Abstract
Abstract Pancreatic cancer is one of the most lethal cancers, with a meager (∼3%) 5-year survival rate. The incidence of pancreatic cancer nearly equals its death rate as this neoplasm is associated with poor responsiveness to conventional chemotherapies. Therefore, it's crucial to identify newer mechanism-based agents and targets to effectively manage pancreatic cancer. Previous studies from our laboratory have demonstrated that the plant alkaloid sanguinarine (13-methyl[1,3]benzodioxolo [5,6-c]-1,3-dioxolo [4,5-i] phenanthridinium possesses strong antiproliferative effects against human pancreatic carcinoma cells (Cancer Letters 249; 198-208, 2007). In this study, employing a large-scale Nano-ESI ultra high resolution label-free quantitative proteomics, we attempted to determine the mechanism of sanguinarine's biological response in human pancreatic cancer cells. Based on pilot experiments, we selected BxPC-3 cells and a 1 microM sanguinarine treatment for 24 hours for our proteomics study. Proteins from control and sanguinarine-treated BxPC-3 cells were digested with trypsin followed by nano-liquid chromatography-tandem mass spectrometry (LC/MS/MS). Following LC/MS/MS acquisition, the data were searched against the Swiss-Prot human proteome database using Sequest HT search engine under the Proteome Discoverer 1.4 software (Thermo Fisher Scientific). Following protein identification, the LC/MS/MS data was aligned using Chromalign and quantitation of peptides was performed on the processed data using Sieve 2.1 (Thermo Fisher Scientific). A cumulative data of a total of 6 replicates (2 biological and 3 technical) were run and analyzed. Upon comparison with untreated control, 37 proteins (from a total of 3108) showed +1.8 fold significant differential expressions (p value <0.05). These proteins can be categorized under distinct biological functions: 1) cellular assembly and organization, 2) cellular function and maintenance, 3) inflammatory response, and 4) cell death and survival. On further data analysis and validation using real-time qPCR and Western blot analyses, we identified the dual specificity phosphatase-4 (DUSP4), as a novel target of sanguinarine in pancreatic cancer cells. In order to determine the clinical relevance of DUSP4 in pancreatic cancer cells, we determined the expression profile of DUSP4 in a tissue microarray (TMA) containing a wide range of pancreatic cancer tissue (different stages) and normal pancreas tissue from autopsy. We found that that DUSP4 is markedly downregulated in cancerous pancreatic tissues. Our data identified, for the first time, DUPS4 as a potential tumor suppressor in pancreatic cancer and a novel target of sanguinarine. However, further studies are needed to validate our findings in detailed in vitro and in vivo studies. Citation Format: Chandra K. Singh, Satwinderjeet Kaur, Jasmine George, Molly C. Pellitteri-Hahn, Cameron O. Scarlett, Nihal Ahmad. Mechanism of anti-proliferative effects of sanguinarine in pancreatic cancer cells: A label-free quantitative proteomics approach. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4533. doi:10.1158/1538-7445.AM2014-4533
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