Proteomic analysis of ubiquitination-associated proteins in a cisplatin-resistant human lung adenocarcinoma cell line

Abstract

Abstract. The objective of this study was to screen for ubiquitination-associated proteins involved in cisplatin resistance in a human lung adenocarcinoma cell strain using a comparative proteomic strategy. We employed 1D SDS-PAGE to separate ubiquitinated proteins isolated and enriched from A549 and A549/CDDP lysates via affinity chromatography. The differentially expressed bands between 45-85 kDa were subsequently hydrolyzed by trypsin and subjected to HPLC-CHIP-MS/MS analysis. Of the 11 proteins identified, 7 proteins were monoubiquitinated or polyubiquitinated substrates and 4 proteins were E3 ubiquitin ligase-associated proteins. The results of western blotting and confocal laser scanning microscopy indicated that the expression levels of the E3 ubiquitin ligases RNF6, LRSAM1 and TRIM25 in A549 cells were significantly lower than those in the A549/ CDDP cell line. The expression levels of the above three ubiquitin ligases in both cell lines were significantly decreased upon treatment with cis-diamminedichloroplatinum (CDDP), and the expression in the A549/CDDP cell after the treatment with CDDP decreased to a lesser extent. The expression of the substrate PKM2 in the A549 cell was higher than that in the A549/CDDP cells. Moreover, the expression of PKM2 increased in the A549 cell line and decreased in the A549/ CDDP cell line upon CDDP treatment. This study suggests that drug resistance is closely correlated with changes in the ubiquitination process at the protein level in a human lung adenocarcinoma cell line.

Label-free quantitative identification of abnormally ubiquitinated proteins as useful biomarkers for human lung squamous cell carcinomas

“…Anti-ubiquitin antibody (specific anti-K-ε-GG group)-based label-free coupled with LC-MS/MS is an effective method to detect, identify, and quantify ubiquitinated proteins and ubiquitination sites, and more than 10,000 ubiquitination sites have been identified and quantified [25]. Currently, ubiquitinomes of lung cancer cells have been studied [26,27]. However, tissue ubiquitinomics has not been reported in LSCC.…”

RNF6 as an Oncogene and Potential Therapeutic Target—A Review

“…Treating cells with cisplatin led to the downregulation of RNF6, but the average decrease was lower in the A549 cisplatin-resistant cells. This points out that RNF6 may take part in drug resistance in lung adenocarcinoma [26]. Figure 1 shows a schematic presentation of the connection between RNF6 and different signaling pathways.…”

E3 Ubiquitin Ligase in Anticancer Drugdsla Resistance: Recent Advances and Future Potential

“…Tripartite motif (TRIM) family proteins, most of which have E3 activities, control important cellular processes such as intracellular signaling, innate immunity, transcription, autophagy, and carcinogenesis (Hatakeyama, 2017). TRIM25 expression was identified to be significantly lower in the cisplatin-resistant non-small cell lung carcer (NSCLC) cell line A549 than in control cell lines (Qin et al, 2012). Overexpression of TRIM32 promoted degradation of Abi2, resulting in enhancement of cell growth, transforming activity, and cell motility.…”