康宁霉素Ⅵ诱导人肺腺癌细胞和肝癌细胞程序性死亡的机制研究
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摘要
癌症一直都是人类健康的重大威胁,近年来有发病率增高的趋势。肝癌和肺癌是两种典型的癌症,患者致病因素复杂、治疗困难、预后不佳。在肿瘤治疗过程中,抗生素是主要化疗药物的来源,而选择性的杀伤作用是其发挥作用的重要方式。康宁霉素是从本实验室保存菌株Trichoderma pseudokoningii SMF2中提取出来的一种peptaibol类抗菌肽,它主要包括三种组分：康宁霉素Ⅵ,Ⅶ,Ⅷ(TKⅥTKⅦ, TKⅧ)。其中康宁霉素Ⅵ具有在牛蛙心肌细胞膜上形成钙离子通道的性质。
在上述研究基础上,本论文对康宁霉素Ⅵ的抗肿瘤机制进行了系统研究,选取人肺腺癌细胞A549和肝癌细胞HepG2作为受试细胞株,通过实验发现康宁霉素Ⅵ可诱导两株细胞都同时发生凋亡与自噬。实验结果证明康宁霉素Ⅵ可诱导HepG2和A549两株肿瘤细胞产生凋亡的一系列特征：细胞膜上磷脂酰丝氨酸外翻、DNA断裂并且电泳产生梯状片段。同时康宁霉素Ⅵ又使肿瘤细胞在凋亡的同时伴随细胞自噬现象：康宁霉素Ⅵ可诱导两株细胞细胞质产生大量空泡,空泡的出现使细胞摄入单丹酰氯戊二胺(MDC)增多,免疫荧光检测到胞内轻链3型微管相关蛋白1(LC3)Ⅰ型被磷脂酰化为Ⅱ型,并锚定于自噬体膜。我们对康宁霉素Ⅵ诱导A549细胞死亡的机制进行了进一步的探究,发现在A549细胞中,上述凋亡与自噬性死亡过程均不依赖于caspases活性,线粒体完整性在此过程中被破坏。在死亡过程中,A549细胞线粒体膜电位去极化,细胞内活性氧物质增多,Bcl-2蛋白家族中抗凋亡蛋白Bcl-xL袤达水平下降,促凋亡蛋白Bax表达水平上升。我们的实验结果首次表明康宁霉素Ⅵ可诱导肝癌细胞和肺腺癌细胞同时发生凋亡与自噬性死亡,并综合分析了康宁霉素Ⅵ所诱导的A549细胞程序死亡信号通路中各主要因子的相互关系,表明线粒体在其中起着重要作用。
Cancer is a major threat to human health and its morbidity is increasing in recent years. Hepatocellular carcinoma and lung cancer are two kinds of typical cancer which are hard to cure. Moreover, the nosogenesis of the cancer is very complicate. In the therapy of cancer, antibiotics are a major source of chemotherapeutic agents. Trichokonins (TKs) are a type of peptaibols secreted by Trichoderma pseudokoningii SMF2 conserved in our lab, which mainly include three kinds:TrichokoninⅥ, TrichokoninⅦand TrichokoninⅦ. TrichokoninⅥhas the ability to form calcium channel on bullfrog cardiac myocyte membrane. Based on the previous studies, the anticancer activity and mechanism of antitumor involved in TrichokoninⅥwere investigated. The findings demonstrated that human lung carcinoma cancer A549 cells and hepatocellular carcinoma HepG2 cells treated with TrichokoninⅥunderwent apoptosis accompanied with autophagy. TrichokoninⅥtriggered features of apoptosis in the two cell lines including phosphoserine exposure, DNA fragmentation and DNA ladder. Meanwhile, features of autophagy were also assessed by formation of autophagosomes and processing of microtube-associated protein 1 light chain 3 (LC3) typeⅠtoⅡin the two cell lines treated with TrichokoninⅥ. In order to investigate the antitumor mechanisms involved in TrichokoninⅥ, we chose A549 cells as a model to detail the process. Our results showed that both the apoptosis and autophagy in A549 were independent of caspases activity. In detail, the expression of Bcl-xL decreased and expression of Bax increased, suggesting that mitochondria inner membrane Bcl-2 protein family may play an important role in this process. At the same time, mitochondria membrane potential dissiapation (△Ψm) and reactive oxygen species (ROS) increase were detected in A549 treated with TrichokoninⅥ. Our results first showed the mechanism involved in peptaibols to induce apoptotic and autophagic cell death in lung cancer cells.
在上述研究基础上,本论文对康宁霉素Ⅵ的抗肿瘤机制进行了系统研究,选取人肺腺癌细胞A549和肝癌细胞HepG2作为受试细胞株,通过实验发现康宁霉素Ⅵ可诱导两株细胞都同时发生凋亡与自噬。实验结果证明康宁霉素Ⅵ可诱导HepG2和A549两株肿瘤细胞产生凋亡的一系列特征：细胞膜上磷脂酰丝氨酸外翻、DNA断裂并且电泳产生梯状片段。同时康宁霉素Ⅵ又使肿瘤细胞在凋亡的同时伴随细胞自噬现象：康宁霉素Ⅵ可诱导两株细胞细胞质产生大量空泡,空泡的出现使细胞摄入单丹酰氯戊二胺(MDC)增多,免疫荧光检测到胞内轻链3型微管相关蛋白1(LC3)Ⅰ型被磷脂酰化为Ⅱ型,并锚定于自噬体膜。我们对康宁霉素Ⅵ诱导A549细胞死亡的机制进行了进一步的探究,发现在A549细胞中,上述凋亡与自噬性死亡过程均不依赖于caspases活性,线粒体完整性在此过程中被破坏。在死亡过程中,A549细胞线粒体膜电位去极化,细胞内活性氧物质增多,Bcl-2蛋白家族中抗凋亡蛋白Bcl-xL袤达水平下降,促凋亡蛋白Bax表达水平上升。我们的实验结果首次表明康宁霉素Ⅵ可诱导肝癌细胞和肺腺癌细胞同时发生凋亡与自噬性死亡,并综合分析了康宁霉素Ⅵ所诱导的A549细胞程序死亡信号通路中各主要因子的相互关系,表明线粒体在其中起着重要作用。
Cancer is a major threat to human health and its morbidity is increasing in recent years. Hepatocellular carcinoma and lung cancer are two kinds of typical cancer which are hard to cure. Moreover, the nosogenesis of the cancer is very complicate. In the therapy of cancer, antibiotics are a major source of chemotherapeutic agents. Trichokonins (TKs) are a type of peptaibols secreted by Trichoderma pseudokoningii SMF2 conserved in our lab, which mainly include three kinds:TrichokoninⅥ, TrichokoninⅦand TrichokoninⅦ. TrichokoninⅥhas the ability to form calcium channel on bullfrog cardiac myocyte membrane. Based on the previous studies, the anticancer activity and mechanism of antitumor involved in TrichokoninⅥwere investigated. The findings demonstrated that human lung carcinoma cancer A549 cells and hepatocellular carcinoma HepG2 cells treated with TrichokoninⅥunderwent apoptosis accompanied with autophagy. TrichokoninⅥtriggered features of apoptosis in the two cell lines including phosphoserine exposure, DNA fragmentation and DNA ladder. Meanwhile, features of autophagy were also assessed by formation of autophagosomes and processing of microtube-associated protein 1 light chain 3 (LC3) typeⅠtoⅡin the two cell lines treated with TrichokoninⅥ. In order to investigate the antitumor mechanisms involved in TrichokoninⅥ, we chose A549 cells as a model to detail the process. Our results showed that both the apoptosis and autophagy in A549 were independent of caspases activity. In detail, the expression of Bcl-xL decreased and expression of Bax increased, suggesting that mitochondria inner membrane Bcl-2 protein family may play an important role in this process. At the same time, mitochondria membrane potential dissiapation (△Ψm) and reactive oxygen species (ROS) increase were detected in A549 treated with TrichokoninⅥ. Our results first showed the mechanism involved in peptaibols to induce apoptotic and autophagic cell death in lung cancer cells.
引文
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