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Integrated proteomic and global phosphoproteomic analysis of cisplatin-induced apoptosis in A549 cells

ABSTRACT: Protein phosphorylation, a widely occurring and significant post-translational modification, is integral to various biological processes. We previously utilized a protein affinity probe to identify genes damaged by cisplatin, revealing that it inflicts substantial damage on protein kinase and protein phosphatase genes. In this study, we investigated cisplatin-induced alterations in the proteome and global phosphoproteome of A549 cells. Employing Fe-IMAC beads and tyrosine phosphorylation enrichment antibodies, we identified 6944 protein groups and 18,274 phosphorylation sites on 4,915 proteins across three biological replicates of both cisplatin-treated A549 cells and control cells. Among these, 730 tyrosine phosphorylation sites were identified—marking the most substantial discovery of such sites in A549 cells following cisplatin treatment. Bioinformatics analysis indicated that the proteins exhibiting significant phosphorylation level changes, which are predominantly involved in RNA processing, modification, transcription, translation, and the spliceosome. This suggests that cisplatin-induced damage to protein kinases and phosphatases may disrupt the normal function of these proteins, consequently impairing DNA replication, RNA translation, and shearing, ultimately culminating in tumor cell death. Moreover, we cross-referenced our proteomic data with our previously obtained cisplatin-damaged genes, observing that the majority of down-regulated proteins derived from cisplatin-induced gene damage.

INSTRUMENT(S): timsTOF Pro

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Malignant Cell, Cell Culture

DISEASE(S): Lung Cancer

SUBMITTER: luyu qi

LAB HEAD: Jianzhong Shao

PROVIDER: PXD053902 | Pride | 2024-10-20

REPOSITORIES: Pride

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			Action	DRS
	ZY001LPPY_Cisplatin_1_Slot2-52_1_4585.d.rar	Other
	ZY001LPPY_Cisplatin_2_Slot2-53_1_4587.d.rar	Other
	ZY001LPPY_Cisplatin_3_Slot2-54_1_4589.d.rar	Other
	ZY001LPPY_Control_1_Slot2-49_1_4579.d.rar	Other
	ZY001LPPY_Control_2_Slot2-50_1_4581.d.rar	Other

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Project description:Understanding the mechanism of resistance in platinum-based regimens for the treatment of high-grade serous ovarian cancer (HGSOC) is important for identifying new therapeutic targets to improve the clinical outcome of ovarian cancer patients. Mass spectrometry-based proteomic strategy was applied to spheroidal cisplatin sensitive and resistant HGSOC generated cell lines in the absence and presence of cisplatin drug. A complete expressed HGSOC proteome and phosphoproteome was characterized in cisplatin sensitive and resistant HGSOC cell lines providing insight into the mechanism of resistance development. PCA analysis showed that phosphorylation of a few proteins provides better classification than the whole proteome of the cellular subtypes. Specifically, a distinctive phosphoproteomic signature between cisplatin sensitive and resistant cell lines in the absence of drug was observed. This same phosphoproteomic signature was observed in our cisplatin sensitive cell line in the absence and presence of drug, indicating a vital role for phosphorylation of proteins in resistance development to cisplatin. The most phosphorylated protein was sequestosome (p62/SQSTM1). Differential expressions of apoptosis by the prognostic factor ratio of Bcl-2/Bax and autophagy, known to be regulated by p62/SQSTM1, was validated in the proteome data and by western blot analysis. A significant increase in apoptosis in the presence of cisplatin was observed in only the sensitive cell line while autophagy revealed increased expression in the resistant relative to sensitive cell line. Furthermore, site specific phosphorylation on 20 modified residues of sequestosome was characterized. Elevated expression of phosphorylation of sequestosome in resistant HGSOC cell lines was validated with western blot analysis. Here, we propose phosphorylation of sequestosome to be a marker and key in cisplatin resistance development in HGOSC ovarian cancers by shuttling ubiquitinated proteins to the autophagy pathway and influencing down-regulation of apoptosis.

Project description:Clinical use of a major cancer chemotherapeutic agent, cisplatin, is restricted to dose-limiting renal toxicity. A transcriptomic approach enabled us to extract putative molecules including Pleckstrin homology-like domain, family A, member-3 (Phlda3) contributing to the renal injury. This study investigated the expression of Phlda3 and its regulatory role in tubular injury induced by cisplatin in vivo and in vitro. Real-time PCR and immunoblot assays confirmed that Phlda3 was highly up-regulated in the kidney of mice after a single dose of cisplatin treatment, which preceded increases in the blood urea nitrogen or serum creatinine levels. Particularly, the level of Phlda3 transcript was substantially increased at an early time than that of kidney injury molecule-1, and remained elevated. The effect of cisplatin or acetaminophen treatment on Phlda3 expression in the liver was minimal. Consistently, treatment of NRK52E, a renal tubular cell line, with cisplatin caused increases in Phlda3 mRNA and protein. Knockdown of Phlda3 reversed the decrease in cell viability by cisplatin, supporting the role of Phlda3 in cell death. Akt phosphorylation initially increased after cisplatin treatment, but decreased thereafter. Cisplatin treatment elicited p53 accumulation via mdm2 repression, inducing its target genes, p21 and cyclin G1. Phlda3 deficiency attenuated a decrease in Akt phosphorylation by cisplatin, and prevented p53 accumulation, supporting the role of Phlda3 for p53-mediated tubular cell death. Other renal toxicants, cyclosporine A and CdCl2, also enhanced Phlda3-dependent cell death. Overall, nephrotoxicants including cisplatin induce Phlda3, which leads to p53-mediated tubular cell death via mdm2 repression caused by Akt inhibition. Since the datasets reported on the effect of cisplatin on the kidney were obtained from animals treated with multiple and relatively high doses of cisplatin, we did our cDNA array analyses day 3 after a single dose of cisplatin administration to mice (i.p., 15 mg/kg) and compared the profiles of kidney microarray data. The transcriptomic approach enabled us to find the putative genes encoding molecules contributing to renal injury. Among those genes represented on the microarray, 33 genes were found to be differentially expressed by cisplatin treatment when a 2-fold change cutoff was used. The regulation of exemplary mRNAs identified in the array analysis was confirmed by qRT-PCR. C57BL/6 mice (9 weeks old) were intraperitoneally injected with a single dose of cisplatin (15 mg/kg body weight) or vehicle (saline). The cDNA microarray experiments were done for the kidney of mice 3 days after treatment. Each group contained 3 males.

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Integrated proteomic and global phosphoproteomic analysis of cisplatin-induced apoptosis in A549 cells

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