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. 2016 Nov 8;7(45):73200-73215.
doi: 10.18632/oncotarget.12298.

Case-specific Potentiation of Glioblastoma Drugs by Pterostilbene

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Free PMC article

Case-specific Potentiation of Glioblastoma Drugs by Pterostilbene

Linnéa Schmidt et al. Oncotarget. .
Free PMC article

Abstract

Glioblastoma multiforme (GBM, astrocytoma grade IV) is the most common malignant primary brain tumor in adults. Addressing the shortage of effective treatment options for this cancer, we explored repurposing of existing drugs into combinations with potent activity against GBM cells. We report that the phytoalexin pterostilbene is a potentiator of two drugs with previously reported anti-GBM activity, the EGFR inhibitor gefitinib and the antidepressant sertraline. Combinations of either of these two compounds with pterostilbene suppress cell growth, viability, sphere formation and inhibit migration in tumor GBM cell (GC) cultures. The potentiating effect of pterostilbene was observed to a varying degree across a panel of 41 patient-derived GCs, and correlated in a case specific manner with the presence of missense mutation of EGFR and PIK3CA and a focal deletion of the chromosomal region 1p32. We identify pterostilbene-induced cell cycle arrest, synergistic inhibition of MAPK activity and induction of Thioredoxin interacting protein (TXNIP) as possible mechanisms behind pterostilbene's effect. Our results highlight a nontoxic stilbenoid compound as a modulator of anticancer drug response, and indicate that pterostilbene might be used to modulate two anticancer compounds in well-defined sets of GBM patients.

Keywords: cancer therapeutics; drug repurposing; glioblastoma; glioblastoma initiating cells; stilbenoids.

Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare that they have no conflicts of interest.

Figures

Figure 1
Figure 1. Combination of pterostilbene with sertraline or gefitinib suppresses glioma cell growth
72 hours viability response to sertraline, gefitinib and pterostilbene, in four GCs treated in triplicates for each dose and combination. (A) Evaluating each compound at three different doses, we obtained pairwise interaction scores (IS, Methods). A negative IS, consistent with a potentiating interaction, was more pronounced at particular dose concentrations. Numbers in the table are mean and SD of IS across GCs (n = 4). (B) Combinations of pterostilbene with sertraline or gefitinib suppressed viability at 72 hours, whereas the single agents did not. (error bars are 95% CI). Both combinations display a significant interaction score at the doses tested in all GCs except for the PS combination in U3017. *p < 0.05, **p < 0.01 (Methods). (C) Images of treated GC U3065 at 24 and 48 hours. (D) Real time cell growth density measurements recorded every 12 hours for 60 hours in U3065, presented as cell growth curves with mean and SD from triplicates for each recorded time point.
Figure 2
Figure 2. Combination of pterostilbene with sertraline or gefitinib affect glioma cell migration and sphere formation
(A) Images of migration trans-well membranes after treatment (U3047). (B) Trans-well migration capacity after 48 hours of treatment in GC cultures. Graphs are plotted from the collected results from experiments in three GC cultures (U3047, U3065 and U3017, a total of 9 ratio data points for each treatment). (C) Gliomasphere formation after 7 days of treatment (experiment in six replicates for each treatment in U3047, U3065 and U3037). All ratios are calculated from vehicle control measurements, and all graphs are presented as means with 95% confidence interval. *p < 0.05, **p < 0.01, ***p < 0.001 (Mann Whitney).
Figure 3
Figure 3. Measurement of pterostilbene induced potentiation across 41 GC cultures
A screening experiment was performed, measuring 11 dose series of each of the drugs both single and as fixed-proportion concentrations for the combinations pterostilbene and gefitinib (PG) and pterostilbene and sertraline (PS) in 41 GCs. (A) Sigmoid dose response curves (dashed lines) were fitted to the mean viability ratio across the 41 GCs (orange, blue and green lines and results for individual GCs in Supplementary Figure S2). Each two sigmoid curves for single drugs (left and middle panel) were used to derive an expected combination response curve under the assumption of no synergy (grey line). The thick black lines (right panel) show the average observed combination response (doses in log10). Blue and red lines represent a synergy or an antagonism, respectively. (B, C) Boxplots of the patientwise average IS (across all overlapping doses tested) and patientwise average CI (effect 40–60%) across all 41 GCs. The results confirm a significant generality of the synergistic response for the drug pairs in a large patient material (p < 0.0001, Methods). (D) Differences in CI across the GC panel annotated by their subtype. CI for PG and PS, left and right, respectively. No statistical difference was obtained between subtypes (ANOVA p = 0.075, main text), although a trend towards a stronger synergy for the PG combination in the Classical subtype was observed.
Figure 4
Figure 4. Predictive markers of synergy between pterostilbene, sertraline and gefitinib in glioblastoma cell (GC) cultures
Using data from 41 GC cultures, we applied elastic net regression to predict the Combination Index (CI) between pterostilbene and sertraline. (A) Prediction of PS CI could be predicted with good accuracy from RNA transcripts (Pearson correlation of 0.63 between X = observed and Y = predicted values as obtained by cross-validation, left). Transcripts selected as predictive transcripts (showing coefficients as bars) included RNF11 and TSPYL1 (right). (B) The same analysis performed using DNA copy number aberration data for the GC cultures. The predictive performance is not as high as for transcripts (left), and the model identifies a set of genes encoded by the same region on chromosome 1p (including the RNF11 locus) as predictive. (C) Association between PS CI and PIK3CA missense somatic mutation status (left) and PG CI and EGFR status (right). PIK3CA missense somatic mutation status (left), PS CI and PTEN missense mutation status (middle) and PG CI and EGFR status (right). (D) Modulation of PS combination effects during simultaneous knockdown of RNF11 (24 and 48 h experiments collected) (p-value obtained by linear model, Methods)
Figure 5
Figure 5. Drug combinations and single drugs affect proliferation, and pterostilbene modulates cell cycle progression
Assessment of proliferation and cell cycle analysis after treatment with single drugs and combinations. (A) EdU incorporation (% positive cells) after 24 and 48 hours. Box and whiskers represent mean and 5–95 percentiles from experiments in three GC cultures (all data plotted in Figure S5B–S5C). *p < 0.05, **p < 0.01, ***p < 0.001 (Mann Whitney) (B) Flow cytometry of 7AAD-stained cells (U3065) to measure the distribution of DNA content in treated cells. An accumulation of cells in the center of the DNA content distribution compared to control sample is indicative of cell cycle arrest with an enriched S-phase population. The pterostilbene and combinations display an evident cell cycle shift in this GC culture. (C) Automated imaging of GC nuclei was used to derive histograms of cell DNA content under treatment with different doses of pterostilbene, upper left. Upper right: to quantify, we fitted GC specific linear regressions in which each of the G0/G1, S and G2/M populations were proportional to the logarithmic dose of pterostilbene (G0/G1 in one GC culture shown). The graph (lower left) shows the distribution of the proportionality (slope) constants across the 14 lines for each population. Lower right: linear mixed effects model result, showing the estimate and 95% confidence interval of a common (‘fixed’) slope effect across the 14 cell cultures (p-values 0.00014, 1.17 × 10−05 and 0.018 for the G1, S and G2/M populations, respectively).
Figure 6
Figure 6. Transcriptional profiling after treatment reveals differentially expressed genes in response to PS and PG combinations
We applied mRNA profiling to measure the response to single drugs and drug pairs after 1 hour of exposure in U3065MG. (A) Transcripts with significant changes for pterostilbene, sertraline and PS (left), and for pterostilbene, gefitinib and PG (right). The circles represent one treatment each and the overlap represents transcripts that are altered in both treatments. The transcripts displayed in the figure represent transcripts with a fold change of at least 0.2 and a significant fold change (adjusted p-value < 0.05) in U3065MG. All transcripts including fold changes and p-values are shown in Supplementary Table S3. Most transcripts are downregulated: all except TXNIP for pterostilbene in the pterostilbene areas and 4/7 (BTG2, SLC3A2, RCAN and HSPA1B) of the ones in the PS area (left). (B) pERK levels after 6 hour treatment using the NanoPro 1000 assay. pERK is significantly decreased after treatment with the PG combination, and significantly increased after treatment with the PS combination. (C) pMEK levels after 6 hour treatment. The PS combination significantly increased the pMEK levels. *p < 0.05, **p < 0.01, ***p < 0.001 (Student's t-test). (D) Modulation of pterostilbene effect during simultaneous knock down of TXNIP (24 and 48h experiments collected) (p-value obtained from linear model, Methods). (E) Pterostilbene effect after simultaneous treatment with NAC. *p < 0.05, **p < 0.01, ***p < 0.001 (Mann Whitney).

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