Dorai Thambi, Shah Ankeeta, Summers Faith, Mathew Rajamma, Huang Jing, Hsieh Tze-Chen, Wu Joseph M
Department of Urology, New York Medical College, Valhalla, New York.
Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, New York.
Prostate. 2018 Nov;78(15):1181-1195. doi: 10.1002/pros.23693. Epub 2018 Jul 15.
In the course of studies aimed at the role of oxidative stress in the development of metastatic potential in the LNCaP-C4-2B prostate cancer progression model system, we found a relative decrease in the level of expression of the cytoplasmic nicotinamide riboside: quinone oxidoreductase (NQO2) and an increase in the oxidative stress in C4-2B cells compared to that in LNCaP or its derivatives C4 and C4-2. It was also found that C4-2B cells specifically shed large extracellular vesicles (LEVs) suggesting that these LEVs and their cargo could participate in the establishment of the osseous metastases. The level of expression of caveolin-1 increased as the system progresses from LNCaP to C4-2B. Since NQO2 RNA levels were not changed in LNCaP, C4, C4-2, and C4-2B, we tested an altered cellular distribution hypothesis of NQO2 being compartmentalized in the membrane fractions of C4-2B cells which are rich in lipid rafts and caveolae. This was confirmed when the detergent resistant membrane fractions were probed on immunoblots. Moreover, when the LEVs were analyzed for membrane associated caveolin-1 as possible cargo, we noticed that the enzyme NQO2 was also a component of the cargo along with caveolin-1 as seen in double immunofluorescence studies. Molecular modeling studies showed that a caveolin-1 accessible site is present in NQO2. Specific interaction between NQO2 and caveolin-1 was confirmed using deletion constructs of caveolin-1 fused with glutathione S-transferase (GST). Interestingly, whole cell lysate and mitochondrial preparations of LNCaP, C4, C4-2, and C4-2B showed an increasing expression of glutaminase (GLS, kidney type). The extrusion of LEVs appears to be a specific property of the bone metastatic C4-2B cells and this process could be inhibited by a GLS specific inhibitor BPTES, suggesting the critical role of a functioning glutamine metabolism. Our results indicate that a high level of expression of caveolin-1 in C4-2B cells contributes to an interaction between caveolin-1 and NQO2 and to their packaging as cargo in the shed LEVs. These results suggest an important role of membrane associated oxidoreductases in the establishment of osseous metastases in prostate cancer.
在旨在研究氧化应激在LNCaP - C4 - 2B前列腺癌进展模型系统中转移潜能发展过程中作用的研究过程中，我们发现与LNCaP或其衍生物C4和C4 - 2相比，C4 - 2B细胞中细胞质烟酰胺核糖：醌氧化还原酶（NQO2）的表达水平相对降低，氧化应激增加。还发现C4 - 2B细胞特异性释放大型细胞外囊泡（LEV），这表明这些LEV及其货物可能参与骨转移的形成。随着系统从LNCaP发展到C4 - 2B，小窝蛋白 - 1的表达水平升高。由于LNCaP、C4、C4 - 2和C4 - 2B中NQO2的RNA水平没有变化，我们测试了NQO2在富含脂筏和小窝的C4 - 2B细胞膜部分中进行区室化的细胞分布改变假说。当在免疫印迹上检测抗去污剂膜部分时，这一假说得到了证实。此外，当分析LEV中作为可能货物的膜相关小窝蛋白 - 1时，我们注意到在双重免疫荧光研究中，酶NQO2与小窝蛋白 - 1一样也是货物的组成部分。分子建模研究表明NQO2中存在一个小窝蛋白 - 1可及位点。使用与谷胱甘肽S - 转移酶（GST）融合的小窝蛋白 - 1缺失构建体证实了NQO2与小窝蛋白 - 1之间的特异性相互作用。有趣的是，LNCaP、C4、C4 - 2和C4 - 2B的全细胞裂解物和线粒体提取物显示谷氨酰胺酶（GLS，肾型）的表达增加。LEV的挤出似乎是骨转移性C4 - 2B细胞的一种特异性特性，并且这个过程可以被GLS特异性抑制剂BPTES抑制，这表明正常谷氨酰胺代谢的关键作用。我们的结果表明，C4 - 2B细胞中小窝蛋白 - 1的高表达有助于小窝蛋白 - 1与NQO2之间的相互作用以及它们作为货物包装在释放的LEV中。这些结果表明膜相关氧化还原酶在前列腺癌骨转移形成中起重要作用。
