The anti-tumor histone deacetylase inhibitor SAHA and the natural flavonoid curcumin exhibit synergistic neuroprotection against amyloid-beta toxicity

Abstract

With the trend of an increasing aged population worldwide, Alzheimer's disease (AD), an age-related neurodegenerative disorder, as one of the major causes of dementia in elderly people is of growing concern. Despite the many hard efforts attempted during the past several decades in trying to elucidate the pathological mechanisms underlying AD and putting forward potential therapeutic strategies, there is still a lack of effective treatments for AD. The efficacy of many potential therapeutic drugs for AD is of main concern in clinical practice. For example, large bodies of evidence show that the anti-tumor histone deacetylase (HDAC) inhibitor, suberoylanilidehydroxamic acid (SAHA), may be of benefit for the treatment of AD; however, its extensive inhibition of HDACs makes it a poor therapeutic. Moreover, the natural flavonoid, curcumin, may also have a potential therapeutic benefit against AD; however, it is plagued by low bioavailability. Therefore, the integrative effects of SAHA and curcumin were investigated as a protection against amyloid-beta neurotoxicity in vitro. We hypothesized that at low doses their synergistic effect would improve therapeutic selectivity, based on experiments that showed that at low concentrations SAHA and curcumin could provide comprehensive protection against Aβ25-35-induced neuronal damage in PC12 cells, strongly implying potent synergism. Furthermore, network analysis suggested that the possible mechanism underlying their synergistic action might be derived from restoration of the damaged functional link between Akt and the CBP/p300 pathway, which plays a crucial role in the pathological development of AD. Thus, our findings provided a feasible avenue for the application of a synergistic drug combination, SAHA and curcumin, in the treatment of AD.

Figure 1. Effect of SAHA and curcumin on cell viability of PC12 cells.

A. PC12 cells were treated with different concentrations of Aβ_25–35 (n = 5). *, p<0.05; ***, p<0.001 vs Ctrl. B. PC12 cells were treated with different concentrations of SAHA (n = 5). *, p<0.05; ***, p<0.001 vs Ctrl. C. PC12 cells were treated with different concentrations of curcumin (n = 5). *, p<0.05; ***, p<0.001 vs Ctrl. D. MTT assay was performed to detect cell viability after treating with SAHA and curcumin against Aβ_25–35-induced cytotoxicity in PC12 cells (n = 5). ***, p<0.001 vs Ctrl; **, p<0.01 vs Aβ_25–35.

Figure 2. Effect of SAHA and curcumin on neuronal integrity and oxidative stress.

A. Result of AO/EB staining. S: SAHA; C: curcumin. B. Result of SOD content measurement. ***, p<0.001 vs Ctrl; #, p<0.05; ##, p<0.01, ###, p<0.001, vs Aβ_25–35.

Figure 3. Effect of SAHA and curcumin on cell apoptosis of PC12 cells.

A. Apoptotic cells were detected by TUNEL assay. Cells were stained with TUNEL positive nuclei (green) and nuclei of PC12 cells (blue). B. The percentage of TUNEL positive cells was determined (n = 5). ***, p<0.001 vs Ctrl; **, p<0.01 vs Aβ_25–35. Ctrl: the control group. C. Western blot of cleaved caspase 3 (n = 3). ***, p<0.001 vs Ctrl; **, p<0.01 vs Aβ_25–35. Ctrl: the control group; CASP3: caspase 3.

Figure 4. Changes of Akt phosphorylation in PC12 cells after SAHA, curcumin or SAHA+curcumin treatment.

A. Representative western blot of the p-Akt (Ser 473) protein expression with SAHA treatment (n = 3). **, p<0.01 vs Ctrl. B. Representative western blot of the p-Akt (Ser 473) protein expression with curcumin treatment (n = 3). **, p<0.01 vs Ctrl. C. Representative western blot of the p-Akt (Ser 473) protein expression with co-treatment of SAHA and curcumin (n = 3) to the PC12 cells (n = 3). *, p<0.05 vs Ctrl; **, p<0.01 vs Aβ_25–35; Ctrl: the control group.

Figure 5. Illustration of gene expression correlation analysis with CREBBP (MMSE score) and EP300 (NFT score) as example.

MMSE score:MiniMental Status Examination score; NFT score: neurofibrillary tangle score. Normalized value: the median-normalized gene expression value (log₂).

Figure 6. Results of network topology calculation for AD-related genes.

A. Statistical result of R² for hub-encoding genes (degree >50) at different thresholds. B. Statistical result of R² for genes encoding proteins of high betweenness centrality (betweenness centrality >5×10⁻⁴) at different thresholds. C. Strong binomial correlation was found between the topological parameters degree and betweenness centrality (R = 0.849). Three AD-related genes AKT1, CREBBP and EP300 were highlighted as blue nodes. R² _e-m, R² _m-m, R² _l-m: R² with MMSE score at early-, medium-, and late-stage of AD, respectively; R² _e-n, R² _m-n, R² _l-n: R² with square root of NFT score at early-, medium-, and late-stage of AD, respectively.

Figure 7. Results of GenePro and expression distance analyses.

The AD-related processes of up- and down-regulation were distinguished using red and green border colors. If gene expression distance was less than 0.20, two nodes representing AD-related processes were connected with edge by solid line. If PPIs was more than 20, them were connected with edge by dashed line.

Figure 8. Network visualization of interactions between proteins encoded by up-regulated AD process genes (red triangle nodes) and those encoded by down-regulated AD process genes (light green rectangle nodes).

Big nodes represent the hBPIN hub proteins that encoded by AD-related genes (degree >50). The black node border represents that the betweenness centrality of the protein is high (betweenness centrality >5×10⁻⁴). Functional interactions between protein Akt and the hub proteins CBP and p300 were highlighted as thick blue edges, which were encoded by AKT1, CREBBP and EP300, respectively.

Figure 9. Network visualization of potential mechanism underlying synergistic cytoprotection by SAHA and curcumin.

SAHA is a non-selective inhibitor of HDACs and along with curcumin synergistically activated Akt. Eight proteins encoded by AD-related genes were found to be simultaneously associated with HDACs-encoding proteins (blue edges) and Akt which is encoded by AKT1 (green edges) in hBPIN. Nodes with dark lines represent hubs of high betweenness centrality in hBPIN.