Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Jul 28;10(7):e0134110.
doi: 10.1371/journal.pone.0134110. eCollection 2015.

Curcumin-Mediated HDAC Inhibition Suppresses the DNA Damage Response and Contributes to Increased DNA Damage Sensitivity

Shu-Huei Wang  1 , Pei-Ya Lin  2 , Ya-Chen Chiu  2 , Ju-Sui Huang  2 , Yi-Tsen Kuo  2 , Jen-Chine Wu  3 , Chin-Chuan Chen  4
Affiliations

Curcumin-Mediated HDAC Inhibition Suppresses the DNA Damage Response and Contributes to Increased DNA Damage Sensitivity

Shu-Huei Wang et al. PLoS One. .

Abstract

Chemo- and radiotherapy cause multiple forms of DNA damage and lead to the death of cancer cells. Inhibitors of the DNA damage response are candidate drugs for use in combination therapies to increase the efficacy of such treatments. In this study, we show that curcumin, a plant polyphenol, sensitizes budding yeast to DNA damage by counteracting the DNA damage response. Following DNA damage, the Mec1-dependent DNA damage checkpoint is inactivated and Rad52 recombinase is degraded by curcumin, which results in deficiencies in double-stand break repair. Additive effects on damage-induced apoptosis and the inhibition of damage-induced autophagy by curcumin were observed. Moreover, rpd3 mutants were found to mimic the curcumin-induced suppression of the DNA damage response. In contrast, hat1 mutants were resistant to DNA damage, and Rad52 degradation was impaired following curcumin treatment. These results indicate that the histone deacetylase inhibitor activity of curcumin is critical to DSB repair and DNA damage sensitivity.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Curcumin sensitizes wild-type cells to the DNA-damaging drugs.
Five-fold serial dilution analysis of the indicated isogenic strains, including WT (BY4741), rpd3∆ (BY4741-rpd3), hda1∆ (BY4741-hda1), sir2∆ (BY4741-sir2), gcn5∆ (BY4741-gcn5), and rtt109∆ (BY4741-rtt109) shows sensitivity to (A) MMS, (B) 4NQO, and (C) HU with or without curcumin. The cells were allowed to grow at 30°C for 3 days and photographed to record colony formation.
Fig 2
Fig 2. Curcumin increases DNA damage sensitivity and inhibits DNA double-strand break repair in SSA strains.
(A) A schematic diagram of the SSA system. Galactose was used to induce HO endonuclease to generate a specific HO lesion. Repair of the HO lesion at the HO cleavage site (black box) requires 5-kb or 30-kb of resection back to the uncleavable HO cleavage site (gray box). Three PCR primers were used to measure the DNA damage and repair. (B) Curcumin inhibits DNA double-strand break repair in the 5-kb resection strains. The repair of the HO lesion was analyzed by PCR. The DSB was induced by the addition of galactose to the 5-kb resection strain (YMV045). After 30 min, cultures were treated with 50 μM and 200 μM curcumin. (C) Curcumin inhibits DNA double-strand break repair in the 30-kb resection strain. PCR analysis of the 30-kb resection strain (YMV002) as described in (B). (D) Five-fold serial dilution analysis of WT-1 (YMV002), rad52∆ (YMV037), WT-2 (YMV045), srs2∆ (YMV057), and rad52∆ (YMV046) shows the sensitivity to galactose and curcumin. The cells were allowed to grow at 30°C for 3 days and photographed to record colony formation.
Fig 3
Fig 3. Curcumin influences the DNA damage response.
(A) Wild-type cells (YMV045) or (B) MRE11-MYC cells (YAY032) were arrested at G2 with 15 μg/ml nocodazole, and HO endonuclease was induced by the addition of galactose to generate a DSB. After 30 min, the cultures were divided equally and treated with or without 200 μM curcumin. The indicated antibodies were used to detect protein expression by western blotting. Amido black staining of total proteins and Pgk1 protein levels serve as loading controls. (C) Schematic of the positions of the primers used for ChIP analyses. (D) MRE11-MYC cells (YAY032), (E) RFA1-MYC cells (YAY022), (F) wild-type cells (YMV045), or (G) DDC1-MYC cells (CCY025) were cultured as in (A), and the recruitment of the indicated proteins flanking the HO lesion was analyzed by ChIP. Error bars represent the standard deviations of at least three independent experiments. Asterisks (*P<0.05) represent a significant difference between curcumin-treated and untreated cells. (n = 3 for (D), (F) and (G). n = 5 for (E)).
Fig 4
Fig 4. Curcumin cooperatively stimulates apoptosis in response to DNA damage.
(A) YMV045 cells were treated with 0.02% MMS for an hour followed by treatment with or without 200 μM curcumin for 3 hours. The fluorescent signals of Annexin V and PI were examined using fluorescence microscopy. Scale bars are 5 μm. The table shows the number of cells expressing Annexin V or PI signals. (B) The panel shows the percentage of fluorescence signals from (A). Error bars represent the standard deviations of three independent experiments. *P<0.05.
Fig 5
Fig 5. Curcumin inhibits MMS-induced autophagy.
(A) The atg8△ cells were transformed with a plasmid containing a GFP fusion of ATG8 (RLY004). The RLY004 cells were treated with 0.02% MMS, 200 μM curcumin, or both for 3 hours and processed for fluorescence microscopy. Arrows indicate the accumulation of GFP-Atg8 in the vacuole. FM 4–64 was used to label the vacuoles. Cells cultured in nitrogen starvation medium (SN) served as positive controls for autophagy. Scale bars are 2.5 μm. The table shows the number of cells expressing GFP foci. The panel shows the percentage of fluorescence signals. Error bars represent the standard deviations of three independent experiments. *P<0.05. (B) As in (A), cells were subjected to immunoblotting analysis using anti-GFP antibodies. The atg1△ cells expressing GFP-Atg8 (RLY005) provide negative controls for autophagy. Amido black staining of total proteins and Pgk1 protein levels serve as loading controls.
Fig 6
Fig 6. Rad52 protein expression was inhibited by curcumin following DNA damage.
RAD52-HA cells (YAY013) were arrested in G2 with nocodazole, and HO endonuclease was induced by the addition of galactose to generate a DSB. After 30 min, the cultures were divided equally and treated with or without 200 μM curcumin. (A) Immunoblotting analyses of Rad52 using HA antibodies at the indicated time points. (B) The recruitment of Rad52 to DSBs was analyzed by ChIP. Error bars represent the standard deviations of three independent experiments. *P<0.05. (C) Samples were processed for reverse transcription to generate cDNA and analyzed by PCR or (D) quantitative PCR. Error bars represent the standard deviations of three independent experiments. (E) The deletion of SEM1 counteracts the disappearance of Rad52 after curcumin treatment following an HO-induced break. Immunoblotting analysis of the RAD52-MYC (YAY028) and sem1△ RAD52-MYC (RLY006) strains was performed as described in Fig 3A. Amido black staining of total proteins and Pgk1 protein levels serve as loading controls.
Fig 7
Fig 7. Curcumin inhibits DNA repair processing in an acetylation dependent manner.
(A) DNA repair is inhibited in rpd3 mutants in response to DNA damage. The same analysis presented in Fig 2B was performed on WT (YMV045) and rpd3∆ (YAY012) strains undergoing SSA with 5 kb resection. (B) The rpd3 mutants mimic curcumin treatment in response to DNA damage. The RAD52-HA (YAY013) and rpd3∆ RAD52-HA (YAY016) cells were cultured as in Fig 3A and processed for immunoblotting analysis using HA antibodies. (C) Curcumin failed to inhibit DSB repair in hat1 mutants. The WT (YMV045) and hat1∆ (ILY001) strains were used for the repair assay. (D) Five-fold serial dilution analysis of WT (BY4741), hat1∆ (BY4741-hat1), and rad6∆ (BY4741-rad6) was performed as described in Fig 1A. (E) The disappearance of Rad52 induced by curcumin is counteracted in hat1 mutants. Immunoblotting analysis of the RAD52-MYC (YAY028) and hat1△ RAD52-MYC (RLY007) strains was performed as described in Fig 3A. Amido black staining of total proteins and Pgk1 protein levels serve as loading controls.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Qin J, Li L. Molecular anatomy of the DNA damage and replication checkpoints. Radiation research. 2003;159(2):139–48. Epub 2003/01/23. . - PubMed
    1. Melo J, Toczyski D. A unified view of the DNA-damage checkpoint. Current opinion in cell biology. 2002;14(2):237–45. Epub 2002/03/14. . - PubMed
    1. Gilbert CS, Green CM, Lowndes NF. Budding yeast Rad9 is an ATP-dependent Rad53 activating machine. Mol Cell. 2001;8(1):129–36. Epub 2001/08/21. . - PubMed
    1. Sanchez Y, Desany BA, Jones WJ, Liu Q, Wang B, Elledge SJ. Regulation of RAD53 by the ATM-like kinases MEC1 and TEL1 in yeast cell cycle checkpoint pathways. Science. 1996;271(5247):357–60. Epub 1996/01/19. . - PubMed
    1. Dietlein F, Reinhardt HC. Molecular pathways: exploiting tumor-specific molecular defects in DNA repair pathways for precision cancer therapy. Clinical cancer research: an official journal of the American Association for Cancer Research. 2014;20(23):5882–7. Epub 2014/12/03. 10.1158/1078-0432.CCR-14-1165 . - DOI - PubMed

Publication types

MeSH terms