The enhancement of antiproliferative and proapoptotic activity of HDAC inhibitors by curcumin is mediated by Hsp90 inhibition

Abstract

Curcumin, a natural polyphenol, has been described to exhibit effects on signaling pathways, leading to induction of apoptosis. In this study, we observed that curcumin inhibited Hsp90 activity causing depletion of client proteins implicated in survival pathways. Based on this observation, this study was designed to investigate the cellular effects of curcumin combination with the pan-HDAC inhibitors, vorinostat and panobinostat, which induce hyperacetylation of Hsp90, resulting in inhibition of its chaperone function. The results showed that, at subtoxic concentrations, curcumin markedly sensitized tumor cells to vorinostat- and panobinostat-induced growth inhibition and apoptosis. The sensitization was associated with persistent depletion of Hsp90 client proteins (EGFR, Raf-1, Akt, and survivin). In conclusion, our findings document a novel mechanism of action of curcumin and support the therapeutic potential of curcumin/HDAC inhibitors combination, because the synergistic interaction was observed at pharmacologically achievable concentrations, which were ineffective when each drug was used alone.

Fig. 1

Antiproliferative effect of curcumin on A431 and STO cells. Cells were treated for 72 h with curcumin and cell growth was evaluated 72 h after the treatment by cell counting. Inverted filled triangle A431 cells (IC₅₀, 2.2 μM); open circle STO cells (IC₅₀, 2.1 μM). Data are the mean of three independent experiments

Fig. 2

a Effect of curcumin on Hsp90 client protein levels in A431 and STO cells. Total cellular extracts were obtained 24 h after treatment with curcumin (5 μM, IC₈₀). Actin is shown as a control for protein loading. C Control, CUR curcumin. b Comparison of the effects of curcumin and 17-AAG on protein levels of Hsp70. Cells were treated as described in (a), with curcumin (5 μM) or 17-AAG (0.1 μM). c Coimmunoprecipitation of Raf-1/Hsp90 in A431 cells treated with 17-AAG or curcumin for 1 h. Cells were treated with equitoxic concentrations of 17-AAG (0.1 μM, IC₈₀) or curcumin (5 μM). Cell lysates were than harvested and immunoprecipitated with anti-Raf-1 rabbit polyclonal antibody. Immunoprecipitates were immunoblotted for Hsp90. Blots were stripped and probed for Raf-1. C Control, AAG 17-AAG, CUR curcumin. d Coimmunoprecipitation of Hsp90/hsc70 and Hsp90/p23 in A431 cells treated with curcumin. Cells were treated with two concentrations of curcumin (2 and 5 μM; i.e., IC₅₀ and IC₈₀) as described above. Immunoprecipitated obtained by anti-Hsp90 antibody were immunoblotted for hsc70 or p23. C Control, CUR2 curcumin 2 μM, CUR5 curcumin 5 μM. e Effect of bortezomib (BOR) on the depletion of Hsp90 client proteins induced by curcumin (CUR) in A431 cells. Total cellular extracts were obtained 24 h after treatment (curcumin 5 μM, bortezomib 0.001 μM). Actin is shown as a control for protein loading. C Control

Fig. 3

Schematic representation of the results obtained from limited proteolysis experiments on recombinant Hsp90. The preferential cleavage sites detected performing enzymatic digestions on recombinant Hsp90, on the Hsp90/curcumin complex, and on the Hsp90/radicicol complex are in dark grey. The Hsp90 N-terminal domain is highlighted in light grey, the middle domain is boxed and the C-terminal domain is highlighted in grey

Fig. 4

Antiproliferative effects of HDAC inhibitors, vorinostat (a) or panobinostat (b), alone or in combination with curcumin. Cells were treated for 72 h with panobinostat or vorinostat combined with two subtoxic concentrations of curcumin (1.5 and 0.5 μM); cell growth, after the treatment, was determined by cell counting. Data are the mean of three independent experiments. Filled square single agent (vorinostat or panobinostat), filled circle curcumin 1.5 μM + vorinostat or panobinostat, filled triangle curcumin 0.5 μM + vorinostat or panobinostat. The effect of the subtoxic concentrations of curcumin alone at the tested concentrations is also shown in each cell line. Open triangle curcumin 0.5 μM, open circle curcumin 1.5 μM. Data from three independent assays were analyzed by t test (vorinostat or panobinostat vs combined treatment): *P < 0.05, ** P < 0.005, ***P < 0.0005

Fig. 5

a Effect of curcumin on apoptosis induced by HDAC inhibitors in A431 cells. Cells were exposed for 72 h to subtoxic concentrations of panobinostat (11.5 nM) or vorinostat (15 nM), alone or in combination with curcumin (1.5 μM). b Apoptotic cell death caused by cytotoxic concentrations of single agent treatment. Cells were exposed for 72 h to cytotoxic concentrations of panobinostat (100 nM), vorinostat (1 μM) and curcumin (5 μM). At the end of the treatment, apoptosis was detected by TUNEL assay and determined by FACS analysis. In a parallel experiment, cells were treated for 24 h and apoptosis was detected after 72 h following drug removal. Data from three independent assays were analyzed by t test (control vs treatment): *P < 0.05, **P < 0.005

Fig. 6

Effects of combination of curcumin/vorinostat (a) or curcumin/panabinostat (b), or single agent treatment (c) on cellular levels of Hsp90 client proteins. a,b Cells were exposed to each drug alone or in combination for 24 h and processed as indicated in legend to Fig. 2. Subtoxic concentrations of each drug were used (i.e. curcumin 1.5 μM, vorinostat 15 nM, panobinostat 11.5 nM). The analysis was also performed after incubation of the drug-treated cells in drug-free medium for 24 h. c Cells were exposed to cytotoxic concentrations (IC₈₀) of each drug (i.e. curcumin 5 μM, vorinostat 1 μM, panobinostat 0.1 μM) for 24 h and processed as indicated in the legend to Fig. 2. The analysis was also performed after incubation of the drug-treated cells in drug-free medium for 24 h. Actin is shown as a control for protein loading

Fig. 7

Coimmunoprecipitation of Raf-1/Hsp90 in A431 cells treated with curcumin, vorinostat, panobinostat alone or in combination. A431 cells were exposed to subtoxic concentrations of panobinostat (11.5 nM), vorinostat (15 nM), and curcumin (1.5 μM) alone or in combination, for 4 h. Cell lysates were than harvested and immunoprecipitated with anti-Raf-1 rabbit polyclonal antibody. Immunoprecipitates were immunoblotted for Raf-1. Blots were stripped and probed for Hsp90 and Hsp70