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  • Shikonin represents naphtoquinonic compound isolated from


    Shikonin represents naphtoquinonic compound isolated from the Chinese plant Lithospermum erythrorhizon[20]. Shikonin treatment inhibits AKT and ERK signaling pathways and induces apoptosis through FOXO transcription factors and mitochondrial pathway [21], [22]. Recent results suggest inhibition of leukemia gssg mg proliferation induced by the shikonin analogue Quambalarine B and this inhibition depends on regulation of the C-MYC protein [23].
    Materials and methods
    Discussion In this work, the natural compound shikonin was characterized as an activator of the Hippo pathway in a human T-lymphoma cell line. Shikonin is known to induce apoptosis in diverse cancer types. This phenomenon is usually ascribed to the inhibition of pro-survival and anti-apoptotic signaling, and reactive oxygen species production [27]. However, recent reports also pointed out the effects of shikonin on cancer cell metabolism, such as the glycolytic pathway [28]. We observed fast shikonin-mediated MST1 activation in a human leukemia cell line, associated with both MST1 phosphorylation on threonine 183 and proteolysis of MST1 resulting in a 36-kDa fragment corresponding to the N-terminal catalytically active kinase domain. Previous works have demonstrated that the cleavage of MST1 is caspase-dependent, and the N-terminal kinase domain translocates into the nucleus, where it participates in the induction of apoptosis [29]. As an important oncogene in diverse cancers and a key factor in the orchestration of cancer cell proliferation and metabolic reprogramming, also known as the Warburg effect, C-MYC was an promising candidate to test the effects of shikonin [17], [23], [30]. Specific anti-cancer properties of shikonin were confirmed by the observation of a decrease in mRNA levels of C-MYC and C-MYC protein levels following shikonin treatment. This decrease correlates with MST1 activation and YAP1 phosphorylation. Moreover, because C-MYC is a regulator of glycolysis in cancer cells, we probed the effect of shikonin on the expression of selected glycolytic proteins. GLUT1 was selected due to its high expression in Jurkat cells and important role in the regulation of glycolytic flux. A decrease in GLUT1 mRNA levels after shikonin treatment was demonstrated by RT-qPCR, but we did not observe any change in the protein levels after the same treatment. This observation could be explained by the long half-life of GLUT1. Because our data indicated possible regulation of C-MYC expression through the MST1-YAP1 pathway, we tested also the effect of the inhibition of MST1 production on C-MYC expression. It is well known that Jurkat cells express only low levels of MST2, and therefore signal transduction through the Hippo pathway depends mainly on the MST1 protein activity [25], [31]. However, we did not detect any significant changes in the mRNA levels of GLUT1 or C-MYC, but we observed significant increases in both C-MYC and GLUT1 during the inhibition of MST1 production. These results highlight an important role of MST1 in the regulation of C-MYC and GLUT1. Our data indicate that this regulation can occur at the transcriptional and translational levels or may involve post-translational inhibition of target proteins through MST1-dependent YAP1 phosphorylation. Similar results were described for phosphorylated TAZ, which binds to the Dishevelled protein in the cytosol and inhibits Wnt signaling, followed by changes in the expression levels of several Wnt target genes [32]. Phosphorylated YAP1 can form a complex with SMAD proteins, inhibiting their transcriptional activity [33]. MST1 can also directly phosphorylate several transcription factors, kinases, or enzymes and regulates their activities [25], [34], [35]. We also characterized the effects of MST1 depletion on the regulation of C-MYC protein and mRNA levels during shikonin treatment in leukemia cells. Cells with inhibited production of MST1 display higher levels of C-MYC and C-MYC mRNA, as well as GLUT1 and GLUT1 mRNA, compared to untreated control cells after shikonin treatment. These results suggest a key role for MST1 in the process of C-MYC and GLUT1 inhibition during shikonin treatment and clarify the regulation of C-MYC and GLUT1 by the Hippo signaling pathway.