Potential of NRF2 Inhibitors—Retinoic Acid, K67, and ML-385—In Overcoming Doxorubicin Resistance in Promyelocytic Leukemia Cells

Drug resistance is one of the major obstacles to the clinical use of doxorubicin, an extensively used chemotherapeutic drug to treat various cancers, including leukemia. Inhibition of the nuclear factor erythroid 2-related factor 2 (NRF2) seems a promising strategy to reverse chemoresistance in cancer cells. NRF2 is a transcription factor that regulates both antioxidant defense and drug detoxification mechanisms. In this study, we investigated the potential of three inhibitors of NRF2—K67, retinoic acid and ML-385—to overcome doxorubicin resistance in promyelocytic leukemia HL-60 cells. For this purpose, low-dose doxorubicin was used to establish doxorubicin-resistant HL-60/DR cells. The expression of NRF2 and its main repressor, Kelch-like ECH-associated protein 1 (KEAP1), at mRNA and protein levels was examined. HL-60/DR cells overexpressed NRF2 at mRNA and protein levels and down-regulated KEAP1 protein compared to drug-sensitive HL-60 cells. The effects of NRF2 inhibitors on doxorubicin-resistant HL-60/DR cell viability, apoptosis, and intracellular reactive oxygen species (ROS) levels were analyzed. We observed that NRF2 inhibitors significantly sensitized doxorubicin-resistant HL-60/DR cells to doxorubicin, which was associated with increased intracellular ROS levels and the expression of CAS-9, suggesting the participation of the mitochondrial-dependent apoptosis pathway. Furthermore, ML-385 inhibitor was used to study the expression of NRF2–KEAP1 pathway genes. NRF2 gene and protein expression remained unchanged; however, we noted the down-regulation of KEAP1 protein upon ML-385 treatment. Additionally, the expression of NRF2-regulated antioxidant and detoxification genes including SOD2, HMOX2, and GSS was maintained upon ML-385 treatment. In conclusion, our results demonstrated that all the studied inhibitors, namely K67, retinoic acid, and ML-385, increased the efficacy of doxorubicin in doxorubicin-resistant HL-60/DR cells, and suggested a potential strategy of combination therapy using NRF2 inhibitors and doxorubicin in overcoming doxorubicin resistance in leukemia.