Drug-sensitivity of resistant acute myeloid leukemia cell line to Doxorubicin is enhanced by Triptolide through down-regulation of HIF-1α and Nrf2

Relapse and treatment-related complication have been the hurdle for the cure of acute myeloid leukemia patients. Finding a new drug-sensitizer could significantly prolong the survival time of patients. Triptolide (TPL) has been shown to enhance the drug-sensitivity of a variety of cancer cells when used in a low-concentration. HL60/DOX is a kind of acute myeloid leukemia cell line which is resistant to a commonly used drug Doxorubicin. This kind of cell line is widely used in the study of drug-sensitization.

This study aims to investigate whether low-dose TPL could enhance the drug-sensitivity of resistant acute myeloid leukemia cell line HL60/DOX to Doxorubicin and related mechanism.

HL60/DOX cells were subjected to different treatments and thereafter MTT assay, flow cytometry and Western blot or RT-PCR were used to determine IC50, apoptotic status and expression of Nrf2, HIF-1α and their target genes.

HL60/DOX cells were exposed to increasing concentrations of Doxorubicin with TPL at a constant IC20 concentration (14nM) for 48h. In comparison with anticancer agent alone, TPL enhanced the cytotoxicity of Doxorubicin (IC50:14.36±2.23 vs. 7.9±0.33μM, 1.82 fold; P=0.008) to HL60/DOX. Results of combination index showed that TPL and anti-cancer agents had synergistic effects when fraction affected was below 60%. Flow cytometry analysis also showed that apoptoticratio of cells treated by Doxorubicin together with TPL was significantly increased compared to cell streated by Doxorubicin alone (19.55±1.70%vs. 72.62±4.83%, P<0.01). Next, we explored the underlying mechanism. Hypoxia-inducible factor- 1α (HIF-1α) is the major mediator of hypoxic responses in malignant cells. High expression of it is correlated with bad clinical outcome in patients. It is also important for the survival of leukemia stem cells which are responsible for relapse. Nrf2 is an important protective factor which is correlated with chemo-resistance in cancer cells. When combined with TPL, Doxorubicin down-regulates Nrf2 and HIF-1α expression at protein and mRNA levels. Down-stream genes of Nrf2 e.g NQO1, GSR and HO-1 as well as target genes of HIF-1α e.g BNIP3, VEGF and CAIX are also down-regulated at mRNA level.

Inconclusion, our study demonstrates that TPL could enhance drug-sensitivity of HL60/DOX in vitro through down-regulation of Nrf2 and HIF-1α, providing the experimental base for further study.

No relevant conflicts of interest to declare.