Thymoquinone Suppresses Cell Proliferation and Enhances Apoptosis of HL60 Leukemia Cells through Re-Expression of JAK/STAT Negative Regulators

Abstract

Objective: The natural compound, thymoquinone (TQ) has demonstrated potential anticancer properties in inhibiting cell proliferation and promoting apoptosis in myeloid leukemia cells, breast cancer cells, and others. However, the effect mechanism of TQ on AML cells still not fully understood. In this study, the authors examined the effects of TQ on the expression of JAK/STAT-negative regulator genes SOCS-1, SOCS-3, and SHP-1, and their consequences on cell proliferation and apoptosis in HL60 leukemia cells.

Methods: MTT and trypan blue exclusion tests were conducted to determine the 50% inhibitory concentration (IC50) and cell proliferation. FITC Annexin and Guava® reagent were used to study the cell apoptosis and examine the cell cycle phases, respectively. The expression of JAK/STAT-negative regulator genes, SOCS-1, SOCS-3, and SHP-1, was investigated using reverse transcriptase- quantitative PCR (RT-qPCR).

Results: TQ demonstrated a potential inhibition of HL60 cell proliferation and a significant increase in apoptotic cells in dose and time-dependent manner. TQ significantly induced cycle arrest at G0-G1 phase (P < 0.001) and enhanced the re-expression of JAK/STAT-negative regulator genes.

Conclusion: TQ potentially inhibited HL60 cell proliferation and significantly increased apoptosis with re-expression of JAK/STAT-negative regulator genes suggesting that TQ could be a new therapeutic candidate for leukemia therapy.<br />.

Keywords: JAK/STAT signaling; Leukemia; negative regulators; thymoquinone.

Figure 1

TQ Reduces the Viability of HL60 Leukemia Cells. Cytotoxicity of TQ and cell viability were assessed by MTT (A) and trypan blue staining (B) for 24, 48, and 72 h. The IC50 were 2, 2, and 1 µM, respectively. The increase in dose concentrations directly relates to the inhibition of HL60 leukemia cells. The values are expressed as mean ± SEM. Experiments were repeated at least three times. **p < 0.002 and ***p < 0.001 indicated statistical significance

Figure 2

Effectiveness of TQ on HL60 Leukemia Cells Apoptosis. The apoptotic activity of HL60 leukemia cells after treatment with 1, 2, and 3 µM of TQ for 24, 48, and 72 h. Cells were stained by FITC Annexin V and PI and analyzed by flow cytometry (A). The percentage of cell death based on the estimation of apoptosis in different treatments (B). Time and dose-dependent increase in apoptotic activity was observed. Data were presented as mean ± SEM. (p < 0.001).

Figure 3

The Cell Cycle Distribution of HL60 Leukemia Cells after Treatment with TQ. Flow cytometric analysis of cell cycle changes after exposure to 1, 2, and 3 μM of TQ for 24, 48, and 72 h. The distribution of the cells was determined using flow Cytometry (A). The percentage of cells in different stages of the cell cycle with respect to control (B). Data were presented as mean ± SEM. (p < 0.001).

Figure 4

RT-qPCR of Negative Regulators of JAK/STAT Signaling in HL60 Leukemia Cells. The relative normalized ratio of RT-qPCR revealed that TQ significantly up-regulates the expression of targeted genes in treated cells. SHP-1 is re-expressed in treated cells more than 3 fold, while SOCS1 and SOCS3 are re-expressed 2-fold higher compared with control. Data were presented as mean ± SEM. (p < 0.001).