Appropriate post-translational processing of collagen requires prolyl hydroxylation, catalyzed by the prolyl 3-(C-P3H) and prolyl 4-(C-P4H) hydroxylases is essential for normal cell function. Here we have investigated the expression, transcriptional regulation and function of the C-P3H and C-P4H families in melanoma. We show that the CP3H family exemplified by Leprel1 and Leprel2 are subject to methylation-dependent transcriptional silencing in primary and metastatic melanoma consistent with a tumour suppressor function. In contrast, although there is transcriptional silencing of P4HA3 in a subset of melanomas, the CP4H family members P4HA1, P4HA2 and P4HA3 are often over-expressed in melanoma, expression being prognostic of worse clinical outcomes. Consistent with tumour suppressor function, ectopic expression of Leprel1 and Leprel2 inhibits melanoma proliferation, whereas P4HA2 and P4HA3 increase proliferation and particularly invasiveness of melanoma cells. Pharmacological inhibition with multiple selective C-P4H inhibitors reduces proliferation and inhibits invasiveness of melanoma cells. Together, our data identify the C-P3H and C-P4H families as potentially important regulators of melanoma growth and invasiveness and suggest that selective inhibition of C-P4H is an attractive strategy to reduce the invasive properties of melanoma cells.

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Abstract. Background: Isothiocyanates are constituents of cruciferous vegetables which have been associated with reduced cancer risk partially through their ability to induce apoptosis in malignant cells including melanoma. Materials and MethodsMalignant melanoma is the fifth most common cancer in UK with its incidence rates being continuously rising faster than any other malignancy (1). Epidemiological studies suggest that an increased dietary consumption of cruciferous vegetables can reduce cancer incidence. These effects can be attributed to the high levels of glucosinolates (GSLs) present which are sulphur-containing glycosides and precursors for a group of compounds called isothiocyanates (ITCs) (2). Briefly, upon mechanical disruption of the plant cell wall (e.g. by chewing), the enzyme myrosinase is released which then catalyses the hydrolysis of GSLs to ITCs, with subsequent release of HSO 4 -and D-glucose (3). Different GSLs can form different ITCs, since glucoraphanin acts as the precursor for sulforaphane (SFN), gluconasturtin for phenethyl isothiocyanate (PEITC) and glucotropaeolin for benzyl isothiocyanate (BITC) (4). There is much speculation as to how ITCs may exhibit their chemotherapeutic effects, but the likelihood is that multiple molecular events are responsible. Potential biochemical mechanisms include (i) inhibition of carcinogen activity via suppression of phase I enzymes in xenobiotic metabolism, (ii) stimulation of phase II enzymes and (iii) induction of apoptosis (5, 6). Despite many reports demonstrating ITCs' effectiveness against different cancers there have been a limited number of studies investigating their ability to induce apoptosis in human malignant melanoma cells (7) which their results are dependent on the utilization of high concentrations of ITCs. 6303

High-resolution I3C N.M.R. Spectroscopy and X-Ray Crystallography of Complexes formed by NN' -Dimethyl-1,7 -diaza -4,lO -dioxacyclododecane