Cells were then spiked with IGF I as indicated for the final 15 min

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 Cells were then spiked with IGF I as indicated for the final 15 min  Empty Cells were then spiked with IGF I as indicated for the final 15 min

Post  huwan123456 on Tue Jun 10, 2014 4:56 am

Although metformin can lower blood glucose, the levels rarely remain within the normal range and as the type 2 diabetes progresses, additional medication such as exogenous insulin is often required to control patients hyperglycaemia. ABT-737 構造 In addition to its anti diabetic effects, metformin has recently been postulated to have a protective role against cancer. Epidemiological and retrospective studies have demonstrated that diabetic patients taking metformin not only have a lower incidence of pancreatic cancer, but also an improved cancer outcome. The indicated anti neoplastic activity of metformin may relate to reduced plasma insulin concentrations or by direct effects on the tumour cells. Recent studies suggest that metformin induced AMPK activation at Thr172 inhibits the central growth control node mam malian target of rapamycin mTOR, thus preventing protein synthesis and cell proliferation.

Metformin has recently been shown to possess anti tumour effects, both in AMPK dependent and independent manners. Although an increasing number of studies demonstrate the anti tumour effects of metformin, relatively little is known about the effects and underlying mechanisms of metformin on pancreatic cancer オーダー AEB071 cells. The goal of this study was to examine the direct effects of metformin on human pancreatic cancer cells in the context of normal or elevated glucose levels. Effects on proliferation, apoptosis, AMPK activation and influence on and by the IGF I pathway were analysed. Methods Materials All chemicals and reagents were purchased from Sigma Aldrich unless stated otherwise.

Cell culture media, penicillin streptomycin and fetal bovine serum were purchased from Invitrogen. IGF I was purchased from GroPep. supplier AG-014699 MTT. Cell Proliferation Kit I was derived from Roche. Anti cleaved PARP, anti phospho AMPKThr172, anti phospho AMPKSer485, anti AMPK, anti IRS 1, anti phospho IGF IRB phospho IRB, anti phospho AktSer473 and anti Akt antibodies were purchased from Cell Signaling Technology Inc. Anti IGF IRB was obtained from Santa Cruz Biotechnology and anti GAPDH from Millipore. Cell culture The human pancreatic adenocarcinoma cell lines AsPC 1, BxPC 3, PANC 1 and MIAPaCa 2 were purchased from Standards. The cells were maintained in RPMI1640 or DMEM supplemented with 10% FBS and antibiotics in a humified 5% CO2 atmosphere at 37 C.

All experiments were performed in glucose free RPMI1640 or DMEM supplemented with 5 mM or 25 mM D glucose, 2 mM L glutamine and antibiotics as above, unless stated otherwise. MTT proliferation assay Cells were plated in 96 well plates in growth media with 5 mM glucose for 24 h before switching to SFM with 5 mM or 25 mM glucose for another 24 h. Cells were subsequently dosed with increasing concentrations of metformin in SFM with 5 mM or 25 mM glucose in sextuplicates. SFM with either 5 mM or 25 mM was used as control. Following incubation for 24 72 h, cell proliferation was assessed by MTT according to the manufacturers instructions. The samples were measured on a Labsystems Multiskan Plus plate reader using the DeltaSoft JV software. Western immunoblotting Cells were cultured in 6 well plates for 24 h. After an additional 24 h in normal glucose SFM, the cells were dosed with metformin in SFM or 1% FBS SFM with 5 or 25 mM glucose for 24 h.

huwan123456

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