Standard therapy for Acute Myeloid Leukemia (AML) is rarely curative, and several suggested improvements have had little success so far. We have reported that in an in vitro model of a potential therapeutic regimen for AML, the activity of cytarabine (AraC) is enhanced by a sequential treatment with a combination of the vitamin D2 analog Doxercalciferol (D2) and the plant-derived antioxidant carnosic acid (CA) Importantly, the enhancement occurred selectively in patient-derived AML blasts, but not in the normal bone marrow cells. We now demonstrate that TXNIP, previously known as Vitamin D up-regulated protein 1 (VDUP1) [PMID 808674] plays a part in signaling cell death (CD) in this regimen. This is shown by the reduced CD when TXNIP protein levels are decreased by the CRISPR/CAS9 or RNAi technology. Further, we show that direct activation of ASK1 kinase by TXNIP is required for the optimal transmission of the CD signal to apoptotic machinery, regulated by JNK and BIM. These studies provide a rationale for a projected clinical trial of this vitamin D-based new therapeutic regimen for AML.

Nobiletin, a dietary citrus flavonoid, has been reported to possess several biological activities, such as anti-oxidant, anti-inflammatory and anti-cancer properties. The aim of this study was to investigate the anti-leukemic effects of nobiletin and its underlying mechanisms on human acute myeloid leukemia (AML) cells. We demonstrated that nobiletin (0-100 μM) significantly reduced cell viability from 100.0±9.6% to 31.1±2.8% in human AML THP-1 cell line. Nobiletin arrested cell cycle progression in G1 phase and induced myeloid cell differentiation in human AML cells. Microarray analysis showed that mRNA expression of the c-KIT gene, a critical proto-oncogene associated with leukemia progression, was dramatically reduced in nobiletin-treated AML cells. Furthermore, we verified that AML cells treated with nobiletin (40 and 80 μM) for 48 h markedly suppressed c-KIT mRNA expression (from 1.00±0.07-fold to 0.62±0.08- and 0.30±0.05-fold) and reduced the level of c-KIT protein expression (from 1.00±0.11-fold to 0.60±0.15- and 0.34±0.05-fold) by inhibition of KIT promoter activity. The knockdown of c-KIT expression by shRNA attenuated cancer cell growth and induced cell differentiation. Moreover, we found that the overexpression of c-KIT abolished nobiletin-mediated cell growth inhibition in leukemia cells. These results indicate that nobiletin exerts anti-leukemic effects through the down-regulation of c-KIT gene expression in AML cells. Finally, we demonstrated that the combination of a conventional AML chemotherapeutic agent, cytarabine, with nobiletin resulted in more reduction of cell viability in AML cells. Our current findings suggest that nobiletin is a novel c-KIT inhibitor and may serve as a chemo-preventive or -therapeutic agent against human AML.