Thymoquinone Inhibits JAK/STAT and PI3K/Akt/ mTOR Signaling Pathways in MV4-11 and K562 Myeloid Leukemia Cells

Constitutive activation of Janus tyrosine kinase-signal transducer and activator of transcription (JAK/STAT) and Phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathways plays a crucial role in the development of acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). Thymoquinone (TQ), one of the main constituents of Nigella sativa, has shown anti-cancer activities in several cancers. However, the inhibitory effect mechanism of TQ on leukemia has not been fully understood. Therefore, this study aimed to investigate the effect of TQ on JAK/STAT and PI3K/Akt/mTOR pathways in MV4-11 AML cells and K562 CML cells. FLT3-ITD positive MV4-11 cells and BCR-ABL positive K562 cells were treated with TQ. Cytotoxicity assay was assessed using WSTs-8 kit. The expression of the target genes was evaluated using RT-qPCR. The phosphorylation status and the levels of proteins involved in JAK/STAT and PI3K/Akt/mTOR pathways were investigated using Jess western analysis. TQ induced a dose and time dependent inhibition of K562 cells proliferation. TQ significantly downregulated PI3K, Akt, and mTOR and upregulated PTEN expression with a significant inhibition of JAK/STAT and PI3K/Akt/mTOR signaling. In conclusion, TQ reduces the expression of PI3K, Akt, and mTOR genes and enhances the expression of PTEN gene at the mRNA and protein levels. TQ also inhibits JAK/STAT and PI3K/Akt/mTOR pathways, and consequently inhibits proliferation of myeloid leukemia cells, suggesting that TQ has potential anti-leukemic effects on both AML and CML cells.


Introduction
Leukemia is a group of blood cancers characterized by accumulated acquired somatic genetic alterations in the hematopoietic stem cells (HSCs) resulting in uncontrolled proliferation and differentiation [1,2].
Acute myeloid leukemia (AML) is characterized by distinct genetic abnormalities, such as FLT3-ITD mutation [3]. FLT3-ITD mutation is the most frequently identified genetic event in AML that results in the activation of several signaling pathways, including Janus activated kinase/signal transducer and activator of transcription (JAK/STAT) and Phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathways [4,5].
Chronic myeloid leukemia (CML) is initiated by a chromosomal translocation t (9; 22), which results in the formation of a Philadelphia (Ph) chromosome with the BCR-ABL  15 μM, and 11 μM, respectively. Wilcoxon signed ranks test was conducted and the results are stated as the median of four independent experiments, * p < 0.05, ** p < 0.01, and *** p < 0.001 are significant versus untreated control cells.
In our previous study, we found that TQ decreased the viability of MV4-11 cells in a dose and time-dependent manner with IC50 values of 7.8 μM, 5.5 μM, and 3.8 μM after 24 h, 48 h, and 72 h of treatment, respectively [27].

Thymoquinone Downregulates Several Genes in PI3K/Akt/mTOR Pathway in MV4-11 and K562 Cells
The mRNA expression levels of PI3K, Akt, mTOR, and PTEN genes in MV4-11 and K562 cells were evaluated by RT-qPCR before and after treatment with TQ. MV4-11 cells were incubated with the IC50 of TQ (5.5 μM) for 48 h. The results of RT-qPCR showed that TQ induced a significant decrease in the mRNA levels of PI3K by 6.3-fold (p < 0.001), Akt by 17.5-fold (p < 0.001), and mTOR by 8.2-fold (p < 0.001) in MV4-11 cells compared to untreated cells. On the other hand, TQ induced a slight increase in the mRNA level of PTEN in MV4-11 cells, but the increase was not significant (p > 0.05) compared to untreated cells (Figure 2A). In our previous study, we found that TQ decreased the viability of MV4-11 cells in a dose and time-dependent manner with IC 50 values of 7.8 µM, 5.5 µM, and 3.8 µM after 24 h, 48 h, and 72 h of treatment, respectively [27].

Thymoquinone Downregulates Several Genes in PI3K/Akt/mTOR Pathway in MV4-11 and K562 Cells
The mRNA expression levels of PI3K, Akt, mTOR, and PTEN genes in MV4-11 and K562 cells were evaluated by RT-qPCR before and after treatment with TQ. MV4-11 cells were incubated with the IC 50 of TQ (5.5 µM) for 48 h. The results of RT-qPCR showed that TQ induced a significant decrease in the mRNA levels of PI3K by 6.3-fold (p < 0.001), Akt by 17.5-fold (p < 0.001), and mTOR by 8.2-fold (p < 0.001) in MV4-11 cells compared to untreated cells. On the other hand, TQ induced a slight increase in the mRNA level of PTEN in MV4-11 cells, but the increase was not significant (p > 0.05) compared to untreated cells ( Figure 2A).
K562 cells were treated with the IC 50 of TQ (15 µM) for 48 h. The results of RT-qPCR showed that TQ induced a significant decrease in the mRNA levels of PI3K by 25-fold (p < 0.001), Akt by 10.5-fold (p < 0.001), and mTOR by 2.7-fold (p < 0.001) compared to untreated K562 cells. On the other hand, TQ increased the mRNA level of PTEN in K562 cells by 1.3-fold (p < 0.05) compared to untreated cells ( Figure 2B). The data represent three separate experiments and are presented as the median and interquartile range (error bars). Wilcoxon signed ranks test was conducted. Where * p < 0.05 and *** p < 0.001 are significant versus untreated control cells.
K562 cells were treated with the IC50 of TQ (15 μM) for 48 h. The results of RT-qPCR showed that TQ induced a significant decrease in the mRNA levels of PI3K by 25-fold (p < 0.001), Akt by 10.5-fold (p < 0.001), and mTOR by 2.7-fold (p < 0.001) compared to untreated K562 cells. On the other hand, TQ increased the mRNA level of PTEN in K562 cells by 1.3-fold (p < 0.05) compared to untreated cells ( Figure 2B).

Thymoquinone Inhibits JAK/STAT Signaling in MV4-11 Cells
The influence of TQ on the protein levels and the phosphorylation status of STAT3, STAT5, and JAK2 were evaluated in MV4-11 cells using Jess simple western analysis before and after treatment with TQ. MV4-11 cells were incubated with the IC50 of TQ (5.5 μM) for 48 h. Jess simple western analysis results showed that TQ significantly inhibited JAK/STAT signaling in MV4-11 cells by reducing the phosphorylation of STAT3 (p < 0.001), STAT5 (p < 0.05), and JAK2 (p < 0.05) compared to untreated cells ( Figure 3 and Table 1). The results also showed that TQ significantly reduced the protein levels of STAT3 (p < 0.05), STAT5 (p < 0.001), and JAK2 (p < 0.01) in MV4-11 cells compared to untreated cells ( Figure 3 and Table 1). The data represent three separate experiments and are presented as the median and interquartile range (error bars). Wilcoxon signed ranks test was conducted. Where * p < 0.05 and *** p < 0.001 are significant versus untreated control cells.

Thymoquinone Inhibits JAK/STAT Signaling in MV4-11 Cells
The influence of TQ on the protein levels and the phosphorylation status of STAT3, STAT5, and JAK2 were evaluated in MV4-11 cells using Jess simple western analysis before and after treatment with TQ. MV4-11 cells were incubated with the IC 50 of TQ (5.5 µM) for 48 h. Jess simple western analysis results showed that TQ significantly inhibited JAK/STAT signaling in MV4-11 cells by reducing the phosphorylation of STAT3 (p < 0.001), STAT5 (p < 0.05), and JAK2 (p < 0.05) compared to untreated cells ( Figure 3 and Table 1). The results also showed that TQ significantly reduced the protein levels of STAT3 (p < 0.05), STAT5 (p < 0.001), and JAK2 (p < 0.01) in MV4-11 cells compared to untreated cells ( Figure 3 and Table 1). The bar graph demonstrates the reduction of STAT3, p-STAT3, STAT5, p-STAT5, JAK2, and p-JAK2 protein levels in MV4-11 cells after TQ treatment. Wilcoxon signed rank test was conducted, and the results are presented as medians and interquartile range (error bars) (n = 3), where * p < 0.05, ** p < 0.01, *** p < 0.001 are significant versus untreated control cells.

Thymoquinone Inhibits PI3K/Akt/mTOR Signaling in MV4-11 Cells
TQ's influence on the protein levels and the phosphorylation status of PI3K, Akt, and the protein expression levels of PTEN were evaluated in MV4-11 cells using Jess simple western analysis. MV4-11 cells were incubated with the IC 50 of TQ (5.5 µM) for 48 h. The results demonstrated that TQ significantly inhibited PI3K/Akt/mTOR signaling in MV4-11 cells by decreasing the phosphorylation of p-PI3K (p < 0.001) and p-Akt (p < 0.001) compared to untreated cells ( Figure 4 and Table 1). The results also demonstrated that TQ significantly reduced the protein levels of PI3K (p < 0.001) and Akt (p < 0.001) in MV4-11 cells compared to untreated cells. On the other hand, TQ induced a significant increase in the protein level of PTEN (p < 0.001) in MV4-11 cells compared to untreated cells ( Figure 4 and Table 1).

Thymoquinone Inhibits PI3K/Akt/mTOR Signaling in MV4-11 Cells
TQ's influence on the protein levels and the phosphorylation status of PI3K, Akt, and the protein expression levels of PTEN were evaluated in MV4-11 cells using Jess simple western analysis. MV4-11 cells were incubated with the IC50 of TQ (5.5 μM) for 48 h. The results demonstrated that TQ significantly inhibited PI3K/Akt/mTOR signaling in MV4-11 cells by decreasing the phosphorylation of p-PI3K (p < 0.001) and p-Akt (p < 0.001) compared to untreated cells ( Figure 4 and Table 1). The results also demonstrated that TQ significantly reduced the protein levels of PI3K (p < 0.001) and Akt (p < 0.001) in MV4-11 cells compared to untreated cells. On the other hand, TQ induced a significant increase in the protein level of PTEN (p < 0.001) in MV4-11 cells compared to untreated cells ( Figure  4 and Table 1).

Thymoquinone Inhibits PI3K/Akt/mTOR Signaling in K562 Cells
The effects of TQ on the protein expression levels and the phosphorylation status of PI3K and Akt, and the protein expression levels of PTEN were evaluated in K562 cells using Jess simple western analysis. K562 cells were incubated with the IC 50 of TQ (15 µM) for 48 h. There was a significant inhibition of PI3K/Akt/mTOR signaling in K562 cells by decreasing the phosphorylation of p-PI3K (p < 0.001) and p-Akt (p < 0.001) compared to untreated cells ( Figure 5 and Table 2). The results also demonstrated that TQ significantly reduced the protein levels of PI3K (p < 0.001) and Akt (p < 0.001) compared to untreated cells. On the other hand, TQ induced a significant increase in the protein level of PTEN (p < 0.001) in comparison with untreated cells ( Figure 5 and Table 2).

Discussion
Despite the significant advances in the treatment of leukemia, the side effects of chemotherapy and the resistance to tyrosine kinase inhibitors (TKIs) represent the main challenges in treating leukemia patients [20,28]. Therefore, searching for new therapeutic alternatives could significantly impact leukemia treatment.
Constitutive activation of JAK/STAT and PI3K/Akt/mTOR signaling pathways are crucial in the pathogenesis of leukemia [16,22,29]. Therefore, proteins involved in

Discussion
Despite the significant advances in the treatment of leukemia, the side effects of chemotherapy and the resistance to tyrosine kinase inhibitors (TKIs) represent the main challenges in treating leukemia patients [20,28]. Therefore, searching for new therapeutic alternatives could significantly impact leukemia treatment.
Constitutive activation of JAK/STAT and PI3K/Akt/mTOR signaling pathways are crucial in the pathogenesis of leukemia [16,22,29]. Therefore, proteins involved in JAK/STAT and PI3K/Akt/mTOR pathways provide vital targets for treating leukemia.
The anti-cancer activities of TQ have been demonstrated in several cancers [24,30]. TQ has been shown to inhibit many signaling pathways, including PI3K/Akt /mTOR and JAK/STAT signaling in several cancer cells [23,31]. However, the underlying mechanisms of TQ's anti-leukemia activities have not been extensively studied.
We have previously reported that TQ exerted anti-leukemia effects by inducing antiproliferative effects and enhancing apoptosis in MV4-11 AML cells and K562 CML cells and through hypomethylation of JAK/STAT and PI3K/Akt/mTOR-negative regulator genes in MV4-11 AML cells [27,32]. In the current study, we have investigated the effect of TQ on JAK/STAT and PI3K/Akt/mTOR signaling pathways in MV4-11 AML cells and K562 CML cells.
The results of the present study showed that TQ induced growth inhibitory effect in K562 cells in a time and dose-dependent manner (Figure 1). Increasing TQ concentration induced a gradual increase in the growth inhibition up to 99% after 72 h of treatment. In addition, the growth inhibitory effect was increased significantly from 57-86% and 97% after incubation with 21 µM TQ for 24 h, 48 h, and 72 h, respectively, indicating a time-dependent course of TQ's growth inhibitory effect. Our results are consistent with our previous findings of TQ's time and dose-dependent course of growth inhibition in MV4-11 cells [27].
Regulation of PI3K/Akt/mTOR signaling has a crucial role in normal hematopoiesis [15]. Aberrant expression of PI3K, Akt, mTOR, and PTEN plays an essential role in the leukemogenesis [33,34]. Therefore, genes involved in PI3K/Akt/mTOR signaling provide critical molecular targets for the treatment of myeloid leukemia. In the current study, to further explore the underlying mechanism behind the antiproliferative and enhancement of apoptotic activities of TQ on MV4-11 and K562 cells, the effect of TQ on the expression of PI3K, Akt, mTOR, and PTEN was evaluated in MV4-11 and K562 cells. The findings revealed a significant decrease in the mRNA levels of PI3K, Akt, and mTOR in both MV4-11 cells (Figure 2A) and K562 cells ( Figure 2B). Additionally, the findings showed that the mRNA level of PTEN was significantly increased in MV4-11 and K562 cells after TQ treatment (Figure 2A,B). The results of this study are in agreement with a recent study that showed that the in vivo administration of TQ downregulated the mRNA levels of PI3K, Akt, and mTOR in hamster oral cancer [35]. The results of this study are also in agreement with a previous study that reported that TQ treatment increased the expression of PTEN at the mRNA level in MCF-7 human breast adenocarcinoma cells [36], and Her2+ and Her2− breast cancer cells [37].
In AML cells, activation of FLT3 tyrosine kinase is frequent and results in dysregulation of PI3K/Akt/mTOR [16]. In addition, BCR-ABL oncogenic protein induces activation of PI3K/Akt/mTOR pathway by several mechanisms in CML cells [22,29]. Moreover, the inactivation of PTEN is associated with leukemogenesis and is frequently inhibited in AML and CML cells [18]. Thus, inhibition of PI3K/Akt/mTOR signaling and re-expression of PTEN protein might provide important therapeutic targets for the treatment of myeloid leukemia.
In the current study, the effect of TQ on the expression of PTEN protein and the activation status and the expression of proteins involved in PI3K/Akt/mTOR signaling were evaluated in MV4-11 and K562 cells. The findings indicated that TQ significantly reduced the phosphorylation of PI3K and Akt proteins in MV4-11 cells (Figure 4 and Table 1), and K562 cells ( Figure 5 and Table 2). The results also showed that TQ dramatically reduced the protein expression level of PI3K and Akt, and increased the protein expression level of PTEN in MV4-11 cells (Figure 4 and Table 1) and K562 cells ( Figure 5 and Table 2). These findings were supported by the results of gene expression analysis, indicating that TQ downregulated PI3K and Akt and upregulated PTEN at the mRNA and protein levels. Similarly, TQ reduced the phosphorylation and the protein expression level of Akt in ML-1, MV4-11, and Kasumi-1 leukemia cells [24], MDA-MB-231, and MDA-MB-436 triple-negative breast cancer (TNBC) cells [25], and Her2+ and Her2− breast cancer cells [37]. Moreover, TQ inhibited the phosphorylation of PI3K and Akt in oral squamous cell carcinoma (KB) cells [38]. Another study has demonstrated that the PTEN protein level was increased, and the PI3K protein level and the phosphorylation of its downstream molecules; Akt and mTOR, were decreased in K562 cells after treatment with ND-09 inhibitory compound [39].
Aberrant activation of JAK/STAT signaling has been associated with the development of hematopoietic malignancies including AML and CML [40,41]. FLT3-ITD and BCR-ABL oncoproteins have constitutive tyrosine kinase activities, which induce hyperactivation of signal transduction pathways, including JAK/STAT signaling [22,42]. Thus, inhibition of JAKs and STATs represents a potential strategy for treating AML and CML [43].
In this study, TQ's effect on JAK/STAT signaling in MV4-11 cells was evaluated. The results showed that TQ significantly decreased the phosphorylation and protein levels of STAT3, STAT5, and JAK2 proteins in MV4-11 cells (Figure 3 and Table 1) and K562 cells (Data not shown) compared to untreated cells.
The results of the current study indicated that TQ induced the inhibition of JAK/STAT signaling, which inhibited growth and enhanced apoptosis in MV4-11 cells as reported previously in our published data [27]. These findings were consistent with our previous findings in which TQ decreased the phosphorylation and the protein levels of JAK2, STAT3, and STAT5 in HL60 AML cells [44]. The results were also supported by the findings of a previous study in which TQ treatment reduced the phosphorylation of JAK2 and STAT3 in SK-MEL-28 melanoma cells [30]. Additionally, Pang et al. 2017 have reported that TQ treatment reduced the phosphorylation and protein expression level of STAT5 in ML-1, MV4-11, and Kasumi-1 leukemia cells [24]. Another study demonstrated that TQ treatment inhibited the phosphorylation of STAT3 in HGC27, SGC7901, and BGC823 gastric cancer cells, and inhibited the phosphorylation of JAK2 in HGC27 cells [14].
Taken together, TQ downregulates the expression of PI3K, Akt, and mTOR, and upregulates the expression of PTEN. In addition, TQ inhibits PI3K/Akt/mTOR and JAK/STAT signaling in myeloid leukemia cells, which was associated with growth inhibition of leukemia cells.

Treatment with Thymoquinone
Thymoquinone (>98% pure), purchased from Sigma-Aldrich (Sigma-Aldrich Corp., Louis, MO, USA). A 30 mM stock solution of TQ was prepared in 1 mL of 100% dimethyl sulfoxide (DMSO) and kept at −80 • C. To prevent cytotoxicity of DMSO, the stock solution of TQ was serially diluted with culture media. After that, treatment concentrations of TQ (15 µM for K562 cells treatment and 5.5 µM for MV4-11 cells treatment) were prepared with final concentrations of DMSO of 0.048% and 0.017% for K562 cells treatment and MV4-11 cells treatment, respectively.
In our previous study, the cytotoxic effect of TQ on MV4-11 cells was investigated, and the IC 50 values of TQ on MV4-11 cells were determined after 24 h, 48 h, and 72 h of treatment [27].

RNA Extraction
MV4-11 and K562 cells were treated with the IC 50 of TQ; 5.5 µM and 15 µM, respectively, for 48 h. Total cellular RNA was extracted from both TQ-treated and untreated cells using ReliaPrep™ RNA Cell Miniprep System RNA extraction kit (Promega, Madison, WI, USA) according to the instructions of the manufacturer. Nanodphotometer (Implen, Weslake Village, CA, USA) was used to measure the purity and concentration of the extracted RNA.

Reverse Transcriptase Quantitative PCR (RT-qPCR)
First, 100 ng of RNA was reverse transcribed into cDNA using GoTaq ® 2-Step RT-qPCR System kit (Promega, USA) according to the manufacturer's instructions. The expression of PI3K, Akt, mTOR, and PTEN was analyzed using SYBR Green-based GoTaq 2-Step RT-qPCR System kit (Promega, Madison, WI, USA). The PCR reactions were performed in triplicates. Each PCR reaction contained 10 µL of GoTaq ® qPCR Master Mix (2×), 0.2 µL of CXR reference dye, 1 µL of forward (20×) and reverse primers (20X), 6.8 µL nuclease-free water, and 2 µL of cDNA template (2 µL of nuclease-free water were added to the no template control (NTC)). The cycling conditions for the RT-qPCR reactions were as follows: activation of GoTaq ® DNA Polymerase, 2 min at 95 • C; denaturation step, 40 cycles at 95 • C for 15 s; annealing and extension, 40 cycles at 60 • C for 1 min. RT-qPCR was analyzed using Applied Biosystem StepOnePlus ™ thermocycler (Applied Biosystems, Foster City, CA, USA). The sequences of the primer sets used for PCR amplification are listed in Table 3. Data were analyzed by StepOne Software v2.3 (ABI step one plus, Foster City, CA, USA). The relative expression levels of the genes were determined using RT-qPCR and the 2 −∆∆Cq method [45] after normalization to the endogenous reference β-actin, and the results were presented as fold changes.

Total Cell Lysates Preparation
The IC 50 of TQ; 5.5 µM and 15 µM were used to treat MV4-11 and K562 cells, respectively, for 48 h. After that, the treated and untreated cells were lysed with RIPA buffer containing a protease inhibitor cocktail (Nacalai Tesque, Kyoto, Japan) and phosphatase inhibitor solution (Nacalai Tesque, Kyoto, Japan). The protein concentrations were measured by Bradford protein assay [49], using bovine serum albumin (BSA) and Coomassie brilliant blue (CBB) solution from Nacalai tesque, Inc. (Kyoto, Japan).

Protein Expression Analysis Using JESS Simple Western Analysis
Protein expression was investigated using the capillary-based Protein Simple Jess system (Jess; Protein Simple, San Jose, CA, USA). Briefly, 1 mg/mL concentration of the cell lysates was prepared after dilution with a sample buffer. Each sample was mixed with a master mix (1× fluorescent standard, 1× sample buffer, and 40 mM dithiothreitol) and heated at 95 • C for 5 min to denature the samples. After that, 3 µL of the denatured proteins and 10 µL of each protein normalization solution, chemiluminescent substrate, primary antibodies, and HRP-conjugated anti-mouse secondary antibodies were pipetted into the appropriate wells of the assay plate. Biotinylated ladder cartridge (12-230 kDa) was integrated for each assay. Subsequently, the plate and the capillaries were subjected to Jess machine for automated protein electrophoresis, blocking, antibody incubation, and signal detection. Within-capillary total protein normalization was performed to account for any differences in protein loading. Normalization reagent allowed the detection of total proteins in the capillary through the binding to the amine group by a biomolecule while also removing housekeeping proteins that might cause inconsistency and unreliability in the protein's expression. With the Jess technology, no loading control is required. Chemiluminescent reactions were analyzed by Compass software (ProteinSimple). Relative protein amounts were assessed using the corrected area of chemiluminescent peaks. The primary antibodies used for Jess simple western analysis and their suppliers are listed in Table 4.

Conclusions
The effect of TQ against MV4-11 AML cells and K562 CML cells was evaluated. Based on our current findings, it can be concluded that TQ-induced growth inhibition of leukemia cells was associated with reduced expression of PI3K, Akt, and mTOR genes and re-expression of PTEN gene at the mRNA and protein levels. Moreover, TQ induced inhibition of PI3K/Akt/mTOR and JAK/STAT signaling pathways, which was associated with inhibition of proliferation of the leukemia cells. The results of this study suggest that TQ has potential anti-leukemic effects on both AML and CML cells. However, further experiments are required to evaluate the molecular mechanisms underlying TQ's antileukemia activities in vivo and in clinical trials.