仅K33突变型泛素通过Akt1的K33泛素化增强A549和NCI-H446细胞对顺铂的化疗耐药性。

K33 only mutant ubiquitin augments cisplatin chemoresistance in A549 and NCI-H446 cells via Akt1 K33 ubiquitination.

作者信息

Yin Yi Zhen, Yang Hong Ying, Liang Yi Yun, Liao Xiao Yan, Jia Jun Jun, Chen Xiao Jie, Wu Jing Yi, Chen Rui Ling, Gao Feng Guang

机构信息

Department of Basic Medicine Science, School of Medicine, Xiamen University, Xiamen, Fujian 361102, China.

Department of Basic Medicine Science, School of Medicine, Xiamen University, Xiamen, Fujian 361102, China; State Key Laboratory of Cellular Stress Biology, School of Medicine, Faculty of Medicine and Life Sciences, Xiamen University, Xiamen, Fujian 361102, China.

出版信息

Cell Signal. 2025 Oct;134:111959. doi: 10.1016/j.cellsig.2025.111959. Epub 2025 Jun 20.

DOI:10.1016/j.cellsig.2025.111959

PMID:40545114

Abstract

BACKGROUND

To probe the function of Akt1 K33 ubiquitination in cisplatin-induced apoptosis in lung cancer.

METHODS

Akt1 K33 ubiquitination modified by MG132/PYR-41, K33 only mutant ubiquitin (K33O), and Akt1 silence was investigated by co-immunoprecipitation in A549 and NCI-H446 cells. Akt phosphorylation was interrogated by western blot. The role of Akt1 K33 ubiquitination in cisplatin-induced apoptosis was assessed by cell viability using crystal violet staining, the changes of mitochondrial membrane potential with Rhodamine 123 (Rho123) and Calcein AM staining, and cell apoptosis with flow cytometric assay via annexin V-PI staining, respectively.

RESULTS

Cisplatin induced apoptosis in A549 and NCI-H446 cells; K33O reversed the cisplatin resistance with elevated K33 ubiquitination; The intervention with Wortmannin induced a significant attenuation in the Akt phosphorylation; Inhibiting Akt1 K33 ubiquitination abolished cisplatin effects on cell viability, manifesting as heightened apoptosis and alterations in mitochondrial membrane potential.

CONCLUSIONS

Akt1 K33 ubiquitination emerges as a promising therapeutic target for lung cancer chemotherapy.

摘要

背景

探讨Akt1 K33泛素化在顺铂诱导肺癌细胞凋亡中的作用。

方法

在A549和NCI-H446细胞中，通过免疫共沉淀研究MG132/PYR-41、仅K33突变的泛素（K33O）修饰的Akt1 K33泛素化以及Akt1沉默情况。通过蛋白质免疫印迹法检测Akt磷酸化。分别使用结晶紫染色通过细胞活力评估Akt1 K33泛素化在顺铂诱导凋亡中的作用，用罗丹明123（Rho123）和钙黄绿素AM染色检测线粒体膜电位变化，通过膜联蛋白V-碘化丙啶染色用流式细胞术检测细胞凋亡。