The Mechanisms Of Synergistically Cytotoxicity Induced By Homoharringtonine And Aclarubicin In Acute Myeloid Leukemia Cells

Section1:The synergistical cytotoxicity in vitro and vivo induced by homoharringtonine and aclarubicin in acute myeloid leukemia cellsObjective:The aim of this section was to investigate the antitumor effect relationship of HHT and ACR against AML cell lines and primary AML cells. And then to determine whether a synergistically cytotoxicity of HHT and ACR were confirmed in vitro and vivo.Methods:MTT assay was used to investigate the effects of HHT, ACR and combination of HHT and ACR on the THP-1cells, Kasumi cells and primary AML cells. Cell apoptosis was measured by Hochest nuclear staining and flow cytometry using the Annexin V-FITC/PI apoptosis assay kit, according to the manufacturer's instructions. The combination index (CI) was calculated using Chou-Talalay method (Calcusyn software, Biosoft) for determining whether the effects of drug combinations were synergistic, additive, or antagonistic. CI<1, CI=1, and CI>1indicate synergism, additive effect, and antagonism, respectively. To examined the in vivo interaction between HHT and ACR THP-1engraft tumor were allowed to grow in female SCID mice before randomization in four groups:placebo, HHT, ACR and HHT plus ACR. Mice were treated with1mg/kg HHT, and/or3mg/kg ACR daily for5days by intraperitoneal injection. Placebo group received equal volume of PBS. The volume of engraft tumor was measured every two days by using the formula:π/6length×width2. Apoptosis of tumor tissue was evaluated by TUNEL in situ hybridization. The metabolic rate of engraft tumor was determined using micro-PET CT. Survival was analyzed by Kaplan-Meier method and compared with x2test.Results:(1) The results of MTT assay showed a dose-and time-depended growth inhibition by both HHT and ACR in THP-1cells and Kasumi cells. The IC50at24h of HHT for THP-1cells and Kasumi-1cells were71.70±8.77nM and57.37±6.97nM respectively. The IC50at24h of ACR for THP-1cells was171.10±6.05nM, and for Kasumi-1cells was146.80±8.65nM.(2) Simultaneously, a dose-depended growth inhibition was observed following treatment with HHT or ACR in primary AML cells for24hours.(3) Compared with single agent, combining HHT and ACR caused significant stronger growth inhibition in THP-1cells, Kasumi cells and primary AML cells. The combination index value at ED50was0.42for THP-1cells,0.69for Kasumi-1cells and0.34-0.55for primary AML cells.(4) Dose-depended apoptosis induced by HHT or ACR was confirmed by annexin V-FITC/PI apoptosis analysis in THP-1cells, Kasumi cells and primary AML cells.(5) Compared with exposure to single agent, more apoptosis positive THP-1cells were detected when cells were simultaneously exposure to HHT and ACR for12hours. The same phenomenon was observed in Kasumi cells and primary AML cells. The CI at ED50was0.32for THP-1cells,0.15for Kasumi-1cells, and within ranges of0.05to0.24for primary AML cells.(6) To evaluate the in vivo anti-tumor effect of HHT alone, ACR alone and combined HHT and ACR, AML xenograft tumor was established by subcutaneous injection of THP-1cells into the dorsal tissue of SCID mice. Significant tumor grow delay (TGD) was observed in mice treated with combined HHT and ACR. In addition, TUNEL assay showed increased apoptosis in groups treated with combining HHT and ACR. Micro-PET analysis showed a stable metabolic rate of xenograft tumor in HHT group and ACR group at3days after the end of injection. A decreased metabolic rate of xenograft tumor was only observed in a mice treated with combining HHT and ACR.(7) The mice received combination of HHT and ACR obtained a statistical longer mean survival time than the placebo mice. Kaplan-Meier survival analysis showed a favorable survival in combined HHT/ACR group. Section2:The mechanisms of synergistically cytotoxity induced by HHT and ACR in acute myeloid leukemia cellsObjective:In this section, we try to explore the targets of synergistically cytotoxity induced by combined HHT/ACR in AML cells. The results will contribute to raise new targeted therapy strategies of AML.Methods:Differential expression of genome genes were detected by Agilent single-channel cDNA microarray screening in THP-1cells exposure to HHT, ACR and combined HHT/ACR respectively. SAS software was used to filter out the distribution of the differential genes in signaling pathways. The level of mRNA expression of significant genes was evaluated using Real-Time PCR. After THP-1and Kasumi cells were treated with HHT, ACR and combined HHT/ACR for the indicate times, whole cell lysates were used for immunoblot. The protein level of caspase9, caspase3, PARP, bcl-2family, wnt3a, β-catenin, P-β-catenin, GSK-3p, PI3K110, P-AKT, c-myc and cyclinDl were detected. The protein levels of wnt3a, β-catenin, PI3K110, P-AKT were also evaluated by western blot in primary AML cells treated with HHT, ACR and HHT plus ACR. To further verify whether targeting wnt3a and AKT could obtain a similar cytotoxity with HHT plus ACR in AML cells, we try to silence the wnt3a gene of THP-1cells by transfecting WNT3a siRNA fragment to THP-1cells using liposomal. Transfection effect of siRNA was evaluated by Flow cytometry. In order to indicate whether the cytotoxity induced by combined HHT/ACR could be partially inhibited by GSK-3β inhibitor, we detected the growth inhibition and apoptosis triggered by HHT, ACR and HHT plus ACR with or without CHIR-99021, a GSK-3β inhibitor. It has been reported that the excessively activation of WNT signal and PI3K/AKT signal, especially β-catenin, had a close relationship with the proliferation of AML leukemia stem cell (LSC). In order to explore whether combined HHT/ACR could synergistically induce apoptosis of AML-LSC, we examined the ratio of Annexin V positive cells in CD34+/CD38" cells by FACS. Results:(1) Microarray analysis showed different gene expression in WNT signaling pathway between THP-1cells treated with HHT and ACR.(2) Obvious down-regulated mRNA levels of WNT3a and β-catenin were observed in THP-1cells and Kasumi cells when they exposure to ACR and combined HHT/ACR for1or3hours. However, HHT failed to inhibit the mRNA level of WNT3a and β-catenin in THP-1cells and Kasumi cells. On the contrary, up-regulating mRNA levels of WNT3a and β-catenin were detected in the two cell lines after exposure to HHT for1hours.(3) Caspase9, caspase3and PARP were activated by HHT, ACR and combined HHT/ACR in THP-1cells and Kasumi cells. These supported that endogenous apoptotic signal was involved in the apoptosis induced by HHT and ACR in AML cell lines.(4) Different effects on Bcl-2family proteins were measured between HHT and ACR. We showed HHT to increase Bax expression and decrease Mcl-1and Bcl-xl expression in THP-1cells and Kasumi cells at3hours. However, there was no modification of Bcl-2expression in the two cell lines treated with HHT. On the contrary, ACR could reduce Bcl-2and Bcl-xl expression as early as3hours, and there was no increased Bax expression in THP-1cells and Kasumi cells before24hours. In addition, down-regulating of Mcl-1expression induced by HHT was earlier than that induced by ACR. Combination of HHT and ACR could simultaneously decrease Bcl-2protein expression and increase Bax protein expression in THP-1cells and Kasumi cells.(5) Decreased expression of PI3K110and P-AKT protein were observed in THP-1and Kasumi cells treated with HHT for3h. In contrast, no significant difference in the expression of two proteins was observed in90nM of ACR-treated cells. Treatment of the two AML cells with HHT resulted in a remarkable inhibition of protein expression of β-catenin, whereas expression of WNT3a remained stable at protein levels. Western Blot analysis also showed ACR could obviously inhibit WNT3a and β-catenin protein levels in THP-1cells and Kasumi cells after3hours exposure. As expected, combined HHT and ACR simultaneously reduced expression of PI3K, P-AKT, WNT3a and β-catenin at protein level in THP-1and Kasumi cells. Similar results have also been proved in primary AML cells.(6) To determine whether silencing of wnt3a enhanced the effects of HHT or AKT inhibitor on apoptosis and growth inhibition, the cytotoxicity of HHT and triciribine(an AKT inhibitor) were evaluated in THP-1cells treated with specific siRNA and cells treated with unrelated siRNA. A significant more growth inhibition triggered by HHT and triciribine was obtained in cells transfected with wnt3a siRNA than in cells transfected with scrambled siRNA. Similarly, silencing of wnt3a could increase cell apoptosis induced by HHT and triciribine in THP-1cells.(7) Similar synergistically growth inhibition and inducing apoptosis were examined in THP-1cells treated with combination of triciribined and ACR.(8) The growth inhibition caused by combined HHT/ACR could be partially inhibited by CHIR-99021in THP-1cells.(9) Combined HHT/ACR may be synergistically induced apoptosis of CD34+/CD38-cells in vitro.Summary:HHT and ACR individually could inhibit cell growth and induce cell apoptosis of AML cells in a dose-dependent manner in vitro. Simultaneous exposure to HHT and ACR on THP-1, Kasumi and primary AML cells resulted in strong synergistic anti-proliferative effect and apoptosis inducing in vitro and in vivo. Combination of HHT and ACR may be result in a favorable survival in AML xenograft mice.Different effects on PI3K/AKT signal and WNT/β-catenin signal were observed in AML cells exposure to HHT and ACR respectively. Combined HHT and ACR simultaneously reduced expression of PI3K, P-AKT, WNT3a and β-catenin at protein level in AML cells. So we speculate that a possible mechanism of synergistically cytotoxity induced combinated HHT/ACR is simultaneously inhibit the activity of PI3K/AKT pathway and WNT/β-catenin pathway in AML cells. As a common factor of PI3K/AKT pathway and WNT/β-catenin pathway, β-catenin could be involved in the synergistically anti-tumor effects induced by combinated HHT/ACR in AML cells.