Comparative Proteomic Profiling of Tumor-Associated Proteins in Human Gastric Cancer Cells Treated with Pectolinarigenin

Abstract

Pectolinarigenin (PEC), a natural flavonoid that is present in citrus fruits, has been reported to exhibit antitumor effects in several cancers. Though the mechanism of PEC-induced cytotoxicity effects has been documented, the proteomic changes that are associated with the cellular response to this flavonoid are poorly understood in gastric cancer cells. In this study, a comparative proteomic analysis was performed to identify proteins associated with PEC-induced cell death in two human gastric cancer cell lines: AGS and MKN-28. Two-dimensional gel electrophoresis (2-DE) revealed a total of 29 and 56 protein spots with significant alteration were screened in AGS and MKN-28 cells respectively. In total, 13 (AGS) and 39 (MKN28) proteins were successfully identified by mass spectrometry from the differential spots and they are known to be involved in signal transduction, apoptosis, transcription and translation, cell structural organization, and metabolism, as is consistent with multiple effects of PEC on tumor cells. Notably, novel target proteins like Probable ATP-dependent RNA helicase DDX4 (DDX4) and E3 ubiquitin-protein ligase LRSAM1 (LRSAM1) along with the commonly differential expressed proteins on both the cell lines that are treated with PEC were confirmed by immunoblotting. The DDX4 accelerates cell cycle progression by abrogating the G2 checkpoint when overexpressed in cancer cells, while the aberrant expression of LRSAM1 may be involved in the cancer pathology. Thus, proteomic analysis provides vital information about target proteins that are important for PEC-induced cell death in gastric cancer cells.

Comparative proteomic analysis uncovers potential biomarkers involved in the anticancer effect of Scutellarein in human gastric cancer cells

“…The synergistic analyses of proteomics data were successful in identifying a unique set of proteins regulated by SCU to induce apoptosis in both treated GC cell lines. Proteins such as PIK3CB, OFD1, CIP2A, BUB1B, ARHGEF2, VCL, EPHB2, IF4G2, CACNA2D1, HIP1R, VAV, and TLR2 have been previously reported to be associated with apoptosis, cell cycle arrest and tumor suppressors induced by another flavonoid in GC cell lines (17). Previous and current data showed that these protein candidates can be altered upon treatment with flavonoids in GC cells.…”

“…GO analysis results showed the greater role of PIK3CB involvement in the maximum category of the biological process as well as being a prominent candidate of all the pathways identified by PANTHER analysis in both cell lines. From our previous study, GC cells treated with Pectolinarigenin, a natural flavonoid, also showed downregulation of PIK3CB protein expression accompanied by a distinct group of cellular functions, including cell growth and proliferation (17). HIP1R is known as an endocytic adaptor with its functional roles in vesicle trafficking and clathrin-mediated endocytosis.…”

“…AGS and SNU484 cells were treated or untreated (control) with SCU (75 µM) for 24 h. Whole proteins were extracted from both groups of cells as previously described ( 17 ). Briefly, after incubation time, the cells were harvested and lysed with lysis buffer on ice for 1 h. The cell lysates were centrifuged at 20,000 × g at 4°C for 15 min and the supernatants were collected.…”

“…Proteins were separated using 2-DE, as reported in our previous study ( 17 ). Briefly, 400 µg of protein per sample (equal quantity) were immobilized onto 18 cm (pH 4–7) DryStrip Gels (GE Healthcare Immobiline™; Amersham Biosciences) for the first dimensional electrophoresis (isoelectric focusing: IEF) using Ettan DALT II system (Amersham Biosciences).…”

“…. In addition to these proteins, we were interested in one more protein, which was found to be expressed in our previous studies and is related to proteomic analysis in GC cells treated with flavonoids (17). We found that protein CIP2A (CIP2A) was downregulated in SCU-treated SNU484 cells compared to the untreated group of cells.…”