Blocking gastrin and CCK-B autocrine loop affects cell proliferation and apoptosis in vitro

Abstract

Gastrin and cholecystokinin-B receptor (CCK-B) were co-expressed in human gastric carcinoma tissues, suggesting that a functional autocrine loop, the gastrin and CCK-B receptor loop, may be presented in gastric cancer cells and play an important role in the pathogenesis and progression of gastric carcinomas. The present study was aimed at studying the effects of blocking the gastrin and CCK-B receptor loop on cell proliferation and apoptosis in gastric cancer cell line SGC-7901 cells (SGC-7901 cells). First, the expression of gastrin and CCK-B receptor mRNAs and gastrin protein in SGC-7901 cells were measured by RT-PCR and immunocytochemistry, respectively. Radioimmunoassay (RIA) was used to detect the concentrations of gastrin in culture medium. The gastrin-CCK-B receptor axis was blocked by using a specific neutralizing antibody against human gastrin and siRNA specifically targeting human CCK-B receptors, respectively. Flow cytometry was used to measure the cell cycle and apoptotic cells, and western blotting was used to measure the expression of CCK-B receptor, caspase-3, and matrix metalloproteinase-2 (MMP-2) in cells. The results showed that SGC-7901 cells not only coexpressed gastrin and CCK-B receptor mRNAs, but also endogenously secreted gastrin protein into the culture medium, thus forming gastrin-CCK-B receptor autocrine loop. Biologically, disrupting gastrin-CCK-B receptor autocrine loop by neutralizing the endogenous gastrin or by knocking down CCK-B receptor expression significantly inhibited the cell proliferation and decreased the percentage of cells residing in the S-phase of the cell cycle, and meanwhile promoted cell apoptosis and increased caspase-3 expression as well as decreased MMP-2 expression. An autocrine loop between endogenously secreted gastrin and CCK-B receptors may play a key role in the regulation of cell proliferation and apoptosis in SGC-7901 cells.