ABSTRACT
The forkhead family members of transcription factors (FoxOs) are expected to be potential cancer-related drug targets and thus are being extremely studied recently. In the present study, FoxO3a, one major member of this family, was identified to be down-regulated in colorectal cancer through micro-array analysis, which was confirmed by RT-PCR and Western blot in 28 patients. Moreover, immunohistochemistry (IHC) showed that the expression levels of FoxO3a were remarkably reduced in 99 cases of primary colorectal cancer, liver metastasis, and even in metaplastic colorectal tissue. IHC also revealed an exclusion of FoxO3a from the nucleus of most cells of tumor-associated tissues. Silencing FoxO3a by siRNA led to elevation of G2-M phase cells. We conclude that the downregulation of FoxO3a may greatly contribute to tumor development, and thus FoxO3a may represent a novel therapeutic target in colorectal cancer.
ABSTRACT
The forkhead family members of transcription factors (FoxOs) are expected to be potential cancer-related drug targets and thus are being extremely studied recently. In the present study, FoxO3a, one major member of this family, was identified to be down-regulated in colorectal cancer through micro-array analysis, which was confirmed by RT-PCR and Western blot in 28 patients. Moreover, immunohistochemistry (IHC) showed that the expression levels of FoxO3a were remarkably reduced in 99 cases of primary colorectal cancer, liver metastasis, and even in metaplastic colorectal tissue. IHC also revealed an exclusion of FoxO3a from the nucleus of most cells of tumor-associated tissues. Silencing FoxO3a by siRNA led to elevation of G2-M phase cells. We conclude that the downregulation of FoxO3a may greatly contribute to tumor development, and thus FoxO3a may represent a novel therapeutic target in colorectal cancer.
Subject(s)
Female , Humans , Male , Cell Cycle Checkpoints , Colon , Metabolism , Pathology , Colorectal Neoplasms , Metabolism , Pathology , Down-Regulation , Forkhead Box Protein O3 , Forkhead Transcription Factors , Metabolism , Liver Neoplasms , Metabolism , Pathology , Metaplasia , Metabolism , Pathology , Rectum , Metabolism , Pathology , Tumor Cells, CulturedABSTRACT
<p><b>OBJECTIVE</b>To elucidate the effect of hSav1 expression on Mst1-mediated apoptosis in HeLa cells.</p><p><b>METHODS</b>Plasmids pCMV-HA-hSav1 and pcDNA/4TO-Flag-Mst1 were constructed and cotransfected into HeLa cells. Triple immunofluorescent labeling of hSav1, Mst1 and nucleus was performed to determine their subcellular localization. Plasmids pCMV-HA-hSav1 and/or pcDNA/4TO-Flag-Mst1 were transfected into HeLa cells, and 36 hours later cisplatin (50 micromol/L) as a pro-apoptotic agent was added for 14 hours. Cell apoptosis was analyzed by annexin V/PI assay.</p><p><b>RESULTS</b>Plasmids pCMV-HA-hSav1 and pcDNA/4TO-Flag-Mst1 were constructed and the authenticity of constructs was verified by sequencing. The binding in vitro showed that hSav1 could be detect from the anti-Mst1 immunoprecipitation complex. The immunofluorescent labeling showed that hSav1 and Mst1 had the same localization in cells. Overexpressed protein hSav1 did not induce a significant cell apoptosis. However, co-expression of hSav1 with Mst1 resulted in a significant increase of apoptosis above the level seen with Mst1 alone (24.5% +/- 2.4% vs. 39.3% +/- 4.0%, P < 0.05).</p><p><b>CONCLUSION</b>Our findings indicate that hSav1 is a newly identified protein that interacts with Mst1 and augments Mst1-mediated apoptosis.</p>
Subject(s)
Humans , Apoptosis , Cell Cycle Proteins , Genetics , Metabolism , Cytoplasm , Metabolism , HeLa Cells , Hepatocyte Growth Factor , Genetics , Metabolism , Plasmids , Proto-Oncogene Proteins , Genetics , Metabolism , TransfectionABSTRACT
<p><b>OBJECTIVE</b>To explore a method which can remove the gastric mucus in order to prepare mucous membrane single cell suspension for the research of cytomics.</p><p><b>METHODS</b>Enzymology was used to remove the mucus gel and to separate mucous layer from the normal fresh gastric tissue. The mucous layer was broken to prepare single cell suspension with machine method. Expression of major cyclins in mucous layer cells was examined by cytoimmunochemistry, flow cytometry(FCM) and confocal microscopy.</p><p><b>RESULTS</b>The 0.1% pepsin could dissolve the mucus gel and 1.2-2.4 U/L dispase could separate the mucous layer completely. The single mucous cell suspension was prepared successfully. FCM results from mucous single cell suspension revealed that expression of cyclin D(3), B(1) was obvious, that of cyclin D(2) was weak and that of cyclin D(1), A, E was the least. Similar results were found with confocal microscopy.</p><p><b>CONCLUSIONS</b>Single cell suspension from mucous layer can be easily prepared by pepsin and dispase. Cyclins schedule expression in vivo is different from cyclins schedule expression in vitro.</p>