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Profiling pancreatic cancer-secreted proteome using 15N amino acids and serum-free media.

ABSTRACT: A new method of determining protein turnover by labeling protein with N amino acids was used in conjunction with serum-free cell culture to profile secreted proteins that are released by MIA PaCa-2 pancreatic cancer cells in culture.MIA PaCa-2 cells were first cultured in Dulbecco modified Eagle medium (Gibco by Invitrogen, Carlsbad, Calif) with 10% fetal bovine serum, then in serum-free modified Eagle medium with or without 50% N algal amino acid mixture. The effect of oxythiamine chloride on secreteome was studied. Secreteome from cell culture media was analyzed by 2-dimensional (2D) gel electrophoresis. Differentially expressed proteins were detected and identified. Protein turnover rates were calculated according to the newly established method. Western blot and enzyme-linked immunosorbent assay were used to validate identified proteins.Among the 14 differentially expressed proteins after oxythiamine treatment, tissue inhibitor of metalloproteases-1 and cytokeratin-10 were identified as 2 newly synthesized secreted proteins caused by substantial N incorporation. The inhibition of tissue inhibitor of metalloproteases-1 expression in MIA PaCa-2 cells by oxythiamine treatment was first demonstrated by 2D gel electrophoresis and further validated by Western blotting and enzyme-linked immunosorbent assay analyses.Our method of labeling protein with N amino acids in conjunction with serum-free cell culture allows the identification of actively secreted proteins from pancreatic cancer cells and is a useful method for serum biomarker discovery.

SUBMITTER: Xiao J

PROVIDER: S-EPMC2835986 | biostudies-literature | 2010 Jan

REPOSITORIES: biostudies-literature

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Profiling pancreatic cancer-secreted proteome using 15N amino acids and serum-free media.

Xiao Jing J Lee Wai-Nang Paul WN Zhao Yingchun Y Cao Rui R Go Vay Liang W VL Recker Robert R RR Wang Qi Q Xiao Gary Guishan GG

Pancreas 20100101 1

<h4>Objectives</h4>A new method of determining protein turnover by labeling protein with N amino acids was used in conjunction with serum-free cell culture to profile secreted proteins that are released by MIA PaCa-2 pancreatic cancer cells in culture.<h4>Methods</h4>MIA PaCa-2 cells were first cultured in Dulbecco modified Eagle medium (Gibco by Invitrogen, Carlsbad, Calif) with 10% fetal bovine serum, then in serum-free modified Eagle medium with or without 50% N algal amino acid mixture. The ...[more]

PMID: 19904223

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Project description:Pigs exposed to heat stress (HS) increase body temperature in which can damage the intestinal epithelia and affect the absorption and availability of amino acids (AA). Protein digestion and metabolism further increase body temperature. An experiment was conducted with six pairs of pigs (of 47.3 ± 1.3 kg initial body weight) exposed to natural HS to assess the effect of substituting dietary protein-bound AA by free AA on morphology and gene expression of intestinal epithelial and serum concentration (SC) of free AA. Treatments were: high protein, 21.9% crude protein (CP) diet (HShp) and low protein, 13.5% CP diet supplemented with crystalline Lys, Thr, Met, Trp, His, Ile, Leu, Phe, and Val (HSaa). The HShp diet met or exceeded all AA requirements. The HSaa diet was formulated on the basis of ideal protein. Pigs were fed the same amount at 0700 and 1900 hours during the 21-d study. Blood samples were collected at 1700 hours (2.0 h before the evening meal), 2030 hours, and 2130 hours (1.5 and 2.5 h after the evening meal). At the end, all pigs were sacrificed to collect intestinal mucosa and a 5-cm section from each segment of the small intestine from each pig. Villi measures, expression of AA transporters (y+L and B0) in mucosa, and SC of AA were analyzed. Ambient temperature fluctuated daily from 24.5 to 42.6 °C. Weight gain and G.F were not affected by dietary treatment. Villi height tended to be larger (P ≤ 0.10) and the villi height:crypt depth ratio was higher in duodenum and jejunum of pigs fed the HSaa diet (P < 0.05). Gene expression of transporter y+L in jejunum tended to be lower (P < 0.10) and transporter B0 in the ileum was lower (P < 0.05) in HSaa pigs. Preprandial (1700 hours) SC of Arg, His, Ile, Leu, Thr, Trp, and Val was higher (P < 0.05), and Phe tended to be higher (P < 0.10) in HShp pigs. At 2030 hours (1.5 h postprandial), serum Lys, Met, and Thr were higher in the HSaa pigs (P < 0.05). At 2130 hours (2.5 h), Arg, His, Ile, Phe, and Trp were lower (P < 0.05); Met was higher (P < 0.05); and Lys tended to be higher (P < 0.10) in HSaa pigs. In conclusion, feeding HS pigs with low protein diets supplemented with free AA reduces the damage of the intestinal epithelia and seems to improve its absorption capacity, in comparison with HS pigs fed diets containing solely protein-bound AA. This information is useful to formulate diets that correct the reduced AA consumption associated with the decreased voluntary feed intake of pigs under HS.

Dataset Information

Profiling pancreatic cancer-secreted proteome using 15N amino acids and serum-free media.

Publications

Profiling pancreatic cancer-secreted proteome using 15N amino acids and serum-free media.

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