鬼臼毒素衍生物GMZ-1体外抗肿瘤作用研究
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摘要
目的:本文研究鬼臼毒素新型衍生物GMZ-1对肿瘤细胞的体外杀伤和抑制生长作用,并对其发挥作用的机制进行初步探索。
方法:
1 MTT法检测GMZ-1的体外抗肿瘤活性
采用MTT法测定GMZ-1对多种肿瘤细胞(Hela、A549、MCF-7、HepG-2、SKOV3、BGC-823、MGC-803、KB、KBV200、K562、K562/A02)及正常细胞(FB)抑制作用的IC_(50)值。
2 GMZ-1诱导肿瘤细胞凋亡的检测
吉姆萨染色(Giemsa staining)和荧光染色(Heochst 33342 staining)观察GMZ-1 K562/A02细胞作用的形态学变化;琼脂糖凝胶电泳观察GMZ-1对KBV200细胞染色体DNA的影响;流式细胞术(FCM)测定经GMZ-1处理后K562/A02细胞凋亡率的变化。
3流式细胞术(FCM)分析GMZ-1对K562/A02细胞周期的影响
4 RT-PCR检测GMZ-1对K562/A02细胞p21、p53、Bax、Caspase-3、PCNA、CyclinA、CyclinB1、Mdr-1基因mRNA表达的影响
5 Western-blot检测GMZ-1对K562/A02细胞Caspase-3、Mdr蛋白表达的影响
6 GMZ-1对HepG-2细胞微管聚合的影响
免疫荧光(Immunofluorescence)观察经GMZ-1处理后HepG-2细胞微管形态变化;荧光半定量测定GMZ-1对HepG-2细胞微管聚合影响的抑制率及IC_(50)值。
7小鼠急性毒性试验初步考察GMZ-1体内毒性
结果:
1 GMZ-1的体外抗肿瘤活性
经MTT法测定发现GMZ-1对多种肿瘤细胞均有较强杀伤作用和抑制生长活性,IC_(50)值范围为0.066μmol/L～0.27μmol/L,明显强于同类已上市抗肿瘤药物依托泊苷(IC_(50)值范围为1.06μmol/L～14.1μmol/L),且对于同一种细胞的耐药株与非耐药株敏感性差别明显弱于依托泊苷,有较好的抗多药耐药作用。同时对人正常成纤维细胞(FB)具有较低毒性,提示GMZ-1对肿瘤细胞有很好选择性。通过绘制K562/A02细胞生长曲线,发现低浓度GMZ-1作用下细胞存活数量高于高浓度作用下的细胞存活数量,提示GMZ-1的作用具有一定的剂量依赖性。
2 GMZ-1诱导肿瘤细胞凋亡
GMZ-1能够诱导K562/A02细胞凋亡,对经过GMZ-1处理后的K562/A02细胞进行染色(所用染料为Giemsa和Hoechst 33342),并利用光学和荧光显微镜观察。光学显微镜下发现细胞核染色质发生聚集固缩,细胞核碎裂成多个染色浓密的凋亡小体;荧光显微镜下发现同正常细胞微弱蓝色荧光相比,GMZ-1处理组细胞出现强亮的蓝色荧光,细胞核呈致密浓染。这些都是细胞发生凋亡的典型形态,且高浓度GMZ-1处理后出现凋亡细胞的数量多于低浓度。提取经GMZ-1处理后KBV200细胞的总DNA,进行琼脂糖凝胶电泳,发现其泳道呈一条一条的梯子状,为细胞发生凋亡所出现的典型的“DNA梯子”(DNA ladder)。通过流式细胞仪对GMZ-1处理后的K562/A02细胞凋亡率的测定,发现在一定浓度和时间范围内,GMZ-1处理后的细胞凋亡率高于正常细胞凋亡率,且凋亡率随着浓度的增大和时间的增多而变大。
3 GMZ-1对K562/A02细胞增值周期的影响
采用流式细胞仪分析了经GMZ-1处理后K562/A02细胞的周期分布情况,结果显示,在一定浓度范围内,GMZ-1作用12h、24h、36h,细胞都发生G_2+M期阻滞。
4 GMZ-1对K562/A02细胞凋亡相关基因p21、p53、Bax、Caspase-3,增
殖细胞核抗原PCNA,细胞周期素CyclinA、CyclinB1,多药耐药基因Mdr-1等mRNA表达的影响RT-PCR分析结果显示,GMZ-1通过上调抑癌基因p21、p53、Bax mRNA的表达而发挥诱导肿瘤细胞凋亡的作用;通过上调CyclinA,下调CyclinB1、PCNA而发挥抑制肿瘤细胞分裂增殖的作用;通过下调Mdr-1基因mRNA的表达而产生抗多药耐药作用。
5 GMZ-1对K562/A02细胞凋亡执行蛋白Caspase-3和耐药蛋白Mdr表达的影响
通过Western-blot分析结果发现, GMZ-1能下调Mdr蛋白的表达,进而发挥抗多药耐药的作用。而对K562/A02细胞Caspase-3的表达影响不明显。
6 GMZ-1对HepG-2细胞微管聚合的影响
通过免疫荧光方法观察到正常HepG-2细胞微管呈致密的网状分布,荧光强度高,细胞立体感强,而经过GMZ-1处理后的微管,网状结构稀疏或消失,呈星点状分布,荧光强度明显减弱,说明HepG-2细胞微管发生了解聚。通过免疫荧光半定量法对微管聚合解聚动态平衡进行测定,结果显示GMZ-1对微管具有很强的抑制作用,其抑制作用的IC50值为0.066±0.007μmol/L,同鬼臼毒素的0.071±0.007μmol/L相差不大,同时GMZ-1各浓度对应抑制率正负值同长春新碱相似,说明GMZ-1可能和长春新碱一样发挥抑制微管聚合的作用。
7 GMZ-1急性毒性试验
昆明小鼠腹腔注射GMZ-1后观察到小鼠出现反应迟钝、蜷缩不活动、相继出现死亡现象,解剖死亡小鼠后发现大肠或小肠有阻塞,通过各组小鼠最后死亡数量计算得GMZ-1腹腔注射的LD50值为97.90±19.21mg/kg (95% confidence interval)。
结论: GMZ-1在体外对多种肿瘤细胞均具有较强的杀伤作用,其对多数肿瘤细胞的IC50值在0.10μmol/L左右,活性强于依托泊苷10-100倍。多项试验证明GMZ-1对肿瘤细胞的杀伤作用可能是通过诱导肿瘤细胞凋亡的方式实现的。GMZ-1能影响肿瘤细胞正常分裂周期,进而抑制肿瘤细胞增殖。GMZ-1发挥这些药理学作用可能与调节多种凋亡相关基因和蛋白、核增殖基因、细胞周期素、多药耐药基因和蛋白的表达有关。腹腔注射GMZ-1显示出一定的胃肠道反应,其它不良反应有待于进一步研究。总之,GMZ-1是一个新的抗肿瘤化合物,其特点为活性高、选择性好、机制明确,有一定的开发前景,有必要进行进一步试验研究,以获得更加全面的评价指标。
Objective: To study the anti-tumor effect in vitro of GMZ-1 which is a novel podophyllotoxin derivative, and the mechanism of its anti-tumor effect. Methods:
1 The anti-tumor activity in vitro of GMZ-1 was measured by MTT assay
MTT assay was used to measure the IC50 values of GMZ-1 on several tumor cell lines, including human cervical cancer cells (Hela), non-small-cell lung carcinoma cells(A549), human breast carcinoma cells (MCF-7), human liver cancer cells(HepG-2) , human ovarian carcinoma cells (SKOV3), human stomach carcinoma cells (BGC-823、MGC-803), human oral squamous carcinoma cells (KB), human oral squamous carcinoma cells resistance to VCR(KBV200), human erythroleukaemia cells (K562), human erythro- leukaemia cells resistance to ADM, and one normal human cell line, human hypertrophic scar fibroblast (FB).
2 Detection of apoptosis of tumor cells induced by GMZ-1
Giemsa staining and Heochst 33342 staining were used to observe the morphological changes of K562/A02 cells after treated with GMZ-1; agarose gel electrophoresis was used to observe the effect of GMZ-1 on DNA of KBV200 cells; the apoptotic rate of K562/A02 cells after treated with GMZ-1 was measured by flow cytometry(FCM).
3 The effect of cell cycle of K562/A02 cells after treated with GMZ-1 was analyzed by FCM.
4 The mRNA expressions of p21、p53、Bax、Caspase-3、PCNA、CyclinA、CyclinB1、Mdr-1 in K562/A02 cells after treated with GMZ-1 were semi-quantified by reverse transcription PCR (RT-PCR).
5 The protein expressions of Caspase-3 and Mdr in K562/A02 after treated with GMZ-1 were detected by Western-blot.
6 Effect of GMZ-1 on microtubule of HepG-2 cells
Immunofluorescence was used to observe the morphological changes of microtubule in HepG-2 cells after treated with GMZ-1 and the inhibition ratio of GMZ-1 on microtubule was measured by fluorescent semi-quantitation.
7 Toxicity indexs in vivo of GMZ-1 was detected initially by Acute Toxicity Tests.
Results:
1 The anti-tumor activity in vitro of GMZ-1 The results of MTT assay showed that GMZ-1 revealed a very intense cytotoxic activity towards many carcinoma cell lines, the range of IC50 values was 0.066 to 0.27μmol/L, which was much bertter than VP-16(range of IC50 values: 1.06μmol/L～14.1μmol/L). GMZ-1 was also more sensitive to multidrug resistant tumor cells than VP-16. What is more, GMZ-1 had low cytotoxic towards normal human cells like FB. According to the growth curve of K562/A02 cells, we found that GMZ-1 inhibited growth of tumor cells in a dose-dependent manner.
2 GMZ-1 induced apoptosis of tumor cells.
After treated with GMZ-1, the typical features of apoptosis such as nucleus shrinkage and splitting, chromatin condensation and deeply dyeing were observed by Giemsa and Heochst 33342 staining in K562/A02 cells. Typical DNA fragmentation pattern (DNA ladder) of apoptosis was observed by Agarose gel electrophoresis in KBV200 cells. FCM analysis indicated that the apoptotic rate of K562/A02 cells accreted consequently with the working concentration of GMZ-1 uprised.
3 Effect of GMZ-1 on cell cycle of K562/A02 cells
After treated with GMZ-1 for 12h, 24h and 36h, the cell cycles of K562/A02 cells were all arrested at G2+M phase.
4 Regulating expressions of p21, p53, Bax, Caspase-3, PCNA, CyclinA, CyclinB1, Mdr-1 in K562/A02 cells
The results of RT-PCR showed that GMZ-1 could up-regulate the mRNA expressions of p21, p53, Bax, CyclinA, and down-regulate the mRNA expressions of CyclinB1, PCNA, Mdr-1, then induced apoptosis of tumor cells, inhibited proliferations of tumor cells and reversed multidrug resistance.
5 Effect on protein expressions of Caspase-3 and Mdr in K562/A02 cells
The Western blotting experiments showed that GMZ-1 could decrease protein expression of Mdr.
6 Immunofluorescence was used to study the effect of GMZ-1 on microtubule of HepG-2 cells
The microtubule was bushy, reticulodromous, fluorescent brightly and spatial in normal HepG-2 cells, while in the cells after treated with GMZ-1 it was discrete, astroid and fluorescent darkly. Like vincristine, the effect of GMZ-1 on microtubule polymerizing was inhibition, not promoting. The IC50 value of inhibitting microtubule polymerizing was 0.066±0.007μmol/L,a little difference compared with podophyllotoxin(0.071±0.007μmol/L).
7 Toxicity indexs of GMZ-1
The reactions of peritoneal injection with GMZ-1 were slow response, scrunch, quiescence, diarrhea and blockage in intestine. The LD50 value was 97.90±19.21mg/kg (95% confidence interval).
Conclusions: GMZ-1 showed a powerful anti-tumor activity against many human tumor cell lines in vitro. The IC50 values were all about at 0.10μmol/L , 10~100 times more than that of VP-16. Inducing apoptosis might be a main mode of actions of GMZ-1, meanwhile, GMZ-1 could interfere cell cycles of tumor cells, and then inhibit proliferation of tumor cells. The mechanisms of these pharmacological effects were regulating apoptotic gene and protein expressions, regulating PCNA and cyclins, regulating expressions of multidrug resistance related gene and protein. GMZ-1 showed a little adverse reaction in intestine or stomach, other adverse effects were planed to study later. In a word, GMZ-1 was a new anti-tumor compound with high activity, high selectivity, definite mechanism. It was worth to study further more to get an overall estimated index.
方法:
1 MTT法检测GMZ-1的体外抗肿瘤活性
采用MTT法测定GMZ-1对多种肿瘤细胞(Hela、A549、MCF-7、HepG-2、SKOV3、BGC-823、MGC-803、KB、KBV200、K562、K562/A02)及正常细胞(FB)抑制作用的IC_(50)值。
2 GMZ-1诱导肿瘤细胞凋亡的检测
吉姆萨染色(Giemsa staining)和荧光染色(Heochst 33342 staining)观察GMZ-1 K562/A02细胞作用的形态学变化;琼脂糖凝胶电泳观察GMZ-1对KBV200细胞染色体DNA的影响;流式细胞术(FCM)测定经GMZ-1处理后K562/A02细胞凋亡率的变化。
3流式细胞术(FCM)分析GMZ-1对K562/A02细胞周期的影响
4 RT-PCR检测GMZ-1对K562/A02细胞p21、p53、Bax、Caspase-3、PCNA、CyclinA、CyclinB1、Mdr-1基因mRNA表达的影响
5 Western-blot检测GMZ-1对K562/A02细胞Caspase-3、Mdr蛋白表达的影响
6 GMZ-1对HepG-2细胞微管聚合的影响
免疫荧光(Immunofluorescence)观察经GMZ-1处理后HepG-2细胞微管形态变化;荧光半定量测定GMZ-1对HepG-2细胞微管聚合影响的抑制率及IC_(50)值。
7小鼠急性毒性试验初步考察GMZ-1体内毒性
结果:
1 GMZ-1的体外抗肿瘤活性
经MTT法测定发现GMZ-1对多种肿瘤细胞均有较强杀伤作用和抑制生长活性,IC_(50)值范围为0.066μmol/L～0.27μmol/L,明显强于同类已上市抗肿瘤药物依托泊苷(IC_(50)值范围为1.06μmol/L～14.1μmol/L),且对于同一种细胞的耐药株与非耐药株敏感性差别明显弱于依托泊苷,有较好的抗多药耐药作用。同时对人正常成纤维细胞(FB)具有较低毒性,提示GMZ-1对肿瘤细胞有很好选择性。通过绘制K562/A02细胞生长曲线,发现低浓度GMZ-1作用下细胞存活数量高于高浓度作用下的细胞存活数量,提示GMZ-1的作用具有一定的剂量依赖性。
2 GMZ-1诱导肿瘤细胞凋亡
GMZ-1能够诱导K562/A02细胞凋亡,对经过GMZ-1处理后的K562/A02细胞进行染色(所用染料为Giemsa和Hoechst 33342),并利用光学和荧光显微镜观察。光学显微镜下发现细胞核染色质发生聚集固缩,细胞核碎裂成多个染色浓密的凋亡小体;荧光显微镜下发现同正常细胞微弱蓝色荧光相比,GMZ-1处理组细胞出现强亮的蓝色荧光,细胞核呈致密浓染。这些都是细胞发生凋亡的典型形态,且高浓度GMZ-1处理后出现凋亡细胞的数量多于低浓度。提取经GMZ-1处理后KBV200细胞的总DNA,进行琼脂糖凝胶电泳,发现其泳道呈一条一条的梯子状,为细胞发生凋亡所出现的典型的“DNA梯子”(DNA ladder)。通过流式细胞仪对GMZ-1处理后的K562/A02细胞凋亡率的测定,发现在一定浓度和时间范围内,GMZ-1处理后的细胞凋亡率高于正常细胞凋亡率,且凋亡率随着浓度的增大和时间的增多而变大。
3 GMZ-1对K562/A02细胞增值周期的影响
采用流式细胞仪分析了经GMZ-1处理后K562/A02细胞的周期分布情况,结果显示,在一定浓度范围内,GMZ-1作用12h、24h、36h,细胞都发生G_2+M期阻滞。
4 GMZ-1对K562/A02细胞凋亡相关基因p21、p53、Bax、Caspase-3,增
殖细胞核抗原PCNA,细胞周期素CyclinA、CyclinB1,多药耐药基因Mdr-1等mRNA表达的影响RT-PCR分析结果显示,GMZ-1通过上调抑癌基因p21、p53、Bax mRNA的表达而发挥诱导肿瘤细胞凋亡的作用;通过上调CyclinA,下调CyclinB1、PCNA而发挥抑制肿瘤细胞分裂增殖的作用;通过下调Mdr-1基因mRNA的表达而产生抗多药耐药作用。
5 GMZ-1对K562/A02细胞凋亡执行蛋白Caspase-3和耐药蛋白Mdr表达的影响
通过Western-blot分析结果发现, GMZ-1能下调Mdr蛋白的表达,进而发挥抗多药耐药的作用。而对K562/A02细胞Caspase-3的表达影响不明显。
6 GMZ-1对HepG-2细胞微管聚合的影响
通过免疫荧光方法观察到正常HepG-2细胞微管呈致密的网状分布,荧光强度高,细胞立体感强,而经过GMZ-1处理后的微管,网状结构稀疏或消失,呈星点状分布,荧光强度明显减弱,说明HepG-2细胞微管发生了解聚。通过免疫荧光半定量法对微管聚合解聚动态平衡进行测定,结果显示GMZ-1对微管具有很强的抑制作用,其抑制作用的IC50值为0.066±0.007μmol/L,同鬼臼毒素的0.071±0.007μmol/L相差不大,同时GMZ-1各浓度对应抑制率正负值同长春新碱相似,说明GMZ-1可能和长春新碱一样发挥抑制微管聚合的作用。
7 GMZ-1急性毒性试验
昆明小鼠腹腔注射GMZ-1后观察到小鼠出现反应迟钝、蜷缩不活动、相继出现死亡现象,解剖死亡小鼠后发现大肠或小肠有阻塞,通过各组小鼠最后死亡数量计算得GMZ-1腹腔注射的LD50值为97.90±19.21mg/kg (95% confidence interval)。
结论: GMZ-1在体外对多种肿瘤细胞均具有较强的杀伤作用,其对多数肿瘤细胞的IC50值在0.10μmol/L左右,活性强于依托泊苷10-100倍。多项试验证明GMZ-1对肿瘤细胞的杀伤作用可能是通过诱导肿瘤细胞凋亡的方式实现的。GMZ-1能影响肿瘤细胞正常分裂周期,进而抑制肿瘤细胞增殖。GMZ-1发挥这些药理学作用可能与调节多种凋亡相关基因和蛋白、核增殖基因、细胞周期素、多药耐药基因和蛋白的表达有关。腹腔注射GMZ-1显示出一定的胃肠道反应,其它不良反应有待于进一步研究。总之,GMZ-1是一个新的抗肿瘤化合物,其特点为活性高、选择性好、机制明确,有一定的开发前景,有必要进行进一步试验研究,以获得更加全面的评价指标。
Objective: To study the anti-tumor effect in vitro of GMZ-1 which is a novel podophyllotoxin derivative, and the mechanism of its anti-tumor effect. Methods:
1 The anti-tumor activity in vitro of GMZ-1 was measured by MTT assay
MTT assay was used to measure the IC50 values of GMZ-1 on several tumor cell lines, including human cervical cancer cells (Hela), non-small-cell lung carcinoma cells(A549), human breast carcinoma cells (MCF-7), human liver cancer cells(HepG-2) , human ovarian carcinoma cells (SKOV3), human stomach carcinoma cells (BGC-823、MGC-803), human oral squamous carcinoma cells (KB), human oral squamous carcinoma cells resistance to VCR(KBV200), human erythroleukaemia cells (K562), human erythro- leukaemia cells resistance to ADM, and one normal human cell line, human hypertrophic scar fibroblast (FB).
2 Detection of apoptosis of tumor cells induced by GMZ-1
Giemsa staining and Heochst 33342 staining were used to observe the morphological changes of K562/A02 cells after treated with GMZ-1; agarose gel electrophoresis was used to observe the effect of GMZ-1 on DNA of KBV200 cells; the apoptotic rate of K562/A02 cells after treated with GMZ-1 was measured by flow cytometry(FCM).
3 The effect of cell cycle of K562/A02 cells after treated with GMZ-1 was analyzed by FCM.
4 The mRNA expressions of p21、p53、Bax、Caspase-3、PCNA、CyclinA、CyclinB1、Mdr-1 in K562/A02 cells after treated with GMZ-1 were semi-quantified by reverse transcription PCR (RT-PCR).
5 The protein expressions of Caspase-3 and Mdr in K562/A02 after treated with GMZ-1 were detected by Western-blot.
6 Effect of GMZ-1 on microtubule of HepG-2 cells
Immunofluorescence was used to observe the morphological changes of microtubule in HepG-2 cells after treated with GMZ-1 and the inhibition ratio of GMZ-1 on microtubule was measured by fluorescent semi-quantitation.
7 Toxicity indexs in vivo of GMZ-1 was detected initially by Acute Toxicity Tests.
Results:
1 The anti-tumor activity in vitro of GMZ-1 The results of MTT assay showed that GMZ-1 revealed a very intense cytotoxic activity towards many carcinoma cell lines, the range of IC50 values was 0.066 to 0.27μmol/L, which was much bertter than VP-16(range of IC50 values: 1.06μmol/L～14.1μmol/L). GMZ-1 was also more sensitive to multidrug resistant tumor cells than VP-16. What is more, GMZ-1 had low cytotoxic towards normal human cells like FB. According to the growth curve of K562/A02 cells, we found that GMZ-1 inhibited growth of tumor cells in a dose-dependent manner.
2 GMZ-1 induced apoptosis of tumor cells.
After treated with GMZ-1, the typical features of apoptosis such as nucleus shrinkage and splitting, chromatin condensation and deeply dyeing were observed by Giemsa and Heochst 33342 staining in K562/A02 cells. Typical DNA fragmentation pattern (DNA ladder) of apoptosis was observed by Agarose gel electrophoresis in KBV200 cells. FCM analysis indicated that the apoptotic rate of K562/A02 cells accreted consequently with the working concentration of GMZ-1 uprised.
3 Effect of GMZ-1 on cell cycle of K562/A02 cells
After treated with GMZ-1 for 12h, 24h and 36h, the cell cycles of K562/A02 cells were all arrested at G2+M phase.
4 Regulating expressions of p21, p53, Bax, Caspase-3, PCNA, CyclinA, CyclinB1, Mdr-1 in K562/A02 cells
The results of RT-PCR showed that GMZ-1 could up-regulate the mRNA expressions of p21, p53, Bax, CyclinA, and down-regulate the mRNA expressions of CyclinB1, PCNA, Mdr-1, then induced apoptosis of tumor cells, inhibited proliferations of tumor cells and reversed multidrug resistance.
5 Effect on protein expressions of Caspase-3 and Mdr in K562/A02 cells
The Western blotting experiments showed that GMZ-1 could decrease protein expression of Mdr.
6 Immunofluorescence was used to study the effect of GMZ-1 on microtubule of HepG-2 cells
The microtubule was bushy, reticulodromous, fluorescent brightly and spatial in normal HepG-2 cells, while in the cells after treated with GMZ-1 it was discrete, astroid and fluorescent darkly. Like vincristine, the effect of GMZ-1 on microtubule polymerizing was inhibition, not promoting. The IC50 value of inhibitting microtubule polymerizing was 0.066±0.007μmol/L,a little difference compared with podophyllotoxin(0.071±0.007μmol/L).
7 Toxicity indexs of GMZ-1
The reactions of peritoneal injection with GMZ-1 were slow response, scrunch, quiescence, diarrhea and blockage in intestine. The LD50 value was 97.90±19.21mg/kg (95% confidence interval).
Conclusions: GMZ-1 showed a powerful anti-tumor activity against many human tumor cell lines in vitro. The IC50 values were all about at 0.10μmol/L , 10~100 times more than that of VP-16. Inducing apoptosis might be a main mode of actions of GMZ-1, meanwhile, GMZ-1 could interfere cell cycles of tumor cells, and then inhibit proliferation of tumor cells. The mechanisms of these pharmacological effects were regulating apoptotic gene and protein expressions, regulating PCNA and cyclins, regulating expressions of multidrug resistance related gene and protein. GMZ-1 showed a little adverse reaction in intestine or stomach, other adverse effects were planed to study later. In a word, GMZ-1 was a new anti-tumor compound with high activity, high selectivity, definite mechanism. It was worth to study further more to get an overall estimated index.
引文
1郭青龙,杨勇,赵万洲.肿瘤药理学.化学工业出版社, 2008(1): 136-145, 127-128
2 Philippe Meresse, Elsa Dechaux, Claude Monneret, et al. Etoposide: Discovery and Medicinal Chemistry[J]. Current Medicinal Chemistry, 2004, 11: 2443-2466
3司徒镇强,吴军正.细胞培养.世界图书出版西安公司, 2004(1)
4韩锐.抗癌药物研究与实验技术.北京医科大学中国协和医科大学联合出版社, 1997: 284-289, 283-283, 399-400
5刘建文,季光,刘成海.药理实验方法学新技术与新方法.化学工业出版社, 2008(2): 67-68, 68-69, 69-70, 76-77
6温进坤,韩梅.医学分子生物学理论与研究技术.科学出版社, 2002(2): 106-141, 204-236
7张红卿,连慕兰等.细胞生物学实验方法与技术.北京师范大学出版社, 1992(1): 73～74
8孙婉,李敏,吴军.以微管蛋白为靶的高通量药物筛选方法的建立与应用[J].中国新药杂志, 2006, 15(21): 1828-1831
9徐淑云.药理实验方法学.人民卫生出版社, 400
10 Herrmannm M, Lorenz HM, Voll R et al. A Rapid and simple method for the isolation of apoptotic DNA fragments[J]. Nucleic Acid Res, 1994, 22: 5506-5507
11 Issell BF et a1. The podophyllotoxin derivatives VP16-213 and VM26[J].Cancer Chemother Pharmacol, 1982, 7(2-3): 73-80
12 Tze-Sing Huang, Chih-Hung Shu, Wen K. Yang, et al. Activation of CDC
25 Phosphatase and CDC 2 Kinase Involved in GL331-induced Apoptosis[J]. CANCER RESEARCH, 1997, 57: 2974-2978
13 Teruhiro Utsugi, Jiro Shibata, Yoshikazu Sugimoto, et al. Antitumor Activity of a Novel Podophyllotoxin Derivative (TOP-53) against Lung Cancer and Lung Metastatic Cancer[J]. CANCER RESEARCH, 1996, 56: 2809-2814
14 Gordaliza M , Garcia P A , del Corral J M M, et al. Podophyllotoxin: distribution, sources, applications and new cytotoxic derivatives[J]. Toxicon, 2004, 44(4): 441-459
15 Hong Chen, Jing Wang, Jingze Zhang, et al. L1EPO, a novel podoph- yllotoxin derivative overcomes P-glycoprotein-mediated multidrug resistance in K562/A02 cell line[J]. Biological pharmaceutical bulletin, 2009, 32(4): 609-613
16 Hong Chen, Wenchao Bi, Bo Cao, et al. A novel podophyllotoxin derivative (YB-1EPN) induces apoptosis and down-regulates express of P-glycoprotein in multidrug resistance cell line KBV200[J]. European Journal of Pharmacology, 2010, 627(1-3): 69-74
17吕晶晶,张予阳,陈虹, et al.鬼臼毒素衍生物CIP-36诱导KBV200细胞凋亡[J].中国药理学通报. 2010, 26(5): 607-610
18 MATERIAL SAFETY DATA SHEET. Sicor, PHARMA- CEUTICALS, INC. 2004, 7
19 Beck WT, et a1. The cell biology of multiple drug resistance[J]. Biochem Pharmacol, 1987, 36(18): 2879-2887
20 Cancer Cell Multidrug Resistance Mechanism of Progress Cell tumor. Knowledge of disease. 2010, 12
21 A. A. Stavrovskaya. Cellular Mechanisms of Multidrug Resistance of Tumor Cells[J]. Biochemistry, 2000, 65(1): 95-106
22 El-Deiry WS, Tokino T, Velculescv VE, et al. WAF1, a potential mediator of p53 tumour suppression [J]. Cell, 1993, 75(19): 817-823
23 Xia HH, Talley NJ. Apoptosis in gastric epithelium induced by helicobacter pylori infection: implications in gastric carcinogenesis [J]. Am J Gastroenterol, 2001, 96 (1): 16-23
24 Xiong Y, Hannon GJ, Zhang H,et al. p21 is a universal inhibitor of cyclin kinases[J]. Nature, 1993, 366(6456): 701-704
25 Raffaella Ravizza, Marzia B Gariboldi, Laura Passarelli, et al. Role of the p53/p21 system in the response of human colon carcinoma cells to Doxorubicin[J]. BMC Cancer, 2004, 4:92
26 Atan Gross, James M. McDonnell, Stanley J. Korsmeyer, et al. BCL-2 family members and the mitochondria in apoptosis[J].Genes, 1999, 13: 1899-1911
27 Steven B. Barlow, Manuel L. Gonzalez-Garay, Fernando Cabral, et al. Paclitaxel-dependent mutants have severely reduced microtubule assembly and reduced tubulin synthesis[J]. Journal of Cell Science, 2002(115): 3469-3478
28 KumarM.R. Bhat, Vijayasaradhi Setaluri. Microtubule-Associated Pro- teins as Targets in Cancer Chemotherapy[J]. Clin Cancer Res, 2007, 13(10): 2849-2854
29 Gerald Bacher, Bernd Nickel, Peter Emig, et al. D-24851, a Novel Synthetic Microtubule Inhibitor, Exerts Curative Antitumoral Activity in Vivo, Shows Efficacy toward Multidrug-resistant Tumor Cells, and Lacks Neurotoxicity[J]. CANCER RESEARCH, 2001, 61: 392-399
1 Longley DB, Johnston PG. Molecular mechanisms of drug resistance[J].J Pathol, 2005, 205: 275-292
2 Rowinsky EK, Calvo E. Novel agents that target tublin and related elements[J]. Semin Oncol, 2006, 33: 421-435
3 Fojo AT, Ueda K, Slamon DJ et al. Expression of a multidrug- resistance gene in human tumors and tissues[J]. Proc Natl Acad Sci U S A, 1987, 84: 265-269
4 Nogales E, Wolf SG, Downing KH. Structure of the alpha beta tubulin dimer by electron crystallography[J]. Nature, 1998, 391: 199-203
5 Verdier-Pinard P, Wang F, Burd B et al. Direct analysis of tubulin expression in cancer cell lines by electrospray ionization mass spectrometry[J]. Biochemistry, 2003, 42: 12019-12027
6 Luduena RF. Multiple forms of tubulin: different gene products and covalent modifications[J]. Int Rev Cytol, 1998, 178: 207-275
7 El-Kareh AW, Labes RE, Secomb TW. Cell cycle checkpoint models for cellular pharmacology of Paclitaxel and platinum drugs[J]. AAPS J, 2008, 10(1): 15-34
8 Childs S, Ling V. The MDR superfamily of genes and its biological implications[J]. Important Adv Oncol, 1994: 21-36
9 Goldman B. Multidrug resistance: can new drugs help chemotherapy score against cancer[J] J Natl Cancer Inst, 2003, 95: 255-257
10 Thomas H, Coley HM. Overcoming multidrug resistance in cancer: an update on the clinical strategy of inhibiting p-glycoprotein[J]. Cancer Control, 2003, 10:159-165
11 Szaka′cs G, Paterson JK, Ludwig JA et al. Targeting multidrug resistance in cancer[J]. Nat Rev Drug Discov, 2006, 5: 219-234
12 Haft M, Wang Y, Veeraraghavan S, et a1. Mutations in alpha and bela tubulin that stabilize microtubules and confer resistance to colcemid and vinblastine[J]. Mol Cancer Ther, 2003, 2(7): 597-605
13 Bhattacharya R, Cabral F. A ubiquitous beta-tubulin disrupts microtubule assembly and inhibits cell proliferation[J]. Mol Biol Cell, 2004, 15(7): 3123-3131
14 Kamath K, Wilson L, Cabral F, et a1. BetaⅢ-tubulin induces paclitaxel resistance in association with reduced effects on microtubule dynamic instability[J]. J Biol Chem, 2005, 280(13): 12902-1290
15 Martello LA, Verdier-Pinard P, Shen HJ et al. Elevated levels of microtubule destabilizing factors in a Taxol-resistant/dependent A549 cell line with an alpha-tubulin mutation[J]. Cancer Res, 2003, 63: 1207-1213
16 Cabral F, Barlow SB. Mechanisms by which mammalian cells acquire resistance to drugs that affect microtubule assembly[J]. FASEB J, 1989, 3: 1593-1599
17 Martello LA, Verdier Pinard P, Shen HJ, et a1. Elevated Ievels of micro- tubule destabilizing factors in a Taxol-reslstant/dependent A549 cell line with an alpha-tubulin mutation[J]. Cancer Res, 2003, 63(6): 1207-1213
18 Dumontet C, Jaffrezou JP, Tsuchiya E, et al. Resistance to microtubule- targeted cytotoxins in a K562 leukemia cell variant associated with altered tubulin expression and polymerization[J]. Bull Cancer, 2004, 91(5): E81-112
19 Gerth K, Bedorf N, Hofle G et al. Epothilons A and B: antifungal and cytotoxic compounds from Sorangium cellulosum (Myxobacteria). Production, physico-chemical and biological properties[J]. J Antibiot (Tokyo), 1996, 49: 560-563
20 Kowalski RJ, Giannakakou P, Hamel E. Activities of the microtubule-stabilizing agents epothilones A and B with purified tubulin and in cells resistant to paclitaxel[J]. J Biol Chem, 1997, 272: 2534-2541
21 Nettles JH, Li H, Cornett B et al. The binding mode of epothilone A on alpha,beta-tubulin by electron crystallography[J]. Science, 2004, 305: 866-869
22 Lee FY, Borzilleri R, Fairchild CR et al. BMS-247550: a novel epothilone analog with a mode of action similar to paclitaxel but possessing superior antitumor efficacy[J]. Clin Cancer Res, 2001, 7: 1429-1437
23 Wartmann M, Altmann KH. The biology and medicinal chemistry of epothilones[J]. Curr Med Chem Anti-Canc Agents, 2002, 2: 123-148
24 Newman RA, Yang J, Raymond M et al. Antitumor efficacy of 26-fluoroepothilone B against human prostate cancer xenografts[J]. Cancer Chemother Pharmacol, 2001, 48: 319-326
25 Bergstralh DT, Taxman DJ, Chou TC et al. A comparison of signaling activities induced by Taxol and desoxyepothilone B[J]. J Chemother, 2004, 16: 563-576
26 Chou TC, Zhang XG, Balog A et al. Desoxyepothilone B: an efficacious microtubule-targeted antitumor agent with a promising in vivo profile relative to epothilone B[J]. Proc Natl Acad Sci U S A, 1998, 95: 9642-9647
27 ter Haar E, Kowalski RJ, Hamel E et al. Discodermolide, a cytotoxic marine agent that stabilizes microtubules more potently than Taxol[J]. Biochemistry, 1996, 35: 243-250
28 Martello LA, McDaid HM, Regl DL et al. Taxol and discodermolide represent a synergistic drug combination in human carcinoma cell lines[J]. Clin Cancer Res, 2000, 6: 1978-1987
29 Honore S, Kamath K, Braguer D et al. Synergistic suppression of microtubule dynamics by discodermolide and paclitaxel in non-small cell lung carcinoma cells[J]. Cancer Res, 2004, 64: 4957-4964
30 Mita A, Lockhart A, Chen T. A phase I pharmacokinetic (PK) of AAA296A (Discodermolide) administered every 3 weeks in adult patients with advanced solid tumors[J]. Proc Am Soc Clin Oncol, 2004, 23: 133 (Abstr 2025)
31 Hamel E, Sackett DL, Vourloumis D, Nicolaou KC. The coral-derived natural products eleutherobin and sarcodictyins A and B: effects on the assembly of purified tubulin with and without microtubule-associated proteins and binding at the polymer taxoid site[J]. Biochemistry, 1999, 38: 5490-5498
32 Mooberry SL, Tien G, Hernandez AH et al. Laulimalide and isolaulima-lide, new paclitaxel-like microtubule-stabilizing agents[J]. Cancer Res, 1999, 59: 653-660
33 Tinley TL, Randall-Hlubek DA, Leal RM et al. Taccalonolides E and A: plantderived steroids with microtubule-stabilizing activity[J]. Cancer Res, 2003, 63: 3211-3220
34 Poncet J. The dolastatins, a family of promising antineoplastic agents[J]. Curr Pharm Des, 1999, 5: 139-162
35 Kavallaris M, Verrills NM, Hill BT. Anticancer therapy with novel tubulininteracting drugs[J]. Drug Resist Updat, 2001, 4: 392-401
2 Philippe Meresse, Elsa Dechaux, Claude Monneret, et al. Etoposide: Discovery and Medicinal Chemistry[J]. Current Medicinal Chemistry, 2004, 11: 2443-2466
3司徒镇强,吴军正.细胞培养.世界图书出版西安公司, 2004(1)
4韩锐.抗癌药物研究与实验技术.北京医科大学中国协和医科大学联合出版社, 1997: 284-289, 283-283, 399-400
5刘建文,季光,刘成海.药理实验方法学新技术与新方法.化学工业出版社, 2008(2): 67-68, 68-69, 69-70, 76-77
6温进坤,韩梅.医学分子生物学理论与研究技术.科学出版社, 2002(2): 106-141, 204-236
7张红卿,连慕兰等.细胞生物学实验方法与技术.北京师范大学出版社, 1992(1): 73～74
8孙婉,李敏,吴军.以微管蛋白为靶的高通量药物筛选方法的建立与应用[J].中国新药杂志, 2006, 15(21): 1828-1831
9徐淑云.药理实验方法学.人民卫生出版社, 400
10 Herrmannm M, Lorenz HM, Voll R et al. A Rapid and simple method for the isolation of apoptotic DNA fragments[J]. Nucleic Acid Res, 1994, 22: 5506-5507
11 Issell BF et a1. The podophyllotoxin derivatives VP16-213 and VM26[J].Cancer Chemother Pharmacol, 1982, 7(2-3): 73-80
12 Tze-Sing Huang, Chih-Hung Shu, Wen K. Yang, et al. Activation of CDC
25 Phosphatase and CDC 2 Kinase Involved in GL331-induced Apoptosis[J]. CANCER RESEARCH, 1997, 57: 2974-2978
13 Teruhiro Utsugi, Jiro Shibata, Yoshikazu Sugimoto, et al. Antitumor Activity of a Novel Podophyllotoxin Derivative (TOP-53) against Lung Cancer and Lung Metastatic Cancer[J]. CANCER RESEARCH, 1996, 56: 2809-2814
14 Gordaliza M , Garcia P A , del Corral J M M, et al. Podophyllotoxin: distribution, sources, applications and new cytotoxic derivatives[J]. Toxicon, 2004, 44(4): 441-459
15 Hong Chen, Jing Wang, Jingze Zhang, et al. L1EPO, a novel podoph- yllotoxin derivative overcomes P-glycoprotein-mediated multidrug resistance in K562/A02 cell line[J]. Biological pharmaceutical bulletin, 2009, 32(4): 609-613
16 Hong Chen, Wenchao Bi, Bo Cao, et al. A novel podophyllotoxin derivative (YB-1EPN) induces apoptosis and down-regulates express of P-glycoprotein in multidrug resistance cell line KBV200[J]. European Journal of Pharmacology, 2010, 627(1-3): 69-74
17吕晶晶,张予阳,陈虹, et al.鬼臼毒素衍生物CIP-36诱导KBV200细胞凋亡[J].中国药理学通报. 2010, 26(5): 607-610
18 MATERIAL SAFETY DATA SHEET. Sicor, PHARMA- CEUTICALS, INC. 2004, 7
19 Beck WT, et a1. The cell biology of multiple drug resistance[J]. Biochem Pharmacol, 1987, 36(18): 2879-2887
20 Cancer Cell Multidrug Resistance Mechanism of Progress Cell tumor. Knowledge of disease. 2010, 12
21 A. A. Stavrovskaya. Cellular Mechanisms of Multidrug Resistance of Tumor Cells[J]. Biochemistry, 2000, 65(1): 95-106
22 El-Deiry WS, Tokino T, Velculescv VE, et al. WAF1, a potential mediator of p53 tumour suppression [J]. Cell, 1993, 75(19): 817-823
23 Xia HH, Talley NJ. Apoptosis in gastric epithelium induced by helicobacter pylori infection: implications in gastric carcinogenesis [J]. Am J Gastroenterol, 2001, 96 (1): 16-23
24 Xiong Y, Hannon GJ, Zhang H,et al. p21 is a universal inhibitor of cyclin kinases[J]. Nature, 1993, 366(6456): 701-704
25 Raffaella Ravizza, Marzia B Gariboldi, Laura Passarelli, et al. Role of the p53/p21 system in the response of human colon carcinoma cells to Doxorubicin[J]. BMC Cancer, 2004, 4:92
26 Atan Gross, James M. McDonnell, Stanley J. Korsmeyer, et al. BCL-2 family members and the mitochondria in apoptosis[J].Genes, 1999, 13: 1899-1911
27 Steven B. Barlow, Manuel L. Gonzalez-Garay, Fernando Cabral, et al. Paclitaxel-dependent mutants have severely reduced microtubule assembly and reduced tubulin synthesis[J]. Journal of Cell Science, 2002(115): 3469-3478
28 KumarM.R. Bhat, Vijayasaradhi Setaluri. Microtubule-Associated Pro- teins as Targets in Cancer Chemotherapy[J]. Clin Cancer Res, 2007, 13(10): 2849-2854
29 Gerald Bacher, Bernd Nickel, Peter Emig, et al. D-24851, a Novel Synthetic Microtubule Inhibitor, Exerts Curative Antitumoral Activity in Vivo, Shows Efficacy toward Multidrug-resistant Tumor Cells, and Lacks Neurotoxicity[J]. CANCER RESEARCH, 2001, 61: 392-399
1 Longley DB, Johnston PG. Molecular mechanisms of drug resistance[J].J Pathol, 2005, 205: 275-292
2 Rowinsky EK, Calvo E. Novel agents that target tublin and related elements[J]. Semin Oncol, 2006, 33: 421-435
3 Fojo AT, Ueda K, Slamon DJ et al. Expression of a multidrug- resistance gene in human tumors and tissues[J]. Proc Natl Acad Sci U S A, 1987, 84: 265-269
4 Nogales E, Wolf SG, Downing KH. Structure of the alpha beta tubulin dimer by electron crystallography[J]. Nature, 1998, 391: 199-203
5 Verdier-Pinard P, Wang F, Burd B et al. Direct analysis of tubulin expression in cancer cell lines by electrospray ionization mass spectrometry[J]. Biochemistry, 2003, 42: 12019-12027
6 Luduena RF. Multiple forms of tubulin: different gene products and covalent modifications[J]. Int Rev Cytol, 1998, 178: 207-275
7 El-Kareh AW, Labes RE, Secomb TW. Cell cycle checkpoint models for cellular pharmacology of Paclitaxel and platinum drugs[J]. AAPS J, 2008, 10(1): 15-34
8 Childs S, Ling V. The MDR superfamily of genes and its biological implications[J]. Important Adv Oncol, 1994: 21-36
9 Goldman B. Multidrug resistance: can new drugs help chemotherapy score against cancer[J] J Natl Cancer Inst, 2003, 95: 255-257
10 Thomas H, Coley HM. Overcoming multidrug resistance in cancer: an update on the clinical strategy of inhibiting p-glycoprotein[J]. Cancer Control, 2003, 10:159-165
11 Szaka′cs G, Paterson JK, Ludwig JA et al. Targeting multidrug resistance in cancer[J]. Nat Rev Drug Discov, 2006, 5: 219-234
12 Haft M, Wang Y, Veeraraghavan S, et a1. Mutations in alpha and bela tubulin that stabilize microtubules and confer resistance to colcemid and vinblastine[J]. Mol Cancer Ther, 2003, 2(7): 597-605
13 Bhattacharya R, Cabral F. A ubiquitous beta-tubulin disrupts microtubule assembly and inhibits cell proliferation[J]. Mol Biol Cell, 2004, 15(7): 3123-3131
14 Kamath K, Wilson L, Cabral F, et a1. BetaⅢ-tubulin induces paclitaxel resistance in association with reduced effects on microtubule dynamic instability[J]. J Biol Chem, 2005, 280(13): 12902-1290
15 Martello LA, Verdier-Pinard P, Shen HJ et al. Elevated levels of microtubule destabilizing factors in a Taxol-resistant/dependent A549 cell line with an alpha-tubulin mutation[J]. Cancer Res, 2003, 63: 1207-1213
16 Cabral F, Barlow SB. Mechanisms by which mammalian cells acquire resistance to drugs that affect microtubule assembly[J]. FASEB J, 1989, 3: 1593-1599
17 Martello LA, Verdier Pinard P, Shen HJ, et a1. Elevated Ievels of micro- tubule destabilizing factors in a Taxol-reslstant/dependent A549 cell line with an alpha-tubulin mutation[J]. Cancer Res, 2003, 63(6): 1207-1213
18 Dumontet C, Jaffrezou JP, Tsuchiya E, et al. Resistance to microtubule- targeted cytotoxins in a K562 leukemia cell variant associated with altered tubulin expression and polymerization[J]. Bull Cancer, 2004, 91(5): E81-112
19 Gerth K, Bedorf N, Hofle G et al. Epothilons A and B: antifungal and cytotoxic compounds from Sorangium cellulosum (Myxobacteria). Production, physico-chemical and biological properties[J]. J Antibiot (Tokyo), 1996, 49: 560-563
20 Kowalski RJ, Giannakakou P, Hamel E. Activities of the microtubule-stabilizing agents epothilones A and B with purified tubulin and in cells resistant to paclitaxel[J]. J Biol Chem, 1997, 272: 2534-2541
21 Nettles JH, Li H, Cornett B et al. The binding mode of epothilone A on alpha,beta-tubulin by electron crystallography[J]. Science, 2004, 305: 866-869
22 Lee FY, Borzilleri R, Fairchild CR et al. BMS-247550: a novel epothilone analog with a mode of action similar to paclitaxel but possessing superior antitumor efficacy[J]. Clin Cancer Res, 2001, 7: 1429-1437
23 Wartmann M, Altmann KH. The biology and medicinal chemistry of epothilones[J]. Curr Med Chem Anti-Canc Agents, 2002, 2: 123-148
24 Newman RA, Yang J, Raymond M et al. Antitumor efficacy of 26-fluoroepothilone B against human prostate cancer xenografts[J]. Cancer Chemother Pharmacol, 2001, 48: 319-326
25 Bergstralh DT, Taxman DJ, Chou TC et al. A comparison of signaling activities induced by Taxol and desoxyepothilone B[J]. J Chemother, 2004, 16: 563-576
26 Chou TC, Zhang XG, Balog A et al. Desoxyepothilone B: an efficacious microtubule-targeted antitumor agent with a promising in vivo profile relative to epothilone B[J]. Proc Natl Acad Sci U S A, 1998, 95: 9642-9647
27 ter Haar E, Kowalski RJ, Hamel E et al. Discodermolide, a cytotoxic marine agent that stabilizes microtubules more potently than Taxol[J]. Biochemistry, 1996, 35: 243-250
28 Martello LA, McDaid HM, Regl DL et al. Taxol and discodermolide represent a synergistic drug combination in human carcinoma cell lines[J]. Clin Cancer Res, 2000, 6: 1978-1987
29 Honore S, Kamath K, Braguer D et al. Synergistic suppression of microtubule dynamics by discodermolide and paclitaxel in non-small cell lung carcinoma cells[J]. Cancer Res, 2004, 64: 4957-4964
30 Mita A, Lockhart A, Chen T. A phase I pharmacokinetic (PK) of AAA296A (Discodermolide) administered every 3 weeks in adult patients with advanced solid tumors[J]. Proc Am Soc Clin Oncol, 2004, 23: 133 (Abstr 2025)
31 Hamel E, Sackett DL, Vourloumis D, Nicolaou KC. The coral-derived natural products eleutherobin and sarcodictyins A and B: effects on the assembly of purified tubulin with and without microtubule-associated proteins and binding at the polymer taxoid site[J]. Biochemistry, 1999, 38: 5490-5498
32 Mooberry SL, Tien G, Hernandez AH et al. Laulimalide and isolaulima-lide, new paclitaxel-like microtubule-stabilizing agents[J]. Cancer Res, 1999, 59: 653-660
33 Tinley TL, Randall-Hlubek DA, Leal RM et al. Taccalonolides E and A: plantderived steroids with microtubule-stabilizing activity[J]. Cancer Res, 2003, 63: 3211-3220
34 Poncet J. The dolastatins, a family of promising antineoplastic agents[J]. Curr Pharm Des, 1999, 5: 139-162
35 Kavallaris M, Verrills NM, Hill BT. Anticancer therapy with novel tubulininteracting drugs[J]. Drug Resist Updat, 2001, 4: 392-401