Insulin and IGF-1 regulate the expression of the pituitary tumor tr

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Insulin and IGF-1 regulate the expression of the pituitary tumor transforming gene (PTTG) in breast tumor cells Andrew Olango, Navpreet Waryah, Naseem Ul-Haq

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Abstract The pituitary tumour transforming gene (PTTG), an oncogene that is highly expressed in most tumours The following report focuses on the effects of Insulin and Insulin like growth factor-1 (IGF-1) as regulators for the expression of the pituitary tumor transforming gene (PTTG) in breast tumour cells. Using a human breast cancer cell line, MCF-7 cells, the effects of insulin and IGF-1 were recorded Insulin and IGF-1 were used to regulate the PTTG mRNA expression

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Introduction PTTG also known as Securin Encodes form a multifunctional protein of 203 amino acids located in the cytoplasm Inspires the expression, and secretion of mitogenic and angiogenic factors 203 amino acids Basic fibroblast growth factor (BFGF) Vascular Endothelial Growth Factor (VEGF). PTTG expression is absent or at extremely low levels in normal healthy tissues with the exception of testis and thymus PTTG is expressed in high amounts in tumors from the pituitary, colon, breast and ovaries Rapid proliferating cells and various tumours from pituitary, colon, thyroid, testis, ovary and breast PTTG is over expressed Normal healthy tissues Testis and Thymus

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Methodology: Cell Culture MCF-7 cells extracted from a patient MCF-7 cells were cultured in Dulbecco ’ s Modified Eagle Medium Cells that merged together were split and placed into 6-well plates and incubated for 18-24 hours.

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Results: The effect of Insulin on PTTG mRNA expression Results suggested Insulin stimulates PTTG expression in a time-dependent manner. The experiment was carried out with a controls serum where MCF-7 cells starved for a set period of time, before treating them with insulin, monitoring them over particular times. Experiment resulted in a 2.5-fold increase in PTTG mRNA levels, treated with 10nM of Insulin over a 24 hour time period and 3.5-fold after 48 hours

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It was also notice that Insulin induced PTTG mRNA and protein expression in a concentration-dependent manner

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Considering that IGF-1 binds to the same receptors, this meant IGF-1 may share the same effects as insulin ’ s ability to increase the expression of PTTG mRNA. Similar to insulin effect, treatment of MCF-7 cells with IGF-1 (20ng/ml) resulted in a 2.5-fold increase in PTTG mRNA expression

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Results suggested both insulin and IGF-1 activate PTTG expression and mediate the two signaling pathways: P13K/AKT and Ras-MAPK To determine which of the two are activated by insulin and IGF-1 the researchers used specific inhibitors for each pathway. MCF-7 treated with 10nM insulin and monitored the short term phosphorylation of AKT and ERK1/2 Within 5 min insulin stimulated AKT Maximum phosphorylation level reached in 30 mins

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Same results were seen in ERK1/2 phosphorylation Within 5 min insulin stimulated AKT Maximum phosphorylation level reached in 30 mins Pretreatment with LY294002 (50µM) blocking insulin induced phosporylation. Complete blocking of insulin induced phosphrylation of AKT Pre-treatment with Ras-MAPK-spesific inhibitor PD98059 (10µM) partially blocked the stimulatory effects of insulin and IGF-1 This suggests that both insulin and IGF-1 regulate the expression of PTTG mRNA through P13K/AKT and Ras-MAPK Actinomycin D, combined with the pretreatment of MCF-7 cells, in the absence of insulin reduced the steady state level of PTTG mRNA Further conformation that insulin stimulated PTTG mRNA synthesis 1.3kb PTTG promoter-luciferase was transfected into MCF-7 cells prior treatment Maximum effect of 1.8-fold was reached Collective results suggest insulin contributes to the synthesis of PTTG mRNA

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Conclusion Physiological concentrations of insulin and IGF-1 increase PTTG expression in MCF-7 cells Insulin activates PTTG promoter and induces the expression of endogenous PTTG in a time and dose dependent manner In the presence of actinomycin D, the results indicate insulin-induced PTTG expression is mainly dependent on new synthesis of new PTTG mRNA Finally the results show that insulin and IGF-1 induced expression of PTTG is through activation of PI3K/AKT signalling pathway in MCF-7 cells