Induction of apoptosis by disturbing mitochondrial-membrane potential and cleaving PARP in Jurkat T cells through treatment with acetoxyscirpenol mycotoxins
Induction of apoptosis by disturbing mitochondrial-membrane potential and cleaving PARP in Jurkat T cells through treatment with acetoxyscirpenol mycotoxins
复制标题
DOI：
10.1248/bpb.29.648
复制
发表时间：
2006-04-01
影响因子：
2
通讯作者：
Kim, HW
中科院分区：
文献类型：
--
作者：
Lee, DH;Park, T;Kim, HW
细脚拟青霉是一种生长在家蚕幼虫体内的著名中药昆虫病原真菌。从细脚拟青霉（Paecilomycestenuipes）中分离得到一种具有细胞毒性的化合物4 β-乙酰氧基三环己二醇（4-MAS）。为了进一步阐明4-MAS的细胞毒性机制，本文报道了其诱导人Jurkat T细胞系凋亡的证据，以及结构相关的乙酰氧基三环醇部分真菌毒素（ASM），如15-乙酰氧基三环醇（15-MAS）、4，15-二乙酰氧基三环醇（4，15-DAS）和30-乙酰基二乙酰氧基三环醇（TAS）。在用于监测细胞活力的MTT减少和时程细胞毒性测定中，测试的所有四种ASM都表现出细胞毒性;在Scirpenol家族的C-4处的单一乙酰氧基化导致相对弱的细胞毒性，而在C-15处的乙酰氧基化导致强的细胞毒性，而不管在C-3和/或C-4位置处的其他乙酰氧基化。磷脂酰丝氨酸外化诱导的所有ASMs在早期阶段在一个时间依赖性的方式处理，表现出典型的凋亡现象，而不是一个坏死。在用DiOC（6）（一种线粒体特异性和电压依赖性染料）染色后，通过流式细胞术分析，ASM还降低了线粒体的内膜电位（A Pm）。ASM在C-15位的乙酰氧基化增加了Δ Psi m的破坏，但在C-3位的乙酰氧基化降低了Δ Psi m。通过蛋白质印迹分析，测试的ASM还将113 kDa PARP切割成89 kDa片段，表明Jurkat T细胞中的半胱天冬酶-3和/或半胱天冬酶-7活化。在Jurkat T细胞中测试的ASM还观察到DNA片段化以时间依赖性方式增加，导致DNA片段化强度顺序为4，15-DAS > 15-MAS > TAS > 4-MAS。这些数据表明，用ASM处理的Jurkat T细胞经历了典型的凋亡性细胞死亡级联。
Paecilomyces tenuipes is a famous Chinese medicinal entomopathogenic fungus that grows within the larvae of silkworms. 4 beta-acetoxyscirpendiol (4-MAS), a cytotoxic compound belonging to the scirpenol subfamily of trichothecene mycotoxin, was isolated from Paecilomyces tenuipes. To further elucidate the cytotoxic mechanism of 4-MAS, evidences of its induction of apoptosis, together with the structurally related acetoxyscirpenol moiety mycotoxins (ASMs) such as, 15-acetoxyscirpenol (15-MAS), 4,15-diacetoxyscirpenol (4,15-DAS), and 30-acetyldiacetoxyscirpenol (TAS), in the human Jurkat T cell line were reported herein. In the MTT reduction and time-course cytotoxicity assays for monitoring cell viability, all the four ASMs that were tested exhibited cytotoxicity; single acetoxylation at C-4 of the scirpenol family resulted in relatively weak cytotoxicity, while acetoxylation at C-15 resulted in strong cytotoxicity regardless of the other acetoxylations at the C-3 and/or C-4 positions. Phosphatidylserine externalization was induced by all the ASMs that were treated at an early phase in a time-dependent manner, showing a typical apoptotic phenomenon, not a necrotic one. The ASMs also reduced the mitochondria's inner-membrane potential (A Pm) through flow cytometry analysis after staining these with DiOC(6), a mitochondria-specific and voltage-dependent dye. Acetoxylation of ASM at C-15 increased Delta Psi m disruption, but that at C-3 reduced the Delta Psi m. The ASMs that were tested also cleaved 113 kDa PARP to an 89-kDa fragment through Western blot assay, suggesting the activation of caspase-3 and/or caspase-7 in the Jurkat T cell. DNA fragmentation was also observed to have been increased in a time-dependent manner by the ASMs that were tested in Jurkat T cells, resulting in the DNA fragmentation intensity order of 4,15-DAS > 15-MAS > TAS > 4-MAS. These data indicate that the Jurkat T cells that were treated with ASMs underwent typical cascades of apoptotic cell death.