briefly centrifuged at 4 C and the cell pellet was resuspended in 100 ll of a single detergent whole cell lysis buffer containing 1% NP 40, 150 mM NaCl, 50 mM Tris, pH 7.4, and 1X anti protease cocktail . The lysate was Fingolimod then sonicated for 23 s on ice and re centrifuged. The protein supernatant was analyzed for protein concentration and equilibrated to equivalent concentrations using a 1X Antarctic phosphatase buffer . Antarctic phosphatase catalyzes the removal of 50 phosphate groups from DNA and RNA and can be used to dephosphorylate proteins. Equivalent concentrations of MCF7 protein lysate were treated with 100 units of AP at 37 C for 1 h. Control lysates were equilibrated with 1X AP buffer only. The reaction was stopped at the end of the incubation period by the application of a 2 mercaptoethanol based loading buffer followed by boiling for 2 min.
The lysates were resolved in a 10% SDSPAGE gel and trans blotted to NC Dopamine Receptor membrane , followed by Western analysis using the immunoprobes as indicated. Blots were stripped and re probed with GAPDH to verify loading accuracy. 2.7. Reagents Troglitazone and Dulbecco’s Modified Eagles Medium were purchased from Sigma Aldrich Canada . Ciglitazone and Trichostatin A were obtained from Biomol , and PXD101 from Cayman Chemicals. Fetal bovine serum and antibiotics were purchased from Invitrogen . Antibodies to unmodified and modified histones, and the HDAC activity assay kit were purchased from Upstate Biotechnology . Methylated histone antibodies were from Abcam. The inhibitors PD98059 and LY294002 were purchased from Biomol.
15PGJ2 was purchased from Cayman Chemical. Antibodies to anthropology AKT and phosphorylated AKT were obtained from Sigma and Santa Cruz Biotechnology, respectively. All other reagents were purchased from Sigma Aldrich Canada. 3. Results 3.1. Killing of MCF7 cells by TRG is associated with changes in histone levels and post translational modifications We previously reported that TRG is a potent killer of DOX resistant K562 myeloid leukemia cells when combined with DOX . We demonstrated that DOX resistant cells had increased histone H3 acetylation and phosphorylation, and that subsequent TRG treatment caused a dosedependent further increase in H3 protein levels and acetylation. In this study, we asked whether TRG had similar effects on a different human cancer line, MCF7 breast cancer cells.
We assayed MCF7 cells for killing using an MTT 2,5 diphenyltetrazolium bromide) cell proliferation assay after treatment with 100 lM of the TZDs TRG and CIG for 48 h. We have used 200 lM TRG with virtually no effect on cultured primary rat hepatocytes . More recently it was shown that 100 lM TRG had no effect on normal bone marrow cells and mobilized peripheral blood progenitors . We also tested a non TZD PPARc agonist, 15PGJ2 . The most potent cell killing was observed with TRG, whereas CIG had only a modest, but non significant, killing effect, and 15PGJ2 had virtually no effect on MCF7 viability . Cell killing by TRG was similar to that induced by the histone deacetylase inhibitors Trichostatin A , sodium butyrate, and PXD101 a potent HDACi used in phase I and II cancer clinical trials . We observed that TRG also induced PARP cleavage in MCF7 cells , as did PXD101, TSA and sodium butyrate.