Comparative proteomic analysis of the ribosomes in 5-fluorouracil resistance of a human colon cancer cell line using the radical-free and highly reducing method of two-dimensional polyacrylamide gel electrophoresis.

Many auxiliary functions of ribosomal proteins (r-proteins) have received considerable attention in recent years. However, human r-proteins have hardly been examined by proteomic analysis. In this study, we isolated ribosomal particles and subsequently compared the proteome of r-proteins between the DLD-1 human colon cancer cell line and its 5-fluorouracil (5-FU)-resistant sub-line, DLD-1/5-FU, using the radical-free and highly reducing method of two-dimensional polyacrylamide gel electrophoresis, which has a superior ability to separate basic proteins, and we discuss the role of r-proteins in 5-FU resistance. Densitometric analysis was performed to quantify modulated proteins, and protein spots showing significant changes were identified by employing matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry. Three basic proteins (L15, L37 and prohibitin) which were significantly modulated between DLD-1 and DLD-1/5-FU were identified. Two proteins, L15 and L37, showed down-regulated expression in DLD-1/5-FU in comparison to DLD-1. Prohibitin, which is not an r-protein and is known to be localized in the mitochondria, showed up-regulated expression in DLD-1/5-FU. These 3 proteins may be related to 5-FU resistance.

Proteomic analysis of the basic proteins in 5-fluorouracil resistance of human colon cancer cell line using the radical-free and highly reducing method of two-dimensional polyacrylamide gel electrophoresis.

5-Fluorouracil (5-FU) is widely used for the treatment of patients with advanced colon cancers and it is the mainstay of chemotherapy. However, the acquisition of resistance to 5-FU is one of the most prominent obstacles to successful chemotherapy. The purpose of this study was to identify the novel biological basis of 5-FU resistance in colon cancer cells. This study is the first comparative proteomic analysis of basic proteins between the DLD-1 human colon cancer cell line and DLD-1/5-FU its 5-FU resistant sub-line using the radical-free and highly reducing method of two-dimensional polyacrylamide gel electrophoresis, which has a superior ability in the separation of basic proteins and the quantification of post-translational modification. A densitometric analysis was performed to quantify the modulated proteins, and protein spots showing significant changes were identified by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry. Six basic proteins significantly modulated between DLD-1 and DLD-1/5-FU were identified. All of them showed up-regulated expression in DLD-1/5-FU in comparison to DLD-1. The six identified spots, corresponding to five different proteins included heterogeneous nuclear ribonucleoprotein G, mitochondrial transcription factor A, histone H2B, histone H4 and ribosomal protein L3. Among the 5 basic proteins, several proteins are potentially related to 5-FU resistance by protecting the cells from DNA damage.