Proteomic Analysis on Anti-Proliferative and Apoptosis Effects of Curcumin Analog, 1,5-bis(4-Hydroxy-3-Methyoxyphenyl)-1,4-Pentadiene-3-One-Treated Human Glioblastoma and Neuroblastoma Cells

Abstract

Curcumin analogs with excellent biological properties have been synthesized to address and overcome the poor pharmacokinetic profiles of curcumin. This study aims to investigate the cytotoxicity, anti-proliferative, and apoptosis-inducing ability of curcumin analog, MS13 on human glioblastoma U-87 MG, and neuroblastoma SH-SY5Y cells, and to examine the global proteome changes in these cells following treatment. Our current findings showed that MS13 induced potent cytotoxicity and anti-proliferative effects on both cells. Increased caspase-3 activity and decreased bcl-2 concentration upon treatment indicate that MS13 induces apoptosis in these cells in a dose- and time-dependent manner. The label-free shotgun proteomic analysis has defined the protein profiles in both glioblastoma and neuroblastoma cells, whereby a total of nine common DEPs, inclusive of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), alpha-enolase (ENO1), heat shock protein HSP 90-alpha (HSP90AA1), Heat shock protein HSP 90-beta (HSP90AB1), Eukaryotic translation initiation factor 5A-1 (EFI5A), heterogenous nuclear ribonucleoprotein K (HNRNPK), tubulin beta chain (TUBB), histone H2AX (H2AFX), and Protein SET were identified. Pathway analysis further elucidated that MS13 may induce its anti-tumor effects in both cells via the common enriched pathways, "Glycolysis" and "Post-translational protein modification." Conclusively, MS13 demonstrates an anti-cancer effect that may indicate its potential use in the management of brain malignancies.

Keywords: anti-proliferation; apoptosis; cytotoxicity; diarylpentanoids; glioblastoma; neuroblastoma; shotgun proteomics.

FIGURE 1

The chemical structure of (A) curcumin and (B) 1,5-bis(4-hydroxy-3-methoxyphenyl)-1,4-pentadiene-3-one (MS13).

FIGURE 2

The cell viability in percentage (%) of (A) U-87 MG glioblastoma and (B) SH-SY5Y neuroblastoma cells upon treatment with MS13 and curcumin. The concentration (ranged from 0 to 100 μM) of MS13 or curcumin was log10 transformed. The cell viability of both cells decreased as the concentration of MS13 or curcumin increased. Results are expressed as the average of percentage of cell viability. Error bars represent mean ± standard error mean. The experiments were performed in triplicates and results were compared between three biological replicates. Statistical analysis was performed using analysis of variance (ANOVA), comparing cell viability of each concentration with the untreated sample. Asterisks indicate the difference in the statistical significance of the mean values between treated and untreated cells. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

FIGURE 3

The anti-proliferative effect of MS13 and curcumin on (A) U-87 MG glioblastoma and (B) SH-SY5Y neuroblastoma cells at 24, 48, and 72 h. Results are expressed as the average of cell viability in percentage (%) against concentration (μM). Error bars represent mean ± standard error mean. All experiments were performed in triplicates and results were compared between three biological replicates. Statistical analysis was performed using ANOVA, comparing cell viability of each concentration with the untreated sample. Asterisks indicate the difference in the statistical significance of the mean values between treated and untreated cells. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

FIGURE 4

Fold-change of caspase-3 activity in MS13-treated (A) U-87 MG and (B) SH-SY5Y cells. The caspase-3 activity was measured relative to control (0.2% DMSO) treatment at 24, 48, and 72 h. All experiments were performed in triplicates and results were compared between three biological replicates. Statistical analysis was performed using ANOVA, comparing the caspase-3 activity upon treatment with each MS13 dose at each time point to the respective untreated control sample. Indication of asterisks: *p < 0.05; **p < 0.01.

FIGURE 5

bcl-2 concentration in MS13-treated (A) U-87 MG and (B) SH-SY5Y cells following 24, 48, and 72 h treatment. All experiments were performed in triplicates and results were compared between three biological replicates. Statistical analysis was performed using ANOVA, comparing the bcl-2 concentration upon treatment with each MS13 dose at each time point to the respective untreated control sample. Indication of asterisks: *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 6

Percentage (%) of up- (red) and downregulated (blue) proteins identified in MS13-treated (A) U-87 MG and (B) SH-SY5Y cells annotated to each statistically enriched GO term for functional categories “Molecular function,” “Cellular component,” and “Biological process.” The number above each bar represents the percentage of protein of the respective GO terms while the asterisks indicate the false-discovery rate (FDR) (p-value) of each GO term upon multiple correction statistical test using the Benjamini–Hochberg procedure. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

FIGURE 7

Protein–protein interaction (PPI) network of all differentially expressed proteins (DEPs) identified in MS13-treated (A) U-87 MG (p-value = 2.64 × 10^–14) and (B) SH-SY5Y (p-value = 1.84 × 10^–14) cells. The nodes represent proteins, wherein the nodes in the same cluster demonstrated the same color. The lines connecting the nodes indicate the association between the proteins. The thicker the line, the higher the degree of confidence prediction of the interaction. Dashed lines represent inter-cluster between the highly connected clusters.