Arsenic Trioxide (ATO) Influences the Gene Expression of Metallothioneins in Human Glioblastoma Cells
Arsenic Trioxide (ATO) Influences the Gene Expression of Metallothioneins in Human Glioblastoma Cells
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DOI：
10.1007/s12011-012-9431-8
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发表时间：
2012-12-01
影响因子：
3.9
通讯作者：
Marc, Janja
中科院分区：
文献类型：
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作者：
Falnoga, Ingrid;Pevec, Andreja Zelenik;Marc, Janja
三氧化二砷（As 2 O3; ATO，TRISENOXA（R））用于治疗难治性或复发性急性早幼粒细胞白血病患者，而其用于治疗胶质母细胞瘤等实体癌的应用仍在评估中。在本研究中，我们研究了三氧化二砷与金属硫蛋白（MT）亚型的相互作用，作为胶质母细胞瘤细胞对砷诱导的细胞毒性的可能（保护性反应）抗性。特别关注的是MT3，主要在大脑中表达的同种型。MT3具有低金属诱导、快速金属结合/释放特性和突出的神经元抑制活性。人类星形细胞瘤用0.6、2和6-7 μ M砷处理胶质母细胞瘤细胞系U87 MG（相当于0.3、1和3-3.5 μ M As 2 O3）处理12、24或48小时，并对不同MT同种型，即MT 2A、MT 1A、MT 1F、MT 1X、MT 1 E和MT3进行基因表达，通过使用SYBR绿色I和TaqmanA（R）基因表达测定的真实的时间qPCR测量。TfR、18 S rRNA、GAPDH和AB作为参考基因进行了测试，最后两个基因被评估为适用于低（GAPDH）和高（AB）砷暴露条件。还使用PvuII通过限制性酶切分析测试并确认了MT 3基因的基因表达。在给定的条件下，在人胶质母细胞瘤细胞系U87 MG中鉴定了六种MT异构体的mRNA。根据砷暴露条件，MT基因表达的增加或减少，观察到每种亚型，最高的增加亚型MT 1X，MT 1F和MT 2A mRNA（高达13倍）和MT 1A，MT 1 E和MT3 mRNA更持久的下降。尽管共同的假设，非诱导的MT3，明显的MT3 mRNA的增加，观察到在高砷暴露（高达4倍）。总之，我们的研究结果清楚地表明，MT亚型基因表达的影响。MT 1X，MT 1F和MT 2A的增加可能代表脑肿瘤获得性耐As细胞毒性，而MT3的增加是更神秘的，其可能参与砷相关的诱导II型细胞死亡。
Arsenic trioxide (As2O3; ATO, TRISENOXA (R)) is used to treat patients with refractory or relapsed acute promyelocytic leukaemia while its application for treatment of solid cancers like glioblastoma is still under evaluation. In the present study, we investigated the interaction of arsenic trioxide with metallothionein (MT) isoforms as a possible (protective response) resistance of glioblastoma cells to arsenic-induced cytotoxicity. Special attention was focused on MT3, the isoform expressed mainly in the brain. MT3 has low metal inducibility, fast metal binding/releasing properties and outstanding neuronal inhibitory activity. The human astrocytoma (glioblastoma) cell line U87 MG was treated with 0.6, 2 and 6-7 mu M arsenic (equivalent to 0.3, 1 and 3-3.5 mu M As2O3) for 12, 24 or 48 h and gene expression for different MT isoforms, namely MT2A, MT1A, MT1F, MT1X, MT1E and MT3, was measured by real time qPCR using SYBR Green I and TaqmanA (R) gene expression assays. TfR, 18S rRNA, GAPDH and AB were tested as reference genes, and the last two evaluated to be appropriate in conditions of low (GAPDH) and high (AB) arsenic exposure. The gene expression of MT3 gene was additionally tested and confirmed by restriction enzyme analysis with PvuII. In the given conditions the mRNAs of six MT isoforms were identified in human glioblastoma cell line U87 MG. Depending on arsenic exposure conditions, an increase or decrease of MT gene expression was observed for each isoform, with the highest increase for isoforms MT1X, MT1F and MT2A mRNA (up to 13-fold) and more persistent decreases for MT1A, MT1E and MT3 mRNA. Despite the common assumption of the noninducibility of MT3, the evident MT3 mRNA increase was observed during high As exposure (up to 4-fold). In conclusion, our results clearly demonstrate the influence of As on MT isoform gene expression. The MT1X, MT1F and MT2A increase could represent brain tumour acquired resistance to As cytotoxicity while the MT3 increase is more enigmatic, with its possible involvement in arsenic-related induction of type II cell death.