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.
Highlights
Acute myeloid leukemia (AML) is the most common acute leukemia in adults [1].AML is a hematological malignancy characterized by multiple acquired mutations that affect cell biological processes such as cell growth, proliferation, and apoptosis [2]
Kruskal–Wallis and Mann–Whitney tests were conducted for statistical analysis usplots of the gene expression data in HL60 associated with TQ treatment (Figure 1) show ing GraphPad Prism 8.4.3 (San Diego, CA, USA), and p < 0.05 was considered as signifisignificant differential gene expression (DEG) at p-adj < 0.05 and fold change with absolute value >1
The results showed that significantly suppressed the expression by inhibiting the expression of JAK/STAT and PI3K/AKT/mTOR genes including JAK2, of JAK2,STAT5a, STAT3,STAT5b, and STAT5 proteins (p < 0.001)
Summary
Acute myeloid leukemia (AML) is the most common acute leukemia in adults [1].AML is a hematological malignancy characterized by multiple acquired mutations that affect cell biological processes such as cell growth, proliferation, and apoptosis [2]. The c-Myc oncogene is overexpressed in HL60 AML cells [3] It mediates multiple tumor cell survival pathways in most human cancers and represents a promising therapeutic target in several cancers [4,5]. Pharmaceuticals 2022, 15, 307 active site for small molecules It is found mainly in the nucleus, and it is impossible to target the nuclear c-Myc by monoclonal antibodies [6]. Targeting the upstream signaling of c-Myc and the PI3K/AKT/mTOR and JAK/STAT pathways could indirectly inhibit c-Myc expression [7]. The JAK/STAT and PI3K/AKT/mTOR pathways are the principal signaling mechanisms for many growth factors and cytokines, and the activation of these pathways stimulates cell proliferation, differentiation, cell migration, and apoptosis [8]. C-Myc was overexpressed by constitutive activation of the JAK/STAT and PI3k/AKT signaling pathways [10]
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