ance of the MEK1/2 ERK1/2 pathway in neoplastic cell survival, MEK1/2 inhibitors have been developed by several pharmaceutical companies and have entered clinical trials, including PD184352, the NVP-TAE684 ALK inhibitor second generation Pfizer MEK1/2 inhibitor PD 0325901 and the Astra Zeneca drug AZD6244 . Heat shock protein 90 is a chaperone protein involved in the proper folding and intracellular disposition of multiple proteins involved in cell signaling and survival. Tumor cells generally have higher rates of protein synthesis than non neoplastic cells and disruption of HSP90 function in tumor cells has been shown to induce improper folding of diverse proteins, including Raf 1, B Raf, AKT, ERBB family receptors, among numerous others, culminating in their proteasomal degradation.
These events have been shown to induce apoptosis or, alternatively, to increase the susceptibility of tumor cells TW-37 877877-35-5 to established cytotoxic agents. Such considerations have led to the development of clinically relevant HSP90 antagonists, such as 17 allylamino 17 demethoxygeldanamycin, which has both superior pharmacokinetic and reduced normal tissue toxicity characteristics compared with geldanamycin. Many studies have argued that inhibition of the PI3 kinase AKT pathway, rather than the Raf MEKl/2 ERKl/2 pathway, represents a key component of 17AAG toxicity and sensitization effects in tumor cells. Free plasma concentrations of 17AAG in patients have been noted to be in the low 1 to 5 mol/L range for up to 12 h after drug Park et al. Page 2 Mol Cancer Ther. Author manuscript, available in PMC 2009 September 1.
NIH PA Author Manuscript NIH PA Author Manuscript NIH PA Author Manuscript infusion, which is significantly higher than the required concentration of drug to inhibit HSP90 function. The goal of the present studies was to determine whether, and by what mechanism, clinically relevant MEK1/2 inhibitors might enhance the activity of clinically relevant geldanamycins against human hepatoma and other GI and GU tumor cells in vitro and in vivo. Our results indicate that clinically relevant MEK1/2 inhibitors interact synergistically with 17AAG and 17DMAGto induce CD95 dependent cell death. Materials and Methods Materials Total BAX, cleaved caspase 3, Phospho /total ERKl/2/5, Phospho /total JNKl 3, Phospho /total p38 MAPK, Anti S473 AKT and total AKT antibodies were purchased from Cell Signaling Technologies.
Active BAX specific antibody for immunoprecipitation was purchased from Sigma. The c FLIP s/L and all the secondary antibodies were purchased from Santa Cruz Biotechnology. The JNK inhibitor peptide, caspase inhibitors and 17AAG was supplied by Calbiochem as powder, dissolved in sterile DMSO, and stored frozen under light protected conditions at�?0. Enhanced chemiluminescence kits were purchased from Amersham Enhanced ChemiLuminescence system and NEN Life Science Products. Trypsin EDTA, RPMI medium, penicillin streptomycin were purchased from GIBCOBRL. BAX/ BAK �?�? BIM �?�?and BID �?�?fibroblasts were kindly provided by Dr. S. Korsmeyer. HuH7, HEPG2 and HEP3B, pancreatic, colorectal, and prostate cancer cells were obtained from the ATCC. Commercially available validated short hairpin RNA molecules to knock down RNA/protein levels were from Qiagen : CD95, FADD, BID. The dominant negative p38 MAPK and activated MEK1 EE recombinant adenoviruses were kindly provided by Drs. K. Valerie, VCU and J. Moltken, respectively. The proprietary drug 17DMAG was supplied by the Dr. David Giu