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. 2016 Jun 7;7(23):35379-89.
doi: 10.18632/oncotarget.9297.

Characterization of the Novel Indolylmaleimides' PDA-66 and PDA-377 Effect on Canine Lymphoma Cells

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Free PMC article

Characterization of the Novel Indolylmaleimides' PDA-66 and PDA-377 Effect on Canine Lymphoma Cells

Wen Liu et al. Oncotarget. .
Free PMC article

Abstract

Protein kinase inhibitors are widely used in chemotherapeutic cancer regimens. Maleimide derivatives such as SB-216763 act as GSK-3 inhibitor targeting cell proliferation, cell death and cell cycle progression.Herein, the two arylindolylmaleimide derivatives PDA-66 and PDA-377 were evaluated as potential chemotherapeutic agents on canine B-cell lymphoma cell lines. Canine lymphoma represents a naturally occurring model closely resembling the human high-grade non-Hodgkin's lymphoma (NHL). PDA-66 showed more pronounced effects on both cell lines. Application of 2.5μM PDA-66 resulted in a significant induction of apoptosis (approx. 11 %), decrease of the metabolic activity (approx. 95 %), anti-proliferative effect (approx. 85 %) and cell death within 48h. Agent induced mode of action was characterized by whole transcriptome sequencing, 12 h and 24 h post-agent exposure. Key PDA-66-modulated pathways identified were cell cycle, DNA replication and p53 signaling. Expression analyses indicated that the drug acting mechanism is mediated through DNA replication and cycle arrest involving the spindle assembly checkpoint.In conclusion, both PDA derivatives displayed strong anti-proliferation activity in canine B-cell lymphoma cells. The cell and molecular PDA-induced effect characterization and the molecular characterization of the agent acting mechanism provides the basis for further evaluation of a potential drug for canine lymphoma serving as model for human NHL.

Keywords: PDA; arylindolylmaleimides; canine lymphoma; transcriptome sequencing.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1. Exposure to PDA-66 and PDA-377 inhibits cell proliferation and metabolic activity in CLBL-1 and CLBL-1M
a. CLBL-1 and CLBL-1M cells were incubated with different concentrations of PDA-66 and PDA-377 for 24 h, 48 h and 72 h. The proliferation was suppressed significantly at the concentration of 2.5 μM. The diagrams show the mean ± SD of three independent counting experiments. Significance of a treatment effect compared to the DMSO control was determined using student's t-test, p<0.05. b. CLBL-1 and CLBL-1M cells were incubated with different concentrations of PDA-66 and PDA-377 ranging from 0.25 μM to 10 μM and incubated for 24 h, 48 h and 72 h. Metabolic activity was determined using WST-1 assay. The results are expressed as percentage of the DMSO-treated cells. The diagrams show the mean ± SD of four independent measurements. Significance of a treatment effect compared to the DMSO control was determined using Dunnett's Multiple Comparison Test with a p value of < 0.05. *: p<0.05; **: p<0.01; ***: p<0.001.
Figure 2
Figure 2. PDA-66 and PDA-377 induce apoptosis
CLBL-1 and CLBL-1M cells were exposed to 0.5 μM, 1.0 μM and 2.5 μM PDA-66 and PDA-377 for 24 h, 48 h and 72 h. Analysis of early apoptosis and late apoptosis was performed using flow cytometry after Annexin V FITC and propidium iodide (PI) staining. As a reference DMSO treated cells were analyzed. Rates of early apoptotic (FITC+, PI) and late apoptotic/dead (FITC+, PI+) cells were determined and displayed as the mean ± SD of three independent measurements. a. Rate of early apoptotic cells after 24 h, 48 h and 72 h. b. Rate of late apoptotic/dead cells after 24 h, 48 h and 72 h. Significance of a treatment effect compared to the DMSO control was determined using student's t-Test, p<0.05. *: p<0.05; **: p<0.01; ***: p<0.001.
Figure 3
Figure 3. The morphological changes after PDA-66 and PDA-377 treatment
CLBL-1 and CLBL-1M cells were incubated with 1.5 μM PDA-66, 2.5 μM PDA-377 and DMSO. Cytospins of treated cells were stained with Pappenheim method after 72 h. Representative pictures are displayed. In DMSO controls, lymphoid round cells show moderately abundant light to dark blue cytoplasm with distinct clear Golgi zones and sometimes distinct vacuolization. The nuclei are single round, indent to cloverleaf-shaped showing coarse chromatin and round eccentric 1-5 nucleoli. In the cells exposed to PDA agents, cells are round to pleomorphic with light to dark blue abundant cytoplasm showing distinct Golgi zones and distinct large vacuolization. Nuclei of round to indent and cloverleaf-shaped (red solid arrow) present with clumped condensed chromatin pattern and rare eccentric round 1-3 nucleoli. Additionally, apoptotic bodies (red dashed arrow), cytoplasmic blebbing (black dashed arrow), increased cytoplasmic and nuclear vacuolization (black solid arrows) and mitotic figures could be observed in analyzed cells.
Figure 4
Figure 4. The MDS plot of the RNA-seq data
The distances correspond to the differences in the biological coefficient of variation between the samples. The analyzed cells were treated with 1.0 μM and 2.5 μM PDA-66 and DMSO respectively for 12 h and 24 h. Four distinct clusters were observed in MDS plot. The biological replicates form close clusters. Cluster A: High-dose (2.5 μM) 24 h treatment CLBL-1; Cluster B: High-dose (2.5 μM) 24 h treatment CLBL-1M; Cluster C: High-dose (2.5 μM) 12 h treatment CLBL-1; Cluster D: High-dose (2.5 μM) 12 h treatment CLBL-1M. S (1-36): Sample names of PDA-66 and DMSO-treated cells.
Figure 5
Figure 5. Heatmap of early (12 h) reacting genes in all high-dose treatment groups
Genes with significantly different expression (FDR<0.001) in any of the high-dose treatments compared to controls were selected and displayed as a heatmap with euclidean distance clustering. Numbers given for each gene are the fold changes expressed as logarithmized ratios (base 2).
Figure 6
Figure 6. Schematic overview of the PDA-66 effect on cell cycle
Schematic overview of the PDA-66 effect on cell cycle-related gene expression. Genes printed in blue are downregulated and genes printed in red are upregulated. Cell cycle-related kinases are printed on the outside of the circle. Genes that are expressed during the G2 phase of the cell cycle are upregulated in the PDA-66 cell cultures as compared to controls, while G1 and S-phase associated genes are significantly downregulated. The expression pattern together with the perturbation in tubulin isoform transcript abundance (insert) suggests activation of the spindle assembly checkpoint (red bolt). Insert: Tubulin isoforms and associated complexes are listed and log-fold changes are given for the two cell lines after 24 h treatment with 2.5 μM PDA-66. Values highlighted in red or blue indicate significant higher or lower abundances, respectively. Greyed values are non-significant changes (FDR>0.05).

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