P28 Identification of a novel slow-releasing hydrogen sulfide donor for cancer therapy

It has previously been reported that hydrogen sulfide (H 2 S) exerts both pro- and anti-apoptotic activity in different cultured cells [1] , [2] , [3] . However, the precise effect and mechanism(s) involved remain unclear. We have previously reported that the slow-releasing H 2 S donor, GYY4137 [4] (morpholin-4-ium 4 methoxyphenyl(morpholino) phosphinodithioate) exerts anti-cancer activity both in vivo and in vitro [5] . In this report, GYY4137 showed a distinct anti-proliferative effect across a broad range of cancer cell lines but did not affect proliferation in normal cell lines. In contrast, NaHS, which releases large amounts of H 2 S over a short time course, failed to affect proliferation of either cancer or normal cell lines. This work raised the possibility that H 2 S donors, with differing H 2 S-releasing kinetics, may be of value to kill cancer (but not normal) cells. To this end, in preliminary compound screening tests, we examined a number of novel compounds all of which are GYY4137 analogues, for the ability to kill both cancer and normal cell lines. We also determined the H 2 S-releasing profiles of these compounds using the conventional methylene blue spectrophotometric assay to monitor H 2 S release. In the course of this study, we identified an additional, slow-releasing H 2 S donor, FW1010. FW1010 can slow release H 2 S more than GYY4137 over a few days. Incubation of either FW1010 or GYY4137 (400 μ M) in culture medium led to the generation of low concentrations of H 2 S over 6 days. Comparing FW1010 to GYY4137, FW1010 exhibited enhanced, concentration-dependent killing of different human cancer cell lines (e.g. MCF7, HepG2, U2OS, HL-60, MDA-MB-231, SKOV3, and OVCAR10) but had little or only minimal killing effect in a normal cell line (WI38).We propose that the ability of GYY4137 and FW1010 to kill cancer cells in vitro is likely due to their ability to release H2S slowly over a period of several days. The precise mechanism of action is not clear but measuring intracellular pH (pHi) by confocal microscopy we noted that FW1010 treatment caused a significant concentration-dependent, decline in intracellular pH in MCF7 cells and that this effect was greater than that of GYY4137 at identical concentrations. In contrast, neither GYY4137 nor FW1010 affected pHi in a normal cell line (i.e. WI38). Moreover, both GYY4137 and FW1010 significantly increased lactate release and glucose uptake in a concentration dependent manner in MCF7 (but not in W138) cells. FW1010 was more potent than GYY4137 in this regard. These experiments provide further evidence of the anti-cancer effect of GYY4137 in vitro and identify a new analogue of this compound with seemingly greater efficacy. Additional studies are ongoing to define further the possible mechanisms of action of both FW1010 and GYY4137.