沙利度胺对人胶质瘤细胞U251的体外作用及其机制的研究
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摘要
目的:通过体外实验观察沙利度胺对人神经胶质瘤细胞U251增殖及对缺氧诱导因子HIF–1、血管内皮生长因子VEGF和对钙离子Ca2+浓度的影响,进一步阐明沙利度胺抗肿瘤、抗血管生成和诱导肿瘤细胞凋亡的作用机制,同时观察沙利度胺联合顺铂对U251细胞的作用。
方法:1、选取人神经胶质瘤U251细胞系进行体外培养,采用四甲基偶氮唑蓝(MTT)法检测沙利度胺不同浓度组(10、25、50、100μg/ml)、不同时间组(24、48、72h)对U251细胞存活和生长的影响情况。流式细胞仪测定沙利度胺对U251细胞周期分布和凋亡的影响。2、根据MTT的结果,设置对照及实验组,分别提取各组细胞总RNA,鉴定其完整性及含量,通过逆转录-聚合酶链反应(revers transcription PCR,RT-PCR)半定量检测沙利度胺处理前后U251细胞中HIF–1和VEGFmRNA的表达水平。采用流式细胞术(flow cytometry,FCM)定量检测沙利度胺处理前后U251细胞周期分布和凋亡及对HIF– 1和VEGF蛋白表达的影响。3、采用MTT比色法检测沙利度胺与抗癌药物顺铂联合应用后对U251细胞的增殖抑制作用,测定其吸光度(OD)值,并采用isobolanalysis公式分析相互作用指数(Iteraction Index),评价两药联合后的作用,如相互作用指数>1为两药拮抗作用;相互作用指数=1为两药相加作用;相互作用指数<1为两药协同作用。4、利用激光共聚焦显微镜观察沙利度胺不同浓度组(10、25、50、100μg/ml)作用72h后U251细胞中Ca2+荧光强度的变化。
结果:1、MTT结果显示:沙利度胺在(10～100)μg/ml浓度范围内能明显抑制U251细胞体外增殖,随着时间、浓度增加抑制率相应升高,以100μg/ml组作用72h抑制率最高。与对照组相比,不同浓度组对U251细胞抑制率差异均具有显著性统计学意义(P<0.01)。沙利度胺处理细胞48、72h,IC50分别为51.62μg/ml、35.76μg/ml。流式细胞仪检测U251细胞周期分布和凋亡显示,10、25、50、100μg/ml沙利度胺作用U251细胞72h,随药物浓度增大, G0/G1期细胞逐渐增多; S期、G2/M期细胞则逐渐减少。即沙利度胺可阻滞U251细胞周期进程于G1期,该作用呈剂量-效应依赖关系。10、25、50、100μg/ml沙利度胺处理细胞72h后,出现典型的亚二倍体凋亡峰,依沙利度胺浓度增大而逐渐升高;凋亡百分率分别为:9.31 %、14.53%、19.36%、28.30%,较对照组有显著性统计学意义(P<0.01)。2、半定量RT-PCR检测结果显示:沙利度胺不同浓度组处理U251细胞72h后,HIF–1和VEGF mRNA都有不同程度的降低。100μg/ml沙利度胺浓度组作用72h对U251细胞HIF– 1和VEGF mRNA表达抑制最显著,比较沙利度胺处理前HIF–1和VEGFmRNA表达水平差异具有显著性统计学意义(P<0.01)。流式细胞术检测结果显示:0、10、25、50、100μg/ml沙利度胺处理U251细胞72h后U251细胞中HIF–1蛋白表达荧光指数(FI值)分别为1.719、1.645、1.329、1.202、1.081,VEGF蛋白表达荧光指数(FI值)分别为2.248、1.976、1.553、1.337、1.097。比较处理组和对照组的FI值,差异具有统计学意义(P<0.05)。以100μg/ml、72h对HIF– 1和VEGF抑制最显著,与RT-PCR结果一致。3、MTT比色法分析沙利度胺联合顺铂对U251细胞的增殖抑制作用,结果显示:沙利度胺可加强顺铂对U251细胞的增殖抑制作用。在10～100μg/ml范围内,随沙利度胺浓度的增大,其对顺铂的协同作用增强。单用顺铂的IC50为22.31μg/ml,联合10、25、50μg/ml沙利度胺后可将顺铂的IC50分别降低至9.62、4.68、2.41μg/mlμg/ml。因此,在一定浓度范围内,沙利度胺可协同顺铂抑制U251细胞的生长增殖。4、Isobolanalysis分析相互作用指数显示,沙利度胺联合顺铂在细胞生长抑制率为50%时的相互作用指数为0.86,证明沙利度胺确实可协同顺铂对U251细胞的增殖抑制作用。5、激光共聚焦显微镜检测结果示:沙利度胺在10～100μg/ml浓度范围内能明显使U251细胞内Ca2+荧光强度逐渐增强,即钙离子逐渐浓度升高,且随着沙利度胺浓度增加Ca2+浓度相应升高,以100μg/ml组作用72h后细胞中Ca2+荧光强度最高。
结论:1、本实验证实在一定浓度范围内,沙利度胺能够抑制U251胶质瘤细胞增殖,并呈浓度-时间依赖关系。2、沙利度胺可阻滞胶质瘤细胞U251于G1期并可诱导该细胞凋亡,诱导细胞凋亡是其抗肿瘤作用机制之一。3、一定浓度的沙利度胺能够在mRNA和蛋白水平抑制U251细胞中HIF– 1和VEGF表达。4、沙利度胺可以使U251细胞内Ca2+浓度升高,证明其具有诱导肿瘤细胞凋亡的作用。5、沙利度胺可协同顺铂对U251细胞的增殖抑制作用。6、本实验证实了沙利度胺具有抗胶质瘤的作用,并通过沙利度胺联合顺铂的研究丰富了胶质瘤的治疗,为胶质瘤的综合治疗提供了新的理论依据。
Objective: This experiment aimed to evaluate the effect of thalidomide on proliferation and the expression of HIF-1 (hyp- oxia inducible factor-1 )、VEGF(vascular endothelial grow- th factor) and the concertration of calcium in glioma cell line in vitro, to investigate possible mechanism of its role as angiogenesis inhibitor and activity of anti-tumor, meanwhile, to investigate the effects of thalidomide combined with anti-cancer agents DDP on human glioma cell line U251 in vitro.
Methods:1 Human glioma cell line U251 was incubated in culture medium in vitro, using MTT assay to detect the growth rate among different thalidomide concentration groups (10、25、50、100μg/ml) and different time groups (24、48、72h). Apoptosis and distribution of cell cycle were examined with flow cytometry. 2 According to the result of MTT, establishing control and experimental group, extracting total RNA of each group, assessing the integrality and content of RNA, the level of HIF-1、VEGFmRNA expression was examined by semiquanti- tative RT-PCR technique in the U251 cells treated before and after with thalidomide. The expression of HIF-1、VEGF protein was semi-quantitately examined by flow cytometry in U251cells treated before and after with thalidomide.3 MTT colorimetric method was performed to evaluate the potential cytostatic effect of combining thalidomide with DDP on human glioma cell line U251 and the OD values were computed. To determine whether the combination of thalidomide with DDP results in a synergistic cytostatic effect, Isobolanalysis formula was performed. In the analysis, there is synergy when the interaction index is less than 1; additivity when the interaction index is equal to 1; and antagonism when the interaction index is more than 1. 4 Confocal laser scanning microscope(LSM)was performed to evaluate the calcium concentration change by different thalidomide concentration groups (10、25、50、100μg/ml) 72h.
Results: 1 MTT assay results:Within the concentration of (10~100)μg/ml, thalidomide can obviously inhibit proliferation of U251 in vitro. The inhibition ratio was stepping up as the concentration increasing, and the highest inhibition ratio was in the group with the concentration of 100μg/ml and 72h. There was significantly statistical significance of inhibition ratio in different concentration groups of U251 cells after being treated thalidomide (P<0.01). Treatment with thalidomide for 48h、72h, the IC50 was 51.62μg/ml、35.76μg/ml,separately. When cells were harvested for the analysis on distribution of cell cycle and apoptosis by flow cytometry, the results were: After treatment with10、25、50、100μg/ml thalidomide for 72h, the number of cells in G0/G1 phase increased gradually, while the number of cells in S phase and G2/M phase decreased grudually. That was, thalidomide could induce an arrest of cell cycle in G1 phase by a dose-dependent manner. In addition, after being treated with 10、25、50、100μg/ml thalidomide for 72h, the typical apoptotic peak which enhanced gradually with the increasing concentration of thalidomide could be observed ,and the apoptotic percentage was 9.31 %、14.53%、19.36%、28.30%.seperately,and there was statistically significant difference between control group and every treatment group(P<0.01). 2、RT-PCR detection results:There was different depression of the HIF-1、VEGF mRNA expression in different concentration groups of U251 cells after being treated thalidomide. Thalidomide obviously repressed the HIF-1、VEGF mRNA expression in U251 at 100μg/ml after treating 72h, and there was significantly statistical significance between before and after treatment (P<0.01). FCM assay results:The expression of HIF-1、VEGF protein had been examined in U251 cells being treated with 0、10、25、50、100μg/ml thalidomide for 72h, the FI-value of HIF-1 in U251 was 1.719、1.645、1.329、1.202、1.081,the FI-value of VEGF in U251 was 2.248、1.976、1.553、1.337、1.097.respectively. The difference was statistically significant (P<0.05). The expression of HIF-1、VEGF protein was significantly inhibited after treament with thalidomide of 100μg/ml、72h. There was consistent with the RT-PCR results. 3 MTT colorimetric method was performed to evaluate the potential cytostatic effect of combining thalidomide with anti-cancer agents cisplatin in U251 cells. The results showed: thalidomide excerted a synergistic cytostatic effect in U251cells when combined with cisplatin. In the range of concentration from 10μg/ml to 100μg/ml, thalidomide had the enhancing synergistic effect combined with cisplatin with the increasing concentration. The IC50 of cisplatin in U251 cells alone was22.31μg/ml,When combined with 10、25、50μg/ml thalidomide, the IC50 decreased to9.62、4.68、2.41μg/ml; Thus, there was synergistic interaction between thalidomide and cisplatin in U251 cells. 4 The analysis of isobolanalysis showed that the interaction index for thalidomide combined with cisplatin used in U251 cells was 0.86 as the inhibition ratio was 50% respectively. It proved that thalidomide indeed had a synergistic effect on inhibiting the proliferation of U251 cells combined with cisplatin. 5、The fluorescence intensity of calcium was increased with the concentration of thalidomide by confocal laser scanning microscope. The fluorescence intensity of calcium was stepping up as the concentration increasing, and the highest intensity was in the group with each concentration and 72h.
Conclusions: 1、We comfirmed that thalidomide had the capability of inhibiting the proliferation of U251 cells in vitro by a dose and time-dependent manner. 2、We confirmed that thalidomide induced an arrest of cell cycle in G1 phase as well as apoptosis in U251 cells, expressing the inducement apoptotic is one of the mechanisms of its anti-tumor. 3、Within a certain drug concentration, thalidomide can inhibit the mRNA and protein expression of HIF–1 and VEGF in U 251 cells. 4、Thalidomide can hoist the calcium concertration of the U251 cell.It was proved that thalidomide has anti-angiogenesis and directly anti-tumor dual effects. 5、The thalidomide combined with cisplatin had a synergistic effect on inhibiting the proliferation of U251 cells.6、This research proved that thalidomide has the effect of anti- glioma and inriched the treatment of glioma through combining thalidomide with cisplatin.This provide a new theoretic way for glioma integrational therapy.
方法:1、选取人神经胶质瘤U251细胞系进行体外培养,采用四甲基偶氮唑蓝(MTT)法检测沙利度胺不同浓度组(10、25、50、100μg/ml)、不同时间组(24、48、72h)对U251细胞存活和生长的影响情况。流式细胞仪测定沙利度胺对U251细胞周期分布和凋亡的影响。2、根据MTT的结果,设置对照及实验组,分别提取各组细胞总RNA,鉴定其完整性及含量,通过逆转录-聚合酶链反应(revers transcription PCR,RT-PCR)半定量检测沙利度胺处理前后U251细胞中HIF–1和VEGFmRNA的表达水平。采用流式细胞术(flow cytometry,FCM)定量检测沙利度胺处理前后U251细胞周期分布和凋亡及对HIF– 1和VEGF蛋白表达的影响。3、采用MTT比色法检测沙利度胺与抗癌药物顺铂联合应用后对U251细胞的增殖抑制作用,测定其吸光度(OD)值,并采用isobolanalysis公式分析相互作用指数(Iteraction Index),评价两药联合后的作用,如相互作用指数>1为两药拮抗作用;相互作用指数=1为两药相加作用;相互作用指数<1为两药协同作用。4、利用激光共聚焦显微镜观察沙利度胺不同浓度组(10、25、50、100μg/ml)作用72h后U251细胞中Ca2+荧光强度的变化。
结果:1、MTT结果显示:沙利度胺在(10～100)μg/ml浓度范围内能明显抑制U251细胞体外增殖,随着时间、浓度增加抑制率相应升高,以100μg/ml组作用72h抑制率最高。与对照组相比,不同浓度组对U251细胞抑制率差异均具有显著性统计学意义(P<0.01)。沙利度胺处理细胞48、72h,IC50分别为51.62μg/ml、35.76μg/ml。流式细胞仪检测U251细胞周期分布和凋亡显示,10、25、50、100μg/ml沙利度胺作用U251细胞72h,随药物浓度增大, G0/G1期细胞逐渐增多; S期、G2/M期细胞则逐渐减少。即沙利度胺可阻滞U251细胞周期进程于G1期,该作用呈剂量-效应依赖关系。10、25、50、100μg/ml沙利度胺处理细胞72h后,出现典型的亚二倍体凋亡峰,依沙利度胺浓度增大而逐渐升高;凋亡百分率分别为:9.31 %、14.53%、19.36%、28.30%,较对照组有显著性统计学意义(P<0.01)。2、半定量RT-PCR检测结果显示:沙利度胺不同浓度组处理U251细胞72h后,HIF–1和VEGF mRNA都有不同程度的降低。100μg/ml沙利度胺浓度组作用72h对U251细胞HIF– 1和VEGF mRNA表达抑制最显著,比较沙利度胺处理前HIF–1和VEGFmRNA表达水平差异具有显著性统计学意义(P<0.01)。流式细胞术检测结果显示:0、10、25、50、100μg/ml沙利度胺处理U251细胞72h后U251细胞中HIF–1蛋白表达荧光指数(FI值)分别为1.719、1.645、1.329、1.202、1.081,VEGF蛋白表达荧光指数(FI值)分别为2.248、1.976、1.553、1.337、1.097。比较处理组和对照组的FI值,差异具有统计学意义(P<0.05)。以100μg/ml、72h对HIF– 1和VEGF抑制最显著,与RT-PCR结果一致。3、MTT比色法分析沙利度胺联合顺铂对U251细胞的增殖抑制作用,结果显示:沙利度胺可加强顺铂对U251细胞的增殖抑制作用。在10～100μg/ml范围内,随沙利度胺浓度的增大,其对顺铂的协同作用增强。单用顺铂的IC50为22.31μg/ml,联合10、25、50μg/ml沙利度胺后可将顺铂的IC50分别降低至9.62、4.68、2.41μg/mlμg/ml。因此,在一定浓度范围内,沙利度胺可协同顺铂抑制U251细胞的生长增殖。4、Isobolanalysis分析相互作用指数显示,沙利度胺联合顺铂在细胞生长抑制率为50%时的相互作用指数为0.86,证明沙利度胺确实可协同顺铂对U251细胞的增殖抑制作用。5、激光共聚焦显微镜检测结果示:沙利度胺在10～100μg/ml浓度范围内能明显使U251细胞内Ca2+荧光强度逐渐增强,即钙离子逐渐浓度升高,且随着沙利度胺浓度增加Ca2+浓度相应升高,以100μg/ml组作用72h后细胞中Ca2+荧光强度最高。
结论:1、本实验证实在一定浓度范围内,沙利度胺能够抑制U251胶质瘤细胞增殖,并呈浓度-时间依赖关系。2、沙利度胺可阻滞胶质瘤细胞U251于G1期并可诱导该细胞凋亡,诱导细胞凋亡是其抗肿瘤作用机制之一。3、一定浓度的沙利度胺能够在mRNA和蛋白水平抑制U251细胞中HIF– 1和VEGF表达。4、沙利度胺可以使U251细胞内Ca2+浓度升高,证明其具有诱导肿瘤细胞凋亡的作用。5、沙利度胺可协同顺铂对U251细胞的增殖抑制作用。6、本实验证实了沙利度胺具有抗胶质瘤的作用,并通过沙利度胺联合顺铂的研究丰富了胶质瘤的治疗,为胶质瘤的综合治疗提供了新的理论依据。
Objective: This experiment aimed to evaluate the effect of thalidomide on proliferation and the expression of HIF-1 (hyp- oxia inducible factor-1 )、VEGF(vascular endothelial grow- th factor) and the concertration of calcium in glioma cell line in vitro, to investigate possible mechanism of its role as angiogenesis inhibitor and activity of anti-tumor, meanwhile, to investigate the effects of thalidomide combined with anti-cancer agents DDP on human glioma cell line U251 in vitro.
Methods:1 Human glioma cell line U251 was incubated in culture medium in vitro, using MTT assay to detect the growth rate among different thalidomide concentration groups (10、25、50、100μg/ml) and different time groups (24、48、72h). Apoptosis and distribution of cell cycle were examined with flow cytometry. 2 According to the result of MTT, establishing control and experimental group, extracting total RNA of each group, assessing the integrality and content of RNA, the level of HIF-1、VEGFmRNA expression was examined by semiquanti- tative RT-PCR technique in the U251 cells treated before and after with thalidomide. The expression of HIF-1、VEGF protein was semi-quantitately examined by flow cytometry in U251cells treated before and after with thalidomide.3 MTT colorimetric method was performed to evaluate the potential cytostatic effect of combining thalidomide with DDP on human glioma cell line U251 and the OD values were computed. To determine whether the combination of thalidomide with DDP results in a synergistic cytostatic effect, Isobolanalysis formula was performed. In the analysis, there is synergy when the interaction index is less than 1; additivity when the interaction index is equal to 1; and antagonism when the interaction index is more than 1. 4 Confocal laser scanning microscope(LSM)was performed to evaluate the calcium concentration change by different thalidomide concentration groups (10、25、50、100μg/ml) 72h.
Results: 1 MTT assay results:Within the concentration of (10~100)μg/ml, thalidomide can obviously inhibit proliferation of U251 in vitro. The inhibition ratio was stepping up as the concentration increasing, and the highest inhibition ratio was in the group with the concentration of 100μg/ml and 72h. There was significantly statistical significance of inhibition ratio in different concentration groups of U251 cells after being treated thalidomide (P<0.01). Treatment with thalidomide for 48h、72h, the IC50 was 51.62μg/ml、35.76μg/ml,separately. When cells were harvested for the analysis on distribution of cell cycle and apoptosis by flow cytometry, the results were: After treatment with10、25、50、100μg/ml thalidomide for 72h, the number of cells in G0/G1 phase increased gradually, while the number of cells in S phase and G2/M phase decreased grudually. That was, thalidomide could induce an arrest of cell cycle in G1 phase by a dose-dependent manner. In addition, after being treated with 10、25、50、100μg/ml thalidomide for 72h, the typical apoptotic peak which enhanced gradually with the increasing concentration of thalidomide could be observed ,and the apoptotic percentage was 9.31 %、14.53%、19.36%、28.30%.seperately,and there was statistically significant difference between control group and every treatment group(P<0.01). 2、RT-PCR detection results:There was different depression of the HIF-1、VEGF mRNA expression in different concentration groups of U251 cells after being treated thalidomide. Thalidomide obviously repressed the HIF-1、VEGF mRNA expression in U251 at 100μg/ml after treating 72h, and there was significantly statistical significance between before and after treatment (P<0.01). FCM assay results:The expression of HIF-1、VEGF protein had been examined in U251 cells being treated with 0、10、25、50、100μg/ml thalidomide for 72h, the FI-value of HIF-1 in U251 was 1.719、1.645、1.329、1.202、1.081,the FI-value of VEGF in U251 was 2.248、1.976、1.553、1.337、1.097.respectively. The difference was statistically significant (P<0.05). The expression of HIF-1、VEGF protein was significantly inhibited after treament with thalidomide of 100μg/ml、72h. There was consistent with the RT-PCR results. 3 MTT colorimetric method was performed to evaluate the potential cytostatic effect of combining thalidomide with anti-cancer agents cisplatin in U251 cells. The results showed: thalidomide excerted a synergistic cytostatic effect in U251cells when combined with cisplatin. In the range of concentration from 10μg/ml to 100μg/ml, thalidomide had the enhancing synergistic effect combined with cisplatin with the increasing concentration. The IC50 of cisplatin in U251 cells alone was22.31μg/ml,When combined with 10、25、50μg/ml thalidomide, the IC50 decreased to9.62、4.68、2.41μg/ml; Thus, there was synergistic interaction between thalidomide and cisplatin in U251 cells. 4 The analysis of isobolanalysis showed that the interaction index for thalidomide combined with cisplatin used in U251 cells was 0.86 as the inhibition ratio was 50% respectively. It proved that thalidomide indeed had a synergistic effect on inhibiting the proliferation of U251 cells combined with cisplatin. 5、The fluorescence intensity of calcium was increased with the concentration of thalidomide by confocal laser scanning microscope. The fluorescence intensity of calcium was stepping up as the concentration increasing, and the highest intensity was in the group with each concentration and 72h.
Conclusions: 1、We comfirmed that thalidomide had the capability of inhibiting the proliferation of U251 cells in vitro by a dose and time-dependent manner. 2、We confirmed that thalidomide induced an arrest of cell cycle in G1 phase as well as apoptosis in U251 cells, expressing the inducement apoptotic is one of the mechanisms of its anti-tumor. 3、Within a certain drug concentration, thalidomide can inhibit the mRNA and protein expression of HIF–1 and VEGF in U 251 cells. 4、Thalidomide can hoist the calcium concertration of the U251 cell.It was proved that thalidomide has anti-angiogenesis and directly anti-tumor dual effects. 5、The thalidomide combined with cisplatin had a synergistic effect on inhibiting the proliferation of U251 cells.6、This research proved that thalidomide has the effect of anti- glioma and inriched the treatment of glioma through combining thalidomide with cisplatin.This provide a new theoretic way for glioma integrational therapy.
引文
1 马廉廷.脑胶质瘤治疗的现状与进展[J] 中华实验外科杂 志,1999 , (16) :388～389
2 李维方. 胶质瘤抗肿瘤血管生成治疗新对策和新途径[ J ]. 国外医学:神经病学神经外科分册, 2000, 27: 200
3 Kenyon B, Browne F, D′Amato R. Effects of thalidomide and relatedmetabolites in a mouse corneal model of neovascu -larization [ J ]. ExpEye Res, 1997, 64: 971 - 978
4 季艳霞, 姜达, 康振桥. 沙利度胺对人乳腺癌细胞增殖及血管内皮生长因子表达的影响 [J]. 肿瘤学杂志, 2005, 11 (1):26~28
5 孔雁,姜达.沙利度胺对人乳腺癌细胞血管内皮生长因子-C 及 其 受 体 表 达 的 影 响 [J]. 肿 瘤 防 治 研 究 , 2007,34(12)921-923
6 姜文华,姜达. 沙利度胺对人肾癌细胞增殖及碱性成纤维胞生长因子表达的影响[J].河北医科大学硕士研究生毕业论文, 2007, 20042341
7 Folkman J. Tumor angiogenesis:therapeutic implications [ J]. N Engl J Med, 1971,285:1182- 1186
8 Millauer B ,Susanne WV ,Haralds ,et al.High affinity VEGF binding and development expression suggest Flk - 1 as a major regulator of vas- culogenesis and angiogenesis Cell ,1993 ; 72 :8835
9 Yuan F ,Chen Y,Dellian M ,et al. Timedependent vascular regression and permeability changes in established humantumor xenografts induced by an antivascular endothelial growth factor/ vascular permeability factor antibody. ProcNatl Acad Sci USA , 1996 ; 93 ( 25 ) :14765
10 Kim KJ ,Li B ,Winer J ,et al. Inhibition of Vascular endothelial growth factor induced angiogenesis suppresses tumor growth in vivo. Nature ,1993 ;362 :841
11 Brekken RA,Thorpe PE.VEGF-VEGF receptor complexes as markers of tumor vascular endothelium[J].J Control Release,2001,74,(1-3):173-181
12 Cooke SP, Boxer GM,Lawrence L,et al A strategy for antitumor vascular therapy by targeting the vascular endothelial growth fator:receptor complex[J].Cancer Res, 2001, 61(9) :3653-3659
13 Buchler P ,Reber HA , Buchler MW, et al . Antiangiogenicactiveity ofgenistein in pancreatic carcinoma cells is mediated by te inhibition of hypoxia - inducible factor - 1 and the down regulateon of VEGF gene expression [J ] . Cancer , 2004 ;100(1) :201～210
14 Semenza GL.HIF-1 and human disease: one highly involved factor.Genes Dec,2000,14:1983-1991
15 Zhong H ,Chiles K.Modulation of hypoxia - inducible factor 1alpha expression by the epidermal growth factor/ phosphatidylinositol 3 - kinase/PTEN/ AKT/ FRAP pathway in human prostate cancer cell s:implicationsfor tumor angiogene sis and therapeutics. Cancer Res ,2000 ,60 (6) : 1541-5
16 Zagzag D, Amirnovin R, Greco MA, et al. Vascular apoptosis and involution in glioma growth and angiogenesis[J]. Lab Invest, 2000,80: 837-849
17 张锐,常义,等. 脑胶质瘤细胞中HIF-1α表达及其与细胞凋亡、增殖关系 [J]. 临床神经外科杂志, 2005, 12(4):153-156
18 D'Amato RJ, Loughnan MS, Flynn E, et al. Thalidomide is an inhibitor of angiogenesis[J]. Proc Natl Acad Sci U S A, 1994, 91(9): 4082~4085
19 Moore TM, Chetham PM, Kelly JJ , et al . Signal transduction andregulation of lung endothelial cell permeability. Interaction betweencalcium and camp[J ] . AmJ Physiol Lung Cell Mol Physiol , 1998 , 275(2 Pt I) : L2032222
20 Smeland E , Bremnes RM, Fuskevag OM, et al . The effect of calcium channel blockers and calcium on methotrexate accumulation in rat hepatocytes[J ] . Anticancer Res , 1995 , 15 (4) : 1221-1225
21 马恩龙,李艳春.沙利度胺与5- 氟尿嘧啶联合应用抗肿瘤作用[J].中国医院药学杂志,2003,23(10):611.
22 丁伟荣, 林茂芳. 沙立度胺对ECV304细胞增生的抑制作用研究[J].实用肿瘤杂志2005 ,20 (1):36-39
23 Hideshima T, Chauhan D , Shima Y, et al. Thalidomide and its analogs overcome drug resistance of human multiple mueloma cells to conventional therapy. Blood , 2000 , 96 : 2943 -2950
24 翟 羽,吕占军. 反应停对小鼠肝癌细胞H22移植瘤生长的影响,癌症, 2003, 22 (12) : 1301 - 6
25 Costa PT, L ealRMLV , MoraisLO, et al. Thalidom ide and pentoxifylline prevent the development of chronic cardiomyopathy provoked by doxorubicin in rats w ithout inhibiting its antitumoral effect [ J ].Blood, 1998, 92 (Supp l 1, part 2) : 235b
26 Amato RJ. Thalidomide therapy for renal cell carcinoma, Critical Reviews in Oncology/Hematology, 2003, 46: S59 - /S65
1 Semenza GL, Wang GL. A nuclear factor induced by hypoxia viadenovoprotein synt hesisbin- ds to t he human eryt hropoietin gene enhance ratasite required for transcriptional activation[J]. Mol CellBiol , 1992 , 12 (12) : 5447
2 Wenger RH. Cellular adaptation to hypoxia : O2-sensing protein hydroxylases , hypoxia-induci -ble transcription factors , andO2- regulated gene expression[J ] . FASEB J ,2002 , 16 (10): 1151
3 Heather ER ,Michelle P ,Wayne MN. Hypoxia-inducible factor-1 alpha is a positive factor in solid tumor growth[J ] . Cancer Res , 2000 , 60 :4010
4 Kruger EA , Blagosklonny MV , Dixon SC ,et al . UCN-01 , a protein kinase C inhibitor , inhi -bits endothelial , cll proliferation and angiogenic hypoxic respose 〔 J 〕 . InvasionMetastasis 1998 -99 ; 18 (4) : 209-18
5 Wenger RH. Cellular adaptation to hypoxia : O2sensing protein Hydr-oxylases , hy -poxia inducible transcription factors ,and O2-regulated gene expression[J ] . FASEB J ,2002 ,16 (10) : 1151
6 Huang LE ,Gu J ,Schau M,et al. Regulation of hypoxia –inducible Factor-1αis mediated by an oxygen-dependent domain via the ubiquitin -proteasome pathway[J ] . Proc Natl Acad Sci , 1998 , 95 : 7987
7 Maxwell PH ,Wiesener MS ,Chang GW, et al . The tumor suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent pro- teolysis[J ] . Nature , 199 -9 ,399 (6733) : 271- 275
8 Norma M,Carsten W,Patrick H ,et al. Independent function of two destruction do -mains in hypo xia-inducible factor-1αchains activated by prolyl hydroxylation [ J ] . EMBO J , 2001 , 20 (18) : 5197
9 Lars OH ,Assaf F ,Stefan F ,et al. DNA-binding site of p53[J ] .Proc Natl Acad Sc -i ,2002 , 99 (16) : 10305
10 Chen D ,Li M,Luo J , et al. Direct interaction between HIF-1alpha and Mdm2 modulate p53 function [ J ] . J Biol Chem , 2003 , 278 (16): 13595
11 Alvarez-Tejado M,Alfranca A ,Aragones J , et al. Lack of evidence for the invo -lveement of the phosphoinositide 3-kinase/ Akt pathway in the activation of hypoxi –a inducible factors by low oxygen tension[J ] . J Biol Chem ,2002 ,277 (16) :13508
12 Zhong H ,Aganni F ,Baccala AA ,et al. Increased expression of hypoxia inducible factor alpha in rat and human prostate cancer [J ] . Cancer Res , 1998 , 58 :5280
13 GradinK,Takasaki C,Fujii-Kuriyama Y,et al. The transcriptional activation function of the HIF-like factor requires phosphorylation at a conserved threonine [J ] . J Biol Chem , 2002 , 277(26) :23508
14 Lando D ,Peet DJ ,Whelan DA ,et al. Asparagine hydroxylation of the HIF trans -activation domain a hypoxic switch [J ] . Science ,2002 , 295(5556) :858
15 Freedman SJ ,Sun ZY,Poy F ,et al. Structural basis for recruitment of CBP/ p300 by hypo -xia-inducible factor-1 alpha[J ] . ProcNatl Acad Sci ,2002 ,99(8) :5367
16 Jorge L ,Ruas H ,Lorenz P ,et al. Functional analysis of hypo-xia inducible fact –or-1 mediated transactivation [ J ] . J Biol Chem ,2003 , 277(41) :38723
17 Carmeliet P, Dor Y, Herbert JM, et al. Role of H IF-1 alpha in Hypoxia mediated apoptosis, cell p roliferation and tumour angio-genesis. Nature. 1998 , 394 ( 6692 ) : 485 -490
18 Fan LF ,Diao LM,Chen DJ ,et al. Expression of HIF-1 alpha and its relationship to apoptosis and proliferation in lung cancer [J ] .Ai Zheng , 2002 ,21(3) : 254
19 Piret JP ,Mottet D ,Raes M,et al. CoCl2 ,a chemical inducer ofHypoxia-inducible factor-1 , and hypoxia reduce apoptotic cell death in hepatoma cell line HepG2[J ] . A nn NYAcad Sci ,2002 , 973 : 443
20 Ghafar MA ,Anastasiadis AG,Chen MW,et al. Acute hypoxia increases the aggr -essive characteristics and survival properties of prostate cancer cells[J].Prostat e, 2003, 54(1) :58
21 Ian Zachary. Molecules in focus vascular endothelial growth factor. The Inter- national Journal of Biochemistry & Cell Biology , 1998 , 30 : 1169-1174
22 Zhong H , De Marzo AM, Laughner E , et al . Overexpression of hypoxia induci ble factor 1 alpha in common human cancers and their metastases.Cancer Res , 1999 , 59 (22) : 5830-5835
23 Maxwell PH. Activation of the HIF pathway in cancer. Curr Opin Genet Dev , 2005,11 : 293 -299
24 Rap isarda A , Zalek J , Hollingshead M, et al. Schedule-dependent inhibition of hypoxia inducible factor-1 alpha p rotein accumulation, angiogenesis, and tumor grow -th by topotecan in U251–HRE glioblastoma xenografts. Cancer Res, 2004 , 64 ( 19 ) : 6845 -6848
2 李维方. 胶质瘤抗肿瘤血管生成治疗新对策和新途径[ J ]. 国外医学:神经病学神经外科分册, 2000, 27: 200
3 Kenyon B, Browne F, D′Amato R. Effects of thalidomide and relatedmetabolites in a mouse corneal model of neovascu -larization [ J ]. ExpEye Res, 1997, 64: 971 - 978
4 季艳霞, 姜达, 康振桥. 沙利度胺对人乳腺癌细胞增殖及血管内皮生长因子表达的影响 [J]. 肿瘤学杂志, 2005, 11 (1):26~28
5 孔雁,姜达.沙利度胺对人乳腺癌细胞血管内皮生长因子-C 及 其 受 体 表 达 的 影 响 [J]. 肿 瘤 防 治 研 究 , 2007,34(12)921-923
6 姜文华,姜达. 沙利度胺对人肾癌细胞增殖及碱性成纤维胞生长因子表达的影响[J].河北医科大学硕士研究生毕业论文, 2007, 20042341
7 Folkman J. Tumor angiogenesis:therapeutic implications [ J]. N Engl J Med, 1971,285:1182- 1186
8 Millauer B ,Susanne WV ,Haralds ,et al.High affinity VEGF binding and development expression suggest Flk - 1 as a major regulator of vas- culogenesis and angiogenesis Cell ,1993 ; 72 :8835
9 Yuan F ,Chen Y,Dellian M ,et al. Timedependent vascular regression and permeability changes in established humantumor xenografts induced by an antivascular endothelial growth factor/ vascular permeability factor antibody. ProcNatl Acad Sci USA , 1996 ; 93 ( 25 ) :14765
10 Kim KJ ,Li B ,Winer J ,et al. Inhibition of Vascular endothelial growth factor induced angiogenesis suppresses tumor growth in vivo. Nature ,1993 ;362 :841
11 Brekken RA,Thorpe PE.VEGF-VEGF receptor complexes as markers of tumor vascular endothelium[J].J Control Release,2001,74,(1-3):173-181
12 Cooke SP, Boxer GM,Lawrence L,et al A strategy for antitumor vascular therapy by targeting the vascular endothelial growth fator:receptor complex[J].Cancer Res, 2001, 61(9) :3653-3659
13 Buchler P ,Reber HA , Buchler MW, et al . Antiangiogenicactiveity ofgenistein in pancreatic carcinoma cells is mediated by te inhibition of hypoxia - inducible factor - 1 and the down regulateon of VEGF gene expression [J ] . Cancer , 2004 ;100(1) :201～210
14 Semenza GL.HIF-1 and human disease: one highly involved factor.Genes Dec,2000,14:1983-1991
15 Zhong H ,Chiles K.Modulation of hypoxia - inducible factor 1alpha expression by the epidermal growth factor/ phosphatidylinositol 3 - kinase/PTEN/ AKT/ FRAP pathway in human prostate cancer cell s:implicationsfor tumor angiogene sis and therapeutics. Cancer Res ,2000 ,60 (6) : 1541-5
16 Zagzag D, Amirnovin R, Greco MA, et al. Vascular apoptosis and involution in glioma growth and angiogenesis[J]. Lab Invest, 2000,80: 837-849
17 张锐,常义,等. 脑胶质瘤细胞中HIF-1α表达及其与细胞凋亡、增殖关系 [J]. 临床神经外科杂志, 2005, 12(4):153-156
18 D'Amato RJ, Loughnan MS, Flynn E, et al. Thalidomide is an inhibitor of angiogenesis[J]. Proc Natl Acad Sci U S A, 1994, 91(9): 4082~4085
19 Moore TM, Chetham PM, Kelly JJ , et al . Signal transduction andregulation of lung endothelial cell permeability. Interaction betweencalcium and camp[J ] . AmJ Physiol Lung Cell Mol Physiol , 1998 , 275(2 Pt I) : L2032222
20 Smeland E , Bremnes RM, Fuskevag OM, et al . The effect of calcium channel blockers and calcium on methotrexate accumulation in rat hepatocytes[J ] . Anticancer Res , 1995 , 15 (4) : 1221-1225
21 马恩龙,李艳春.沙利度胺与5- 氟尿嘧啶联合应用抗肿瘤作用[J].中国医院药学杂志,2003,23(10):611.
22 丁伟荣, 林茂芳. 沙立度胺对ECV304细胞增生的抑制作用研究[J].实用肿瘤杂志2005 ,20 (1):36-39
23 Hideshima T, Chauhan D , Shima Y, et al. Thalidomide and its analogs overcome drug resistance of human multiple mueloma cells to conventional therapy. Blood , 2000 , 96 : 2943 -2950
24 翟 羽,吕占军. 反应停对小鼠肝癌细胞H22移植瘤生长的影响,癌症, 2003, 22 (12) : 1301 - 6
25 Costa PT, L ealRMLV , MoraisLO, et al. Thalidom ide and pentoxifylline prevent the development of chronic cardiomyopathy provoked by doxorubicin in rats w ithout inhibiting its antitumoral effect [ J ].Blood, 1998, 92 (Supp l 1, part 2) : 235b
26 Amato RJ. Thalidomide therapy for renal cell carcinoma, Critical Reviews in Oncology/Hematology, 2003, 46: S59 - /S65
1 Semenza GL, Wang GL. A nuclear factor induced by hypoxia viadenovoprotein synt hesisbin- ds to t he human eryt hropoietin gene enhance ratasite required for transcriptional activation[J]. Mol CellBiol , 1992 , 12 (12) : 5447
2 Wenger RH. Cellular adaptation to hypoxia : O2-sensing protein hydroxylases , hypoxia-induci -ble transcription factors , andO2- regulated gene expression[J ] . FASEB J ,2002 , 16 (10): 1151
3 Heather ER ,Michelle P ,Wayne MN. Hypoxia-inducible factor-1 alpha is a positive factor in solid tumor growth[J ] . Cancer Res , 2000 , 60 :4010
4 Kruger EA , Blagosklonny MV , Dixon SC ,et al . UCN-01 , a protein kinase C inhibitor , inhi -bits endothelial , cll proliferation and angiogenic hypoxic respose 〔 J 〕 . InvasionMetastasis 1998 -99 ; 18 (4) : 209-18
5 Wenger RH. Cellular adaptation to hypoxia : O2sensing protein Hydr-oxylases , hy -poxia inducible transcription factors ,and O2-regulated gene expression[J ] . FASEB J ,2002 ,16 (10) : 1151
6 Huang LE ,Gu J ,Schau M,et al. Regulation of hypoxia –inducible Factor-1αis mediated by an oxygen-dependent domain via the ubiquitin -proteasome pathway[J ] . Proc Natl Acad Sci , 1998 , 95 : 7987
7 Maxwell PH ,Wiesener MS ,Chang GW, et al . The tumor suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent pro- teolysis[J ] . Nature , 199 -9 ,399 (6733) : 271- 275
8 Norma M,Carsten W,Patrick H ,et al. Independent function of two destruction do -mains in hypo xia-inducible factor-1αchains activated by prolyl hydroxylation [ J ] . EMBO J , 2001 , 20 (18) : 5197
9 Lars OH ,Assaf F ,Stefan F ,et al. DNA-binding site of p53[J ] .Proc Natl Acad Sc -i ,2002 , 99 (16) : 10305
10 Chen D ,Li M,Luo J , et al. Direct interaction between HIF-1alpha and Mdm2 modulate p53 function [ J ] . J Biol Chem , 2003 , 278 (16): 13595
11 Alvarez-Tejado M,Alfranca A ,Aragones J , et al. Lack of evidence for the invo -lveement of the phosphoinositide 3-kinase/ Akt pathway in the activation of hypoxi –a inducible factors by low oxygen tension[J ] . J Biol Chem ,2002 ,277 (16) :13508
12 Zhong H ,Aganni F ,Baccala AA ,et al. Increased expression of hypoxia inducible factor alpha in rat and human prostate cancer [J ] . Cancer Res , 1998 , 58 :5280
13 GradinK,Takasaki C,Fujii-Kuriyama Y,et al. The transcriptional activation function of the HIF-like factor requires phosphorylation at a conserved threonine [J ] . J Biol Chem , 2002 , 277(26) :23508
14 Lando D ,Peet DJ ,Whelan DA ,et al. Asparagine hydroxylation of the HIF trans -activation domain a hypoxic switch [J ] . Science ,2002 , 295(5556) :858
15 Freedman SJ ,Sun ZY,Poy F ,et al. Structural basis for recruitment of CBP/ p300 by hypo -xia-inducible factor-1 alpha[J ] . ProcNatl Acad Sci ,2002 ,99(8) :5367
16 Jorge L ,Ruas H ,Lorenz P ,et al. Functional analysis of hypo-xia inducible fact –or-1 mediated transactivation [ J ] . J Biol Chem ,2003 , 277(41) :38723
17 Carmeliet P, Dor Y, Herbert JM, et al. Role of H IF-1 alpha in Hypoxia mediated apoptosis, cell p roliferation and tumour angio-genesis. Nature. 1998 , 394 ( 6692 ) : 485 -490
18 Fan LF ,Diao LM,Chen DJ ,et al. Expression of HIF-1 alpha and its relationship to apoptosis and proliferation in lung cancer [J ] .Ai Zheng , 2002 ,21(3) : 254
19 Piret JP ,Mottet D ,Raes M,et al. CoCl2 ,a chemical inducer ofHypoxia-inducible factor-1 , and hypoxia reduce apoptotic cell death in hepatoma cell line HepG2[J ] . A nn NYAcad Sci ,2002 , 973 : 443
20 Ghafar MA ,Anastasiadis AG,Chen MW,et al. Acute hypoxia increases the aggr -essive characteristics and survival properties of prostate cancer cells[J].Prostat e, 2003, 54(1) :58
21 Ian Zachary. Molecules in focus vascular endothelial growth factor. The Inter- national Journal of Biochemistry & Cell Biology , 1998 , 30 : 1169-1174
22 Zhong H , De Marzo AM, Laughner E , et al . Overexpression of hypoxia induci ble factor 1 alpha in common human cancers and their metastases.Cancer Res , 1999 , 59 (22) : 5830-5835
23 Maxwell PH. Activation of the HIF pathway in cancer. Curr Opin Genet Dev , 2005,11 : 293 -299
24 Rap isarda A , Zalek J , Hollingshead M, et al. Schedule-dependent inhibition of hypoxia inducible factor-1 alpha p rotein accumulation, angiogenesis, and tumor grow -th by topotecan in U251–HRE glioblastoma xenografts. Cancer Res, 2004 , 64 ( 19 ) : 6845 -6848