论文部分内容阅读
目的:探讨锰在调节人类前列腺癌细胞系 PC-3细胞系中线粒体顺乌头酸酶(mACON)的活性过程中可能发挥的作用。方法:用还原性烟草酰酸腺嘌呤二核苷酸配对法测量 PC-3细胞中的 mACON 酶活性。运用免疫印迹法与暂时基因表达实验测定 mACON 的基因表达。用定点突变报告基因分析法与核酸胶体位移法确认以前公认的基因反应元件。结果:体外实验确认二氯化锰(MnCl_2)处理16 h 后可抑制 mACON 的酶活性而影响了柠檬酸的有效利用并抑制了 PC-3细胞的细胞增殖。尽管暂时基因表达实验结果显示 MnCl_2通过铁反应元件路使 mACON 基因的转译增加了五倍,但免疫印迹法报告基因分析结果却显示 MnCl_2处理抑制了 mACON 的蛋白与基因表达。若同时加入柠檬酸氨铁,MnCl_2的抑制效果可被逆转。定点突变报告基因分析法与核酸胶体位移法实验结果表明一个以前公认的位于 mACON 基因的促进子序列上的金属反应元件参与了 MnCl_2对 mACON 基因表达的调控。结论:本研究结果指出锰可作为铁的拮抗剂破坏 mACON 的酶活性和基因表达,进而干扰前列腺细胞的柠檬酸代谢。
Objective: To investigate the possible role of manganese in the regulation of mitochondrial aconitase (mACON) activity in human prostate cancer cell line PC-3. Methods: The mACONase activity in PC-3 cells was measured using the reductive tobacco acyl adenine dinucleotide pairing method. The mACON gene expression was determined by Western blotting and transient gene expression assays. Previously accepted gene response elements were identified by site-directed reporter gene assay and colloidal DNA gel shift. Results: In vitro experiments confirmed that manganese chloride (MnCl2) treatment 16 h after inhibition of mACON enzyme activity affect the effective use of citric acid and inhibited PC-3 cell proliferation. Although transient gene expression experiments showed that MnCl 2 increased the translation of the mACON gene five-fold through the iron-responsive elemental pathway, the immunoblot reporter assay showed that MnCl 2 inhibited mACON protein and gene expression. The inhibitory effect of MnCl 2 can be reversed if ammonium ferric citrate is added simultaneously. Site-directed mutagenesis reporter assay and colloidal nucleic acid displacement assay showed that a previously accepted metal-responsive element located on the promoter of the mACON gene was involved in the regulation of mACON gene expression by MnCl 2. CONCLUSIONS: The results of this study indicate that manganese can act as an iron antagonist to disrupt mACON enzyme activity and gene expression, thereby disrupting citrate metabolism in prostate cells.