Transformation of MSC induces transcriptional down regulation of antioxidant

However, although many inhibi tors of IGF receptors have been developed and promising antitumor activity has been demonstrated in pre clinical studies, clinical studies showed very limited efficacy of IGFR inhibitors as single agent therapy in solid tumors, including HCC. An important mechanism of resistance ABT-888 臨床試験 to IGFR inhibi tors is the compensatory activation of related signaling pathways. The PI3KAktmTOR pathway is the most extensively studied and Akt signaling has been demon strated to be a critical mechanism of drug resistance in HCC cells. Targeting a single point in these pivotal signaling pathways will induce compensatory up or down stream activation and result in drug resistance.<br><br> To overcome this compensatory activation, オーダー Afatinib combination of agents targeting more than one molecule in a signaling pathway, known as vertical blockade, were extensively tested in multiple preclinical models and synergistic anti cancer efficacy has been demonstrated. In this study, we sought to clarify whether combining inhibitors that target the IGFR and the PI3KAktmTOR signaling pathway could enhance therapeutic efficacy in HCC. The optimal combination of the potential syner gistic antitumor activities between IGFR and PI3KAkt mTOR inhibitors were explored. Survivin, an apoptosis inhibitory protein that is over expressed in multiple cancer types and plays critical roles in regulating apoptosis, cell proliferation and survival, was found to be a direct downstream target of the PI3KAktmTOR pathway. The role of survivin in mediating the anti tumor efficacy of IGFRPI3KAktmTOR vertical blockade was also examined.<br><br> Methods and materials Cell culture and reagents The HCC cell lines tested in this study included PLC 価格 AG-1478 PRF5, Hep3B, and Huh7. Cells were cultured in Dulbeccos modified Eagles medium supplemented with 10% fetal bovine serum, penicillin, and strep tomycin. Primary human umbilical venous endothelial cells, which were used to test the anti angiogenic activity of the drug combinations, were cultured as previously described. These cells were maintained in a humidified incubator under 5% CO2 at 37 C. The molecular targeted agents tested in this study included NVP AEW541, MK2206, BEZ235, and RAD001. For in vitro experiments, the drugs were dissolved in DMSO and the final DMSO concentration was kept below 0. 1%.<br><br> For in vivo experiments, the following the optimal dosage were usedNVP AEW541 once per day for 25 days based on previous studies, BEZ235 PEG300 90%, 25 mgkg, gavage once per day for 25 days based on previous studies, and MK2206 once per week for 4 weeks based on previous studies. The antibodies used for Western blotting, and immunohistochemical staining included anti Myc Tag, p Akt, caspase 3, p GSK3B, GSK3B, p P70S6K, P70S6K, p 4EBP 1, Mcl 1, Bim, DR 5, FADD, Bcl 2, Bad, Bax, survivin, GAPDH, and actin. Tests of in vitro anti tumor efficacy Cell viability was assessed by MTT 2, 5 diphenyltetrazolium bromide assay. The IC50 values after drug treatment were calculated using CompuSyn software based on changes in absorbance as determined by spec trophotometry. Apoptotic cell death was measured by flow cytom etry and Western blotting analysis. The poten tial synergistic antitumor effects between different drug treatments were determined by median dose effect ana lysis using the combination index isobologram method, as previously described.