Inhibition of c-MYC with involvement of ERK/JNK/MAPK and AKT pathways as a novel mechanism for shikonin and its derivatives in killing leukemia cells
Despite significant advances in chemotherapy, leukemia remains a life-threatening condition, with poor prognosis and drug resistance posing major challenges to treatment. There is an urgent need for new drugs. Our previous research demonstrated that shikonin, a natural naphthoquinone, is particularly effective against various leukemia cell lines compared to solid tumors, though the mechanisms behind its action are not fully understood. In this study, we evaluated shikonin and 14 of its derivatives on U937 leukemia cells, identifying four derivatives (isobutyrylshikonin, 2-methylbutyrylshikonin, isovalerylshikonin, and β,β-dimethylacrylshikonin) as more potent than shikonin itself. AnnexinV-PI analysis indicated that shikonins induce apoptosis.
Our investigation revealed that G1/S cell cycle checkpoint regulation and the transcription factor c-MYC, which is essential for cell cycle regulation and proliferation, are frequently downregulated following shikonin treatment, as shown by mRNA microarray analyses. Western blotting and DNA-binding assays further confirmed that shikonins inhibit c-MYC expression and its transcriptional activity. This reduction in c-MYC expression was linked to altered activity of ERK, JNK MAPK, and AKT pathways, suggesting their role in shikonin-induced c-MYC inactivation. Molecular docking studies indicated that shikonin and its derivatives bind to the same DNA-binding domain of c-MYC as known c-MYC inhibitors 10058-F4 and 10074-G5, supporting a direct interaction with c-MYC.
The effects observed in U937 cells were validated in additional leukemia cell lines (Jurkat, Molt4, CCRF-CEM, and multidrug-resistant CEM/ADR5000), where shikonin similarly inhibited c-MYC expression and influenced the phosphorylation of AKT, ERK1/2, and SAPK/JNK. In summary, shikonin and its derivatives exert anti-leukemic effects through the inhibition of c-MYC and associated pathways, highlighting a novel mechanism for their activity.