05. Figure 2 Immunohistochemical detection of GKN1 protein in gastric tissue specimens. Paraffin sections were immunostained with anti-GKN1 antibody and reviewed for GKN1 levels. GKN1 progressively decreased from normal gastric mucosa, atrophic gastritis, intestinal metaplasia, and dysplasia to gastric cancer. A: normal gastric mucosa; B: atrophic gastritis; C: intestinal metaplasia; D: dysplasia; E, gastric cancer; F, the corresponding distant non-cancerous tissue. Transfection
of GKN1 reduced gastric cell proliferation Next, we determined whether restoration of GKN1 expression would suppress gastric cancer AGS cells viability. To this end, we generated AGS cells that stably expressed GKN1 expression was confirmed by RT-PCR and Weston blotting. Cell viability (MTT) assays showed that AGS cells stably expressing GKN1 grew at click here a much slower rate compared to the vector-transfected control cells in both 24 hour and 48 hour cultures (Figure 3). This data clearly indicate Quizartinib concentration that restoration of GKN1 expression inhibits AGS cell proliferation. Figure 3 Suppression of cancer cell viability by GKN1. The GKN1 or vector transfected gastric cancer cells were grown and subjected to MTT assay. The data showed that viability of AGS cells with GKN1 transfection was significantly decreased compared to the cells with vector transfection in 24 h (74.6%) and 48 h
(71.7%). Effect of GKN1 on AGS cell apoptosis and cell cycle re-distribution We examined whether inhibition of cell proliferation by GKN1 was due to the induction of apoptosis. To this end, we examined the levels of apoptotic cells using flow cytometry, and found that compared to the vector transfected cells, GKN1 transfected AGS cells were apoptotic (Figure 4A). The TUNEL assay demonstrated that endogenous GKN1 significantly induced apoptosis in AGS cells, and examination of morphology demonstrated that the nuclei of GKN1 transfected tumor cells exhibited condensation and fragmentation RVX-208 (Figure 4B). Figure 4 Apoptosis induction of gastric cancer cell
by GKN1. A: Flow cytometric assay. The GKN1 or vector transfected gastric cancer AGS cells were grown and subjected to flow cytometry assay for detection of apoptosis; B: TUNEL assay. The GKN1 or vector transfected gastric cancer cells were grown on glass slides and then subjected to TUNEL assay. Next, we examined cell cycle changes in these tumor cells, because suppression of cell viability is closely related to regulation of the cell cycle. Olomoucine, a purine derivative, is a cyclin-dependent kinase (CDK) inhibitor, thus we used it to enrich parental AGS cells in the G1 phase. Specifically, cells were arrested in the cell cycle with 1 h olomoucine treatment and continued to incubate for another 1 h without olomoucine. The cell cycle distribution of GKN1 transfected cells Selleck SHP099 changed from 41.9% of G1 and 35.0% of S phase to 41.