Transcriptomics-Based Investigation of Molecular Mechanisms Underlying Apoptosis Induced by ZnO Nanoparticles in Human Diffuse Large B-Cell Lymphoma

Abstract

Introduction: Zinc oxide nanoparticles (ZnO NPs) show anti-cancer activity. Diffuse Large B-cell Lymphoma (DLBCL) is a type of B-cell malignancies with unsatisfying treatment outcomes. This study was set to assess the potential of ZnO NPs to selectively induce apoptosis in human DLBCL cells (OCI-LY3), and to describe possible molecular mechanisms of action.

Methods: The impact of ZnO NPs on DLBCL cells and normal peripheral blood mononuclear cells (PBMCs) was studied using cytotoxicity assay and flow-cytometry. Transcriptomics analysis was conducted to identify ZnO NPs-dependent changes in the transcriptomic profiles of DLBCL cells.

Results: ZnO NPs selectively induced apoptosis in DLBCL cells, and caused changes in their transcriptomes. Deferential gene expression (DGE) with fold change (FC) ≥3 and p ≤ 0.008 with corrected p ≤ 0.05 was identified for 528 genes; 125 genes were over-expressed and 403 genes were under-expressed in ZnO NPs-treated DLBCL cells. The over-expressed genes involved in biological processes and pathways like stress response to metal ion, cellular response to zinc ion, metallothioneins bind metals, oxidative stress, and negative regulation of growth. In contrast, the under-expressed genes were implicated in DNA packaging complex, signaling by NOTCH, negative regulation of gene expression by epigenetic, signaling by WNT, M phase of cell cycle, and telomere maintenance. Setting the FC to ≥1.5 with p ≤ 0.05 and corrected p ≤ 0.1 showed ZnO NPs to induce over-expression of anti-oxidant genes and under-expression of oncogenes; target B-cell receptor (BCR) signaling pathway and NF-κB pathway; and promote apoptosis by intrinsic and extrinsic pathways.

Discussion: Overall, ZnO NPs selectively induced apoptosis in DLBCL cells, and possible molecular mechanisms of action were described.

Keywords: DLBCL; ZnO NPs; apoptosis; mechanism of action; transcriptomics.

Figure 1

Evaluation of the cytotoxicity of ZnO NPs against DLBCL cells and normal cells. The viability of DLBCL cells (A) and PBMCs (B) following treatment with different concentrations of ZnO NPs for 120 hours was determined using MTT assay. The type of cellular death caused by ZnO NPs (30 μg/mL) in DLBCL cells (C) was identified using annexin V-FITC and propidium iodide (PI) with flow cytometer. The data of flow cytometer were presented using column graph (D). ZnO NPs-induced production of ROS was measured in DLBCL cells using DCFH-DA (E). All experiments were performed in triplicate. Asterisk symbol was used to define p values; p ≤ 0.05 is *p ≤ 0.01 is **p ≤ 0.001 is*** and p ≤ 0.0001 is****.

Figure 2

ZnO NPs induced significant changes in the transcriptomic profile of DLBCL cells. Volcano plot demonstrates differentially expressed genes (DEGs: FC≥3; p≤0.008; corrected p≤0.05) in the ZnO NPs-treated DLBCL cells compared with untreated DLBCL cells (A). Hierarchical cluster analysis with heatmap presentation were done for the DEGs (B). The color range represents the normalized signal value of probes (log2 transformation and 75 percentile shift normalization).

Figure 3

Genes identified with over-expression in the ZnO NPs-treated DLBCL cells compared with the untreated DLBCL cells. Hierarchical cluster analysis with heatmap presentation was conducted on the over-expressed genes (FC≥3; p≤0.008; corrected p≤0.05) that significantly over-represented the biological processes and pathways: cellular response to zinc ions (A), response to chemicals (B) and negative regulator of growth (C). The color range represents the normalized signal value of probes (log2 transformation and 75 percentile shift normalization).

Figure 4

Genes that were under-expressed in the ZnO NPs-treated DLBCL cells compared with the untreated DLBCL cells. Hierarchical cluster analysis with heatmap presentation was conducted on the under-expressed genes (FC≥3; p≤0.008; corrected p≤0.05) that significantly over-represented the biological processes and pathways: negative regulation of transcription (A), chromatin assembly (B), telomere maintenance (C) and NOTCH signaling (D). The color range represents the normalized signal value of probes (log2 transformation and 75 percentile shift normalization).

Figure 5

Expression profile of oxidative stress responsive gene. Hierarchical cluster analysis with heatmap presentation was done on oxidative stress responsive gene (FC ≥1.5 with p≤0.05 and corrected p≤0.1) in ZnO NPs treated DLBCL cells versus untreated DLBCL cells. The color range represents the normalized signal value of probes (log2 transformation and 75 percentile shift normalization).

Figure 6

Expression profile of apoptotic gene. Hierarchical cluster analysis with heatmap presentation were constructed for anti-apoptotic genes (A) and pro-apoptotic genes (B) (FC ≥1.5 with p≤0.05 and corrected p≤0.1) in ZnO NPs-treated DLBCL cells compared with untreated DLBCL cells. The color range represents the normalized signal value of probes (log2 transformation and 75 percentile shift normalization).

Figure 7

Expression profile of oncogenes. Hierarchical cluster analysis with heatmap presentation were constructed for oncogenes (FC ≥1.5 with p≤0.05 and corrected p≤0.1) in ZnO NPs treated DLBCL cells compared with untreated DLBCL cells. The color range represents the normalized signal value of probes (log2 transformation and 75 percentile shift normalization).

Figure 8

Expression profile of B-cell receptor signaling genes. Hierarchical cluster analysis with heatmap presentation were constructed for the genes (FC ≥1.5 with p≤0.05 and corrected p≤0.1) in ZnO NPs-treated DLBCL cells compared with untreated DLBCL cells. The color range represents the normalized signal value of probes (log2 transformation and 75 percentile shift normalization).

Figure 9

Expression profile of nuclear factor kappa B signaling genes. Hierarchical cluster analysis with heatmap presentation were constructed for the genes (FC ≥1.5 with p≤0.05 and corrected p≤0.1) in ZnO NPs-treated DLBCL cells compared with untreated DLBCL cells. The color range represents the normalized signal value of probes (log2 transformation and 75 percentile shift normalization).