羟基喜树碱和去甲斑蝥素对肿瘤及其宿主细胞区别杀伤作用的研究
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摘要
肿瘤是人类健康的大敌,其治疗也主要是手术切除、放疗与化疗。近年来,中药及其活性成分在临床上用于抗肿瘤的治疗越来越受到各界学者的关注。喜树碱(camptothecin,CPT)是分离自珙桐科植物喜树(Camptotheca acuminate Decne)的一种五环生物碱,是迄今发现的唯一有选择性抑制DNA拓扑异构酶Ⅰ(TopoⅠ)的抗癌植物药。10-羟基喜树碱(Hydroxycamptothecine HCPT)的化学结构与CPT相似,是后来分离自喜树的另一种抗癌活性成分。HCPT与CPT相比,毒性小,疗效更好,现已广泛用于临床,主要用于非小细胞肺癌、肝癌和膀胱癌等肿瘤的治疗。斑蝥(mylabris)系鞘翅目芫青科昆虫,南方大斑蝥或黑黄小斑蝥的干虫体均可入药,在我国用于肿瘤的治疗已有两千余年的历史,具攻毒蚀疽、破血散结的作用。去甲斑蝥素(norcantharidin,NCTD)是在对斑蝥抗肿瘤有效成分斑蝥素(canthaidin)的构效关系研究基础上,由人工合成而来。NCTD在具有显著抗肿瘤活性的同时能升高外周血白细胞数,现广泛用于临床,主要用于肝癌、肺癌等的治疗。
目前,大多数抗癌药物在对癌细胞有效杀伤的同时,对其正常宿主细胞的杀伤作用也不可忽视,甚至一些药物对正常细胞的杀伤大于肿瘤细胞。因此,研究药物对肿瘤和宿主细胞的区别杀伤作用显得尤为重要和迫切。本文选用同一组织来源的人肺腺癌细胞(A549)和宿主人胚肺成纤维细胞(MRC-5),用不同浓度HCPT和NCTD作用不同时间,研究这两种药物对肿瘤和宿主的区别杀伤作用。结果显示:50-100μmol/L的HCPT在0-72h对A549杀伤作用均大于MRC-5,但作用72h时,对MRC-5杀伤作用也很大,HCPT对两种细胞的半数抑制浓度均＜50μmol/L;30-60μmol/L的NCTD在作用24h时,对A549的杀伤作用强于MRC-5,而作用48h和72h时对MRC-5的杀伤超过A549。通过药物脉冲作用细胞24h后恢复培养5天发现,实验剂量范围内HCPT对A549和MRC-5的生长抑制作用在停止药物作用后1天之内仍然存在,但杀伤A549强于MRC-5的区别杀伤作用在恢复培养中不明显,两种细胞生长与对照相比均未恢复;实验剂量范围内NCTD对A549和MRC-5的生长抑制作用在停止药物作用后马上消失,但杀伤A549强于MRC-5的区别杀伤作用在恢复培养时仍然存在,表现为30μmol/LNCTD剂量组MRC-5生长恢复较快,恢复培养3天时与对照组相比无显著性差异,而A549需要5天。用30μmol/L的NCTD和50μmol/L的HCPT作用细胞,收集细胞进行流式细胞术,结果显示:50μmol/L的HCPT引起A549细胞S期阻滞,且48h后凋亡率明显增加,而MRC-5则只显示明显S期阻滞;30μmol/L的NCTD能引起A549和MRC-5的G2-M期阻滞,A549强于MRC-5,但NCTD作用48h后MRC-5凋亡率有小幅度升高。因此,认为HCPT和NCTD对A549和MRC-5的细胞周期和凋亡影响不同,是这两种药物在一定时间和剂量范围内对两种细胞存在区别杀伤作用的原因之一。
为使抗癌药敏试验研究更贴近体内环境,在体外建立了一种肿瘤和宿主细胞的共培养体系,并在此条件下研究A549和MRC-5对HCPT和NCTD的敏感性。结果显示:A549在单独培养和与MRC-5共培养时对HCPT和NCTD的敏感性没有区别;MRC-5在与A549共培养12h和72h时对HCPT较单独培养时敏感,共培养12h和24h时对NCTD较单独培养时敏感。
Camptothecin(CPT)is an alkaloid extracted from Camptotheca acuninata Decne,the only selective inhibitor of DNA topoisomeraseⅠ(TopoⅠ). Hydroxyamptothecin(HCPT)is another alkaloid that has effective anti-cancer activity, and was extracted from Camptotheca acuninata after CPT.Compared with CPT, HCPT has stronger anti-cancer effect and lower toxicity,and be widely used in clinical,mainly used in the treatment of cancer,such as non small cell lung cancer, liver cancer,bladder cancer and so on.Cantharidin is the effective ingredient of Chinese medicine Mylabris.Norcantharidin(NCTD)is the demethylated analogue of cantharidin,and has significant anti-cancer activity and small side effects,and be used widely in the treatment of cancer,such as liver cancer,lung cancer and so on.Most of the anti-caner drugs can kill normal host cell when they reacted,and even kill more normal host cell than tumor cell.So,it is very necessary and urgent to study the different effect about anti-cancer drug between tumor and its normal host cell.
This study choose lung cancer cell(A549)and its normal host cell embryo lung fibroblasts(MRC-5),these two cell lines are come from the same human tissue.In order to research the distinctions between the role of anti-A549 and MRC-5 of HCPT and NCTD,this study use different concentrations of HCPT and NCTD to affect these two cell lines.The result shows that:HCPT induced significant proliferation inhibition in A549 and MRC-5 cells when the concentration upon 50um/L.In 0-72 hours,MRC-5 cell was more resistant to HCPT induce cytotoxicity in the concentration range of 50-100μm/L.The IC50 of 24h HCPT treatment for A549 and MRC-5 were 155.97 and＞200μmol/L,48h were A54μ9＜50μmol/L and MRC-5 133.96μmol/L,72h were A549＜50μmol/L and MRC-550μmol/L;NCTD induced proliferation inhibition in A549 and MRC-5 cells when the concentration upon 30μm/L,and MRC-5 cells were more resistant to NCTD induce cytotoxicity in the concentration range of 30-60μm/L and the time range of 0-24h.The IC50 of 24h NCTD treatment for A549 and MRC-5 were 97.71 and＞120μmol/L,48h were A549 79.66μmol/L and MRC-5 68.05μol/L,72h were A549 50.04μol/L and MRC-5 36.98μol/L.
A549 and MRC-5 were exposed to HCPT and NCTD for a period of time(24h), and then cultured 5 days with normal culture medium.The result shows that:HCPT also inhibited subsequent cell proliferation in both two cells within 5 day,but the different cytotoxic effects of HCPT that A549 was stronger than MRC-5 is not significantly;NCTD inhibited subsequent cell proliferation in both A549 and MRC-5, but the different cytotoxic effects of NCTD that A549 was stronger than MRC-5 is also exist in the 3-4 day of the concentration of 30μmol/L.
Flow cytometry analysis indicates 50μmol/L HCPT delays cell cycle progression of A549 and MRC-5 with apparent effect on S phase,and a marked increase in apoptosis after 48h of A549 only;30μmol/L NCTD delays cell cycle progression of A549 with apparent effect on G2-M phase,and weakly effect of MRC-5.The apoptosis of MRC-5 was slightly higher after 48h.
In order to study anti-cancer drug sensitivity in-vivo environment,this study established a tumor and host cells co-culture system in vitro specially,researched the growth characteristics of A549 and MRC-5 and the sensitivities of these tow cell line about HCPT and NCTD.The result shows that there are no difference in drug sensitivities of HCPT and NCTD between A549 cultured alone and be co-cultured with MRC-5;However,MRC-5 cell was more sensitive with HCPT when be co-cultured with A549 in 12h and 72h,and also more sensitive with NCTD when be co-cultured with A549 in 12h and 24h.
目前,大多数抗癌药物在对癌细胞有效杀伤的同时,对其正常宿主细胞的杀伤作用也不可忽视,甚至一些药物对正常细胞的杀伤大于肿瘤细胞。因此,研究药物对肿瘤和宿主细胞的区别杀伤作用显得尤为重要和迫切。本文选用同一组织来源的人肺腺癌细胞(A549)和宿主人胚肺成纤维细胞(MRC-5),用不同浓度HCPT和NCTD作用不同时间,研究这两种药物对肿瘤和宿主的区别杀伤作用。结果显示:50-100μmol/L的HCPT在0-72h对A549杀伤作用均大于MRC-5,但作用72h时,对MRC-5杀伤作用也很大,HCPT对两种细胞的半数抑制浓度均＜50μmol/L;30-60μmol/L的NCTD在作用24h时,对A549的杀伤作用强于MRC-5,而作用48h和72h时对MRC-5的杀伤超过A549。通过药物脉冲作用细胞24h后恢复培养5天发现,实验剂量范围内HCPT对A549和MRC-5的生长抑制作用在停止药物作用后1天之内仍然存在,但杀伤A549强于MRC-5的区别杀伤作用在恢复培养中不明显,两种细胞生长与对照相比均未恢复;实验剂量范围内NCTD对A549和MRC-5的生长抑制作用在停止药物作用后马上消失,但杀伤A549强于MRC-5的区别杀伤作用在恢复培养时仍然存在,表现为30μmol/LNCTD剂量组MRC-5生长恢复较快,恢复培养3天时与对照组相比无显著性差异,而A549需要5天。用30μmol/L的NCTD和50μmol/L的HCPT作用细胞,收集细胞进行流式细胞术,结果显示:50μmol/L的HCPT引起A549细胞S期阻滞,且48h后凋亡率明显增加,而MRC-5则只显示明显S期阻滞;30μmol/L的NCTD能引起A549和MRC-5的G2-M期阻滞,A549强于MRC-5,但NCTD作用48h后MRC-5凋亡率有小幅度升高。因此,认为HCPT和NCTD对A549和MRC-5的细胞周期和凋亡影响不同,是这两种药物在一定时间和剂量范围内对两种细胞存在区别杀伤作用的原因之一。
为使抗癌药敏试验研究更贴近体内环境,在体外建立了一种肿瘤和宿主细胞的共培养体系,并在此条件下研究A549和MRC-5对HCPT和NCTD的敏感性。结果显示:A549在单独培养和与MRC-5共培养时对HCPT和NCTD的敏感性没有区别;MRC-5在与A549共培养12h和72h时对HCPT较单独培养时敏感,共培养12h和24h时对NCTD较单独培养时敏感。
Camptothecin(CPT)is an alkaloid extracted from Camptotheca acuninata Decne,the only selective inhibitor of DNA topoisomeraseⅠ(TopoⅠ). Hydroxyamptothecin(HCPT)is another alkaloid that has effective anti-cancer activity, and was extracted from Camptotheca acuninata after CPT.Compared with CPT, HCPT has stronger anti-cancer effect and lower toxicity,and be widely used in clinical,mainly used in the treatment of cancer,such as non small cell lung cancer, liver cancer,bladder cancer and so on.Cantharidin is the effective ingredient of Chinese medicine Mylabris.Norcantharidin(NCTD)is the demethylated analogue of cantharidin,and has significant anti-cancer activity and small side effects,and be used widely in the treatment of cancer,such as liver cancer,lung cancer and so on.Most of the anti-caner drugs can kill normal host cell when they reacted,and even kill more normal host cell than tumor cell.So,it is very necessary and urgent to study the different effect about anti-cancer drug between tumor and its normal host cell.
This study choose lung cancer cell(A549)and its normal host cell embryo lung fibroblasts(MRC-5),these two cell lines are come from the same human tissue.In order to research the distinctions between the role of anti-A549 and MRC-5 of HCPT and NCTD,this study use different concentrations of HCPT and NCTD to affect these two cell lines.The result shows that:HCPT induced significant proliferation inhibition in A549 and MRC-5 cells when the concentration upon 50um/L.In 0-72 hours,MRC-5 cell was more resistant to HCPT induce cytotoxicity in the concentration range of 50-100μm/L.The IC50 of 24h HCPT treatment for A549 and MRC-5 were 155.97 and＞200μmol/L,48h were A54μ9＜50μmol/L and MRC-5 133.96μmol/L,72h were A549＜50μmol/L and MRC-550μmol/L;NCTD induced proliferation inhibition in A549 and MRC-5 cells when the concentration upon 30μm/L,and MRC-5 cells were more resistant to NCTD induce cytotoxicity in the concentration range of 30-60μm/L and the time range of 0-24h.The IC50 of 24h NCTD treatment for A549 and MRC-5 were 97.71 and＞120μmol/L,48h were A549 79.66μmol/L and MRC-5 68.05μol/L,72h were A549 50.04μol/L and MRC-5 36.98μol/L.
A549 and MRC-5 were exposed to HCPT and NCTD for a period of time(24h), and then cultured 5 days with normal culture medium.The result shows that:HCPT also inhibited subsequent cell proliferation in both two cells within 5 day,but the different cytotoxic effects of HCPT that A549 was stronger than MRC-5 is not significantly;NCTD inhibited subsequent cell proliferation in both A549 and MRC-5, but the different cytotoxic effects of NCTD that A549 was stronger than MRC-5 is also exist in the 3-4 day of the concentration of 30μmol/L.
Flow cytometry analysis indicates 50μmol/L HCPT delays cell cycle progression of A549 and MRC-5 with apparent effect on S phase,and a marked increase in apoptosis after 48h of A549 only;30μmol/L NCTD delays cell cycle progression of A549 with apparent effect on G2-M phase,and weakly effect of MRC-5.The apoptosis of MRC-5 was slightly higher after 48h.
In order to study anti-cancer drug sensitivity in-vivo environment,this study established a tumor and host cells co-culture system in vitro specially,researched the growth characteristics of A549 and MRC-5 and the sensitivities of these tow cell line about HCPT and NCTD.The result shows that there are no difference in drug sensitivities of HCPT and NCTD between A549 cultured alone and be co-cultured with MRC-5;However,MRC-5 cell was more sensitive with HCPT when be co-cultured with A549 in 12h and 72h,and also more sensitive with NCTD when be co-cultured with A549 in 12h and 24h.
引文
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