Gene Expression of GSK3, TGF ?-1 and PI3 Kinase in Oral Squamous Cell Carcinoma – A Real Time PCR Based Approach

Background: Oral squamous cell carcinoma (OSCC) is the most predominant type of oral cancer which has a poor prognosis, with 5-year survival rates less than 50%. Clinical characteristics such as tumor position, TNM classification and method of treat- ment, as well as histological grades have all been studied as OSCC prognostic factors but evaluating the genetic expression is the evolving trend in early diagnosis. Aim: To compare the gene expression of TGF-?-1, GSK3, Pi3 kinase in OSCC and normal tissue samples and to correlate the expression levels of these molecules with the pathological grading and survival in OSCC patients. Also to understand the role of GSK3 in Pi3 kinase pathway and TGF-? signaling pathway in OSCC progression thereby attempting targeted therapy in OSCC patients. Materials and Methods: 10 OSCC samples as well as normal healthy samples were collected and RNA isolation was done us- ing RNA easy kit from Qiagen (Valencia, CA), and thensubjected to cDNA synthesis using Human TGF-?1, Human GSK3? and Human Pi3 kinase primers. Real time PCR was performed using gene specific primers at 40 cycles. The results were retrieved, tabulated and analyzed. Results: The current research results revealed that there were up regulation of mRNA expression in GSK3, TGF ?-1 and Pi3 kinase in OSCC patients than in healthy individuals. On comparison, Pi3 kinase showed highest mRNA expression levels than GSK3 and TGF ?-1. Conclusion: The expression of GSK3 and its role in activation of Pi3 kinase pathway plays a crucial role in progression of oral cancer and targeting GSK3?could be a novel and targeted approach for treating OSCC.

Insulin Signaling Network in Cancer.

The primary function of insulin is viewed as a hormone that controls blood glucose level. However, there is growing evidence that aberrant insulin level and insulin-mediated signaling can lead to cancer development and progression. The insulin-cancer relationship has stemmed from various observational and epidemiological studies, which linked higher incidence of cancer with central obesity, type II diabetes and other conditions associated with increased levels of circulating insulin, insulin resistance and hyperinsulinemic states. Increased risk of developing a range of cancers is also seen with a certain treatment options used to lower blood glucose level in diabetic patients. While metformin monotherapy has the lowest risk of developing cancer, in comparison, treatment with insulin or insulin secretagogues shows more likelihood to develop solid cancers. Cellular signaling initiated by insulin provides a clue regarding these diverse cellular outcomes. This review discusses how the insulin enacts such diverse physiological effects and the insulin-cancer relationship, with focus on the role of insulin signaling in cancer.

Effect of PI3K Inhibitor on Proliferation of Human Glomerular Mesangial Cells / 天津医药

Objective To investigate the effect of LY294002 on proliferation of cultured human glomerular mesan-gial cells, and to study on the mechanism underlying this proliferation. Methods Different concentrations of LY294002 (0.02,0.2,2,20,100 mg/L) were administrated to the cultured human glomerular mesangial cells. The effects of mesangial cell proliferation were measured using CCK-8 colorimetric assay. Under appropriate concentrations, proliferation of cultured mesangial cells were measured using CCK-8 at 0.5, 1, 24, 48 hours of drug administration time. Results LY294002(0.02 mg/L)didn’t obviously inhibited the growth of mesangial cells(P>0.05), the inhibition rate was 5.05%. The effect of LY294002 on the cells decreased with rising concentration (0.2 to 20 mg/L) in a dose-dependent manner (P0.05), but inhibited proliferation gradually from 1 to 48 h (P<0.05). Conclu-sion Within the certain range of concentration and time, the LY294002 inhibits the proliferation of human glomerular me-sangial cells, and PI3K/Akt/mTOR signaling pathway may regulate the proliferation of human mesangial cell.

Gecko Proteins Exert Anti-Tumor Effect against Cervical Cancer Cells Via PI3-Kinase/Akt Pathway

Anti-tumor activity of the proteins from Gecko (GP) on cervical cancer cells, and its signaling mechanisms were assessed by viable cell counting, propidium iodide (PI) staining, and Western blot analysis. GP induced the cell death of HeLa cells in a dose-dependent manner while it did not affect the viability of normal cells. Western blot analysis showed that GP decreased the activation of Akt, and co-administration of GP and Akt inhibitors synergistically exerted anti-tumor activities on HeLa cells, suggesting the involvement of PI3-kinase/Akt pathway in GP-induced cell death of the cancer cells. Indeed, the cytotoxic effect of GP against HeLa cells was inhibited by overexpression of constituvely active form of Akt in HeLa cells. The candidates of the functional proteins in GP were analyzed by Mass-spectrum. Taken together, our results suggest that GP elicits anti-tumor activity against HeLa cells by inhibition of PI3-kinase/Akt pathway.

Brain Tumor Stem Cells as Therapeutic Targets in Models of Glioma

At this time, brain tumor stem cells remain a controversial hypothesis while malignant brain tumors continue to present a dire prognosis of severe morbidity and mortality. Yet, brain tumor stem cells may represent an essential cellular target for glioma therapy as they are postulated to be the tumorigenic cells responsible for recurrence. Targeting oncogenic pathways that are essential to the survival and growth of brain tumor stem cells represents a promising area for developing therapeutics. However, due to the multiple oncogenic pathways involved in glioma, it is necessary to determine which pathways are the essential targets for therapy. Furthermore, research still needs to comprehend the morphogenic processes of cell populations involved in tumor formation. Here, we review research and discuss perspectives on models of glioma in order to delineate the current issues in defining brain tumor stem cells as therapeutic targets in models of glioma.

Emerging Molecular Targets for the Treatment of Asthma.

Current therapeutic approaches for the treatment of asthma have limitations in their ability to target all the features of the disease. Indeed, existing pharmacological asthma therapies are based on decades old strategies that were developed prior to the rapid growth in knowledge stemming from cell and molecular biology in the past decade. Thus, there is an unmet need for developing new drugs to target these features along with improved efficacy and safety. In the present review, the limitations of prevalent pharmacological asthma therapy are discussed briefly, and some explanations are suggested as to why new therapeutic targets are required to treat asthma, and finally directions for novel asthma therapies are proposed.

Apoptotic effect in combination of Cyclosporin A and Taxol on oral squamous cell carcinoma cell line through the PI-3 kinase/Akt1 Pathway

Oral cancer take up 2-6% of all carcinomas and squamous cell carcinoma, which is the most common type in oral cancer, has a poor prognosis due to its high metastasis and recurrence rates. In treating oral cancer, chemotherapy to the primary, metastasized and recurrent lesion is a very important and useful treatment, even though its widespread usage is limited due to high general toxicity and local toxicity to other organs. Taxol, a microtubule stabilizing agent, is an anticancer drug that induces cell apoptosis by inhibiting depolymerization of microtubules in between the metaphase and anaphase of the cell mitosis. Recently, its effectiveness and mechanism on various tumor has been reported. However, not much research has been done on the application of Taxol to oral squamous cell carcinoma. Cyclosporin A, which is an immunosuppressant, is being used on cancers and when co-administered with Taxol, effectiveness of Taxol is enhanced by inhibition of Taxol induced multidrug resistance. In this study, Cyclosporin A with different concentration of Taxol was co-administered to HN22, the oral squamous cell carcinomacell line. To observe the cell apoptosis and the mechanisms that take part in this process, mortality evaluation of tumor cell using wortmannin, c-DNA microarray, RT-PCR analysis, cytometry analysis and western blotting were used, and based upon the observation on the effect and mechanism of the agent, the following results were obtained: 1. The HN22 cell line viability was lowest when 100micrometer of Wortmannin and 5microgram/ml of Taxol were co-administered, showing that Taxol participates in P13K-AKT1 pathway. 2. In c-DNA microarray, where 1microgram/ml of cyclosporine A and 3mg/ml of Taxol were co-administered, no up regulation of AKT1, PTEN and BAD c-DNA that participate in cell apoptosis was observed. 3. When 1microgram/ml of Cyclosporin A was applied alone to HN22 cell line,no difference was found in AKT1, PTEN and BAD mRNA expression. 4. Increased AKT1, mRNA expression was observed when 3microgram/ml of Taxol was applied alone to HN22 cell line. 5. When 1microgram/ml of Cyclosporin A and Taxol (3microgram/ml and 5microgram/ml) were co-administered to HN22 cell line, PTEN mRNA expression increased, whereas AKT1 and BAD mRNA decreased. 6. As a result of cytometry analysis, in the group of Cyclosporin A(1microgram/ml) and Taxol(3microgram/ml) co-administration, increased Annxin V was observed, which shows that apoptosis occurred by deformation of plasma membrane. However, no significant difference was observed with varying concentration. 7. In western blot analysis, no caspase 3 was observed in the group of Cyclosporin A(1microgram/ml) and Taxol(3microgram/ml) co-administration. From the results of this study, it can be concluded that synergistic effect can be observed in combination therapy of Taxol and Cyclosporin A on oral squamous cell carcinoma cell line, where decreased activity of the cell line was observed. This resulted in decreased AKT1 and BAD mRNA and increased PTEN mRNA expression and when wortmannin and Taxol were co-administered, the viability decreased which confirms that Taxol decreases the viability of tumor cell line. Hence, when Taxol and cyclosporine A are co-administered, it can be assumed that cell apoptosis occurs through AKt1 pathway.

Phosphatidylinositol-3-kinase Inhibitor Enhances Nitric Oxide Synthesis and Apoptosis in LPS-Stimulated Macrophages / 대한해부학회지

Phosphatidylinositol-3-kinase (PI3-kinase) is critical player in cell proliferation and death. The mechanism by which bacterial lipopolysaccharide (LPS) induces synthesis of nitric oxide (NO) in murine macrophages is incompletely understood. In this study we examined the effects of wortmannin and LY294002, two inhibitors of PI3-kinase, on the induction of NO synthesis and NO-mediated apoptosis in LPS-stimulated macrophages. Treatment of wortmannin and LY294002 markedly potentiated NO synthesis and inducible NO synthase (iNOS) expression in LPS-stimulated cells. Furthermore, wortmannin also enhanced apoptosis in LPS-stimulated cells. These results suggest that PI3-kinase might play an important role in transducing the signal that involved in LPS-induced macrophage activation.

The Effects of Wortmannin on Leukemia Cells Proliferation Cycle and BCL-2 Expression / 中国医师杂志

Objective To investigate the effects of wortmannin (WT) on leukemia cells cycle and BCL-2 protein expression. Methods 0 25?mol/L PI-3 kinase inhibitor wortmanin was used to treat K562 cells for 24 hours, and Arac was simultaneously used to induce the cell apoptosis. The specific fluoresent staining and FCM analysis were adopted to measure the cell cycle distribution and BCL-2 expression. Results Compared with the control cells, the proliferation index(PI) of the K562 cells treated by wortmannin was significantly lower, the cell number of G1 phase and the percentage of cell apoptosis increased, and the BCL-2 protein expression significantly decreased. Conclusion Wortmannin could inhibitedtheproliferationofK5 6 2cells ,andwascellcyclespecificagent (CCSA) .

Role of Shc and phosphoinositide 3-kinase in heregulin-induced mitogenic signaling via ErbB3

ErbB3/HER3 is a cell surface receptor which belongs to the ErbB/HER subfamily of receptor protein tyrosine kinases. When expressed in NIH/3T3 cells, ErbB3 can form heterodimeric coreceptor with endogenous ErbB2. Among known intracellular effectors of the ErbB2/ErbB3 are mitogen-activated protein kinase (MAPK) and phosphoinositide (PI) 3-kinase. In the present study, we studied relative contributions of above two distinct signaling pathways to the heregulin-induced mitogenic response via activated ErbB3. For this, clonal NIH-3T3 cell lines expressing wild-type ErbB3 and ErbB3 mutants were stimulated with heregulin beta1. While cyclin D1 level was markedly high and further increased by treatment of heregulin in cells expressing wild-type ErbB3, the elimination of either Shc binding or PI 3-kinase binding lowered both levels. This result was supported by the reduction of cyclin D1 expression by preteatment with MAPK kinase inhibitor or PI 3-kinase inhibitor before stimulation with heregulin. In accordance with the cyclin D1 expression, elimination of either Shc binding or PI 3-kinase binding reduced the heregulin-induced DNA synthesis and cell growth rate. Our results obtained by the comparison of wild-type and ErbB3 mutants indicate that the full induction of the cell cycle progression through G1/S phase by ErbB3 activation is dependent on both Shc/MAPK and PI 3-kinase signal transduction pathways.

Apoptotic Cell Death Induced by Disruption of Epithelial Cell and Extracellular Matrix Interactions in Mouse Inner Medullary Collection Duct Cells / 대한신장학회잡지

Cell-matrix interactions have major effects upon phenotypic features such as gene regulation, cytoskeletal structure, differentiation, and aspects of cell growth control. Upon detachment from the matrix epithelial cells enter into programmed cell death and this cell detachment-induced apoptosis has been referred to as "anoikis". This study was undertaken to determine whether apoptosis is induced by inhibition of contact with extracellular matrix in mouse inner medullary collecting duct cells (mIMCD-3), what are signaling mechanisms of the process and whether EGF protects detachment-induced apoptosis. Upon detachment from the extracellular matrix, MDCK and mIMCD-3, which were derived from inner medulla of SV40 transgenic mouse, entered into programmed cell death as a time-dependent manner. Apoptotic cell death induced by cell detachment increased in serum-free medium, which was partly protected by the addition of epidermal growth factor (EGF). Ly294002 and wortmannin, inhibitors of phosphatidylinositol 3-kinase (PI 3-kinase), negated the EGF effect, whereas PD98059, a MEK inhibitor, did not. The addition of SB203580, an inhibitor of p38 kinase, did not protect apoptosis in suspended mIMCD-3 cells. These results indicate that apoptosis is induced by inhibition of contact with extracellular matrix in mouse inner medullary collecting duct cells and that PI 3-kinase, not MAPK, is a key mediator of the EGF-induced survival of renal epithelial cells in the absence of attachment.

The regulatory mechanism of phosphatidylinositol 3-kinase by insulin in 3T3 L1 fibroblasts: phosphorylation-independent activation of phosphatidylinositol 3-kinase

Phosphatidylinositol (PI) 3-kinase plays an important role in transducing the signals of various growth factor receptors. However, the regulatory mechanism of PI3-kinase activity by these growth factor receptors is not completely understood. Therefore, we attempted to clarify the regulatory mechanism of PI3-kinase using insulin and 3T3 L1 fibroblasts. Our results showed that insulin stimulated PI3-kinase activity seven-fold and concomitantly phosphorylated a p85 subunit at the tyrosine residue. However, this tyrosine phosphorylation was not significant in the activation of PI3-kinase as the PI3-kinase pulled down by the overexpressed GST-p85 fusion protein showed as high an activity as the immunoprecipitated one. The p110 subunit was phosphorylated at both serine and tyrosine residues without insulin treatment. Since the phosphorylation state was not changed by insulin. The results suggested that phosphorylation of the p110 subunit does not control PI3-kinase activity. Finally, it was shown that the insulin receptor substrate-1 (IRS-1) binding to PI3-kinase was not sufficient for full activation because the amount of IRS-1 pulled down by the GST-p85 fusion protein reached almost maximum, after incubation with insulin-treated cell lysates for 20 min, whereas PI3-kinase activity reached its maximum only after incubation for 5 h. All results suggest that the phosphorylation of p85 subunit at tyrosine residues and phosphorylation of p110 subunit at tyrosine or serine residues are not functionally significant in the regulation of PI3-kinase activity. They also suggest that P13-kinase is needed to bind to other protein(s) as well as the insulin receptor substrate-1 for full activation.