Gene Expression Profiling and Protein Analysis Reveal Suppression of the C-Myc Oncogene and Inhibition JAK/STAT and PI3K/AKT/mTOR Signaling by Thymoquinone in Acute Myeloid Leukemia Cells

Abstract

Overexpression of c-Myc plays an essential role in leukemogenesis and drug resistance, making c-Myc an attractive target for cancer therapy. However, targeting c-Myc directly is impossible, and c-Myc upstream regulator pathways could be targeted instead. This study investigated the effects of thymoquinone (TQ), a bioactive constituent in Nigella sativa, on the activation of upstream regulators of c-Myc: the JAK/STAT and PI3K/AKT/mTOR pathways in HL60 leukemia cells. Next-generation sequencing (NGS) was performed for gene expression profiling after TQ treatment. The expression of c-Myc and genes involved in JAK/STAT and PI3K/AKT/mTOR were validated by quantitative reverse transcription PCR (RT-qPCR). In addition, Jess assay analysis was performed to determine TQ’s effects on JAK/STAT and PI3K/AKT signaling and c-Myc protein expression. The results showed 114 significant differentially expressed genes after TQ treatment (p < 0.002). DAVID analysis revealed that most of these genes’ effect was on apoptosis and proliferation. There was downregulation of c-Myc, PI3K, AKT, mTOR, JAK2, STAT3, STAT5a, and STAT5b. Protein analysis showed that TQ also inhibited JAK/STAT and PI3K/AKT signaling, resulting in inhibition of c-Myc protein expression. In conclusion, the findings suggest that TQ potentially inhibits proliferation and induces apoptosis in HL60 leukemia cells by downregulation of c-Myc expression through inhibition of the JAK/STAT and PI3K/AKT signaling pathways.

Thymoquinone Inhibits JAK/STAT and PI3K/Akt/ mTOR Signaling Pathways in MV4-11 and K562 Myeloid Leukemia 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].…”

SKAP1 Is a Novel Biomarker and Therapeutic Target for Gastric Cancer: Evidence from Expression, Functional, and Bioinformatic Analyses

“…Subsequent qPCR analysis demonstrated downregulation of JAK1 mRNA levels in MKN45 cells, as well as decreased mRNA levels of PIK3CA and PIK3CD, following SKAP1 silencing (Figure S2). As JAK can activate the PI3K-AKT signaling pathway directly without STAT [28], we inferred that SKAP1 can modulate the JAK/PI3K/AKT signaling pathway to influence the biological function of GC cells. Further Western blot analysis confirmed that silencing SKAP1 led to significantly reduced protein expression of JAK1 and P-JAK1, as well as decreased expression of P-PI3K and P-AKT compared to that in the control group (Figure 6A).…”

The effects of MYC on tumor immunity and immunotherapy

“…The mTORC1-dependent phosphorylation of eukaryotic translation initiation factor 4E (eIF4E) binding protein 1 (4EBP1) can block the ability of mTOR to negatively regulate the translation initiation factor eIF4E, thus promoting the translation of mRNA containing complex RNA secondary structures (such as MYC) [ 94 ]. Thymoquinone, a bioactive constituent in Nigella sativa, can potentially inhibit proliferation and induces apoptosis in HL60 leukemia cells by downregulation of c-Myc expression through inhibition of the PI3K/AKT/mTOR signaling pathways [ 95 ]. In addition, in several studies, these inhibitors exhibited significant therapeutic effects in MYC-driven cancers (including neuroblastoma, small-cell lung cancer, and multiple hematopoietic cancers) [ 89 , 96 ].…”