Transcriptomics-Based Characterization of the Toxicity of ZnO Nanoparticles Against Chronic Myeloid Leukemia Cells

Figures & data

Figure 1 Cytotoxicity assessment of ZnO NPs on leukemic and normal cells; and dissolution analysis of the NPs in culture medium. MTT assay was used to measure the viability of K562 cells (A) and PBMCs (B) post treatment with different concentrations of ZnO NPs for 120 hours. MTT assay was also used to determine the viability of K562 cells post incubation with 10 μg/mL for five different periods of time (C). Dissolution analysis was conducted to determine the concentration of released Zn ions from ZnO NPs to the culture medium RPMI 1640 (D). Annexin V-FITC and propidium iodide (PI) with flow cytometer were used to identify the kind of death caused by ZnO NPs (40 μg/mL) in K562 cells (E). The data of flow cytometer were presented using column graph (F). All experiments were conducted in triplicate. PBMCs: peripheral blood mononuclear cells. Asterisk symbol was used to define p values; p ≤ 0.01 is ** and p ≤ 0.001 is ***.

Figure 2 A transcriptomic signature was associated with ZnO NPs-treated K562 cells. Volcano plot shows differentially expressed genes (DEGs: FC≥4; p≤0.008; corrected p≤0.05) in the ZnO NPs-treated K562 cells versus untreated K562 cells (A). Hierarchical cluster analysis with heatmap presentation was performed on the DEGs (B). The color range represents the normalized signal value of probes (log2 transformation and 75 percentile shift normalization).

Table 1 Pathway Enrichment Analysis of the Differentially Expressed Genes (DEGs: FC≥4; p≤0.008; Corrected p≤0.05) in the ZnO NPs-Treated K562 Compared with the Untreated K562 Cells

GO Accession No	GO Term	Reg	Corr. p value	Count in Selection	Count in Total	Source
GO:0097501	Stress response to metal ion	Up	5.23E-17	10	16	GP
GO:0061687	Detoxification of inorganic compound	Up	5.23E-17	10	16	GP
GO:0006882	Cellular zinc ion homeostasis	Up	4.44E-15	11	35	GP
GO:0071294	Cellular response to zinc ion	Up	1.06E-11	8	12	GS
GO:0009636	Response to toxic substance	Up	2.55E-11	21	88	GP
GO:0071276	Cellular response to cadmium ion	Up	7.14E-07	6	14	GS
GO:0071241	Cellular response to inorganic substance	Up	2.26E-06	10	105	GS
GO:0010035	Response to inorganic substance	Up	6.80E-06	15	360	GS
GO:0010038	Response to metal ion	Up	6.80E-06	13	250	GS
GO:0045926	Negative regulation of growth	Up	0.007	9	210	GS
GO:0009636	Response to toxic substance	Up	0.01	11	356	GS
GO:0042221	Response to chemical	Up	0.01	41	3716	GS
GO:0001816	Cytokine production	Down	8.15E-08	31	811	GP
GO:0002376	Immune system process	Down	0.000012	54	2004	GS
GO:0002684	Positive regulation of immune system process	Down	0.00002	29	723	GS
GO:0002274	Myeloid leukocyte activation	Down	0.00005	11	94	GS
GO:0001775	Cell activation	Down	0.00006	26	624	GS
GO:0032944	Regulation of mononuclear cell proliferation	Down	0.0001	13	170	GS
GO:0046427	Positive regulation of receptor signaling pathway via JAK-STAT	Down	0.007	6	91	GP
GO:0002275	Myeloid cell activation involved in immune response	Down	0.0002	7	36	GS
GO:1903037	Regulation of leukocyte cell-cell adhesion	Down	0.0005	14	227	GS
GO:0032946	Positive regulation of mononuclear cell proliferation	Down	0.0009	10	114	GS
GO:0007166	Cell surface receptor signaling pathway	Down	0.001	46	1948	GS
GO:0097529	Myeloid leukocyte migration	Down	0.001	8	73	GS
GO:0042531	Positive regulation of tyrosine phosphorylation of STAT protein	Down	0.002	6	72	GP
GO:000716	Signal transduction	Down	0.003	81	4557	GS
GO:0022409	Positive regulation of cell-cell adhesion	Down	0.004	12	212	GS
GO:0014068	Positive regulation of phosphatidylinositol 3-kinase signaling	Down	0.007	6	90	GP
GO:0006935	Chemotaxis	Down	0.01	20	617	GS
GO:1903708	Positive regulation of hemopoiesis	Down	0.03	7	187	GP
GO:0045861	Negative regulation of proteolysis	Down	0.04	10	361	GP
GO:0008593	Regulation of Notch signaling pathway	Down	0.04	5	107	GP
GO:0002696	Positive regulation of leukocyte activation	Down	0.04	11	248	GS
GO:0098657	Import into cell	Down	0.05	16	483	GS
GO:0043410	Positive regulation of MAPK cascade	Down	0.05	13	552	GP
GO:0045766	Positive regulation of angiogenesis	Down	0.05	7	206	GP
GO:0030335	Positive regulation of cell migration	Down	0.05	12	297	GS
GO:0002757	Immune response-activating signal transduction	Down	0.05	12	297	GS

Note: The source of the findings shown in this table is (GS) GeneSpring GX version 12.1 software package (Agilent Technologies, Inc.) and (GP) gProfiler.

Abbreviations: GO, gene ontology; Reg, expression regulation of the genes that enriched GO terms; Corr p, corrected p value that was calculated on the basis of Benjamini–Hochberg false discovery rate (FDR). Count in selection, number of genes that were assigned to a GO term; Count in total, number of all genes that have been known to function in a GO term.

Figure 3 Over-expressed genes in the ZnO NPs-treated K562 cells compared with the untreated K562 cells. Hierarchical cluster analysis with heatmap presentation was conducted on the over-expressed genes (FC≥4; p≤0.008; corrected p≤0.05) that significantly over-represented the biological processes “cellular response to zinc ions and negative regulation of growth” (A) and “response to chemicals” (B). The color range represents the normalized signal value of probes (log2 transformation and 75 percentile shift normalization).

Figure 4 Down-expressed genes in the ZnO NPs-treated K562 cells compared with the untreated K562 cells. Hierarchical cluster analysis with heatmap presentation was applied on the down-expressed genes (FC≥4; p≤0.008; corrected p≤0.05) that significantly enriched the biological processes “cell activation” (A), “positive regulation of mononuclear cell proliferation” (B), “positive regulation of cell adhesion” (C) and “positive regulation of cell migration” (D). The color range represents the normalized signal value of probes (log2 transformation and 75 percentile shift normalization).

Figure 5 ZnO NPs targeted pathways that promote cellular proliferation and survival. ZnO NPs reduced the expression of genes (FC≥4; p≤0.008; corrected p≤0.05) that significantly enriched “positive regulation of tyrosine phosphorylation of STAT protein” (IGF1, IL18, IL23A, KIT, CCL5 and HES1), “positive regulation of receptor signaling pathway via JAK-STAT” (IGF1, IL18, IL23A, KIT, CCL5 and HES1) and “positive regulation of phosphatidylinositol 3-kinase signaling” (IGF1, PIK3CG, IL18, KIT, CCL5 and UNC5B). The gene expression data were shown from three ZnO NPs-treated K562 samples and three untreated K562 samples. The normalized signal value of probes was generated by log2 transformation and 75 percentile shift normalization of the raw data.

Figure 6 Expression of cytokine and cytokine receptor genes in the ZnO NPs-treated K562 cells compared with the untreated K562 cells. The fold change (FC) of the gene expression was based on the data that were recorded in three ZnO NPs-treated K562 samples and three untreated K562 samples. All FC data had p≤0.007 and corrected p≤0.05.

Figure 7 Network of differentially expressed genes (DEGs) is associated with molecular transport and inflammatory response. A network of 35 genes was constructed to understand the cross-talk between genes and functional relevance of this network. Red colored nodes indicated genes from the DEGs, whereas nodes with no color were not from the DEGs that was used for generating this network. Arrows show the direction of effect. The numbers under the genes are as follows from the top to bottom: p value, corrected p value, fold change (FC) of the DEGs, and log FC.

Figure 8 K562 cells altered the expression of their oxidative stress responsive genes in response to the treatment with ZnO NPs. Hierarchical cluster analysis with heatmap presentation demonstrated changes in the expression of oxidative stress responsive genes in the ZnO NPs-treated K562 cells as opposed to the untreated K562 cells. The altered gene expression was identified with fold change ≥1.5, p≤0.05 and corrected p≤0.1. The color range represents the normalized signal value of probes (log2 transformation and 75 percentile shift normalization)

Figure 9 ZnO NPs induced changes in the expression of apoptotic genes in K562 cells. Hierarchical cluster analysis and heatmap presentation showed altered expression of anti-apoptotic genes (A) and pro-apoptotic genes (B) in the ZnO NPs-treated K562 cells compared with the untreated K562 cells. All differentially expressed genes were found with fold change ≥1.5, p≤0.05 and corrected p≤0.1. The color range represents the normalized signal value of probes (log2 transformation and 75 percentile shift normalization).

Figure 10 ZnO NPs targeted NF-κB pathway in K562 cells. The treatment with ZnO NPs reduced the expression of NF-κB pathway activator IKBKB (FC=3.4, p=0.007; corrected p=0.05) and the NF-κB subunits: NFKB2 (FC=2.42; p=0.001; corrected p=0.03), RELB (FC=2; p=0.01; corrected p=0.06) and REL (FC=1.42; p=0.03; corrected p=0.09). The gene expression data were obtained from three samples of ZnO NPs-treated K562 cells and three samples of untreated K562 cells. The normalized signal value of probes was generated by log2 transformation and 75 percentile shift normalization of the raw data.