Sorafenib Induces Leukemic Stem Cells Apoptosis Through Blocking Hedgehog/AKT Signaling Pathway

BackgroundLeukemic stem cells (LSCs) play a central role in the relapse and refractory of acute myeloid leukemia (AML). As known, LSCs are only present from0.01%to0.09%in AML patients and have biological characteristics as stem cells including differentiation, proliferation and self-renewal. Moreover, LSCs fail to triggerring programmed cell death, express drug transporters (p-gp et al.) and possess DNA repair systems, which result in LSCs resistant to current anti-leukemic strategies. So are abnormal signaling pathways (Hedgehog/AKT Signaling Pathway) detected in leukemic stem cells. It is important to identify and study LSCs for directly targeting LSCs to ultimately cure leukemia. We have observed that AML cell line KG1a cells contain CD34+CD38-LSCs, and isolated these cells from KGla cells successfully, which also had characteristics like stem cells. Based on our previous research, it is necessary to look for more effective methods breaking down the chemoresistance of LSCs and killing them.Sorafenib is a small molecule kinase inhibitor, which was approved by the USA Food and Drug Administration for the first-line treatment of renal cell carcinoma and hepatocellular carcinoma. It was initially developed to target Raf kinase and subsequently found inhibiting vascular endothelial growth factor receptor (VEGFR), platelet derived growth factor receptor (PDGFR), fibroblast growth factor receptor (FGFR) and FMS-like tyrosine kinase3(FLT3). Sorafenib also relates to signaling pathways involved in leukemogenesis and becomes a potential agent for AML patients, while sparing normal hematopoietic stem cells. Therefore, we had a hypothesis that sorafenib could be the one to kill leukemic stem cells effectively.MethodsChapter Ⅰ The identification and isolation of leukemic stem cells from leukemia cell line KGla cellsCD34+CD38-cells were separated from AML cell line KGla cells through Magnetic activated cell sorting (MACS) using CD38-FITC and anti-FITC microbeads. The cell surface phenotype of CD34+CD38-cells were analyzed by flow cytometry. Chapter Ⅱ Mitochondrial apoptosis in leukemic stem cells induced by SorafenibTo analyze the viabilities of LSCs after different concentration sorafenib treatments was by MTT assay. To detect apoptosic cells in LSCs was through Propidium Iodide. To observe morphological apoptosis of LSCs was trough laser scanning confocal microscopy with Hochest33342. To measure the expressions of Caspase9, Caspase3, PARP proteins was using western blotting. Chapter Ⅲ The mechanisms of apoptosis in leukemic stem cells induced by sorafenibTo detect the expressions of Bcl2family proteins (Bcl2, Bcl-xl, Mcl-1and Bax), Gli1, Gli2, p-Akt and Akt proteins was by western blotting.ResultsChapter Ⅰ The identification and isolation of leukemic stem cells from leukemia cell line KGla cellsThe purification of CD34+CD38-cells were more than93.950%after separated from KGla cells.Chapter Ⅱ Mitochondrial apoptosis in leukemic stem cells induced by Sorafenib1. The cytotoxicity effect of different concentrations sorafenib on leukemic stem cellsLSCs were sensitive to sorafenib, and the cytotoxicity effect of sorafenib on LSCs exsisted a dose-dependent manner. Exposed LSCs in different concentrations sorafenib (0～80μM) for72h, the cellular viability differences of each concentration were significant (F=125.595, P=0.000). From the dose-response curve (Figure2.1), we could find40μM sorafenib brought LSCs the most serious suppression.2. Apoptosis in leukemic stem cells treated with sorafenibPart1Apoptosis in LSCs detected by flow cytometryExposed LSCs to40μM sorafenib for0h,8h,12h,16h,24h, apoptotic rates of each time point catched by Propidium Iodide through flow cytometry. At12h, LSCs began surferring apoptosis, and apoptotic rates were present gradually increasing with time. The differences of apoptosic rates among time points were significant (F=50.599, P=0.000).Part2Morphological apoptosis in leukemic stem cells observed through laser scanning confocal microscopySorafenib with (0～40μM) having treated LSCs for72h, the typical morphology of apoptosis-apoptotic bodies were observed in intervention groups, as there were no changes in control groups. Part3Mitochondrial apoptosis of leukemic stem cells were measured by western blottingLeukemic stem cells were treated with sorafenib for0h,8h,12h,16h,24h, and the activation of Caspase9, Caspase3and PARP gradually increased. Figure2.3revealed that pro-Caspase9and pro-Caspase3proteins decreased with time, as cleave-PARP protein increased.Chapter Ⅲ The mechanisms of apoptosis in leukemic stem cells induced by sorafenib1. Sorafenib induced leukemic stem cells apoptosis mainly by downregulating anti-apoptotic Bcl2proteinsAfter intervented with Sorafenib40μM for0h,8h,12h,16h,24h, LSCs were collected to extract total protein for Western blotting. The results revealed that the expressions of anti-apoptotic proteins Bcl2, Bcl-xl and Mcl-1decreasing with time, while pro-apoptotic protein Bax did not change. Looking at Figure3.1A, Mcl-1decreased in the first (8h), Bcl-xl was the second one (12h), and Bcl2was the last of three (16h).2. Sorafenib suppressed Hedgehog/AKT signaling pathway in leukemic stem cellsWhile different time points of Sorafenib40μM having treated LSCs, the key proteins of Hedgehog/AKT signaling pathway were detected by Western blotting. We found that Gli1, Gli2, p-Akt and Akt proteins showed decreasing trends with time.Conclusions1. Acute myeloid leukemia cell line KGla cells contain CD34+CD38-leukemic stem cells and are novel cell model for leukemic stem cells research.2. Leukemic stem cells were sensitive to sorafenib and the cytotoxicity effect on them induced by sorafenib exsisted a dose-dependent manner.3. Sorafenib could induce leukemic stem cells mitochondrial apoptosis.4. Sorafenib induced leukemic stem cells apoptosis mainly through downregulating anti-apoptotic Bcl2proteins.5. Sorafenib could suppress Hedgehog/AKT signaling pathway in leukemic stem cells.