Ong Li-Chu, Tan Yuen-Fen, Tan Boon Shing, Chung Felicia Fei-Lei, Cheong Soon-Keng, Leong Chee-Onn
Center for Cancer and Stem Cell Research, International Medical University, Bukit Jalil, 57000 Kuala Lumpur, Malaysia; School of Postgraduate Studies, International Medical University, Bukit Jalil, 57000 Kuala Lumpur, Malaysia.
Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.
Toxicol Appl Pharmacol. 2017 Aug 15;329:347-357. doi: 10.1016/j.taap.2017.06.024. Epub 2017 Jul 1.
Single-walled carbon nanotubes (SWCNTs) are carbon-based nanomaterials that possess immense industrial potential. Despite accumulating evidence that exposure to SWCNTs might be toxic to humans, our understanding of the mechanisms for cellular toxicity of SWCNTs remain limited. Here, we demonstrated that acute exposure of short (1-3μm) and regular-length (5-30μm) pristine, carboxylated or hydroxylated SWCNTs inhibited cell proliferation in human somatic and human stem cells in a cell type-dependent manner. The toxicity of regular-length pristine SWCNT was most evidenced in NP69>CYT00086>MCF-10A>MRC-5>HaCaT > HEK-293T>HepG2. In contrast, the short pristine SWCNTs were relatively less toxic in most of the cells being tested, except for NP69 which is more sensitive to short pristine SWCNTs as compared to regular-length pristine SWCNTs. Interestingly, carboxylation and hydroxylation of regular-length SWCNTs, but not the short SWCNTs, significantly reduced the cytotoxicity. Exposure of SWCNTs also induced caspase 3 and 9 activities, mitochondrial membrane depolarization, and significant apoptosis and necrosis in MRC-5 embryonic lung fibroblasts. In contrast, SWCNTs inhibited the proliferation of HaCaT human keratinocytes without inducing cell death. Further analyses by gene expression profiling and Connectivity Map analysis showed that SWCNTs induced a gene expression signature characteristic of heat shock protein 90 (HSP90) inhibition in MRC-5 cells, suggesting that SWCNTs may inhibit the HSP90 signaling pathway. Indeed, exposure of MRC-5 cells to SWCNTs results in a dose-dependent decrease in HSP90 client proteins (AKT, CDK4 and BCL2) and a concomitant increase in HSP70 expression. In addition, SWCNTs also significantly inhibited HSP90-dependent protein refolding. Finally, we showed that ectopic expression of HSP90, but not HSP40 or HSP70, completely abrogated the cytotoxic effects of SWCNTs, suggesting that SWCNT-induced cellular toxicity is HSP90 dependent. In summary, our findings suggest that the toxic effects of SWCNTs are mediated through inhibition of HSP90 in human lung fibroblasts and keratinocytes.
单壁碳纳米管（SWCNTs）是具有巨大工业潜力的碳基纳米材料。尽管越来越多的证据表明接触SWCNTs可能对人类有毒，但我们对SWCNTs细胞毒性机制的了解仍然有限。在此，我们证明，短（1 - 3μm）和常规长度（5 - 30μm）的原始、羧化或羟基化SWCNTs的急性暴露以细胞类型依赖的方式抑制人类体细胞和人类干细胞的细胞增殖。常规长度原始SWCNT的毒性在NP69>CYT00086>MCF - 10A>MRC - 5>HaCaT > HEK - 293T>HepG2中最为明显。相比之下，短原始SWCNTs在大多数测试细胞中毒性相对较小，除了NP69，与常规长度原始SWCNTs相比，它对短原始SWCNTs更敏感。有趣的是，常规长度SWCNTs的羧化和羟基化，而不是短SWCNTs，显著降低了细胞毒性。SWCNTs的暴露还诱导了MRC - 5胚胎肺成纤维细胞中半胱天冬酶3和9的活性、线粒体膜去极化以及显著的凋亡和坏死。相比之下，SWCNTs抑制了HaCaT人角质形成细胞的增殖而未诱导细胞死亡。通过基因表达谱分析和连接图谱分析进一步表明，SWCNTs在MRC - 5细胞中诱导了热休克蛋白90（HSP90）抑制特征的基因表达特征，表明SWCNTs可能抑制HSP90信号通路。事实上，MRC - 5细胞暴露于SWCNTs会导致HSP90客户蛋白（AKT、CDK4和BCL2）剂量依赖性降低以及HSP70表达随之增加。此外，SWCNTs还显著抑制了HSP90依赖性蛋白重折叠。最后，我们表明HSP90的异位表达，而不是HSP40或HSP70，完全消除了SWCNTs的细胞毒性作用，表明SWCNT诱导的细胞毒性是HSP90依赖性的。总之，我们的研究结果表明，SWCNTs的毒性作用是通过抑制人肺成纤维细胞和角质形成细胞中的HSP90介导的。
