Journal: PLoS ONE
Article Title: Synergistic Antitumor Effects of Novel HDAC Inhibitors and Paclitaxel In Vitro and In Vivo
Figure Lengend Snippet: Induction of apoptosis and analysis of apoptosis-related factors in IGROV-1 and IGROV-1/Pt1 and A431 cells exposed to the IC 50 PTX or vinorelbine (VIN) or ST2782 (ST) or SAHA or their combination. A) Cleavage of caspase 3 (CPP32) and PARP-1 and cytochrome C release. Western blot analysis was performed after 24 h exposure to drug concentrations used in experiments of Fig. 2A . β-Tubulin is shown as a control of protein loading. Release of cytochrome C was examined in cytosolic and mitochondrial extracts, prepared after 24 h exposure. B) Apoptosis determined by TUNEL assay after 72 h of treatment (ST2782 10 µM, PTX 1.2 µM and SAHA 10 µM). The percentages of TUNEL-positive cells are indicated in each panel. One representative experiment of at least three is shown. The results, expressed as percentage of TUNEL-positive cells, represent the mean±SD of three independent experiments. C) Cleavage of CPP32 and PARP-1 in IGROV-1 cells treated with SAHA and/or PTX to the same concentrations used for TUNEL assay. D) Analysis of cytochrome C release in cytosol and in mitochondrial extract prepared after 48 h exposure to PTX or SAHA alone or their combination in IGROV-1 cells. E) Cleavage of CPP32 and PARP-1 in IGROV-1 cells treated with vinorelbine (0.047 µM) or ST2782 (10 µM) alone or in combination. F) Cleavage of CPP32 in A431 cells treated with ST2782 (ST, 3 µM) or PTX (0.0035 µM) alone or in combination. β-Tubulin is shown as a control of protein loading when whole-cell lysates are used. The size of CPP32 and of their cleavage products are indicated. An antibody against a mitochondrial marker and an antibody against actin were used as protein control to ensure a correct subcellular fractionation process.
Article Snippet: After dilution, the drug was kept in ice and administered i.v. We used primary antibodies against p53 (Dako, Glostrup, Denmark); p21WAF1/Cip1 (Neomarker, Union City, CA); caspase-3 (CPP32), cleaved caspase-3 (Asp175 ), acetylated p53 (lys382 ) (Cell Signalling Technology, Beverly, MA); PARP-1 (Oncogene Science, Uniondale, NY); cytochrome C (BD Pharmingen, Becton Dickinson, Franklin Lakes, NJ); actin, acetylated tubulin and β-tubulin (Sigma, St. Louis, MO); mitotic protein monoclonal 2 (MPM-2) (Upstate Biotechnology, Lake Placid, NY); Raf-1 (sc-7267), cyclin-B1 (sc-245), cdc-25C (sc-327) (Santa Cruz Biotechnology, Santa Cruz, CA); mitochondrial marker (Abcam, Cambridge, UK); topoisomerase II (Transduction Laboratories, Lexington).
Techniques: Western Blot, TUNEL Assay, Marker, Fractionation