高三尖杉酯碱联合阿克拉霉素协同杀伤急性髓细胞白血病细胞的机制研究
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摘要
第一部分：高三尖杉酯碱联合阿克拉霉素在体内外对急性髓细胞白血病细胞的协同杀伤作用研究
目的：
探讨高三尖杉酯碱与阿克拉霉素在体内外对AML细胞株细胞及原代AML细胞的杀伤作用及作用关系。
方法：
采用MTT法检测不同浓度HHT、ACR及按一定浓度比联合HHT和ACR对AML细胞生长的抑制作用。采用流式细胞仪Annexin V/PI双染色法、Hochest染色法检测不同浓度HHT、ACR及按一定浓度比联合HHT和ACR对AML细胞株细胞及原代AML细胞的诱导凋亡作用。采用CalcuSyn软件分析HHT与ACR在抑制AML细胞增殖及诱导凋亡上的作用关系。构建AML细胞荷瘤小鼠模型用于体内研究。当瘤体体积达到100-120mm3时随机分入对照组、HHT治疗组、ACR治疗组及HHT联合ACR治疗组。入组后监测瘤体大小。采用原位细胞凋亡检测试剂盒进行TUNEL染色检测瘤体组织原位凋亡。采用Micro-PET扫描对对照组及药物处理组小鼠体内瘤体活性进行实时观察。统计学分析,单一变量两组间比较采用t检验,多组间比较采用单因素方差分析。Kaplan-Meier法进行生存分析。以p值<0.05认为具有统计学差异。
结果：
(1)实验浓度HHT对THP-1细胞(急性单核细胞白血病细胞株)、Kasumi细胞(急性粒细胞白血病细胞株)的生长均具有明显的抑制作用,并呈浓度依赖性。(2)实验浓度ACR对THP-1细胞、Kasumi细胞的生长亦具有明显抑制作用,并呈浓度依赖性。(3)实验浓度HHT、ACR均能抑制原代AML细胞生长,随浓度增加,抑制作用增强。(4)1：2.5摩尔浓度比联合HHT与ACR能协同抑制THP-1细胞、Kasumi细胞及原代AML细胞的生长,并能协同诱导其凋亡。(5)采用THP-1细胞能成功构建荷瘤小鼠模型。HHT治疗、ACR治疗及联合HHT和ACR治疗均能延长THP-1荷瘤小鼠瘤体倍增时间。联合治疗较单药治疗能更明显地延长THP-1荷瘤小鼠瘤体倍增时间。联合HHT与ACR治疗组小鼠瘤体组织细胞凋亡较单药治疗组明显增加。(6)Micro-PET扫描结果显示HHT联合ACR治疗组THP-1荷瘤小鼠瘤体代谢活性较用药前有所下降。(7)仅联合HHT和ACR治疗后能延长THP-1荷瘤小鼠的生存时间。小结：
(1)一定浓度比联合HHT和ACR在体外可协同抑制THP-1细胞、Kasumi细胞和原代AML细胞增殖及诱导其凋亡。(2)与单药相比,HHT联合ACR在体内可更明显地抑制THP-1荷瘤小鼠瘤体增殖及诱导凋亡,并可能更明显地抑制荷瘤小鼠体内的肿瘤活性。(3)体内研究显示HHT与ACR联合治疗可改善THP-1荷瘤小鼠的生存。
第二部分：高三尖杉酯碱联合阿克拉霉素协同杀伤急性髓细胞白血病细胞的分子机制研究
目的：
探讨高三尖杉酯碱联合阿克拉霉素协同杀伤急性髓细胞白血病细胞的分子机制,为AML靶向联合治疗方案的设计提供实验依据。
方法：
采用Agilent单通道表达谱芯片筛选对照组、HHT、ACR、HHT联合ACR作用THP-1细胞后差异表达的基因。Real-Time PCR检测目的基因m RNA表达水平对芯片结果进行验证。Western Blot检测Caspase家族蛋白、Bcl-2家族蛋白表达变化、WNT通路关键蛋白、PI3K/AKT通路关键蛋白表达变化。为进一步验证靶向抑制WNT3a及AKT后能否模拟HHT与ACR对THP-1细胞的协同杀伤作用,我们采用脂质体转染技术转染WNT3a的si RNA干扰片段以沉默THP-1细胞WNT3a基因。再分别采用MTT法及流式细胞术Annexin V/PI双染色法检测WNT3a干扰后联合HHT或AKT抑制剂后对THP-1细胞生长抑制及诱导凋亡作用。进一步我们采用GSK-3p抑制剂上调THP-1细胞P-catenin表达,观察是否能逆转HHT联合ACR对THP-1细胞的生长抑制及诱导凋亡作用。由于WNT通路及PI3K/AKT通路在AML-LSC中存在异常活化,尤其是WNT通路中的β-catenin在AML-LSC自我更新中具有尤为重要的作用。我们的研究中HHT与ACR联合可协同抑制WNT信号及PI3K/AKT信号,并可共同下调β-catenin,因此我们采用流式细胞术初步分析了HHT、ACR单用及联合对AML患者骨髓单个核细胞中CD34+／CD38-干／祖细胞群的诱导凋亡作用,并采用CalcuSyn软件进行协同效应分析。
结果：
(1)芯片筛选结果显示多种WNT基因及PIK3CA基因在HHT组、ACR组和联合用药组表达存在差异。(2)定量PCR结果也证实36nM HHT作用THP-1细胞和Kasumi细胞1h后WNT3A基因m RNA表达先上调随后回落至用药前水平,而90nM ACR作用THP-1细胞和Kasumi细胞1h-12h后WNT3A基因m RNA表达明显下调,联合HHT和ACR作用1h-12h后WNT3A基因m RNA表达也明显下调。P-catenin基因m RNA表达变化也呈类似结果。HHT、ACR、HHT联合ACR作用THP-1、Kasumi细胞1h-12h后GSK-3p基因m RNA表达变化无明显规律。(3)实验浓度HHT及ACR单独作用均可激活Caspase9、Caspase3并引起PARP剪切。HHT联合ACR可更明显激活Caspase9、Caspase3及PARP。(4)实验浓度HHT作用3h即可下调THP-1细胞和Kasumi细胞Bcl-x1、Mcl-1蛋白表达并上调Bax蛋白,但HHT对两种细胞Bcl-2蛋白表达无明显影响。ACR则在短时间内明显下调Bcl-2蛋白和Bcl-x1蛋白表达,而在作用较长时间才下调Mcl-1蛋白及上调Bax蛋白。(5)36nM HHT作用3h-24h后对THP-1细胞和Kasumi细胞WNT3a蛋白表达无明显影响,但作用3h即可明显下调PI3K110、P-AKT和β-catenin蛋白并上调GSK-3p蛋白。90nM ACR作用两种AML细胞株细胞3h后即明显下调WNT3a蛋白表达及(3-catenin蛋白,但对P13K110、P-AKT及GSK-3p蛋白表达无明显影响。36nM HHT联合90nM ACR可同时抑制THP-1细胞、Kasumi细胞WNT3a. β-catenin、PI3K110及P-AKT蛋白表达。(6)沉默THP-1细胞WNT3a基因后再联合HHT或AKT抑制剂能增加HHT及AKT抑制剂对THP-1细胞的生长抑制及诱导凋亡作用。一定浓度比联合AKT抑制剂与ACR亦能协同抑制THP-1细胞生长并协同诱导其发生凋亡。(7)采用GSK-3β抑制剂部分上调THP-1细胞β-catenin后能部分逆转实验浓度HHT联合ACR对THP-1细胞的生长抑制作用,但对HHT、ACR单药引起的生长抑制作用无明显改善。GSK-3p抑制剂作用对HHT、ACR、HHT联合ACR对THP-1细胞的诱导凋亡作用无明显逆转。(8)实验浓度HHT联合ACR可能可以协同诱导AML患者骨髓中CD34+／CD38-细胞凋亡。
小结：
(1)实验浓度HHT作用AML细胞株细胞不同时间对WNT3a基因、β-catenin基因表达无明显下调,而ACR及HHT联合ACR作用后明显下调WNT3a基因和β-catenin基因mRNA的表达,与芯片结果一致。(2)HHT及ACR最终可能通过Caspase9激活的内源性凋亡途径而在体外诱导AML细胞株细胞发生凋亡。HHT联合ACR能更明显激活Caspase9.Caspase3。(3)HHT可上调THP-1细胞及Kasumi细胞Bax蛋白表达但对Bcl-2蛋白无明显影响；ACR则可明显下调THP-1细胞及Kasumi细胞Bcl-2蛋白表达。HHT联合ACR能更明显地下调AML细胞株细胞Bcl-2蛋白、Bcl-xl蛋白表达,同时上调Bax蛋白表达。(4)HHT可抑制AML细胞株细胞及原代AML细胞的PI3K/AKT通路活性,并可能进一步抑制其下游信号分子如Mcl-1,同时上调Bax蛋白而诱导AML细胞株细胞发生凋亡。HHT还可能通过减低AKT活性后解除其对GSK-3β的抑制而间接下调P-catenin蛋白表达。(5)ACR可抑制AML细胞株细胞和原代AML细胞WNT3a、β-catenin蛋白的表达,并可能通过进一步抑制WNT通路下游靶分子CyclinDl、C-myc而发挥对AML细胞株细胞的生长抑制作用及诱导凋亡作用。(6)HHT联合ACR可同时抑制AML细胞株细胞及原代AML细胞的P13K/AKT信号及WNT/β-catenin信号。(7)HHT可抑制THP-1荷瘤小鼠瘤体细胞PI3K、P-AKT、β-catenin蛋白表达。ACR可抑制THP-1荷瘤小鼠瘤体细胞WNT3a、β-catenin蛋白表达。HHT联合ACR可同时抑制THP-1荷瘤小鼠瘤体细胞P13K、P-AKT、WNT3a、β-catenin蛋白表达。(8)沉默WNT3a可明显增加HHT或AKT抑制剂对THP-1细胞的杀伤作用。采用GSK-3β抑制剂上调P-catenin表达后可能部分逆转HHT联合ACR对THP-1细胞生长的抑制作用。(8)HHT联合ACR在体外也可能协同诱导AML患者骨髓中的白血病干／祖细胞凋亡。
结论：
实验浓度HHT与ACR联合在体外能协同抑制AML细胞株细胞及原代AML细胞生长,并协同诱导其凋亡。
实验浓度HHT与ACR联合在体内也可能通过诱导AML细胞荷瘤小鼠瘤体细胞凋亡而减慢瘤体增长速度,从而延长小鼠生存时间。
HHT可能通过抑制AML细胞的PI3K/AKT活性而抑制其生长并诱导凋亡。ACR可能通过抑制AML细胞的WNT/β-catenin通路活性而抑制其生长并诱导凋亡。
HHT联合ACR可能通过同时抑制PI3K/AKT通路及WNT/β-catenin通路而发挥协同杀伤AML细胞的效应。β-catenin作为上述两条信号通路的共同作用分子可能是HHT与ACR协同杀伤AML细胞的分子靶点之一。
Section1:The synergistical cytotoxicity in vitro and vivo induced by homoharringtonine and aclarubicin in acute myeloid leukemia cells
Objective:
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 cells
Objective:
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.
目的：
探讨高三尖杉酯碱与阿克拉霉素在体内外对AML细胞株细胞及原代AML细胞的杀伤作用及作用关系。
方法：
采用MTT法检测不同浓度HHT、ACR及按一定浓度比联合HHT和ACR对AML细胞生长的抑制作用。采用流式细胞仪Annexin V/PI双染色法、Hochest染色法检测不同浓度HHT、ACR及按一定浓度比联合HHT和ACR对AML细胞株细胞及原代AML细胞的诱导凋亡作用。采用CalcuSyn软件分析HHT与ACR在抑制AML细胞增殖及诱导凋亡上的作用关系。构建AML细胞荷瘤小鼠模型用于体内研究。当瘤体体积达到100-120mm3时随机分入对照组、HHT治疗组、ACR治疗组及HHT联合ACR治疗组。入组后监测瘤体大小。采用原位细胞凋亡检测试剂盒进行TUNEL染色检测瘤体组织原位凋亡。采用Micro-PET扫描对对照组及药物处理组小鼠体内瘤体活性进行实时观察。统计学分析,单一变量两组间比较采用t检验,多组间比较采用单因素方差分析。Kaplan-Meier法进行生存分析。以p值<0.05认为具有统计学差异。
结果：
(1)实验浓度HHT对THP-1细胞(急性单核细胞白血病细胞株)、Kasumi细胞(急性粒细胞白血病细胞株)的生长均具有明显的抑制作用,并呈浓度依赖性。(2)实验浓度ACR对THP-1细胞、Kasumi细胞的生长亦具有明显抑制作用,并呈浓度依赖性。(3)实验浓度HHT、ACR均能抑制原代AML细胞生长,随浓度增加,抑制作用增强。(4)1：2.5摩尔浓度比联合HHT与ACR能协同抑制THP-1细胞、Kasumi细胞及原代AML细胞的生长,并能协同诱导其凋亡。(5)采用THP-1细胞能成功构建荷瘤小鼠模型。HHT治疗、ACR治疗及联合HHT和ACR治疗均能延长THP-1荷瘤小鼠瘤体倍增时间。联合治疗较单药治疗能更明显地延长THP-1荷瘤小鼠瘤体倍增时间。联合HHT与ACR治疗组小鼠瘤体组织细胞凋亡较单药治疗组明显增加。(6)Micro-PET扫描结果显示HHT联合ACR治疗组THP-1荷瘤小鼠瘤体代谢活性较用药前有所下降。(7)仅联合HHT和ACR治疗后能延长THP-1荷瘤小鼠的生存时间。小结：
(1)一定浓度比联合HHT和ACR在体外可协同抑制THP-1细胞、Kasumi细胞和原代AML细胞增殖及诱导其凋亡。(2)与单药相比,HHT联合ACR在体内可更明显地抑制THP-1荷瘤小鼠瘤体增殖及诱导凋亡,并可能更明显地抑制荷瘤小鼠体内的肿瘤活性。(3)体内研究显示HHT与ACR联合治疗可改善THP-1荷瘤小鼠的生存。
第二部分：高三尖杉酯碱联合阿克拉霉素协同杀伤急性髓细胞白血病细胞的分子机制研究
目的：
探讨高三尖杉酯碱联合阿克拉霉素协同杀伤急性髓细胞白血病细胞的分子机制,为AML靶向联合治疗方案的设计提供实验依据。
方法：
采用Agilent单通道表达谱芯片筛选对照组、HHT、ACR、HHT联合ACR作用THP-1细胞后差异表达的基因。Real-Time PCR检测目的基因m RNA表达水平对芯片结果进行验证。Western Blot检测Caspase家族蛋白、Bcl-2家族蛋白表达变化、WNT通路关键蛋白、PI3K/AKT通路关键蛋白表达变化。为进一步验证靶向抑制WNT3a及AKT后能否模拟HHT与ACR对THP-1细胞的协同杀伤作用,我们采用脂质体转染技术转染WNT3a的si RNA干扰片段以沉默THP-1细胞WNT3a基因。再分别采用MTT法及流式细胞术Annexin V/PI双染色法检测WNT3a干扰后联合HHT或AKT抑制剂后对THP-1细胞生长抑制及诱导凋亡作用。进一步我们采用GSK-3p抑制剂上调THP-1细胞P-catenin表达,观察是否能逆转HHT联合ACR对THP-1细胞的生长抑制及诱导凋亡作用。由于WNT通路及PI3K/AKT通路在AML-LSC中存在异常活化,尤其是WNT通路中的β-catenin在AML-LSC自我更新中具有尤为重要的作用。我们的研究中HHT与ACR联合可协同抑制WNT信号及PI3K/AKT信号,并可共同下调β-catenin,因此我们采用流式细胞术初步分析了HHT、ACR单用及联合对AML患者骨髓单个核细胞中CD34+／CD38-干／祖细胞群的诱导凋亡作用,并采用CalcuSyn软件进行协同效应分析。
结果：
(1)芯片筛选结果显示多种WNT基因及PIK3CA基因在HHT组、ACR组和联合用药组表达存在差异。(2)定量PCR结果也证实36nM HHT作用THP-1细胞和Kasumi细胞1h后WNT3A基因m RNA表达先上调随后回落至用药前水平,而90nM ACR作用THP-1细胞和Kasumi细胞1h-12h后WNT3A基因m RNA表达明显下调,联合HHT和ACR作用1h-12h后WNT3A基因m RNA表达也明显下调。P-catenin基因m RNA表达变化也呈类似结果。HHT、ACR、HHT联合ACR作用THP-1、Kasumi细胞1h-12h后GSK-3p基因m RNA表达变化无明显规律。(3)实验浓度HHT及ACR单独作用均可激活Caspase9、Caspase3并引起PARP剪切。HHT联合ACR可更明显激活Caspase9、Caspase3及PARP。(4)实验浓度HHT作用3h即可下调THP-1细胞和Kasumi细胞Bcl-x1、Mcl-1蛋白表达并上调Bax蛋白,但HHT对两种细胞Bcl-2蛋白表达无明显影响。ACR则在短时间内明显下调Bcl-2蛋白和Bcl-x1蛋白表达,而在作用较长时间才下调Mcl-1蛋白及上调Bax蛋白。(5)36nM HHT作用3h-24h后对THP-1细胞和Kasumi细胞WNT3a蛋白表达无明显影响,但作用3h即可明显下调PI3K110、P-AKT和β-catenin蛋白并上调GSK-3p蛋白。90nM ACR作用两种AML细胞株细胞3h后即明显下调WNT3a蛋白表达及(3-catenin蛋白,但对P13K110、P-AKT及GSK-3p蛋白表达无明显影响。36nM HHT联合90nM ACR可同时抑制THP-1细胞、Kasumi细胞WNT3a. β-catenin、PI3K110及P-AKT蛋白表达。(6)沉默THP-1细胞WNT3a基因后再联合HHT或AKT抑制剂能增加HHT及AKT抑制剂对THP-1细胞的生长抑制及诱导凋亡作用。一定浓度比联合AKT抑制剂与ACR亦能协同抑制THP-1细胞生长并协同诱导其发生凋亡。(7)采用GSK-3β抑制剂部分上调THP-1细胞β-catenin后能部分逆转实验浓度HHT联合ACR对THP-1细胞的生长抑制作用,但对HHT、ACR单药引起的生长抑制作用无明显改善。GSK-3p抑制剂作用对HHT、ACR、HHT联合ACR对THP-1细胞的诱导凋亡作用无明显逆转。(8)实验浓度HHT联合ACR可能可以协同诱导AML患者骨髓中CD34+／CD38-细胞凋亡。
小结：
(1)实验浓度HHT作用AML细胞株细胞不同时间对WNT3a基因、β-catenin基因表达无明显下调,而ACR及HHT联合ACR作用后明显下调WNT3a基因和β-catenin基因mRNA的表达,与芯片结果一致。(2)HHT及ACR最终可能通过Caspase9激活的内源性凋亡途径而在体外诱导AML细胞株细胞发生凋亡。HHT联合ACR能更明显激活Caspase9.Caspase3。(3)HHT可上调THP-1细胞及Kasumi细胞Bax蛋白表达但对Bcl-2蛋白无明显影响；ACR则可明显下调THP-1细胞及Kasumi细胞Bcl-2蛋白表达。HHT联合ACR能更明显地下调AML细胞株细胞Bcl-2蛋白、Bcl-xl蛋白表达,同时上调Bax蛋白表达。(4)HHT可抑制AML细胞株细胞及原代AML细胞的PI3K/AKT通路活性,并可能进一步抑制其下游信号分子如Mcl-1,同时上调Bax蛋白而诱导AML细胞株细胞发生凋亡。HHT还可能通过减低AKT活性后解除其对GSK-3β的抑制而间接下调P-catenin蛋白表达。(5)ACR可抑制AML细胞株细胞和原代AML细胞WNT3a、β-catenin蛋白的表达,并可能通过进一步抑制WNT通路下游靶分子CyclinDl、C-myc而发挥对AML细胞株细胞的生长抑制作用及诱导凋亡作用。(6)HHT联合ACR可同时抑制AML细胞株细胞及原代AML细胞的P13K/AKT信号及WNT/β-catenin信号。(7)HHT可抑制THP-1荷瘤小鼠瘤体细胞PI3K、P-AKT、β-catenin蛋白表达。ACR可抑制THP-1荷瘤小鼠瘤体细胞WNT3a、β-catenin蛋白表达。HHT联合ACR可同时抑制THP-1荷瘤小鼠瘤体细胞P13K、P-AKT、WNT3a、β-catenin蛋白表达。(8)沉默WNT3a可明显增加HHT或AKT抑制剂对THP-1细胞的杀伤作用。采用GSK-3β抑制剂上调P-catenin表达后可能部分逆转HHT联合ACR对THP-1细胞生长的抑制作用。(8)HHT联合ACR在体外也可能协同诱导AML患者骨髓中的白血病干／祖细胞凋亡。
结论：
实验浓度HHT与ACR联合在体外能协同抑制AML细胞株细胞及原代AML细胞生长,并协同诱导其凋亡。
实验浓度HHT与ACR联合在体内也可能通过诱导AML细胞荷瘤小鼠瘤体细胞凋亡而减慢瘤体增长速度,从而延长小鼠生存时间。
HHT可能通过抑制AML细胞的PI3K/AKT活性而抑制其生长并诱导凋亡。ACR可能通过抑制AML细胞的WNT/β-catenin通路活性而抑制其生长并诱导凋亡。
HHT联合ACR可能通过同时抑制PI3K/AKT通路及WNT/β-catenin通路而发挥协同杀伤AML细胞的效应。β-catenin作为上述两条信号通路的共同作用分子可能是HHT与ACR协同杀伤AML细胞的分子靶点之一。
Section1:The synergistical cytotoxicity in vitro and vivo induced by homoharringtonine and aclarubicin in acute myeloid leukemia cells
Objective:
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 cells
Objective:
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.
引文
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