Heme-Mediated Change in Sensitivity to Anti-Leukemia Reagents in BCR/ABL-Positive Cell Lines.

Heme modulates various cellular functions as a component of hemoproteins. Recently, heme-mediated direct regulation of activity of transcription factors such as Bach1 has been shown. We reported in the last meeting that heme affects sensitivity to imatinib through, at least in part, regulation of Nrf2 transcription factor activity. In this study, we performed further analysis of the mechanisms of heme-mediated regulation of imatinib-sensitivity and the role of heme in the sensitivity to other small molecule compounds and conventional anti-leukemia drugs. IC50 values of imatinib against the BCR/ABL-positive cell line KCL22 were increased by addition of 1–100μM hemin, which eventually becomes heme in cells, in a dose-dependant manner. In the presence of hemin, imatinib-mediated increases in the levels of cleaved caspase 3, cleaved caspase 7, cleaved caspase 9 and cleaved PARP as well as the number of annexin V-positive cells were abrogated, suggesting that heme has an inhibitory effect on imatinib-induced apoptosis signaling. Luciferase reporter assay demonstrated that hemin increased MARE (Maf recognition element)-mediated γ-glutamylcystein synthetase (γ-GCS) light subunit gene promoter activity. Accordingly, the level of γ-GCS light subunit protein was increased after hemin treatment. We next examined the effect of suppression of Nrf2 expression on hemin-mediated change in imatinib sensitivity because Nrf2 might regulate MARE-mediated gene expressions. Transfection of Nrf2 siRNA resulted in partial abrogation of hemin-mediated upregulation of IC50 values of imatinib with suppression of hemin-mediated induction of γ-GCS light subunit expression, suggesting that suppression of Nrf2 partially restored the sensitivity to imatinib in hemin-treated cells. We also examined the effect of hemin on the sensitivity to conventional anti-leukemia drugs such as cytarabine, eoposide, hydroxyurea daunorubicin, idarubicin, doxorubicin and mitoxantrone in KCL22 cells. While IC50 values of cytarabine, etoposide and hydroxyurea were not changed by hemin treatment, addition of hemin resulted in significant increase in IC50 values of four anthracyclin anti-leukemia drugs. Furthermore, hemin also abrogated the sensitivity to U0126, which is a small molecule compound targeting MEK1/2. These results suggest that heme is an important molecule in the regulation of sensitivity to various anti-leukemia reagents.