Lapatinib, after 16 hours of incubation, was further demonstrated by western blotting to simultaneously suppress, in a dose-dependent manner, the phosphorylation of p170 ErbB1 at

Tyr1173 and/or p185 ErbB2 at Tyr1248 in both cultured rat (BDEneu or C611B) and human (HuCCT1) cholangiocarcinoma cells (Fig. 4B). In each case, however, and as expected, total ErbB1 and ErbB2 protein levels were not affected by the lapatinib treatment. Western blotting also showed that after a 24-hour to 36-hour incubation, the phosphorylation levels of the downstream signaling proteins Akt and p42/44 MAPK were also concomitantly decreased in both the rat and human cholangiocarcinoma cell lines, but that total Akt and p42/44 MAPK protein levels were unaffected. NVP-BKM120 supplier Lapatinib treatment after 36 hours of incubation also resulted in a dose-dependent inhibition of the key cell cycle regulator cyclin D1 in the cultured BDEneu, C611B, and HuCCT1 cells (Fig. 4B), and after 72 hours of incubation, significantly increased the activation of caspase-3 in

these cholangiocarcinoma cell lines, as demonstrated by western blotting (Fig. 4B) and ELISA (Fig. 5A-C). Caspase-3 activation in cultured BDEneu cells was associated with significant MLN8237 ic50 apoptosis induced by lapatinib after 72 hours of incubation, as demonstrated by DNA laddering and DAPI immunofluorescence staining (Fig. 5D). The therapeutic efficacy of lapatinib to suppress cholangiocarcinoma growth in vivo was tested in our syngeneic rat orthotopic intrahepatic cholangiocarcinoma model.4 As demonstrated in Fig. 6, lapatinib treatment, when initiated beginning 2 days after initial bile duct inoculation of the BDEneu cells into liver, resulted in a significant suppression of intrahepatic tumor growth, as reflected by an approximately 70% reduction in mean liver tumor wet weight (Fig. 6A), together with a marked reduction

in serum bilirubin levels in the treated animals at the time of sacrifice (Fig. 6B) when Methamphetamine compared to vehicle control values. Both rat groups exhibited a transient weight loss following surgery to orthotopically transplant BDEneu cells into liver, but as shown in Fig. 6C, the 2-day lapatinib-treated group steadily gained weight during the remaining treatment period, whereas the vehicle-treated control group exhibited a progressive decline in mean body weight beginning at day 20 after BDEneu cell inoculation (Fig. 6C). During the period of 20-26 days following BDEneu cell inoculation, the vehicle control rats became icteric and exhibited significantly larger liver tumors than those of the lapatinib-treated group (Fig. 6B). When the lapatinib treatment was delayed until day 8 after BDEneu cell inoculation, it was without significant effect in suppressing intrahepatic tumor growth over that of the vehicle-treated control animals (Fig. 6D). As further demonstrated in Supporting Fig.