高表达OAZI-1增加小鼠黑素瘤B16-F1细胞对多胺类似物CPENSpm的敏感性

OAZI-1 overexpression increases sensitivity of mouse melanoma B16-F1 cells to polyamine analog CPENSpm

目的检测高表达OAZI-1的小鼠黑素瘤B16-F1细胞对抗癌药物多胺类似物CPENSpm敏感性的影响并探讨其作用机制。方法高表达OAZI-1的B16-F1细胞经CPENSpm处理后,MTT法检测细胞增殖;QT-RT-PCR检测细胞内OAZI-1、ODC和OAZ的mRNA表达水平;反相高效液相色谱法检测细胞内多胺含量;化学发光法分析细胞内SMO酶活性。结果高表达OAZI-1显著增加B16-F1细胞对CPENSpm的敏感性,QT-RT-PCR结果显示,OAZI-1高表达增加ODCmRNA但降低OAZ mRNA水平,CPENSpm处理对ODC和OAZ的mRNA水平无进一步影响。与对照细胞相比,CPENSpm处理能更显著性地降低OAZI-1高表达细胞中的多胺含量,同时更强地诱导SMO酶的活性。结论 CPENSpm处理能在更大程度上耗竭OAZI-1高表达的B16-F1细胞中的多胺,由此对该肿瘤细胞显示出更大的细胞毒性。

Objective To detect the influence of ornithine antizyme inhibitor-1（OAZI-1） overexpression on the sensitivity of mouse melanoma B16-F1 cells to polyamine analog CPENSpm,and to study the underlying mechanism.Methods B16-F1 cells with OAZI-1 overexpression were treated with CPENSpm.MTT assay was used to detect cell proliferation.QT-RT-PCR was used to determine the mRNA expression levels of OAZI-1,ornithine decarboxylase（ODC） and ornithine decarboxylase antizyme（OAZ） in B16-F1 cells.RP-HPLC was used to determine the polyamine content in B16-F1 cells.Chemiluminescence assay was used to determine the activity of spermine oxidase（SMO） in B16-F1 cells.Results The MTT results showed that OAZI-1 overexpression increased the sensitivity of B16-F1 cells to CPENSpm significantly.The QT-RT-PCR results showed an increased mRNA expression level of ODC but a decreased mRNA expression level of OAZ in B16-F1 cells with OAZI-1 overexpression（B16/over）.No further changes of the mRNA expression levels of ODC and OAZ were detected in both B16/over cells and control cells（B16/3.1） after being treated with CPENSpm for 24 h.Compared with those in B16/3.1 cells,the polyamine content was lowered significantly,and the SMO activity was increased in B16/over cells after 24-h CPENSpm treatment.Conclusion CPENSpm treatment can largely deplete intracellular polyamine in B16-F1 cells with OAZI-1 overexpression,resulting in increased cytotoxicity on those tumor cells.