Mono-galloyl glucose derivatives are potent poly(ADP-ribose) glycohydrolase (PARG) inhibitors and partially reduce PARP-1-dependent cell death

Abstract

Background and purpose: Maintenance of poly(ADP-ribose) (PAR) polymers at homoeostatic levels by PAR glycohydrolase (PARG) is central in cell functioning and survival. Yet the pharmacological relevance of PARG inhibitors is still debated. Gallotannin, a complex mixture of hydrolysable tannins from oak gall, inhibits PARG but which of its constituents is responsible for the inhibition and whether the pharmacodynamic properties are due to its antioxidant properties, has not yet been established.

Experimental approach: A structure-activity relationship study was conducted on different natural and synthetic tannins/galloyl derivatives as potential PARG inhibitors, using a novel in vitro enzymic assay. Cytotoxicity was assayed in cultured HeLa cells.

Key results: Mono-galloyl glucose compounds were potent inhibitors of PARG, with activities similar to that of ADP-(hydroxymethyl) pyrrolidinediol, the most potent PARG inhibitor yet identified. When tested on HeLa cells exposed to the PAR polymerase (PARP)-1-activating compound 1-methyl-3-nitro-1-nitrosoguanidine (MNNG), 3-galloyl glucose weakly inhibited PAR degradation. Conversely, the more lipophilic, 3-galloyl-1,2-O-isopropylidene glucose, despite being inactive on the pure enzyme, efficiently prolonged the half-life of the polymers in intact HeLa cells. Also, PARG inhibitors, but not radical scavengers, reduced, in part, cell death caused by MNNG.

Conclusions and implications: Taken together, our findings identify mono-galloyl glucose derivatives as potent PARG inhibitors, and emphasize the active function of this enzyme in cell death.

Figure 1

Synthesis and degradation of poly(ADP-ribose) (PAR) by pure poly(ADP-ribose) polymerase (PARP-1) and poly(ADP-ribose) glycohydrolase (PARG). (a) Time-dependent incorporation of radioactivity from ³H-NAD into trichloroacetic acid (TCA)-precipitated proteins in the absence or presence of 30 μM phenanthridinone (PHE; a PARP-1 inhibitor) at time 0 or 60 min. (b) Effect of pure PARG (0.05 U) added after 60 min on incorporation of radioactivity from ³H-NAD into TCA-precipitated proteins in the absence or presence of 30 μM PHE. (c) PARG-dependent reduction of radioactivity incorporation is concentration dependent. Each point/column represents the mean±s.e.mean of three (a, b) and two (c) experiments conducted in duplicate.

Figure 2

Chemical constituents of gallotannin and other galloyl-containing molecules tested as poly(ADP-ribose) glycohydrolase (PARG) inhibitors. (a) Representative HPLC/DAD chromatographic profile of gallotannin, reported at 280 nm. The presence and % of the different galloyl derivatives are shown. a–g represent the esa-, epta-, octa-, ena-, deca-, endeca-, dodeca- and galloyl glucose derivatives, respectively. (b) Structures of galloyl derivatives tested as PARG inhibitors.

Figure 3

Mono-galloyl glucose derivatives are potent poly(ADP-ribose) glycohydrolase (PARG) inhibitors. (a) List with chemical names and numbers of the compounds tested as PARG inhibitors. (b) Histogram showing the relative inhibitory potency of the compounds tested at 10 μM on PARG activity. (c) Inhibitory activity on PARG of compounds (5), (6), (7) and (16). In (b, c), each column/point represents the mean±s.e.mean of three experiments conducted in duplicate. ^*P<0.05 vs no. 1 (ANOVA+Tukey's post hoc test).

Figure 4

Effects of mono-galloyl glucose on poly(ADP-ribose) glycohydrolase (PARG) activity in intact cells. (a) HeLa cells were exposed to the poly(ADP-ribose) polymerase (PARP-1)-activating compound 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) (100 μM) in the absence or presence of 10 μM 3-galloyl-glucose (5) and poly(ADP-ribose) (PAR) levels were evaluated by western blotting at different times. β-Actin is shown as a loading control. (b) HeLa cells were exposed to the PARP-1-activating compound MNNG (100 μM) in the absence or presence of 3-galloyl-1,2-O-isopropylidene glucose (4) (10 or 100 μM) and PAR levels were evaluated by western blotting at different time points. β-Actin is shown as a loading control. Compound (4) was pre-incubated for 30 min. (c) Immunocytochemical evaluation of intracellular PAR contents in cells exposed to MNNG in the absence or presence of 3-galloyl-1,2-O-isopropylidene glucose (4, 100 μM). (d) Nicotinimade adenine dinucleotide (NAD) levels in cells exposed 30 min to 30 μM 6(5H)-phenanthridinone (PHE) or 30 and 60 min to 100 μM 3-galloyl-1,2-O-isopropylidene glucose (4). (e) NAD contents in cells exposed to 100 μM MNNG in the presence or absence of PHE (30 μM) or 3-galloyl-1,2-O-isopropylidene glucose (4, 100 μM). A blot/experiment representative of two (a–c) or three (d) is shown. Bar=20 μm. Columns are the mean±s.e.mean of two experiments (e). ^*P<0.05, ^**P<0.01 vs control (C) (ANOVA+Tukey's post hoc test).

Figure 5

Effect of gallotannin or 3-galloyl-1,2-O-isopropylidene glucose on cell death triggered by 24 h exposure to 1-methyl-3-nitro-1-nitrosoguanidine (MNNG). Cells were exposed to 100 μM gallotannin (1) or 100 μM 3-galloyl-1,2-O-isopropylidene glucose (4) in the presence or absence of 100 μM MNNG for 1 h and cell death was evaluated 24 h later by means of methylthiazolyl tetrazolium (MTT) reduction or lactate dehydrogenase (LDH) release assays. Both PARG inhibitors were present in the incubating media for 1 (a), 3 (b), 6 h (c) (see time lines on right of a–c). Bars are the mean±s.e.mean of three experiments conducted in duplicate. ^*P<0.05 vs control (ctrl), ^#P<0.05 vs MNNG (ANOVA+Tukey's post hoc test).

Figure 6

Effect of gallotannin, 3-galloyl-1,2-O-isopropylidene glucose, 4-hydroxy-TEMPO (TMP) or epigallocatechin gallate on cell death triggered by 12 h exposure to 1-methyl-3-nitro-1-nitrosoguanidine (MNNG). (a) HeLa cells were exposed to 100 μM gallotannin (1), 100 μM 3-galloyl-1,2-O-isopropylidene glucose (4), 1 mM TMP or 100 μM epigallocatechin gallate (15) for 12 h and cell death was evaluated by methylthiazolyl tetrazolium (MTT) reduction and lactate dehydrogenase (LDH) release. Columns represent the mean±s.e.mean of three experiments conducted in duplicate. ^*P<0.05, ^**P<0.01 vs ctrl. ^§P<0.05, ^§§P<0.01 vs MNNG (ANOVA+Tukey's post hoc test). (b) Phase-contrast micrograph visualization of cells exposed with or without 100 μM MNNG for 1 h in the presence or absence of 100 μM gallotannin (1) or 100 μM 3-galloyl-1,2-O-isopropylidene glucose (4). (c) Cell counting of cultures exposed to 100 μM gallotannin (1) or 100 μM 3-galloyl-1,2-O-isopropylidene glucose (4) in the presence or absence of MNNG. ^**P<0.01 vs ctrl (ANOVA+Tukey's post hoc test). (d) Cell nuclei visualized with the comet assay of cells exposed for 30 min to MNNG or 3-galloyl-1,2-O-isopropylidene glucose (4) both at 100 μM. (e) Measurement of DNA damage (% of DNA damage in tail) in cells treated as in (d). Bars represent the mean±s.e.mean of three (a, c) and two (e) experiments. Images are representative of three (b) and two (d) experiments. Bar=20 μm.

Figure 7

Effect of gallotannin (1) or 3-galloyl-1,2-O-isopropylidene glucose (4) on mitochondrial apoptosis-inducing factor (AIF) release and nuclear morphology of cells exposed to 1-methyl-3-nitro-1-nitrosoguanidine (MNNG). (a) AIF immunostaining of cells under control conditions or 4 h after exposure to 100 μM MNNG for 1 h in the absence or presence of 100 μM gallotannin (1) or 100 μM 3-galloyl-1,2-O-isopropylidene glucose (4). The effect of the two compounds alone is shown. Note the loss of punctate staining and AIF redistribution into the cytoplasm and nucleus in MNNG-exposed cells. (b) Hoechst 33258 staining of nuclei of cells under control conditions or 8 h after exposure to 100 μM MNNG for 1 h in the absence or presence of gallotannin (1) or 3-galloyl-1,2-O-isopropylidene glucose (4) both at 100 μM. The effect of gallotannin (1) or 3-galloyl-1,2-O-isopropylidene glucose (4) alone is also shown. Note nuclear shrinkage, pyknosis and chromatin condensation (arrows) in MNNG-exposed cells, and partial prevention of these nuclear alterations by 3-galloyl-1,2-O-isopropylidene glucose (4). Arrowheads point to partially shrunken nuclei with irregular surfaces. Images are representative of three experiments. Bar=20 μm (a) and 10 μm (b).