Untargeted Metabolomics Identifies Key Metabolic Pathways Altered by Thymoquinone in Leukemic Cancer Cells

Abstract

Thymoquinone (TQ), a naturally occurring anticancer compound extracted from Nigella sativa oil, has been extensively reported to possess potent anti-cancer properties. Experimental studies showed the anti-proliferative, pro-apoptotic, and anti-metastatic effects of TQ on different cancer cells. One of the possible mechanisms underlying these effects includes alteration in key metabolic pathways that are critical for cancer cell survival. However, an extensive landscape of the metabolites altered by TQ in cancer cells remains elusive. Here, we performed an untargeted metabolomics study using leukemic cancer cell lines during treatment with TQ and found alteration in approximately 335 metabolites. Pathway analysis showed alteration in key metabolic pathways like TCA cycle, amino acid metabolism, sphingolipid metabolism and nucleotide metabolism, which are critical for leukemic cell survival and death. We found a dramatic increase in metabolites like thymine glycol in TQ-treated cancer cells, a metabolite known to induce DNA damage and apoptosis. Similarly, we observed a sharp decline in cellular guanine levels, important for leukemic cancer cell survival. Overall, we provided an extensive metabolic landscape of leukemic cancer cells and identified the key metabolites and pathways altered, which could be critical and responsible for the anti-proliferative function of TQ.

Keywords: DNA damage; LC-MS/MS; leukemia; metabolism; metabolites; thymoquinone.

Figure 1

Cell viability was determined by using trypan blue exclusion test. Cells were cultured in the presence of different concentrations of TQ. TQ treated cell viability in (A) Jurkat, (B) HL-60 and (C) K-562 exposed for 24 h viability was determined and compared to untreated cells. Values are shown as mean ± S.E.M. Where * p < 0.05 and ** p < 0.01.

Figure 2

Cell growth was determined by WST-1 assay that measuring the growth rate on (A) Jurkat, (B) HL-60 and (C) K-562 cell. Cells were cultured in the presence of different concentrations of TQ. Value are shown as mean ± S.E.M. Values are shown as mean ± S.E.M. Where ** p < 0.01.

Figure 3

Representative dot blot of Annexin-V and PI on leukemic cells Data represented as dot blot where the lower left quadrant shows the live cells (Ann−, PI−), the lower right quadrant represents the early apoptotic cells (Ann+, PI−), the upper left quadrant represents necrotic cells (Ann−, PI+), and the upper right quadrant represents the late apoptotic cells (Ann+, PI+). Annexin-V and PI expression on Jurkat, HL-60 and K-562 cells as determined by flow cytometry.

Figure 4

Histograms for cell cycle from analysis of (A) Jurkat, (B) HL-60 and (C) K-562 were treated with increasing concentration of TQ for 24 h. Cells were exposed to TQ, stained with PI and were analysed by using flow cytometry.

Figure 5

Different appearance of Leukemic cells Morphologic studies of leukemic cell line using the light microscope were carried out to observe the morphologic changes in Jurkat, HL-60 and K-562 cell line treated with TQ (5 µM and 10 µM) compare with un treated cells (control), positive control H₂O₂ and negative control after 24 h. The P-value for (A) Jurkat, (B) HL-60 and (C) K-562 measurement by one-way ANOVA and denoted as P; * ≤0.01, ** ≤0.005 and *** ≤0.0001. The figure showed P-value between control and TQ dose 5 µM, Control and 10 µM and control and positive and negative control by t-test analysis.

Figure 6

Metabolomics of TQ treated leukemic cell lines. (A) PCA analysis of six group it represents samples in the groups were closely cluster to one another. (B) Total Ion Chromatogram of all six groups and significant metabolic features are marked in respective retention time and spot size indicate its abundance. (C) Total Ion Chromatogram of all HL-60 and Jurkat cells. (D) Heatmap of pairwise correlation values of 356 metabolites along with depiction of major metabolic pathway of HL-60 and Jurkat cells affected by TQ. The Pearson correlation coefficients were calculated for log2-transformed ratios of the median values of CTRL with treated Jurkat and HL-60. For a better overview only, metabolites with the highest reproducibility (>95%) were displayed.

Figure 6

Metabolomics of TQ treated leukemic cell lines. (A) PCA analysis of six group it represents samples in the groups were closely cluster to one another. (B) Total Ion Chromatogram of all six groups and significant metabolic features are marked in respective retention time and spot size indicate its abundance. (C) Total Ion Chromatogram of all HL-60 and Jurkat cells. (D) Heatmap of pairwise correlation values of 356 metabolites along with depiction of major metabolic pathway of HL-60 and Jurkat cells affected by TQ. The Pearson correlation coefficients were calculated for log2-transformed ratios of the median values of CTRL with treated Jurkat and HL-60. For a better overview only, metabolites with the highest reproducibility (>95%) were displayed.

Figure 7

Identified pathways altered by TQ treatment in both (A) HL-60 and (B) Jurkat cell lines.

Figure 8

Lipids specifically sphingosine and ceramide pathway are key targets of TQ (A) The synthesis of sphingosine and ceramide started in the presence of serine palmitoyltransferase (SPT), a rate limiting enzyme of the pathway. (B) Metabolites related to lipid metabolism altered by TQ treatment in HL-60 cell line. (C) Metabolites related to lipid metabolism altered by TQ treatment in Jurkat cell lines.

Figure 9

TQ alters TCA cycle (A) TCA cycle and cellular metabolism. (B) Variation between TCA metabolite observed due to effect of TQ treatment on HL-60 cells. (C) Variation between TCA metabolite observed due to effect of TQ treatment on Jurkat cells.

Figure 10

TQ treatment modulates epigenetic regulatory metabolite levels (A) DNA methylation pathway, where SAM is S-Adenosylmethionine and SAH is S-Adenosylhomocysteine (B–E) Metabolite variation in DNA methylation pathway.

Figure 11

TQ alters metabolite levels associated with one carbon metabolism (A) One carbon Metabolism pathway, where THF is Tetrahydrofolate. (B,C) Metabolites related to one carbon metabolism altered by TQ treatment in HL-60 cell line. (D,E) Metabolites related to one carbon metabolism altered by TQ treatment in Jurkat cell lines.