Jung Ji Hoon, Lee Hyemin, Kim Ju-Ha, Sim Deok Yong, Ahn Hyojin, Kim Bonglee, Chang Suhwan, Kim Sung-Hoon
College of Kyung Hee Medicine, Kyung Hee University, Seoul 02447, Korea.
Department of Biomedical Sciences, University of Ulsan, College of Medicine, Asan Medical Center, Seoul 05505, Korea.
Cancers (Basel). 2019 Apr 24;11(4):582. doi: 10.3390/cancers11040582.
Among ribosomal proteins essential for protein synthesis, the functions of ribosomal protein L5 (RPL5) and RPL11 still remain unclear to date. Here, the roles of RPL5 and RPL11 were investigated in association with p53/p21 signaling in the antitumor effect of puromycin mainly in HCT116 and H1299 cancer cells. Cell proliferation assays using 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assays and colony formation assays, cell cycle analysis, Reverse transcription polymerase chain reaction (RT-PCR) and Western blotting were performed in cancer cells. Puromycin exerted cytotoxic and anti-proliferative effects in p53 wild-type HCT116 more than in p53 null H1299 cells. Consistently, puromycin increased sub-G1, cleaved Poly (ADP-ribose) polymerase (PARP), activated p53, p21, and Mouse double minute 2 homolog (MDM2), and attenuated expression of c-Myc in HCT116 cells. Notably, puromycin upregulated the expression of RPL5 and RPL11 to directly bind to MDM2 in HCT116 cells. Conversely, deletion of RPL5 and RPL11 blocked the activation of p53, p21, and MDM2 in HCT116 cells. Also, puromycin enhanced the antitumor effect with reactivating p53 and inducing tumor apoptosis (RITA) or doxorubicin in HCT116 cells. These findings suggest that puromycin induces p53-dependent apoptosis via upregulation of RPL5 or RPL11 for binding with MDM2, and so can be used more effectively in p53 wild-type cancers by combination with RITA or doxorubicin.
在蛋白质合成所必需的核糖体蛋白中，核糖体蛋白L5（RPL5）和RPL11的功能至今仍不清楚。在此，主要在HCT116和H1299癌细胞中，研究了RPL5和RPL11在嘌呤霉素抗肿瘤作用中与p53/p21信号传导相关的作用。在癌细胞中进行了使用3-[4,5-二甲基噻唑-2-基]-2,5-二苯基四氮唑溴盐（MTT）试验和集落形成试验的细胞增殖测定、细胞周期分析、逆转录聚合酶链反应（RT-PCR）和蛋白质免疫印迹法。嘌呤霉素对p53野生型HCT116细胞的细胞毒性和抗增殖作用比对p53缺失的H1299细胞更强。一致地，嘌呤霉素增加了亚G1期、裂解的聚（ADP-核糖）聚合酶（PARP），激活了p53、p21和小鼠双微体2同源物（MDM2），并减弱了HCT116细胞中c-Myc的表达。值得注意的是，嘌呤霉素上调了RPL5和RPL11的表达，使其在HCT116细胞中直接与MDM2结合。相反，RPL5和RPL11的缺失阻断了HCT116细胞中p53、p21和MDM2的激活。此外，嘌呤霉素通过重新激活p53和诱导肿瘤凋亡（RITA）或阿霉素增强了对HCT116细胞的抗肿瘤作用。这些发现表明，嘌呤霉素通过上调RPL5或RPL11与MDM2结合来诱导p53依赖性凋亡，因此与RITA或阿霉素联合使用时，在p53野生型癌症中可以更有效地发挥作用。
