硼替佐米联合三尖杉酯碱或三氧化二砷对耐药急性髓系白血病细胞增殖、凋亡的实验研究
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摘要
背景与目的:
尽管急性白血病的治疗效果近年来有了较明显的提高,但仍然有15～30%的病人发生原发性耐药,60～80%的患者完全缓解后因继发性耐药复发而死亡。随着生物学技术快速发展,人们发现真核细胞内蛋白质降解大部分是通过泛素—蛋白酶体通路(Ubiquitin-Protease Pathway,UPP)完成,人白血病细胞含有异常高水平的蛋白酶体活性,且在诱导终末白血病细胞分化时蛋白酶体迅速被下调,表明蛋白酶体的活性与白血病细胞的恶性增殖有着密切相关性,因此蛋白酶体可能成为白血病治疗的新靶点。
目前研究证实,蛋白酶体在细胞内蛋白降解、MHC-I类抗原递呈、细胞内部错误折叠蛋白的修复上发挥着重要的功能,多种与细胞周期调控、信号转导、细胞凋亡相关的蛋白的正常代谢,都与其功能的正确执行密切相关。特异性阻断蛋白酶体功能显著改变以上蛋白在细胞内的表达水平,导致细胞内蛋白质代谢发生紊乱,从而诱导细胞发生凋亡,是目前认识的蛋白酶体抑制剂主要的抗肿瘤作用机制。
硼替佐米(Bortezomib)是第一个被美国FDA批准用于临床的蛋白酶体抑制剂,唯一的适应症是难治、复发性多发性骨髓瘤(Multiple Myeloma,MM)的治疗。此外,初步的多项临床试验表明硼替佐米单独或联合其他药物治疗非何杰金氏淋巴瘤、慢性淋巴细胞性白血病、成人T细胞白血病、肺癌、卵巢癌等也具有良好效果。有关硼替佐米治疗急性髓系白血病的研究至今很少报道,我们前期工作中发现硼替佐米体外能够抑制急性髓系白血病细胞株HL60的增殖并诱导其凋亡,与三尖杉酯碱(HT)或三氧化二砷(As_2O_3)联合后作用明显增强,但它们对产生多药耐药的急性髓系白血病细胞的作用如何至今尚缺乏报道,此乃是本研究立题的主要动机。
研究方法:
首先选择急性髓系白血病多药耐药细胞株HL60/ADM为研究对象。应用MTT法比较HL60/ADM和HL60细胞之间对阿霉素杀伤作用的敏感性。以耐药倍数大于30倍定义为耐药,此后将HL60/ADM细胞培养于含有10%小牛血清的RPMI-1640培养基中进行培养。经过预实验在0～1000nM之间筛选硼替佐米对HL60/ADM细胞作用的最佳浓度,在0～72h之间选择处理的最佳时间,在0～752nM之间确定HT的IC_(50)浓度,在0～40μM之间确定As_2O_3的IC_(50)浓度,依据台盼蓝拒染法确定各实验组处理后细胞存活情况。
确定硼替佐米0～50nM为后续实验浓度,作用48h为最佳处理时间。硼替佐米的IC_(50)浓度约为20nM,HT的IC_(50)浓度为752nM,As_2O_3的IC_(50)浓度为15μM。在上述预实验的基础上,将HL60/ADM细胞分为硼替佐米、As_2O_3或HT单药处理组、硼替佐米联合As_2O_3处理组和硼替佐米联合HT处理组,每组处理的细胞数一致,每组均设置空白对照组。各组检测指标相同,即用MTT法检测细胞增殖、应用AnnexinV-FITC/PI染色流式细胞术和Hoechst33342染色检测细胞凋亡,并应用流式细胞仪检测HL60/ADM细胞摄取阿霉素的情况。
在完成上述实验研究之后,选择9例复发、难治性急性髓系白血病患者原代细胞应用MTT法研究其增殖变化。硼替佐米不同浓度间增殖活性差异采用单因素方差分析;硼替佐米联合HT或As_2O_3对HL60/ADM白血病细胞增殖活性影响比较使用析因分析;细胞凋亡改变采用方差分析,两两比较采用LSD法;阿霉素阳性率比较采用两样本t检验。药物联合后对原代细胞的细胞活性差异采用随机区组方差分析,两两比较采用LSD法检验。
结果:
1.HL60/ADM细胞的耐药性确定
通过MTT法检测HL60/ADM或白血病细胞对阿霉素的敏感性发现,阿霉素作用于HL60/ADM细胞的IC_(50)值约为16.283μg/ml,作用于HL60细胞的IC_(50)值仅为0.087μg/ml,HL60/ADM细胞对阿霉素高度耐药,耐药倍数为187倍。
2.硼替佐米对HL60/ADM白血病细胞增殖、凋亡的影响
台盼蓝拒染计数显示:0、10、20、30、40、50nM硼替佐米处理HL60/ADM白血病细胞12小时,各浓度间活细胞数无统计学差异(P＞0.05),延长时间至24h,各浓度硼替佐米对细胞的抑制效应差异开始显现。随着时间的延长和浓度增加,处理组细胞增殖抑制率逐渐增加。
MTT法检测结果显示:10～50nM硼替佐米处理12～60h对HL60/ADM白血病细胞均具有增殖抑制作用,随处理时间延长和药物剂量增加,细胞增殖抑制率逐渐提高,呈明显时间—剂量依赖关系。其48h IC_(50)值约为20nM,其中40nM处理48h达最大抑制效果(抑制率61%),继续增加剂量或者延长作用时间至60h,抑制效果无明显增强(P=0.478,P=0.161)。
Hoechst33342染色形态学观察显示:未加硼替佐米的空白对照组细胞,核呈弥漫均匀蓝色荧光,而10nM、20nM和50nM硼替佐米处理组细胞均出现核固缩、凝集,并有凋亡小体,表现为明亮的颗粒状蓝染,随着浓度增加凋亡小体逐渐增多。
AnnexinV-FITC/PI双染法流式细胞仪检测凋亡发现:10nM硼替佐米处理HL60/ADM细胞24h,其平均细胞凋亡率为9.58%,对照组为8.85%(P＜0.05);同样处理时间下,20nM硼替佐米处理组平均细胞凋亡率为16.45%,显著高于10nM处理组(P均＜0.01);延长作用时间至48小时,10nM硼替佐米处理组细胞凋亡率增加至17.22%,20nM处理组细胞凋亡率则明显增高达73.46%,均显著高于对照组(14.84%,P＜0.05),与同剂量下作用24h相比,差异具有显著统计学意义(P均＜0.01)。
3.硼替佐米联合三尖杉酯碱或三氧化二砷对HL60/ADM白血病细胞增殖、凋亡的影响
MTT法检测结果显示:HT或As_2O_3单独应用对HL60/ADM白血病细胞均具有剂量依赖性增殖抑制作用,其中94、188、376、564、752nM HT和2、4、10、20、30、40μM As_2O_3处理HL60/ADM白血病细胞48h,细胞增殖抑制率分别为9%、20%、31%、39%、52%和25%、33%、40%、58%、64%、69%;HT及As_2O_3对HL60/ADM白血病细胞48h的IC_(50)浓度分别为752nM和15μM。10～50nM的硼替佐米对HL60/ADM白血病细胞增殖同样表现出抑制作用,其48h的IC_(50)浓度约为20nM。
将752nM HT或15μM As_2O_3分别与10～50nM的硼替佐米联合,可观察到联合处理后细胞抑制效果进一步增强;在达到药物最大抑制效果前,硼替佐米联合组对细胞增殖的抑制效果显著高于硼替佐米单药组(P均＜0.05);析因分析显示:10nM硼替佐米与752nM HT联合处理的效果显著比硼替佐米单药处理组高,但与相同剂量HT单独处理组相比,其差异无统计学意义,P＞0.05;而10nM硼替佐米与15μM As_2O_3联合处理组的抑制率均显著高于二者相同剂量单药处理组,P＜0.05;Hoechst33342染色结果显示:经48h培养空白对照组细胞未见明显凋亡发生,细胞核呈弥漫均匀蓝色荧光;10nM硼替佐米单药处理组细胞可见核固缩、凝集,并伴凋亡小体,其表现为明亮的颗粒状蓝染。硼替佐米与As_2O_3或HT联合处理组凋亡小体数量均多于各单药处理组,以硼替佐米与As_2O_3联合组更明显。
流式细胞仪检测结果显示:各实验组细胞经药物处理后细胞变化以凋亡为主。As_2O_3与硼替佐米联合处理组24、48h后的细胞凋亡率均显著高于硼替佐米单药组:其中处理48h二者平均细胞凋亡率分别为81.89%和17.22%(P＜0.01);同样,HT联合硼替佐米处理组24、48h凋亡率亦高于硼替佐米单药处理组;在48h二者平均细胞凋亡率分别为80.64%和17.22%(P＜0.01)。检测细胞阿霉素阳性率显示:在相同浓度阿霉素培养体系中,加入硼替佐米处理组细胞的荧光阳性率显著高于对照组(P均＜0.05),提示硼替佐米可能促使HL60/ADM白血病细胞摄取阿霉素或使其在细胞内排除延迟。
4.硼替佐米联合三尖杉酯碱或三氧化二砷对复发、难治性急性髓系白血病原代细胞增殖影响
10～200nM硼替佐米均能抑制原代白血病细胞增殖,抑制率随浓度增加而逐渐增高,其中10nM、20nM、40nM、80nM、100nM和200nM剂量处理48h,平均抑制率分别为8.7%、18.1%、26.8%、39.2%、48.06%、51.44%;与对照组相比差异具有统计学意义(P＜0.05);10～100nM剂量组之间差异具有统计学意义(P＜0.05);100nM～200nM剂量组抑制率无显著性差异(P=0.290)。20nM硼替佐米联合752nM HT处理48h,原代细胞平均增殖抑制率与两药单独作用时比较无明显差异(P均＞0.05):而与15μM As_2O_3联合处理48h,对原代白血病细胞平均增殖抑制率均显著高于两药单独作用时(P均＜0.05);相同条件下处理HL60/ADM多药耐药细胞发现,联合处理组细胞增殖抑制率均高于各单药处理组(P均＜0.05)。
结论:
1、硼替佐米单药对HL60/ADM多药耐药细胞具有抑制增殖及诱导凋亡的作用,其效果呈时间-剂量依赖性,随着时间的延长和剂量的增加作用显著增强。
2、三尖杉酯碱和三氧化二砷对HL60/ADM多药耐药细胞均具有抑制增殖作用,效果呈剂量依赖性,各自的IC_(50)剂量下分别与不同浓度硼替佐米联合处理后,与硼替佐米单药处理组相比,其抑制增殖和诱导凋亡作用明显增强。
3、硼替佐米能够促进HL60/ADM细胞对阿霉素的摄取,提高细胞内阿霉素浓度,从而降低细胞对化疗药物的耐药性,逆转耐药。
4、硼替佐米、三尖杉酯碱、三氧化二砷单药均能在体外抑制急性髓系白血病原代细胞的增殖,但其抑制率明显低于HL60/ADM细胞。
5、硼替佐米与三尖杉酯碱或三氧化二砷联合作用均能抑制急性髓系白血病原代细胞的增殖,与各单药作用相比,与HT联合其作用无明显提高,而与三氧化二砷联合效果均比二者单药处理时显著增强。
Background and objectives
Although the therapeutic efficacy of acute leukemia has been greatly improved in the recent years, but there are still up to 15～30% patients present original drug resistance. And 60～80% relapsed after the first CR, some of them die soon after relapsed.
With great progress in Molecular Biology, it has been found that the maintenance of homeostasis and the ability of cells to respond to their environment depend on the orderly degradation of key regulatory proteins and their inhibitors. The proteasome plays an essential role in the targeted degradation of such proteins and is therefore involved in the activation and inactivation of many cellular processes. In fact, studies using proteasome inhibitors have shown that the proteasome is responsible for the elimination of more than 80% of all cellular proteins. Specific targets of proteasome include cell-cycle proteins, tumor suppressors, and transcription factors, as well as mutant and damaged proteins. The proteasome has been identified as an excellent target for cancer therapy because of its critical metabolic function. In 2003, bortezomib became first-in-class proteasome inhibitor that received approval from the U.S. Food and Drug Administration (FDA) for the treatment of multiple myeloma patients who have received at least two prior therapies and have demonstrated disease progression on the last therapy. However, bortezomib has demonstrated significant activity against a broad range of human tumor cells. In the earlier stage of our research, we found that bortezomib has a dose- and time- dependent antiproliferation and proapoptotic effect on HL60 leukemia cells in vitro, when combined with IC_(50) HT or IC_(50) As_2O_3, the effect has been abviously improved. In this study, we assume to investigate the effect of bortezomib in combination with harringtonine or arsenic trioxide on proliferation and apoptosis of multidrug resistant leukemia cells.
Methods
HL60/ADM leukemia cells were selected in this study and were cultured in the RPMI-1640 medium supplemented with 10% calf serum. First of all we design a MTT assay experiment to ensure that HL60/ADM is highly multidrug resistant(compare with HL60, with at least 30 multiples). Then , in order to determine the best concentration and time of experiment, 0-1000nM bortezomib was used to treat cells for 0-72h in vitro and proliferation activity of HL60/ADM leukemia cells was detected by trypan blue dye exclusion method. We chose 0-50nM bortezomib as the experiment concentration. The change of cell activity after treated by bortezomib was analyzed by MTT assay and apoptosis of HL60/ADM leukemia cells were detected by by fluorescence microscopy (hoechst33342) and flow cytometry (AnnexinV-FITC/PI). Intracellular concentration of Adriamycin(ADM) was determined by flow cytometry. We determine the optimal time of culture is 48h and use 0-752nM HT and 0-40μM As_2O_3 to treat HL60/ADM leukemia cells for 48h respectively. The half maximal inhibitory concentration (IC_(50)) of HT and As_2O_3 was confirmed then(752nM and 15μM). Bortezomib combined with IC_(50) concentration of HT or As_2O_3 treating, the proliferation and apoptosis changes of HL60/ADM leukemia cells and primary cells which come from 9 patients were detected by the same methods above. The differences of effect between groups were done by statistics anylysis.
Results
1. The multidrug resistance of HL60/ADM leukemia cells
From the ADM toxicity experiment which was detected by MTT assay, we found that the IC_(50) of ADM to HL60/ADM is 16.283μg/ml, while the IC_(50) of ADM to HL60 is 0.087μg/ml; HL60/ADM is highly multidrug resistance (187 times).
2. Effect of bortezomib alone on the proliferation and apoptosis of HL60/ADM cells
Dates from trypan blue dye exclusion method reveal that there is no significant difference between 0-50nM bortezomib treating for 12 hours(P>0.05). after 24 h, the number of cells in different concentration has gradually decreased in a dose- and time- manner. Dates from MTT assays show that In 10-50nM bortezomib treated cells, inhibition rates enhanced in a time- and dose-dependently manner , 40nM bortezomib can best inhibit the proliferation activity of HL60/ADM cells when incubated for 48 hours, with the IC50 dose 20nM. When we raised the dose to 50nM or prolonged the incubated time to 60 h, the inhibition rates had no significant enhancement (P=0.478, P=0.161); Normal cells and apoptotic cells could be distinguished under the fluorescence microscope after staining with Hochest 33342. Normal nuclei kept intact and stained evenly. Apoptotic cells show strong chromatin condensation and nuclear fragmentation. apoptosis cells shows in 10nM,20nM or 50nM, and increased in dose maner.
After detecting the apoptosis of HL60/ADM cells with flow cytometry, there were apoptotic cells in 10nM or 20nM bortezomib treating groups.10nM bortezomib treating for 24h provide a apoptosis rate 9.58%,while 20nM treating group with 16.45% apoptosis rate which is significant higher then 10nM group. Prolong the treating time to 48h, the apoptosis rate in 10nM or 20nM group increased to 17.22% and 73.46%, respectively, which were significant higher then those in the same concentration of 24h.
3. Effect of bortezomib in combination with harringtonine or arsenic trioxide on the proliferation and apoptosis of HL60/ADM cells
As_2O_3 alone can also inhibit the proliferation of HL60/ADM. When cultured for 48 hours, 2、4、10、20、30、40μM As_2O_3 brought inhibition rates of 25%、33%、40% > 58% , 64% , 69%, respectively, with the IC50 dose as 15μM. When cultured for 48 hours, 94、188、376、564、752 nM HT brought inhibition rates of 9%、20%、31 %、39%、52%, respectively, with the IC_(50) dose as 752nM. 15μM As_2O_3 or 752nM HT which is combined with 10-50nM of bortezomib can inhibit the proliferation of HL60/ADM, and the dual inhibition of bortezomib plus As_2O_3 is significant higher then that of either above. While there is no different between bortezomib plus HT group and HT treating group. fluorescence microscope show that The apoptotic cells in both compounds combined groups are more than those in the single-agent groups. flow cytometry show that there were apoptotic cells in all groups. After they were cultured for 24 or 48 hours, the apoptotic rates of the agents combined groups were higher than the single-agent groups. After the cells had been cultured for 48 hours, the apoptotic rates of the bortezomib plus As_2O_3 , bortezomib plus HT group and the bortezomib group were 81.89% , 80.64% and 17.22%(P<0.01), respectively. And mostly, apoptosis in the combined group present in a late-stage. The intraceflular accumulation of ADM of HL60/ADM cells in the botezomib group is stronger than the bortezomib-free group(P<0.05 ).10nM Bortezomib could significantly enhance the intracellular accumulation of ADM in HL60/ADM cells which indicated that bortezomib might have the ability to reverse multidrug resistant.
4. Effect of brotezomib in combination with harringtonine or arsenic trioxide on the proliferation of primary leukemia cells
10-200nM bortezomib can inhibit proliferation of primary cells, inhibition rates increased in a dose-dependently manner; 10nM、20nM、40nM、80nM、100nM、200nM bortezomib treating for 48h provide inhibition rates as 8.7%、18.1%、26.8 %、39.2%、48.06%、51.44%, respectively. 100nM bortezomib could best inhibit the proliferation activity. Higher doses made no significant increase of the inhibition rate. 20nM bortezomib combined with 752nM HT treating for 48h, make no difference with either of above treating alone(P>0.05). while the inhibiton rate of 20nM bortezomib combined with 15μM As_2O_3 is significantly higher then those of either treating alone.(P<0.05)
Conclusiones
1. Bortezomib has a dose- and time- dependent antiproliferation and proapoptotic effect on H60/ADM leukemia cells in vitro. With prolongation of time and increase of concentration of bortezomib, the effect has gradually increased.
2. HT and As_2O_3 also have a dose dependent antiproliferation and proapoptic effects on HL60/ADM cells, while combined with bortezomib, the effect is obviously improved when compared with bortezomib used alone.
3. Bortezomib could significantly enhance the intracellular accumulation of ADM in HL60/ADM cells which indicated that bortezomib might have the ability to reverse multidrug resistant.
4. Bortezomib、HT and As_2O_3 have a dose-dependent antiproliferation effect on relapsed/refractory AML primary cells in vitro. And the effect was obviously inferior to HL60/ADM leukemia cells.
5. Bortezomib in combination with HT or As_2O_3 has antiproliferation on relapsed/refractory AML primary cells in vitro. Compared with the effect of which drugs used alone, the effect of bortezomib with HT group has no improved, while the effect of bortezomib in combination As_2O_3 group is superior to the effects of either group which drugs used alone.
尽管急性白血病的治疗效果近年来有了较明显的提高,但仍然有15～30%的病人发生原发性耐药,60～80%的患者完全缓解后因继发性耐药复发而死亡。随着生物学技术快速发展,人们发现真核细胞内蛋白质降解大部分是通过泛素—蛋白酶体通路(Ubiquitin-Protease Pathway,UPP)完成,人白血病细胞含有异常高水平的蛋白酶体活性,且在诱导终末白血病细胞分化时蛋白酶体迅速被下调,表明蛋白酶体的活性与白血病细胞的恶性增殖有着密切相关性,因此蛋白酶体可能成为白血病治疗的新靶点。
目前研究证实,蛋白酶体在细胞内蛋白降解、MHC-I类抗原递呈、细胞内部错误折叠蛋白的修复上发挥着重要的功能,多种与细胞周期调控、信号转导、细胞凋亡相关的蛋白的正常代谢,都与其功能的正确执行密切相关。特异性阻断蛋白酶体功能显著改变以上蛋白在细胞内的表达水平,导致细胞内蛋白质代谢发生紊乱,从而诱导细胞发生凋亡,是目前认识的蛋白酶体抑制剂主要的抗肿瘤作用机制。
硼替佐米(Bortezomib)是第一个被美国FDA批准用于临床的蛋白酶体抑制剂,唯一的适应症是难治、复发性多发性骨髓瘤(Multiple Myeloma,MM)的治疗。此外,初步的多项临床试验表明硼替佐米单独或联合其他药物治疗非何杰金氏淋巴瘤、慢性淋巴细胞性白血病、成人T细胞白血病、肺癌、卵巢癌等也具有良好效果。有关硼替佐米治疗急性髓系白血病的研究至今很少报道,我们前期工作中发现硼替佐米体外能够抑制急性髓系白血病细胞株HL60的增殖并诱导其凋亡,与三尖杉酯碱(HT)或三氧化二砷(As_2O_3)联合后作用明显增强,但它们对产生多药耐药的急性髓系白血病细胞的作用如何至今尚缺乏报道,此乃是本研究立题的主要动机。
研究方法:
首先选择急性髓系白血病多药耐药细胞株HL60/ADM为研究对象。应用MTT法比较HL60/ADM和HL60细胞之间对阿霉素杀伤作用的敏感性。以耐药倍数大于30倍定义为耐药,此后将HL60/ADM细胞培养于含有10%小牛血清的RPMI-1640培养基中进行培养。经过预实验在0～1000nM之间筛选硼替佐米对HL60/ADM细胞作用的最佳浓度,在0～72h之间选择处理的最佳时间,在0～752nM之间确定HT的IC_(50)浓度,在0～40μM之间确定As_2O_3的IC_(50)浓度,依据台盼蓝拒染法确定各实验组处理后细胞存活情况。
确定硼替佐米0～50nM为后续实验浓度,作用48h为最佳处理时间。硼替佐米的IC_(50)浓度约为20nM,HT的IC_(50)浓度为752nM,As_2O_3的IC_(50)浓度为15μM。在上述预实验的基础上,将HL60/ADM细胞分为硼替佐米、As_2O_3或HT单药处理组、硼替佐米联合As_2O_3处理组和硼替佐米联合HT处理组,每组处理的细胞数一致,每组均设置空白对照组。各组检测指标相同,即用MTT法检测细胞增殖、应用AnnexinV-FITC/PI染色流式细胞术和Hoechst33342染色检测细胞凋亡,并应用流式细胞仪检测HL60/ADM细胞摄取阿霉素的情况。
在完成上述实验研究之后,选择9例复发、难治性急性髓系白血病患者原代细胞应用MTT法研究其增殖变化。硼替佐米不同浓度间增殖活性差异采用单因素方差分析;硼替佐米联合HT或As_2O_3对HL60/ADM白血病细胞增殖活性影响比较使用析因分析;细胞凋亡改变采用方差分析,两两比较采用LSD法;阿霉素阳性率比较采用两样本t检验。药物联合后对原代细胞的细胞活性差异采用随机区组方差分析,两两比较采用LSD法检验。
结果:
1.HL60/ADM细胞的耐药性确定
通过MTT法检测HL60/ADM或白血病细胞对阿霉素的敏感性发现,阿霉素作用于HL60/ADM细胞的IC_(50)值约为16.283μg/ml,作用于HL60细胞的IC_(50)值仅为0.087μg/ml,HL60/ADM细胞对阿霉素高度耐药,耐药倍数为187倍。
2.硼替佐米对HL60/ADM白血病细胞增殖、凋亡的影响
台盼蓝拒染计数显示:0、10、20、30、40、50nM硼替佐米处理HL60/ADM白血病细胞12小时,各浓度间活细胞数无统计学差异(P＞0.05),延长时间至24h,各浓度硼替佐米对细胞的抑制效应差异开始显现。随着时间的延长和浓度增加,处理组细胞增殖抑制率逐渐增加。
MTT法检测结果显示:10～50nM硼替佐米处理12～60h对HL60/ADM白血病细胞均具有增殖抑制作用,随处理时间延长和药物剂量增加,细胞增殖抑制率逐渐提高,呈明显时间—剂量依赖关系。其48h IC_(50)值约为20nM,其中40nM处理48h达最大抑制效果(抑制率61%),继续增加剂量或者延长作用时间至60h,抑制效果无明显增强(P=0.478,P=0.161)。
Hoechst33342染色形态学观察显示:未加硼替佐米的空白对照组细胞,核呈弥漫均匀蓝色荧光,而10nM、20nM和50nM硼替佐米处理组细胞均出现核固缩、凝集,并有凋亡小体,表现为明亮的颗粒状蓝染,随着浓度增加凋亡小体逐渐增多。
AnnexinV-FITC/PI双染法流式细胞仪检测凋亡发现:10nM硼替佐米处理HL60/ADM细胞24h,其平均细胞凋亡率为9.58%,对照组为8.85%(P＜0.05);同样处理时间下,20nM硼替佐米处理组平均细胞凋亡率为16.45%,显著高于10nM处理组(P均＜0.01);延长作用时间至48小时,10nM硼替佐米处理组细胞凋亡率增加至17.22%,20nM处理组细胞凋亡率则明显增高达73.46%,均显著高于对照组(14.84%,P＜0.05),与同剂量下作用24h相比,差异具有显著统计学意义(P均＜0.01)。
3.硼替佐米联合三尖杉酯碱或三氧化二砷对HL60/ADM白血病细胞增殖、凋亡的影响
MTT法检测结果显示:HT或As_2O_3单独应用对HL60/ADM白血病细胞均具有剂量依赖性增殖抑制作用,其中94、188、376、564、752nM HT和2、4、10、20、30、40μM As_2O_3处理HL60/ADM白血病细胞48h,细胞增殖抑制率分别为9%、20%、31%、39%、52%和25%、33%、40%、58%、64%、69%;HT及As_2O_3对HL60/ADM白血病细胞48h的IC_(50)浓度分别为752nM和15μM。10～50nM的硼替佐米对HL60/ADM白血病细胞增殖同样表现出抑制作用,其48h的IC_(50)浓度约为20nM。
将752nM HT或15μM As_2O_3分别与10～50nM的硼替佐米联合,可观察到联合处理后细胞抑制效果进一步增强;在达到药物最大抑制效果前,硼替佐米联合组对细胞增殖的抑制效果显著高于硼替佐米单药组(P均＜0.05);析因分析显示:10nM硼替佐米与752nM HT联合处理的效果显著比硼替佐米单药处理组高,但与相同剂量HT单独处理组相比,其差异无统计学意义,P＞0.05;而10nM硼替佐米与15μM As_2O_3联合处理组的抑制率均显著高于二者相同剂量单药处理组,P＜0.05;Hoechst33342染色结果显示:经48h培养空白对照组细胞未见明显凋亡发生,细胞核呈弥漫均匀蓝色荧光;10nM硼替佐米单药处理组细胞可见核固缩、凝集,并伴凋亡小体,其表现为明亮的颗粒状蓝染。硼替佐米与As_2O_3或HT联合处理组凋亡小体数量均多于各单药处理组,以硼替佐米与As_2O_3联合组更明显。
流式细胞仪检测结果显示:各实验组细胞经药物处理后细胞变化以凋亡为主。As_2O_3与硼替佐米联合处理组24、48h后的细胞凋亡率均显著高于硼替佐米单药组:其中处理48h二者平均细胞凋亡率分别为81.89%和17.22%(P＜0.01);同样,HT联合硼替佐米处理组24、48h凋亡率亦高于硼替佐米单药处理组;在48h二者平均细胞凋亡率分别为80.64%和17.22%(P＜0.01)。检测细胞阿霉素阳性率显示:在相同浓度阿霉素培养体系中,加入硼替佐米处理组细胞的荧光阳性率显著高于对照组(P均＜0.05),提示硼替佐米可能促使HL60/ADM白血病细胞摄取阿霉素或使其在细胞内排除延迟。
4.硼替佐米联合三尖杉酯碱或三氧化二砷对复发、难治性急性髓系白血病原代细胞增殖影响
10～200nM硼替佐米均能抑制原代白血病细胞增殖,抑制率随浓度增加而逐渐增高,其中10nM、20nM、40nM、80nM、100nM和200nM剂量处理48h,平均抑制率分别为8.7%、18.1%、26.8%、39.2%、48.06%、51.44%;与对照组相比差异具有统计学意义(P＜0.05);10～100nM剂量组之间差异具有统计学意义(P＜0.05);100nM～200nM剂量组抑制率无显著性差异(P=0.290)。20nM硼替佐米联合752nM HT处理48h,原代细胞平均增殖抑制率与两药单独作用时比较无明显差异(P均＞0.05):而与15μM As_2O_3联合处理48h,对原代白血病细胞平均增殖抑制率均显著高于两药单独作用时(P均＜0.05);相同条件下处理HL60/ADM多药耐药细胞发现,联合处理组细胞增殖抑制率均高于各单药处理组(P均＜0.05)。
结论:
1、硼替佐米单药对HL60/ADM多药耐药细胞具有抑制增殖及诱导凋亡的作用,其效果呈时间-剂量依赖性,随着时间的延长和剂量的增加作用显著增强。
2、三尖杉酯碱和三氧化二砷对HL60/ADM多药耐药细胞均具有抑制增殖作用,效果呈剂量依赖性,各自的IC_(50)剂量下分别与不同浓度硼替佐米联合处理后,与硼替佐米单药处理组相比,其抑制增殖和诱导凋亡作用明显增强。
3、硼替佐米能够促进HL60/ADM细胞对阿霉素的摄取,提高细胞内阿霉素浓度,从而降低细胞对化疗药物的耐药性,逆转耐药。
4、硼替佐米、三尖杉酯碱、三氧化二砷单药均能在体外抑制急性髓系白血病原代细胞的增殖,但其抑制率明显低于HL60/ADM细胞。
5、硼替佐米与三尖杉酯碱或三氧化二砷联合作用均能抑制急性髓系白血病原代细胞的增殖,与各单药作用相比,与HT联合其作用无明显提高,而与三氧化二砷联合效果均比二者单药处理时显著增强。
Background and objectives
Although the therapeutic efficacy of acute leukemia has been greatly improved in the recent years, but there are still up to 15～30% patients present original drug resistance. And 60～80% relapsed after the first CR, some of them die soon after relapsed.
With great progress in Molecular Biology, it has been found that the maintenance of homeostasis and the ability of cells to respond to their environment depend on the orderly degradation of key regulatory proteins and their inhibitors. The proteasome plays an essential role in the targeted degradation of such proteins and is therefore involved in the activation and inactivation of many cellular processes. In fact, studies using proteasome inhibitors have shown that the proteasome is responsible for the elimination of more than 80% of all cellular proteins. Specific targets of proteasome include cell-cycle proteins, tumor suppressors, and transcription factors, as well as mutant and damaged proteins. The proteasome has been identified as an excellent target for cancer therapy because of its critical metabolic function. In 2003, bortezomib became first-in-class proteasome inhibitor that received approval from the U.S. Food and Drug Administration (FDA) for the treatment of multiple myeloma patients who have received at least two prior therapies and have demonstrated disease progression on the last therapy. However, bortezomib has demonstrated significant activity against a broad range of human tumor cells. In the earlier stage of our research, we found that bortezomib has a dose- and time- dependent antiproliferation and proapoptotic effect on HL60 leukemia cells in vitro, when combined with IC_(50) HT or IC_(50) As_2O_3, the effect has been abviously improved. In this study, we assume to investigate the effect of bortezomib in combination with harringtonine or arsenic trioxide on proliferation and apoptosis of multidrug resistant leukemia cells.
Methods
HL60/ADM leukemia cells were selected in this study and were cultured in the RPMI-1640 medium supplemented with 10% calf serum. First of all we design a MTT assay experiment to ensure that HL60/ADM is highly multidrug resistant(compare with HL60, with at least 30 multiples). Then , in order to determine the best concentration and time of experiment, 0-1000nM bortezomib was used to treat cells for 0-72h in vitro and proliferation activity of HL60/ADM leukemia cells was detected by trypan blue dye exclusion method. We chose 0-50nM bortezomib as the experiment concentration. The change of cell activity after treated by bortezomib was analyzed by MTT assay and apoptosis of HL60/ADM leukemia cells were detected by by fluorescence microscopy (hoechst33342) and flow cytometry (AnnexinV-FITC/PI). Intracellular concentration of Adriamycin(ADM) was determined by flow cytometry. We determine the optimal time of culture is 48h and use 0-752nM HT and 0-40μM As_2O_3 to treat HL60/ADM leukemia cells for 48h respectively. The half maximal inhibitory concentration (IC_(50)) of HT and As_2O_3 was confirmed then(752nM and 15μM). Bortezomib combined with IC_(50) concentration of HT or As_2O_3 treating, the proliferation and apoptosis changes of HL60/ADM leukemia cells and primary cells which come from 9 patients were detected by the same methods above. The differences of effect between groups were done by statistics anylysis.
Results
1. The multidrug resistance of HL60/ADM leukemia cells
From the ADM toxicity experiment which was detected by MTT assay, we found that the IC_(50) of ADM to HL60/ADM is 16.283μg/ml, while the IC_(50) of ADM to HL60 is 0.087μg/ml; HL60/ADM is highly multidrug resistance (187 times).
2. Effect of bortezomib alone on the proliferation and apoptosis of HL60/ADM cells
Dates from trypan blue dye exclusion method reveal that there is no significant difference between 0-50nM bortezomib treating for 12 hours(P>0.05). after 24 h, the number of cells in different concentration has gradually decreased in a dose- and time- manner. Dates from MTT assays show that In 10-50nM bortezomib treated cells, inhibition rates enhanced in a time- and dose-dependently manner , 40nM bortezomib can best inhibit the proliferation activity of HL60/ADM cells when incubated for 48 hours, with the IC50 dose 20nM. When we raised the dose to 50nM or prolonged the incubated time to 60 h, the inhibition rates had no significant enhancement (P=0.478, P=0.161); Normal cells and apoptotic cells could be distinguished under the fluorescence microscope after staining with Hochest 33342. Normal nuclei kept intact and stained evenly. Apoptotic cells show strong chromatin condensation and nuclear fragmentation. apoptosis cells shows in 10nM,20nM or 50nM, and increased in dose maner.
After detecting the apoptosis of HL60/ADM cells with flow cytometry, there were apoptotic cells in 10nM or 20nM bortezomib treating groups.10nM bortezomib treating for 24h provide a apoptosis rate 9.58%,while 20nM treating group with 16.45% apoptosis rate which is significant higher then 10nM group. Prolong the treating time to 48h, the apoptosis rate in 10nM or 20nM group increased to 17.22% and 73.46%, respectively, which were significant higher then those in the same concentration of 24h.
3. Effect of bortezomib in combination with harringtonine or arsenic trioxide on the proliferation and apoptosis of HL60/ADM cells
As_2O_3 alone can also inhibit the proliferation of HL60/ADM. When cultured for 48 hours, 2、4、10、20、30、40μM As_2O_3 brought inhibition rates of 25%、33%、40% > 58% , 64% , 69%, respectively, with the IC50 dose as 15μM. When cultured for 48 hours, 94、188、376、564、752 nM HT brought inhibition rates of 9%、20%、31 %、39%、52%, respectively, with the IC_(50) dose as 752nM. 15μM As_2O_3 or 752nM HT which is combined with 10-50nM of bortezomib can inhibit the proliferation of HL60/ADM, and the dual inhibition of bortezomib plus As_2O_3 is significant higher then that of either above. While there is no different between bortezomib plus HT group and HT treating group. fluorescence microscope show that The apoptotic cells in both compounds combined groups are more than those in the single-agent groups. flow cytometry show that there were apoptotic cells in all groups. After they were cultured for 24 or 48 hours, the apoptotic rates of the agents combined groups were higher than the single-agent groups. After the cells had been cultured for 48 hours, the apoptotic rates of the bortezomib plus As_2O_3 , bortezomib plus HT group and the bortezomib group were 81.89% , 80.64% and 17.22%(P<0.01), respectively. And mostly, apoptosis in the combined group present in a late-stage. The intraceflular accumulation of ADM of HL60/ADM cells in the botezomib group is stronger than the bortezomib-free group(P<0.05 ).10nM Bortezomib could significantly enhance the intracellular accumulation of ADM in HL60/ADM cells which indicated that bortezomib might have the ability to reverse multidrug resistant.
4. Effect of brotezomib in combination with harringtonine or arsenic trioxide on the proliferation of primary leukemia cells
10-200nM bortezomib can inhibit proliferation of primary cells, inhibition rates increased in a dose-dependently manner; 10nM、20nM、40nM、80nM、100nM、200nM bortezomib treating for 48h provide inhibition rates as 8.7%、18.1%、26.8 %、39.2%、48.06%、51.44%, respectively. 100nM bortezomib could best inhibit the proliferation activity. Higher doses made no significant increase of the inhibition rate. 20nM bortezomib combined with 752nM HT treating for 48h, make no difference with either of above treating alone(P>0.05). while the inhibiton rate of 20nM bortezomib combined with 15μM As_2O_3 is significantly higher then those of either treating alone.(P<0.05)
Conclusiones
1. Bortezomib has a dose- and time- dependent antiproliferation and proapoptotic effect on H60/ADM leukemia cells in vitro. With prolongation of time and increase of concentration of bortezomib, the effect has gradually increased.
2. HT and As_2O_3 also have a dose dependent antiproliferation and proapoptic effects on HL60/ADM cells, while combined with bortezomib, the effect is obviously improved when compared with bortezomib used alone.
3. Bortezomib could significantly enhance the intracellular accumulation of ADM in HL60/ADM cells which indicated that bortezomib might have the ability to reverse multidrug resistant.
4. Bortezomib、HT and As_2O_3 have a dose-dependent antiproliferation effect on relapsed/refractory AML primary cells in vitro. And the effect was obviously inferior to HL60/ADM leukemia cells.
5. Bortezomib in combination with HT or As_2O_3 has antiproliferation on relapsed/refractory AML primary cells in vitro. Compared with the effect of which drugs used alone, the effect of bortezomib with HT group has no improved, while the effect of bortezomib in combination As_2O_3 group is superior to the effects of either group which drugs used alone.
引文
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6.孙启鑫:孟儿义:扶云碧:李利.硼替佐米单用或联合三尖杉酯碱体外诱导HL-60细胞凋亡实验研究.中国实验血液学杂志,2007:15(2):233-236.
7.孙启鑫:孟凡义:扶云碧:李利:田帅.硼替佐米或联合三氧化二砷诱导HL-60细胞凋亡实验研究.南方医科大学学报,2007:27(7):1022-1025.
8.扶云碧:孙启鑫:孟凡义:谢军:周光飚.蛋白酶体抑制剂硼替佐米诱导髓系白血病细胞株HL60凋亡的机制研究.中华医学杂志,2006,Vol 86,No.34:(2413-2416).
9.李利:孟凡义:扶云碧:蔡艳霞:孙启鑫.硼替佐米或联合三氧化二砷治疗HL-60白血病细胞移植瘤裸鼠的实验研究.南方医科大学学报2007;27(10):1504-1506.
10.马军.白血病的治疗进展.癌症进展杂志2005;3(2):94-97.
11.Rudiger Hehlmann,Ute Berger,Andreas Hochhaus.Chronic myeloid leukemia:a model for oncology.Ann Hematol.2005,84:487-497.
12.Adams J.Development of the p roteasome inhibitor PS-341[J].Oncologist,2002,7(1):9-16.
13. Hideshima T, Richardson P, Chauhan D, et al. The proteasome inhibitor PS-341 inhibits growth, induces apoptosis, and over comes drug resistance in human multiple myeloma cells [ J ]. Cancer Res, 2001, 61 (7) : 3071 - 3076.
14. An J, Sun Y, FisherM, et al. Maximal apoptosis of renal cell carcinoma by the p roteasome inhibitor bortezomib is nuclear factor-κB dependent [ J ]. M ol Cancer Ther, 2004, 3(6) : 727 -736.
15. Bentires2AljM, Barbu V , FilletM, et al. NF-κB transcription factor induces drug resistance through MDRl expression in cancer cells[ J ]. Oncogene, 2003, 22 (1) : 90 - 97.
16. Fekete MR, McBride WH, Pajonk F. Anthracyclines, p roteasome activity and multi-drug resistance[ J ]. BMC Cancer, 2005,5: 114.
17. Stapnes C, Doskeland AP, Hatfield K. The proteasome inhibitors bortezomib and PR-171 have antiproliferative and proapoptotic effects on primary human acute myeloid leukemia cells. Br J Haematol. 2007, 136(6):814-828.
18. Cortes J, Thomas D, Roller C, et al. Phase I study of bortezomib in refractory or relapsed acute leukemias. Clin Cancer Res, 2004 10:3371-3376.
19. Lightcap ES, McCormack TA, Pien CS, et al. Proteasome inhibition measurements: clinical application. Clin Chem 2000;46:673 - 83.
20. Tallman MS. New Strategies for the Treatment of Acute Myeloid Leukemia Including Antibodies and Other Novel Agents. Hematology (Am Soc Hematol Educ Program), 2005, 143-150.
21. Adams J, Palombella VJ, Sausville EA, et al. Proteasome inhibitors: a novel class of potent and effective antitumor agents.Cancer Res 1999,59:2615-2622.
22.Adams J:The development of proteasome inhibitors as anticancer drugs.Cancer Cell 2004,5:417-421.
23.Shah SA,Potter MW,McDade TP,et al:26S Proteasome inhibition induces apoptosis and limits growth of human pancreatic cancer.J Cell Biochem 82:110-122,2001.
24.Richardson PG,Hideshima T,Anderson KC:Bortezomib(PS-341):A novel,first-inclass proteasome inhibitor for the treatment of multiple myeloma and other cancers.Cancer Control 10:361-369,2003.
25.Germann UA,Ford PJ,Shlyakhter D,et al.Chemo sensitization and drug accumulation effects of VS-710,verapanmil,cyclosporine A,MS-209 and GF120918 in multidrug resistant HL60/ADM cells expressing the multidrug resistance-associated protein MRP.Anticancer Drugs,1997,8:141-151.
26.李素霞:乔振华。人类白血病多药耐药细胞株K562/ADM、HL60/ADM的耐药性及耐药逆转的研究.白血病.淋巴瘤2003:12(2):93-96.
27.Shuichi Miyawai.Treatment of myelocytic leukemia:past,present,and future.Education Program.Ann.meeting,JSH/JSCH,2004,2-8
28.Kaspers GJ,Veerman AJ,Pieters R,et al.Drug combination testing in acute lymphoblastic leukemia using the MTT assay.Leuk Res,1995,19:175-181.
29.Cortes J,Thomas D,Koller C,et al.Phase I study of bortezomib in refractory or relapsed acute leukemias.Clin Cancer Res,2004,10:3371-3376.
30.Papandreou C,Daliani D,Millikan RE,et al:Phase I study of intravenous(I.V.) proteasome inhibitor PS-341 in patients(pts) with advanced malignancies(abstract 340).Proc Am Soc Clin Oncol 20:86,2001.
31.Attar EC,Learner E,Amrein PC.Phase I dose-escalating trial of bortezomib(VELCADE) in combination with idarubicin and cytosine arabinoside in patients with acute myeloid leukemia.Blood 2004:104(11):498a.
32.梅文莉,吴娇,戴好富.三尖杉属植物化学成分与药理活性研究进展.中草药,2006,37(3):452-458.
33.马军.三氧化二砷在白血病治疗中的临床应用.中国处方药,2004,31:17-20.
34.Fang J Y,Chen YX,Lu J,et al.Epigenetic modification regu-lates both expression of tumor-associated genes and cell cycle progressing in human colon cancer cell lines:Colo-320 and SW1116[J].Cell Res,2004,14(3):217-226.
35.Baker EKEl-Osta A.MDR1,chemotherapy and chromatin re-modeling[J].Cancer Biol Ther,2004,3(9):819-824.
36.Adams J,Palombella VJ,Elliott PJ.Proteasome inhibition:a new strategy in cancer treatment.Invest New Drugs 2000;18(2):109-21.
37.Leith CP,Kopecky KJ,Chen.IM,et al.Frequency and clinical significance of the expression of the multidrug resistance proteins MDR1/P2glycoprotein,MRP1,and LRP in acute myeloid leukemia.Blood,1999;94:1086-1099.
38.Wu H,Hair WN,Yang JM.Small interfering RNA-induced suppression of MDR1(P-glycoprotein) restores sensitivity to multidrugresistant cancer cells[J].Cancer Res,2003,63(7):1515-1519.
39.张之南等.《血液病学》第一版,人民卫生出版社.2003.
40.Baker EKE1-Osta A.The rise of DNA methylation and the importance of chromatin on multidrug resistance in cancer[J].Ex pCell Res,2003,290(2):177-394.
41.Orlowski RZ,Stinchcombe TE,Mitchell BS,et al.Phase Ⅰ trial of the proteasome inhibitor PS-341 in patients with refractory hematologic malignancies.J Clin Oncol 2002;20:4420-7.
42.Tallman MS.New Strategies for the Treatment of Acute Myeloid Leukemia Including Antibodies and Other Novel Agents.Hematology(Am Soc Hematol Educ Program),2005,143-150.
43.Gatto S,Scappini B,Pham L,et al.The proteasome inhibitor PS-341inhibits growth and induces apoptosis in Bcr/Abl-positive cell lines sensitive and resistant to imatinib mesylate.Haematologica,2003,88:853-863.
1.Cheson BD.Radioimmunotherapy of non-Hodgkin lymphomas[J].Blood.2003,101(2):391-398.
2.Wiseman GA,White CA,Witzig TE,et al.Radioimmunotherapy of relapsed non-Hodgkin' s lymphoma with Zevalin,a 90 Y-labeled anti-CD20monoclonal antibody.1999 Oct;5(10 Suppl):3281s-3286s.
3.Jurcic JG,Larson SM,Sgouros G,et al.Targeted alpha particle immunotherapy for myeloid leukemia.Blood.2002,100{4):1233-9.
4 Goldenberg DM,Horowitz JA,Sharkey RM,et al Targeting,dosimetry,and radioimmunotherapy of B-cell lymphomas with iodine-131-labeled LL2 monoclonal antibody.J Clin Oncol.1991 9(4):548-64.
5 Crossbard ML,Multani PS,Freedman AS,et al.A Phase Ⅱ Study of Adjuvant Therapy with Anti-B4-blocked Ricin after Autologous Bone Marrow Transplantation for Patients with Relapsed B-Cell Non-Hodgkin' s Lymphoma.Clin Cancer Res.1999,5(9):2392-8.
6.Matthews DC,Badger CC,Fisher DR,et al.Selective radiation of hematolymphoid tissue deliverd by anti-CD45 antibody.Cancer Res.1992,52(5):1228-34.
7.Matthews DC,Appelbaum FR,Eary JF,et al.Phase Ⅰ study of 131-Ⅰ-anti-CD45 antibodies plus cyclophosphamide and total body irradiation for advanced acute leukemia and myelodysplastic syndrome. Blood.1999;94(4):1237-47.
8.Eneida R.Nemecek,Donald K.et al Biodistribution of Yttrium-90-labeled Anti-CD45 Antibody in a Nonhuman Primate Model.Clinical Cancer Res.2005;11(2 Pt 1):787-94.
9.纪方 苗积生 沈茜等。~(90)Y标记抗人成骨肉瘤单抗体内生物分布.中国矫形外科杂.1991,7(3):258-260.
10.蒋长英,陈镇华,钟高仁等.人-鼠嵌合单抗~(131)Ⅰ-chLym-1/biotin治疗恶性B细胞淋巴瘤初探[J].中华核医学杂志,1997,17(4):244-245.
2.Adams J.The development of proteasome inhibitors as anticancer drugs.Cancer Cell 5:417-421,2004.
3.Hershko A.Lessons from the discovery of the ubiquitin system.Trends Biochem Sci 21:445-449,1996.
4.Voorhees PM,Dees EC,O' Neil B,Orlowski RZ:The proteasome as a target for cancer therapy.Clin Cancer Res 9:6316-6325,2003.
5.Adams J:The development of proteasome inhibitors as anticancer drugs.Cancer Cell 2004,5:417-421.
6.孙启鑫:孟儿义:扶云碧:李利.硼替佐米单用或联合三尖杉酯碱体外诱导HL-60细胞凋亡实验研究.中国实验血液学杂志,2007:15(2):233-236.
7.孙启鑫:孟凡义:扶云碧:李利:田帅.硼替佐米或联合三氧化二砷诱导HL-60细胞凋亡实验研究.南方医科大学学报,2007:27(7):1022-1025.
8.扶云碧:孙启鑫:孟凡义:谢军:周光飚.蛋白酶体抑制剂硼替佐米诱导髓系白血病细胞株HL60凋亡的机制研究.中华医学杂志,2006,Vol 86,No.34:(2413-2416).
9.李利:孟凡义:扶云碧:蔡艳霞:孙启鑫.硼替佐米或联合三氧化二砷治疗HL-60白血病细胞移植瘤裸鼠的实验研究.南方医科大学学报2007;27(10):1504-1506.
10.马军.白血病的治疗进展.癌症进展杂志2005;3(2):94-97.
11.Rudiger Hehlmann,Ute Berger,Andreas Hochhaus.Chronic myeloid leukemia:a model for oncology.Ann Hematol.2005,84:487-497.
12.Adams J.Development of the p roteasome inhibitor PS-341[J].Oncologist,2002,7(1):9-16.
13. Hideshima T, Richardson P, Chauhan D, et al. The proteasome inhibitor PS-341 inhibits growth, induces apoptosis, and over comes drug resistance in human multiple myeloma cells [ J ]. Cancer Res, 2001, 61 (7) : 3071 - 3076.
14. An J, Sun Y, FisherM, et al. Maximal apoptosis of renal cell carcinoma by the p roteasome inhibitor bortezomib is nuclear factor-κB dependent [ J ]. M ol Cancer Ther, 2004, 3(6) : 727 -736.
15. Bentires2AljM, Barbu V , FilletM, et al. NF-κB transcription factor induces drug resistance through MDRl expression in cancer cells[ J ]. Oncogene, 2003, 22 (1) : 90 - 97.
16. Fekete MR, McBride WH, Pajonk F. Anthracyclines, p roteasome activity and multi-drug resistance[ J ]. BMC Cancer, 2005,5: 114.
17. Stapnes C, Doskeland AP, Hatfield K. The proteasome inhibitors bortezomib and PR-171 have antiproliferative and proapoptotic effects on primary human acute myeloid leukemia cells. Br J Haematol. 2007, 136(6):814-828.
18. Cortes J, Thomas D, Roller C, et al. Phase I study of bortezomib in refractory or relapsed acute leukemias. Clin Cancer Res, 2004 10:3371-3376.
19. Lightcap ES, McCormack TA, Pien CS, et al. Proteasome inhibition measurements: clinical application. Clin Chem 2000;46:673 - 83.
20. Tallman MS. New Strategies for the Treatment of Acute Myeloid Leukemia Including Antibodies and Other Novel Agents. Hematology (Am Soc Hematol Educ Program), 2005, 143-150.
21. Adams J, Palombella VJ, Sausville EA, et al. Proteasome inhibitors: a novel class of potent and effective antitumor agents.Cancer Res 1999,59:2615-2622.
22.Adams J:The development of proteasome inhibitors as anticancer drugs.Cancer Cell 2004,5:417-421.
23.Shah SA,Potter MW,McDade TP,et al:26S Proteasome inhibition induces apoptosis and limits growth of human pancreatic cancer.J Cell Biochem 82:110-122,2001.
24.Richardson PG,Hideshima T,Anderson KC:Bortezomib(PS-341):A novel,first-inclass proteasome inhibitor for the treatment of multiple myeloma and other cancers.Cancer Control 10:361-369,2003.
25.Germann UA,Ford PJ,Shlyakhter D,et al.Chemo sensitization and drug accumulation effects of VS-710,verapanmil,cyclosporine A,MS-209 and GF120918 in multidrug resistant HL60/ADM cells expressing the multidrug resistance-associated protein MRP.Anticancer Drugs,1997,8:141-151.
26.李素霞:乔振华。人类白血病多药耐药细胞株K562/ADM、HL60/ADM的耐药性及耐药逆转的研究.白血病.淋巴瘤2003:12(2):93-96.
27.Shuichi Miyawai.Treatment of myelocytic leukemia:past,present,and future.Education Program.Ann.meeting,JSH/JSCH,2004,2-8
28.Kaspers GJ,Veerman AJ,Pieters R,et al.Drug combination testing in acute lymphoblastic leukemia using the MTT assay.Leuk Res,1995,19:175-181.
29.Cortes J,Thomas D,Koller C,et al.Phase I study of bortezomib in refractory or relapsed acute leukemias.Clin Cancer Res,2004,10:3371-3376.
30.Papandreou C,Daliani D,Millikan RE,et al:Phase I study of intravenous(I.V.) proteasome inhibitor PS-341 in patients(pts) with advanced malignancies(abstract 340).Proc Am Soc Clin Oncol 20:86,2001.
31.Attar EC,Learner E,Amrein PC.Phase I dose-escalating trial of bortezomib(VELCADE) in combination with idarubicin and cytosine arabinoside in patients with acute myeloid leukemia.Blood 2004:104(11):498a.
32.梅文莉,吴娇,戴好富.三尖杉属植物化学成分与药理活性研究进展.中草药,2006,37(3):452-458.
33.马军.三氧化二砷在白血病治疗中的临床应用.中国处方药,2004,31:17-20.
34.Fang J Y,Chen YX,Lu J,et al.Epigenetic modification regu-lates both expression of tumor-associated genes and cell cycle progressing in human colon cancer cell lines:Colo-320 and SW1116[J].Cell Res,2004,14(3):217-226.
35.Baker EKEl-Osta A.MDR1,chemotherapy and chromatin re-modeling[J].Cancer Biol Ther,2004,3(9):819-824.
36.Adams J,Palombella VJ,Elliott PJ.Proteasome inhibition:a new strategy in cancer treatment.Invest New Drugs 2000;18(2):109-21.
37.Leith CP,Kopecky KJ,Chen.IM,et al.Frequency and clinical significance of the expression of the multidrug resistance proteins MDR1/P2glycoprotein,MRP1,and LRP in acute myeloid leukemia.Blood,1999;94:1086-1099.
38.Wu H,Hair WN,Yang JM.Small interfering RNA-induced suppression of MDR1(P-glycoprotein) restores sensitivity to multidrugresistant cancer cells[J].Cancer Res,2003,63(7):1515-1519.
39.张之南等.《血液病学》第一版,人民卫生出版社.2003.
40.Baker EKE1-Osta A.The rise of DNA methylation and the importance of chromatin on multidrug resistance in cancer[J].Ex pCell Res,2003,290(2):177-394.
41.Orlowski RZ,Stinchcombe TE,Mitchell BS,et al.Phase Ⅰ trial of the proteasome inhibitor PS-341 in patients with refractory hematologic malignancies.J Clin Oncol 2002;20:4420-7.
42.Tallman MS.New Strategies for the Treatment of Acute Myeloid Leukemia Including Antibodies and Other Novel Agents.Hematology(Am Soc Hematol Educ Program),2005,143-150.
43.Gatto S,Scappini B,Pham L,et al.The proteasome inhibitor PS-341inhibits growth and induces apoptosis in Bcr/Abl-positive cell lines sensitive and resistant to imatinib mesylate.Haematologica,2003,88:853-863.
1.Cheson BD.Radioimmunotherapy of non-Hodgkin lymphomas[J].Blood.2003,101(2):391-398.
2.Wiseman GA,White CA,Witzig TE,et al.Radioimmunotherapy of relapsed non-Hodgkin' s lymphoma with Zevalin,a 90 Y-labeled anti-CD20monoclonal antibody.1999 Oct;5(10 Suppl):3281s-3286s.
3.Jurcic JG,Larson SM,Sgouros G,et al.Targeted alpha particle immunotherapy for myeloid leukemia.Blood.2002,100{4):1233-9.
4 Goldenberg DM,Horowitz JA,Sharkey RM,et al Targeting,dosimetry,and radioimmunotherapy of B-cell lymphomas with iodine-131-labeled LL2 monoclonal antibody.J Clin Oncol.1991 9(4):548-64.
5 Crossbard ML,Multani PS,Freedman AS,et al.A Phase Ⅱ Study of Adjuvant Therapy with Anti-B4-blocked Ricin after Autologous Bone Marrow Transplantation for Patients with Relapsed B-Cell Non-Hodgkin' s Lymphoma.Clin Cancer Res.1999,5(9):2392-8.
6.Matthews DC,Badger CC,Fisher DR,et al.Selective radiation of hematolymphoid tissue deliverd by anti-CD45 antibody.Cancer Res.1992,52(5):1228-34.
7.Matthews DC,Appelbaum FR,Eary JF,et al.Phase Ⅰ study of 131-Ⅰ-anti-CD45 antibodies plus cyclophosphamide and total body irradiation for advanced acute leukemia and myelodysplastic syndrome. Blood.1999;94(4):1237-47.
8.Eneida R.Nemecek,Donald K.et al Biodistribution of Yttrium-90-labeled Anti-CD45 Antibody in a Nonhuman Primate Model.Clinical Cancer Res.2005;11(2 Pt 1):787-94.
9.纪方 苗积生 沈茜等。~(90)Y标记抗人成骨肉瘤单抗体内生物分布.中国矫形外科杂.1991,7(3):258-260.
10.蒋长英,陈镇华,钟高仁等.人-鼠嵌合单抗~(131)Ⅰ-chLym-1/biotin治疗恶性B细胞淋巴瘤初探[J].中华核医学杂志,1997,17(4):244-245.