Low-dose Triptolide in combination with Idarubicin could induce apoptosis in leukemia stem and progenitor cells via affecting the intrinsic and extrinsic factors

Abstract

BackgroundRelapse has been a major hurdle for the success of acute myeloid leukemia chemotherapy, whereas leukemia stem cells (LSCs) have been considered to be responsible for relapse recently. Thus new drug targeting LSCs is urgently needed. Triptolide (TPL), a diterpenoid triepoxide derived from Tripterygium wilfordii, has been used in Traditional Chinese Medicine (TCM) for centuries. Recently, it has been reported that TPL has the potential of depleting quiescent CD34+ primitive CML progenitor cells. Moreover, normal CD34+ hemopoietic stem cells are less sensitive than AML blasts to TPL, suggesting the seclectivity of TPL in targeting LSCs. AimsIn this study, the ability of triptolide to induce apoptosis in leukemia stem and progenitor cells was investigated with the underlying mechanism being explored. MethodsLeukemia stem and progenitor cells were sorted from KG1a cells using flow cytometry with cell cycle being analyzed and then were subjected to different treatments and thereafter MTT assay, flow cytometry and Western blot or RT-PCR, intracellular ROS measurement colony forming assay were used to determine IC50, apoptotic status and expression of Nrf2, HIF-1α and their target genes, ROS level and colony forming ability. ResultsTPL is highly cytotoxic to leukemia stem and progenitor cells with IC20 of 5.0±0.81nM and IC50 of 20.48±1.6nM after 72h exposure. Leukemia stem and progenitor cells were also exposed to series concentrations of IDA with or without TPL(IC20F5.0nM). TPL significantly enhanced cytotoxicity of IDA to LSCs (IC50-IDA: 285.20±13.7 nM vs. IC50-IDA+TPL: 27.01±0.73 nM, P<0.001) Combination index was also analyzed. Results of combination index showed that synergism effect was observed over a wide range of IC50-IC90 concentrations of IDA and TPL in combination. Moreover, apoptotic ratio of cells treated by IDA with TPL was significantly increased compared to cells treated by IDA alone (24.85±1.70% vs. 76.87±8.34%, P<0.001). What's more, TPL in combination with IDA significantly inhibited the clonogenicity of leukemia stem and progenitor cells (-VE: 2097.3±106.07/well; TPL: 1387.7±40.05/well; IDA: 1252±46.87/well; TPL+IDA: 211±93.18/well, P<0.001). Next, we explore the underlying mechanism of TPL-induced apoptosis in LSCs. The survival of LSCs are dependent of intrinsic factors (Nrf2 and ROS) and extrinsic factors(HIF-1α, VLA4). Induction of ROS while inhibition of protective factor Nrf2 could lead to the apoptosis of LSCs. ROS activation was concentration-dependent when IDA was combined with a constant concentration of TPL (IC20:5nM). The highest activity of intracellular ROS appeared in IDA 200nM+TPL5nM group. Down-regulation of Nrf2 as well as its target genes such as NQO1, GSR and HO-1 was also observed in our study. Moreover, extrinsic factors such as HIF-1α and its down-stream genes such as BNIP3, VEGF and CAIX were also down-regulated in TPL with IDA group. Important extrinsic factors affecting the adhesion of LSCs such as VLA4 and CXCR4 were also down-regulated in TPL with IDA group. ConclusionOur findings indicate that the cytotoxicity of triptolide and the enhancing effect of triptolide on the cytotoxicity of idarubicin in leukemia stem and progenitor cells may be caused by affecting the intrinsic and extrinsic factors. Disclosures:No relevant conflicts of interest to declare.