Differential effects of garcinol and curcumin on histone and p53 modifications in tumour cells

Abstract

Background: Post-translational modifications (PTMs) of histones and other proteins are perturbed in tumours. For example, reduced levels of acetylated H4K16 and trimethylated H4K20 are associated with high tumour grade and poor survival in breast cancer. Drug-like molecules that can reprogram selected histone PTMs in tumour cells are therefore of interest as potential cancer chemopreventive agents. In this study we assessed the effects of the phytocompounds garcinol and curcumin on histone and p53 modification in cancer cells, focussing on the breast tumour cell line MCF7.

Methods: Cell viability/proliferation assays, cell cycle analysis by flow cytometry, immunodetection of specific histone and p53 acetylation marks, western blotting, siRNA and RT-qPCR.

Results: Although treatment with curcumin, garcinol or the garcinol derivative LTK-14 hampered MCF7 cell proliferation, differential effects of these compounds on histone modifications were observed. Garcinol treatment resulted in a strong reduction in H3K18 acetylation, which is required for S phase progression. Similar effects of garcinol on H3K18 acetylation were observed in the osteosarcoma cells lines U2OS and SaOS2. In contrast, global levels of acetylated H4K16 and trimethylated H4K20 in MCF7 cells were elevated after garcinol treatment. This was accompanied by upregulation of DNA damage signalling markers such as γH2A.X, H3K56Ac, p53 and TIP60. In contrast, exposure of MCF7 cells to curcumin resulted in increased global levels of acetylated H3K18 and H4K16, and was less effective in inducing DNA damage markers. In addition to its effects on histone modifications, garcinol was found to block CBP/p300-mediated acetylation of the C-terminal activation domain of p53, but resulted in enhanced acetylation of p53K120, and accumulation of p53 in the cytoplasmic compartment. Finally, we show that the elevation of H4K20Me3 levels by garcinol correlated with increased expression of SUV420H2, and was prevented by siRNA targeting of SUV420H2.

Conclusion: In summary, although garcinol and curcumin can both inhibit histone acetyltransferase activities, our results show that these compounds have differential effects on cancer cells in culture. Garcinol treatment alters expression of chromatin modifying enzymes in MCF7 cells, resulting in reprogramming of key histone and p53 PTMs and growth arrest, underscoring its potential as a cancer chemopreventive agent.

Figure 1

Curcumin, garcinol and LTK-14 impede MCF7 cell proliferation. (A) MCF7 cells were seeded at a density of approximately 5 × 10³ cells per well in microtitre plates and allowed to adhere overnight. The initial cell density was determined in a control plate prior to addition of curcumin, garcinol or LTK-14 at the indicated concentrations, or vehicle. After 24 hours, the change in the number of viable cells was estimated using MTT assays (see Methods). The data shown are the means of 5 replicates ± standard deviations. (B) Cell cycle analyses of MCF7 cells after 24 hours in culture in the presence of garcinol or LTK14 (10 μM) or vehicle control. The bar charts show a representative experiment indicating the percentage of cells in G1, S or G2/M phases, and the subG1 population as determined by BrdU incorporation and propidium iodide staining. (C) Cell cycle analyses of MCF7 cells after 24 hours culture in the presence of curcumin (10 μM or 20 μM) or vehicle control, as described in (B).

Figure 2

Reprogramming of global histone modifications by garcinol and curcumin. (A) Nuclear staining of MCF7 breast cancer cells with antibodies detecting pan acetyl H3 (top panels) or pan acetyl H4 (bottom panels). Control shows typical staining in the absence of inhibitors (vehicle only), and the effect of treatment with HAT inhibitors at the indicated concentrations are also shown. Scalebar: 10 μm. (B&C) Immunostaining of MCF7 cells following treatment with the indicated concentrations of curcumin or garcinol for 24 hours. Vehicle control is also shown. Histone PTM-specific antibodies were used to reveal H3K18Ac, H4K16Ac and H3K9Ac levels in response to treatment. Scalebar: 10μm. (D) Western blots on whole cell extracts of MCF7 cells. Cell extracts were prepared following 24 hours culture in the presence of HAT inhibitors (or vehicle control) at the indicated concentrations. Specific antibodies were used to detect bulk levels of H3K18Ac, H4K16Ac and H3K9Ac. Densitometry measurements were performed using Image J software [21]. The level of each histone PTM in controls (vehicle only, normalised to a loading control) was set to 1. (E) Western blots showing bulk levels of H3K18Ac in whole cell extracts of U2OS and SaOS2 osteosarcoma cells following exposure to 10 μM or 20 μM curcumin or garcinol (as indicated in increasing scale). Actin loading controls are also shown, and the data were quantified by Image J as in (D).

Figure 3

Garcinol induces DNA repair pathways and alters p53 acetylation. (A) Immunostaining of MCF7 cells for the DNA damage marker γH2A.X following treatment with curcumin, garcinol or vehicle for 24 hours. (B) Western blots of acid extracted histones prepared from MCF7 cells following treatment for 24 hours with HAT inhibitors at the indicated concentrations. Specific antibodies were used to reveal γH2A.X and H3K56Ac. Immunodetection of histone H3 and Coomassie staining of extracted histones are shown as loading controls. (C) Western blots showing the levels of hMOF, TIP60 and SIRT1 proteins in whole cell extracts of MCF7 cells following curcumin or garcinol treatment or control (DMSO). (D) Western blots detecting p53 expression levels and selected p53 acetylation PTMs (K373/382Ac or K120Ac) following garcinol treatment of MCF7 cells for 24 hours. (E) Immunostaining of MCF7 cells as treated in (D) showing the detection level and subcellular distribution of p53 and TIP60 proteins in MCF7 cells. Scalebar is 10 μm. (F) Higher magnification of boxed insets in (E) showing localisation of p53K120Ac and p53K373/382Ac proteins to the cytoplasm and nucleus, respectively.

Figure 4

Garcinol reprograms H4K20 trimethylation by inducing SUV420H2. (A) Immunodetection of H4K20Me3 in MCF7 cells in response to garcinol treatment. Scalebar: 10 μm. (B) Western blots showing relative levels of H4K20Me3 and H3K9Me3 in acid-extracted histones prepared from MCF7 cells following treatment for 24 hours with HAT inhibitors at the indicated concentrations or control (DMSO). (C) Western blots showing induction of SUV420H2 and H4K20Me3 in MCF7 cells in response to garcinol exposure as in (B). (D) Quantitative analysis of the relative levels of SUV420H2 and H4K20Me3 in MCF7 cells in response to garcinol treatment as detected by flow cytometry. Cells were exposed to garcinol as in (B) and then fixed and permeabilised before incubation with primary and secondary (543 fluorophore-conjugated) antibodies. The data shown is the number of cells scoring positive for the indicated antigens from a total of 4000 scanned cells. (E) Relative levels of SUV420H2 transcripts in MCF7 cells at 24 hours post-transfection with siRNA duplexes targeting SUV420H2 (siSUV420H2) or a scramble control (siCTRL) siRNA mediated knockdown. (F) Western blots on MCF7 cell extracts following siRNA targeting as in (E) followed by exposure to garcinol (20 μM) for a further 24 hrs. The blot shows detection of H4K20Me3 levels following garcinol treatment in the control or SUV420H2 depleted cells.