Vanillins--a novel family of DNA-PK inhibitors - PubMed (original) (raw)
Vanillins--a novel family of DNA-PK inhibitors
Stephen Durant et al. Nucleic Acids Res. 2003.
Abstract
Non-homologous DNA end-joining (NHEJ) is a major pathway of double strand break (DSB) repair in human cells. Here we show that vanillin (3-methoxy-4-hydroxybenzaldehyde)--a naturally occurring food component and an acknowledged antimutagen, anticlastogen and anticarcinogen--is an inhibitor of NHEJ. Vanillin blocked DNA end-joining by human cell extracts by directly inhibiting the activity of DNA-PK, a crucial NHEJ component. Inhibition was selective and vanillin had no detectable effect on other steps of the NHEJ process, on an unrelated protein kinase or on DNA mismatch repair by cell extracts. Subtoxic concentrations of vanillin did not affect the ATM/ATR-dependent phosphorylation of Chk2 or the S-phase checkpoint response after ionising radiation. They significantly potentiated the cytotoxicity of cisplatin, but did not affect sensitivity to UVC. A limited screen of structurally related compounds identified two substituted vanillin derivatives that were 100- and 50-fold more potent than vanillin as DNA-PK inhibitors. These compounds also sensitised cells to cisplatin. The inhibition of NHEJ is consistent with the antimutagenic and other biological properties of vanillin, possibly altering the balance between DSB repair by NHEJ and homologous recombination.
Figures
Figure 1
Structures of vanillin and wortmannin.
Figure 2
DNA end-joining inhibition by vanillin. (a) End-joining activity of 10 µg GM00558 extract. Lane 1, extract alone; lanes 2–4, extract pre-incubated with vanillin for 10 min at 4°C; lane 5, extract pre-incubated with 0.25 µM wortmannin for 10 min at 4°C; lane 6, 1 kb DNA ladder. The right panel shows end-joining activity as measured by intensity of ligated products (measured using Storm 840 IQ software) as a percentage of no vanillin treatment plotted against vanillin concentration. Vanillin has an IC50 of 300 µM. (b) Mismatch repair activity. Activity of HeLa cell extract in repair of an A.C mismatched plasmid substrate. Lane 1, marker; lane 2, no extract; lane 3, extract; lanes 4–7, extract pre-incubated for 5 min at room temperature with a range of concentrations of vanillin shown.
Figure 3
DNA-PK activity. (a) Purified DNA-PK activity. Left panel, dose response of increasing concentrations of purified DNA-PK. Open box represents 0.25 µM wortmannin incubated with 5 ng DNA-PK. Right panel, activity of 1.5 ng DNA-PK pre-incubated for 5 min at 4°C with vanillin at varying concentrations. (b) DNA-PK activity using GM00558 extract. Left panel, dose response of increasing concentrations of extract. Right panel, activity of 1 µg extract pre-incubated with vanillin at increasing concentrations.
Figure 4
Ligase adenylation and Ku-IP6 binding. (a) Ligase adenylation. (i) Incorporation of [α-32P]ATP by a purified Ligase I titration. (ii) Effect of vanillin on Ligase I at 190 ng. A weak signal was generated, therefore (iii) shows effect of vanillin titration on 760 ng Ligase I. (iv) Incorporation of [α-32P]ATP by GM00558 extract. (v) Ten microgram extract pre-incubated (10 min at 4°C) with vanillin. (b) Ku binding to IP6. Effect of vanillin on Ku binding to 3H-labelled IP6. Counts per minute (c.p.m.) of labelled IP6 eluted plotted against vanillin concentration.
Figure 5
Kinase activity. (a) Effect of DNA-PK inhibitors on PKC activity. Using 10 ng of PKC vanillin, DMNB, wortmannin (wort) and myristoylated peptide inhibitor was added at increasing concentrations. (b) Phosphoryl ation of Chk2. Extracts made from GM00558 cells treated with 0, 10 and 20 Gy IR and incubated with 300 µM vanillin or 15 µM DMNB were used to measure Phospho-Chk2 by western blot. (c) Radio-resistant DNA synthesis. GM00558 and D5037 (MRE11-deficient) cells incubated with 3H TdR for 3 h. Vanillin was added as a continuous exposure while wortmannin was added during the first hour of incubation. Curves show levels of DNA synthesis with and without 300 µM vanillin. Single points indicate levels of DNA synthesis after exposure to 10 Gy IR at higher concentrations of vanillin and wortmannin.
Figure 6
Cytotoxic and growth responses to vanillin. A2780 responses to: (a) vanillin titrations and combinations of vanillin with (b) cisplatin, (c) UV-C and (d) IR exposure. (e) TK6 and WTK1 (p5-defective) growth responses to cisplatin with and without (300 µM) vanillin. (f) Growth responses of D5037 (MRE11-deficient) cells to cisplatin and 15 µM DMNB.
Figure 7
Confocal microscopy. RPA/Rad51 foci formation in the nuclei of A2780 cells: (a) untreated, (b) treated with 300 mM vanillin, (c) exposed for 1 h to 25 µM wortmannin followed by 20 h incubation, (d) exposed to 5 µM cisplatin for 1 h followed by 20 h incubation, (e) exposed to cisplatin while in continuous exposure to vanillin (300 µM) and (f) exposed to cisplatin followed by a 1 h exposure to wortmannin 20 h later. RPA (red), Rad51 (green) and merge (yellow).
Figure 8
Isolation and characterisation of vanillin-derived DNA-PK inhibitors. (a) Left panel, DNA-PK inhibition by vanillin derivatives. Each compound was included in the DNA-PK assay at 100 µM. Structures of active compounds 4 and 11 shown in the right panel. (b) DNA-PK inhibition by vanillin and DMNB. (c) Growth inhibition of D5037 cells exposed for 1 h to 15 µM DMNB 20 h after a 1-h exposure to 2.5 µM cisplatin.
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