBS, CSE, 3-MST was assayed by quantitative real time RT-PCR, expression of Bcl-2, XIAP, c-FLIP, caspase 3, PARP, IKBa and Akt/p-Akt was determined by western blotting. Levels of H2S in the supernatant and in total cellular extracts were assayed by colorimetric assay. Results: Diallyl trisulfide (DATS) is a garlic-derived polysulfide able to release H2S . Our results demonstrate that DATS greatly suppressed, in a time and concentration-dependent manner, proliferation of the three human melanoma cell lines used. The most striking effect was obtained on the A375 cell line whose proliferation was inhibited, following incubation with DATS (10–30–100μM, 72h) by 30%, 70%, and 78% respectively (p <0.001). This effect well correlated with the significant increase in H2S levels found in both supernatants and cellular lysates. Conversely, DATS up to 300 μM did not affect proliferation of HaCat. DATS-induced inhibition of A375 proliferation (10–100μM, 72h) was almost completely reversed by haemoglobin (10μM; p <0.001) a scavenger of H2S. Moreover, DATS-induced inhibition of A375 proliferation was due to the induction of apoptosis as demonstrated by FACS analysis with Annexin V/PI staining and further confirmed by the inhibition of Bcl-2, XIAP, FLIP, as well as by the cleavage and consequent activation of caspase-3 and inactivation of poly (ADP ribose) polymerase (PARP-1). Constitutive NF-kB and activated Akt expression have been described in melanoma. We also demonstrated that H2S released by DATS suppressed both constitutive NF-kB/DNA-binding activity and Akt phosphorylation suggesting that the apoptotic effect observed following exposure to H2S was consistent with the signal transduction pathways activated. Conclusion: In conclusion, we demonstrate that H2S triggers, in relevant human melanoma cell lines, an apoptotic effect. This effect, in turn, activates downstream a signal pattern that candidates H2S as a possible novel therapeutic/target or diagnostic tool. [Copyright &y& Elsevier]