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

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Abstract
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 1,25D3 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.