Discovery of tumor markers for gastric cancer by proteomics

Abstract

Gastric cancer (GC) has a high rate of morbidity and mortality among various cancers worldwide. The development of noninvasive diagnostic methods or technologies for tracking the occurrence of GC is urgent, and searching reliable biomarkers is considered.This study intended to directly discover differential biomarkers from GC tissues by two-dimension-differential gel electrophoresis (2D-DIGE), and further validate protein expression by western blotting (WB) and immunohistochemistry (IHC).Pairs of GC tissues (gastric cancer tissues and the adjacent normal tissues) obtained from surgery was investigated for 2D-DIEG.Five proteins wereconfirmed by WB and IHC, including glucose-regulated protein 78 (GRP78), glutathione s-transferase pi (GSTpi), apolipoprotein AI (ApoAI), alpha-1 antitrypsin (A1AT) and gastrokine-1 (GKN-1). Among the results, GRP78, GSTpi and A1ATwere significantlyup-regulated and down-regulated respectively in gastric cancer patients. Moreover, GRP78 and ApoAI were correlated with A1AT for protein expressions.This study presumes these proteins could be candidates of reliable biomarkers for gastric cancer.

Figure 1. The 2D-DIGE results paired tissue samples.

The differential proteins presented on gels. (left): normal; (right): cancer. Thirty-six proteins were picked by DeCyder statistical software, but only fifteen proteins were identified by PMF or PFF technique. The gel conditions: pI: 4–7; gel: 4–12%.The analytical ranges were from pI 4 to 7 forisoelectric focusing and from 4% to 12% gradient gel for sodium dodecyl sulfate- polyacrylamide gelelectrophoresis.

Figure 2. The detailed comparison between normal and tumorous tissue from 2D-DIGE.

The selected putative proteins were presented as stereopictures and enlarged scales gel images. These proteins were selected according to the significant difference below 0.05 (p<0.05). In addition, the duplicated identification of ATPB, PDIRP5, ApoAI and GSTpi on adjacent spots indicated that they had a different modification.

Figure 3

(A) By Western blotting, 12 paired of gastric cancer and normal tissues was used to validate the putative proteins including GRP78, GSTpi, A1AT, ApoAI and GKN-1. (B) The GSTpi and GRP78 were significantly over-expressed in gastric cancer tissues.Down-expressions of A1AT, ApoAI and GKN-1 were noted. **p<0.01. *p<0.05.

Figure 4. The correlations between clinical stages of gastric cancer and the expressions of proteins.

Although GRP78 and GSTpi were increased in different stages of gastric cancer, no statistical significance could be found. A trend of increase with clinical stage was found in GRP78.

Figure 5. The correlation among the selected proteins.

The GRP78 in these 12 pairs of tissues was expressed correlatively with that of ApoAI (r2: −0.61) and A1AT (r2: −0.49) individually. Meanwhile, ApoAI was expressed correlatively with that of A1AT (r2: 0.62). However, GSTpi and GKN-1 seemed to be independent. **p<0.01. *p<0.05.

Figure 6. Immunohistochemistric stains were used to identify the location of the putative proteins.

The up-regulated proteins, GRP78 and GSTpi, were specifically located on the gastric adenocarcinoma cells. Contrarily, the down-regulated proteins including ApoAI and A1AT were present on the normal gastric glands. Another down-regulated protein, GKN-1, was shown to appear on the mucosa of gastric tissue.