Yonsei Med J.  2015 Mar;56(2):563-571. 10.3349/ymj.2015.56.2.563.

Differentially Expressed Proteins in Nitric Oxide-Stimulated NIH/3T3 Fibroblasts: Implications for Inhibiting Cancer Development

• Shim DH
• Lim JW
• Kim H

• KMID: 2070039
• DOI: http://doi.org/10.3349/ymj.2015.56.2.563

Abstract

PURPOSE
Recent evidence shows that nitric oxide (NO) may exhibit both pro-cancer and anti-cancer activities. The present study aimed to determine the differentially expressed proteins in NO-treated NIH/3T3 fibroblasts in order to investigate whether NO induces proteins with pro-cancer or anti-cancer effects.
MATERIALS AND METHODS
The cells were treated with 300 microM of an NO donor 3,3-bis-(aminoethyl)-1-hydroxy-2-oxo-1-triazene (NOC-18) for 12 h. The changed protein patterns, which were separated by two-dimensional electrophoresis using pH gradients of 4-7, were conclusively identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of the peptide digests.
RESULTS
Seventeen differentially expressed proteins were identified in NOC-18-treated cells. Nine proteins [vinculin protein, keratin 19, ubiquitous tropomodulin, F-actin capping protein (alpha1 subunit), tropomyosin 3, 26S proteasome-associated pad1 homolog, T-complex protein 1 (epsilon subunit) N(G)-dimethylarginine dimethylaminohydrolase, and heat shock protein 90] were increased and eight proteins (heat shock protein 70, glucosidase II, lamin B1, calreticulin, nucleophosmin 1, microtubule-associated protein retinitis pigmentosa/end binding family member 1, 150 kD oxygen-regulated protein precursor, and heat shock 70-related protein albino or pale green 2) were decreased by NOC-18 in the cells. Thirteen proteins are related to the suppression of cancer cell proliferation, invasion, and metastasis while two proteins (heat shock protein 90 and N(G)-dimethylarginine dimethylaminohydrolase) are related to carcinogenesis. The functions of 150 kD oxygen-regulated protein precursor and T-complex protein 1 (epsilon subunit) are unknown in relation to carcinogenesis.
CONCLUSION
Most proteins differentially expressed by NOC-18 are involved in inhibiting cancer development.

Keyword

Nitric oxide; proteomic analysis; cancer; NIH/3T3 cells

MeSH Terms

Animals
Electrophoresis, Gel, Two-Dimensional/*methods
Fibroblasts/*metabolism/pathology
HSP70 Heat-Shock Proteins
Humans
Mice
NIH 3T3 Cells
Neoplasms/*metabolism/pathology
Nitric Oxide Donors
Nitroso Compounds
Proteins/analysis/*metabolism
Proteomics/*methods
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
HSP70 Heat-Shock Proteins
Nitric Oxide Donors
Nitroso Compounds
Proteins

Figure

• 

  Fig. 1 2-DE gel map derived from non-treated cells (none) and NOC-18-treated cells (NOC-18). (A) Increased proteins in non-treated cells (none) compared to NOC-18-treated cells (NOC-18) in a 12-h culture. (B) Increased proteins in NOC-18-treated cells (NOC-18) compared to non-treated cells (none) in a 12-h culture. NOC-18, NO donor 3,3-bis-(aminoethyl)-1-hydroxy-2-oxo-1-triazene; 2-DE, two-dimensional electrophoresis; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis; APG, albino or pale green; RP/EB, retinitis pigmentosa/end binding.

• 

  Fig. 2 Segments of a 2-DE gel map derived from non-treated cells (none) and NOC-18-treated cells (NOC-18). Arrowed spots are the proteins that increased in NOC-18-treated cells when compared to non-treated cells (none) in a 12-h culture. Protein expression level is expressed as a percentage of the volume, which is determined by the relative spot volume of each protein compared to the total spot volume (total amount of proteins/sum of each protein spot) in the gel. NOC-18, NO donor 3,3-bis-(aminoethyl)-1-hydroxy-2-oxo-1-triazene; 2-DE, two-dimensional electrophoresis.

• 

  Fig. 3 Segments of a 2-DE gel map derived from non-treated cells (none) and NOC-18-treated cells (NOC-18). Arrowed spots are the proteins that decreased in NOC-18-treated cells when compared to non-treated cells (none) in a 12-h culture. Protein expression level is expressed as a percentage of the volume, which is determined by the relative spot volume of each protein compared to the total spot volume (total amount of proteins/sum of each protein spot) in the gel. NOC-18, NO donor 3,3-bis-(aminoethyl)-1-hydroxy-2-oxo-1-triazene; 2-DE, two-dimensional electrophoresis.

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