Methoxylated but not hydroxylated flavones elicit significant activity against Parp-1-mediated cell death (Parthanatos)

Abstract

Flavonoids, of which flavones are a sub-group, are plant secondary metabolites found in a variety of natural food sources (e.g., vegetables) and wines. They elicit beneficial roles in health and disease through their antioxidant activity, but some of them have also now been found to exert specific effects on cell signalling. We recently showed that methoxylation of the flavone structure at the 4ʹ position, or additionally at the 3ʹ position, to produce 4ʹ-methoxyflavone (4MF) and 3ʹ,4ʹ-dimethoxyflavone (DMF), respectively, significantly enhanced activity against the cell death (“parthanatos”) mediated by poly (ADP-ribose) polymerase (PARP). We report here our attempt to correlate the antioxidant and parthanatos-inhibitory activities of these methoxylated flavones with those of the hydroxylated flavonoids. Cultures of HeLa and HaCaT cells were exposed to MNNG (50µM, up to 25min), which induces parthanatos, and the oxidant hydrogen peroxide (100µM – 2mM, up to 24h). The effects (up to 20µM) of the methoxylated flavones 4MF and DMF, the hydroxylated flavone luteolin (LN), and the non-flavone flavonoids quercetin (QE), naringin (NG) and epigallocatechin gallate (EGCG) on the reduction in viability (indicative of cell death) and morphological changes induced by MNNG or peroxide were then investigated. Both alamar blue and MTT assays were used to quantify viability. MNNG induced significant reduction in cell viability, which was not affected by the pan-caspase inhibitor Z-VAD-fmk but significantly blocked by DPQ, a PARP-1 inhibitor, consistent with the biochemical profile of parthanatos. Hydrogen peroxide also elicited a significant decrease in cell viability, with partial or no protection afforded by either Z-VAD-fmk or DPQ (dependent on peroxide concentration and treatment duration). 4MF and DMF demonstrated significant protection against MNNG-induced cell death but LN, QE, NG and EGCG showed little or no protection. On the other hand, 4MF and DMF elicited mostly negligible effects against hydrogen peroxide, whereas LN, QE, NG and EGCG elicited various levels of protection against it. We conclude that methoxylation at the 4ʹ or 3ʹ, 4ʹ positions of flavones favours anti-parthanatos but not antioxidant activity, whereas hydroxylation enhances antioxidant but not anti-parthanatos activity.