, Hercules, CA). Caspase-3 activity was measured by enzyme-linked immunosorbent assay (ELISA) using the ApoAlert Caspase-3 colorimetric assay kit from Clontech Laboratories, Inc. (Mountain View, CA).6 In addition, apoptotic cell death was assessed in the cultured cholangiocarcinoma cell lines by DNA laddering, as described,6 and by detection with fluorescence microscopy of nuclear fragmentation in 4′,6-diamidino-2-phenylindole (DAPI) nuclear stained preparations. Animal experimentation was performed in accordance with criteria outlined in the Guide for the Care and Use of Laboratory Animals,9 using protocols

approved by the Institutional Animal selleck chemical Care and Use Committee at Virginia Commonwealth University. Briefly, 4 × 106 BDEneu cells (viability ≥ 90%) were inoculated into the bile ducts of syngeneic young adult male Fischer 344 rats as described.4 Rats were randomized twice before being designated as either “treated” or “control”. The treated group was administered lapatinib at 75 mg/kg of body weight given by gavage twice a day for a period of 24-26 days. The control group received the

vehicle only, composed SCH772984 solubility dmso of 0.5% (wt/wt) hydroxypropyl methylcellulose and 0.1% (wt/wt) Tween-80 in distilled water, according to the same treatment schedule as the lapatinib-treated group. Treatment was initiated at either 2 days (early treatment) or 8 days (late treatment) after bile duct inoculation of the BDEneu cells. All animals were weighed once a day in the morning before the start of a daily treatment. At the conclusion of the treatment period, the rats were sacrificed, and gross hepatic tumor incidence and liver tumor wet weights were determined for

both the vehicle-treated control and lapatinib-treated groups. Serum samples were obtained from blood collected by cardiac puncture at the time of sacrifice and flash frozen for total bilirubin assessment using the QuantiChrome Bilirubin Assay Kit (DIBR-80) purchased from BioAssay Systems (Hayward, CA). Flash frozen cryopreserved tumor samples were also obtained at sacrifice and used to assess, by immunohistochemistry and western blotting, the effect of Oxalosuccinic acid lapatinib treatment on ErbB2 tyrosine 1248 (Tyr1248) phosphorylation, as described.4 Mean values ± standard deviation (SD) were calculated and the Student two-tailed t test was used to determine P values, with a P value of ≤ 0.05 considered significant. The synergistic effects of AG879 in combination with AG1517 on in vitro cell growth inhibition was determined by the combination index (CI) method.10 A CI value < 1 indicated synergism. Relative expression levels of ErbB1 and ErbB2 proteins in the two rat and two human cholangiocarcinoma cell lines employed in this study were determined by western blotting. As shown in Fig. 1, the four different cholangiocarcinoma cell lines analyzed expressed variable levels of p170 ErbB1 and p185 ErbB2.