cyclin-d1 and Insulinoma

Insulinoma-associated protein 1 (INSM1), a zinc finger transcriptional factor, is proven to be deregulated in several types of cancers. However, comprehension of the molecular mechanism of INSM1-mediated tumor progression remains poor. Here, we show that the radioresistant nasopharyngeal carcinoma (NPC) patients have higher expressions of INSM1 that correlated with poor prognosis. Genetic manipulation of INSM1 expression sufficiently controls the response of NPC cells to irradiation (IR). Mechanistically, cells exposed to IR, increased intracellular INSM1 competitively disrupts the interaction of cyclin D1 and CDK4 resulting in cell survival by the cyclin D1-dependent DNA repair machinery. Moreover, knockdown of INSM1 sensitives NPC cells to IR in vivo and protects xenograft mice from mortality. Taken together, these results indicate that INSM1 modulates NPC to radiotherapy by controlling cyclin D1-dependent DNA repair machinery that could be manipulated as a novel molecular target for NPC therapy.

Topics: Animals; Biopsy; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase 4; DNA Repair; Female; Gene Expression Regulation, Neoplastic; Heterografts; Humans; Insulinoma; Male; Mice; Nasopharyngeal Carcinoma; Radiation Tolerance; Repressor Proteins

Up-regulation of phosphatidylinositol-3-kinase (PI3K)-AKT signaling facilitates tumor cell growth and inhibits cell demise. The AKT-pathway also plays an important role in cytostatic therapy resistance and response to hypoxia and angiogenesis. Using real-time cell proliferation assay we examined the potency of triciribine in three distinct neuroendocrine gastrointestinal tumor cell lines. Also we investigated triciribine's induction of apoptosis and effects on a broad range of cancer-associated gene products. Furthermore, we characterized the role of PTEN as a possible predictor of sensitivity to triciribine in GEP-NETs. We also looked for additive anti-neoplastic effects of triciribine when combined with conventional cytostatic drugs or other targeted drugs, affecting different molecules of the PI3K-AKT-pathway and we assessed the potency of triciribine to inhibit tumor growth in vivo, by using the chick chorioallantoic membrane assay. Treatment of insulinoma (CM) or gut neuroendocrine tumor cells (STC-1) with triciribine significantly reduced tumor cell growth by 59% and 65%, respectively. By contrast, the highly expressing PTEN carcinoid cell line BON did not respond, even at higher doses. Combinations of triciribine with classic cytostatic drugs as well as drugs targeting other molecules of the PI3K-AKT-pathway led to synergistic anti-proliferative effects. Additional in vivo-evaluations confirmed the anti-neoplastic potency of triciribine. Thus, our data show that inhibition the AKT-pathway potently reduces the growth of GEP-NET cells alone or in combination therapies. AKT inhibition may provide a rationale for future evaluations.

Topics: Aging; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Gastrointestinal Neoplasms; Gene Expression Regulation, Neoplastic; Humans; Insulinoma; Neuroendocrine Tumors; Pancreatic Neoplasms; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Ribonucleosides; Signal Transduction

Regenerating gene (Reg) family protein Reg3alpha is normally expressed in pancreatic acinar and endocrine cells. In order to explore its effect on islet beta-cell replication, insulinoma MIN6 cells were stably transfected with murine Reg3alpha cDNA. Determined using real-time PCR and Western blots, the levels of Reg3alpha mRNA and protein in Reg3alpha-transfected clones were increased 10- and 6-fold, respectively. Western blots also revealed that the protein was released into the culture medium, consistent with an endocrine effect. In MTT cell proliferation assay, Reg3alpha-overexpressing cells exhibited a 2-fold increase in the rate of cell growth. In order to investigate the intracellular mechanism, we studied cell cycle regulatory proteins. In Reg3alpha-expressing cells, we detected 2.2- and 2.5-fold increased levels of cyclin D1 and CDK4, respectively, which paralleled a 1.8-fold increase in the rate of Akt phosphorylation. It is established that beta-cell replication is associated with increased cyclin D1 and CDK4 levels; deficiency in CDK4 or cyclin D2 results in reduced beta-cell mass and diabetes. Our results suggest that Reg3alpha stimulates beta-cell replication, by activating Akt kinase and increasing the levels of cyclin D1/CDK4.

Topics: Animals; Antigens, Neoplasm; Biomarkers, Tumor; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Insulin-Secreting Cells; Insulinoma; Lectins, C-Type; Mice; Pancreatitis-Associated Proteins; Phosphorylation; Proteins; Proto-Oncogene Proteins c-akt

Rodent insulinoma cell lines may serve as a model for designing continuously replicating human beta-cell lines and provide clues as to the central cell cycle regulatory molecules in the beta-cell.. We performed a comprehensive G1/S proteome analysis on the four most widely studied rodent insulinoma cell lines and defined their flow cytometric profiles and growth characteristics.. 1) Despite their common T-antigen-derived origins, MIN6 and BTC3 cells display markedly different G1/S expression profiles; 2) despite their common radiation origins, RINm5F and INS1 cells display striking differences in cell cycle protein profiles; 3) phosphorylation of pRb is absent in INS1 and RINm5F cells; 4) cyclin D2 is absent in RINm5F and BTC3 cells and therefore apparently dispensable for their proliferation; 5) every cell cycle inhibitor is upregulated, presumably in a futile attempt to halt proliferation; 6) among the G1/S proteome members, seven are pro-proliferation molecules: cyclin-dependent kinase-1, -2, -4, and -6 and cyclins A, E, and D3; and 7) overexpression of the combination of these seven converts arrested proliferation rates in primary rat beta-cells to those in insulinoma cells. Unfortunately, this therapeutic overexpression appears to mildly attenuate beta-cell differentiation and function.. These studies underscore the importance of characterizing the cell cycle at the protein level in rodent insulinoma cell lines. They also emphasize the hazards of interpreting data from rodent insulinoma cell lines as modeling normal cell cycle progression. Most importantly, they provide seven candidate targets for inducing proliferation in human beta-cells.

Topics: Animals; Blotting, Western; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin A; Cyclin D1; Cyclin E; Cyclin-Dependent Kinases; Flow Cytometry; G1 Phase; Gene Expression Regulation, Neoplastic; Humans; Insulin-Secreting Cells; Insulinoma; Islets of Langerhans; Mice; Mice, Inbred C57BL; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; S Phase; Transfection

Sex-determining region Y-box (SOX) 6 negatively regulates glucose-stimulated insulin secretion from beta-cells and is a down-regulated transcription factor in the pancreatic islet cells of hyperinsulinemic obese mice. To determine the contribution of SOX6 to insulin resistance, we analyzed the effects of SOX6 on cell proliferation. Small interfering RNA-mediated attenuation of SOX6 expression stimulated the proliferation of insulinoma INS-1E and NIH-3T3 cells, whereas retroviral overexpression resulted in inhibition of cell growth. Quantitative real time-PCR analysis revealed that the levels of cyclin D1 transcripts were markedly decreased by SOX6 overexpression. Luciferase-reporter assay with beta-catenin showed that SOX6 suppresses cyclin D1 promoter activities. In vitro binding experiments showed that the LZ/Q domain of SOX6 physically interacts with armadillo repeats 1-4 of beta-catenin. Furthermore, chromatin immunoprecipitation assay revealed that increased SOX6 expression significantly reduced the levels of acetylated histones H3 and H4 at the cyclin D1 promoter. By using a histone deacetylase (HDAC) inhibitor and co-immunoprecipitation analysis, we showed that SOX6 suppressed cyclin D1 activities by interacting withbeta-catenin and HDAC1. The data presented suggest that SOX6 may be an important factor in obesity-related insulin resistance.

Topics: Animals; beta Catenin; Cell Division; Cell Line, Tumor; Cyclin D1; DNA-Binding Proteins; Down-Regulation; High Mobility Group Proteins; Histone Deacetylase 1; Histone Deacetylases; Histones; Humans; Hyperinsulinism; Insulin Resistance; Insulin-Secreting Cells; Insulinoma; Kidney; Leucine Zippers; Mice; NIH 3T3 Cells; Obesity; Pancreatic Neoplasms; Promoter Regions, Genetic; Protein Structure, Tertiary; Rats; SOXD Transcription Factors; Transcription Factors; Transduction, Genetic

To investigate the effect of baicalin on the proliferation of insulinoma cell line and the molecular mechanism involved.. Such methods as light microscope, MTT assay, flow cytometry and Western blotting were applied to investigate the effects of baicalin (0, 100, 200, and 400 microg/ml baicalin treated for 24 h or 200 microg/ml baicalin treated at different time points) on the cell proliferation, cell survival rate, the cell cycle and related molecular mechanisms.. The number of proliferating cells obviously decreased with the increase of baicalin under the light microscope, and the survival rate of cells decreased as determined by MTT assay. After being treated with baicalin, the number of insulinoma cells in S-phase obviously decreased from 38.2% (0 microg/ml) to 9.4% (400 microg/ml), and the number of cells in phase G1 increased from 56.4% (0 microg/ml) to 85.9% (400 microg/ml). In the meantime, the expression of cyclin D1 was obviously declined by Western blotting.. Baica-lin can inhibit the proliferation of insulinoma cells, and the down-regulation of the expression of cyclin D1 might also be involved in these events.

Topics: Antineoplastic Agents, Phytogenic; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Flavonoids; Humans; Insulinoma; Pancreatic Neoplasms