Cooperation between antioxidants and 1,25-dihydroxyvitamin D₃ in induction of leukemia HL60 cell differentiation through the JNK/AP-1/Egr-1 pathway

Vitamin D derivatives have demonstrated anti-cancer activity, but their clinical use is precluded by hypercalcemia, Previously, we found that carnosic acid potentiates differentiation of human leukemia cells induced by low concentrations of 1alpha,25-dihydroxyvitamin D₃ (1,25D₃). In this study, we investigated if this effect is a general property of antioxidants, and whether there is a common mechanism whereby antioxidants potentiate monocytic differentiation. We found that all antioxidants tested enhanced differentiation-related cell cycle arrest induced by a low (1 nM) concentration of 1,25D₃. Addition of antioxidants to 1,25D ₃ activated the JNK pathway as indicated by increased phosphorylation of c-jun and ATF-2, although each compound alone had a minimal effect. Antioxidants also enhanced the 1,25D₃-induced AP-1 DNA binding and transactivation ability. Expression of Egr-1 and c-fos was increased by combinations of antioxidants and 1,25D₃, in parallel with the activation of the JNK pathway. The potentiation of differentiation by antioxidants was inhibited by JNK inhibitor SP600125 and a dominant negative JNK 1/2 construct, and Egr-1 and c-fos expression was proportionally decreased, suggesting that JNK pathway regulates these transcription factors. While potentiating the prodifferentiation effect of 1,25D₃, antioxidants did not promote the elevation of basal levels of intracellular calcium by 1,25D₃. The results indicate that JNK-AP1 pathway has an important role in the potentiation of 1,25D₃-induced differentiation by antioxidants, and regulates expression of Egr-1 and c-fos. Combinations of antioxidants with 1,25D₃ should be further evaluated for use in cancer chemoprevention and therapy.