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. 2019 Apr 26:7:e6716.
doi: 10.7717/peerj.6716. eCollection 2019.

A combination of curcumin, vorinostat and silibinin reverses A β-induced nerve cell toxicity via activation of AKT-MDM2-p53 pathway

Jia Meng  1 , Yan Li  2 , Mingming Zhang  1 , Wenjing Li  1 , Lin Zhou  1 , Qiujun Wang  1 , Lin Lin  1 , Lihong Jiang  1 , Wenliang Zhu  3
Affiliations

A combination of curcumin, vorinostat and silibinin reverses A β-induced nerve cell toxicity via activation of AKT-MDM2-p53 pathway

Jia Meng et al. PeerJ. .

Abstract

Alzheimer's disease (AD) is a significant health issue for the elderly and becoming increasingly common as the global population ages. Although many efforts have been made to elucidate its pathology, there is still a lack of effective clinical anti-AD agents. Previous research has shown the neuroprotective properties of a combination of curcumin and vorinostat. In this study, nine other neuroprotective agents were investigated to examine whether a three-drug combination of curcumin, vorinostat, and a new drug is more advantageous than the previous two-drug combination in alleviating amyloid beta (Aβ)-induced nerve cell toxicity. Cell viability assay was performed to screen these agents, and further validation tests, including determination of cellular oxidative stress, apoptosis, and activity of the AKT/MDM2/p53 pathway, were performed. Among the nine candidate compounds, only silibinin at 1 µM reduced Aβ25-35-induced toxicity in PC12 cells. The neuroprotective effects of 1 µM silibinin in combination with 5 µM curcumin and 0.5 µM vorinostat (CVS) was shown in PC12 cells, in which it decreased apoptosis and oxidative stress marker levels that were increased by 20 µM Aβ25-35. Western blotting results showed that CVS pretreatment significantly increased the phosphorylation of AKT, BAD, and MDM2, which resulted in decreased intracellular expression of p53. Further, immunofluorescence results showed reduced p53 levels in the nuclei of PC12 cells following CVS pretreatment, indicating a reduction in the p53-mediated transcriptional activity associated with Aβ25-35 exposure. In conclusion, our findings suggested that pretreatment with CVS protected PC12 cells from Aβ25-35-induced toxicity through modulation of the AKT/MDM2/p53 pathway. Thus, CVS may present a new therapeutic option for treating AD.

Keywords: Alzheimer’s disease; Amyloid beta; Curcumin; Silibinin; Vorinostat.

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Conflict of interest statement

The authors declare there are no competing interests.

Figures

Figure 1
Figure 1. CVS treatment inhibits Aβ25−35-induced PC12 cell apoptosis.
(A) Individual effects of 9 candidate compounds on cell viability of Aβ25−35-treated PC12 cells. (B–F) Effects of CVS on cell viability (B), reversion of CASP3 expression (C, D), and apoptotic cell levels (E, F) in Aβ25−35-treated PC12 cells. Each experiment was completed with a minimum of five replicates. Statistical significance is presented as; ##p < 0.001 versus Ctrl; *p < 0.01, **p < 0.001 versus Aβ. Abbreviations: Aβ, Aβ25−35 treatment group; C, curcumin and Aβ25−35 treatment group; CASP3, Caspase 3, Ctrl, control group; CV, curcumin, vorinostat and Aβ25−35 treatment group; CVS, curcumin, vorinostat, silibinin and Aβ25−35 treatment group.
Figure 2
Figure 2. CVS pretreatment prevents Aβ25−35-induced oxidative stress in PC12 cells.
(A–H) Effects of CVS on ROS (A–F), SOD (G), and MDA levels (H). Each experiment was completed with a minimum of five replicates. Statistical significance is presented as; ##p < 0.001 versus Ctrl; *p < 0.01, **p < 0.001 versus Aβ. Ctrl: control group; Aβ: Aβ25−35 treatment group. Abbreviations: Aβ, Aβ25−35 treatment group; C, curcumin and Aβ25−35 treatment group; Ctrl, control group; CV, curcumin, vorinostat and Aβ25−35 treatment group; CVS, curcumin, vorinostat, silibinin and Aβ25−35 treatment group.
Figure 3
Figure 3. Effects of CVS pretreatment on the expression of critical proteins.
(A–H) Effects of CVS on the expression of p-AKT (A, B), p-BAD (C, D), p-MDM2 (E, F), and p53 (G, H) in activated Aβ25−35-treated PC12 cells. Each experiment was completed with a minimum of five replicates. Statistical significance is presented as; ##p < 0.001 versus Ctrl; * italicp < 0.01, **p < 0.001 versus Aβ. Abbreviations: Aβ, Aβ25−35 treatment group; AKT, AKT serine/threonine kinase; BAD, BCL2 associated agonist of cell death; C, curcumin and Aβ25−35 treatment group; Ctrl, control group; CV, curcumin, vorinostat and Aβ25−35 treatment group; CVS, curcumin, vorinostat, silibinin and Aβ25−35 treatment group; MDM2, MDM2 proto-oncogene 2; p53, tumor protein p53.
Figure 4
Figure 4. CVS reduced the intranuclear content of p53 in Aβ25−35-treated PC12 cells.
(A) Effects of CVS on the intranuclear content of p53. (B) Results of immunofluorescence staining assay. Each experiment was completed with a minimum of five replicates. Statistical significance is presented as; ##p <  0.001 versus Ctrl; *p < 0.01, **p < 0.001 versus Aβ. Abbreviations: Aβ, Aβ25−35 treatment group; C, curcumin and A β25−35 treatment group; Ctrl, control group; CV, curcumin, vorinostat and Aβ25−35 treatment group; CVS, curcumin, vorinostat, silibinin and Aβ25−35 treatment group.
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