HIF-1α(+/+; −/−) mouse embryonic fibroblast cell lines were kindly provided by Dr. Randall Johnson (University of California, San Diego). Cells were cultured in α-modified Eagle medium or in Dulbecco’s Modified Eagle’s medium, both of which were supplemented with 10% heat-inactivated fetal calf serum, in a 5% CO2 humidified atmosphere at 37°C. The oxygen tension in the chamber was either 20% (normoxic) or 1% (hypoxic). Chaetocin and other chemicals were administered to medium 1 hour before normoxic or hypoxic incubation. Male nude mice (BALB/cAnNCrj-n/n) were purchased from Charles buy Bortezomib River Japan (Shin-Yokohama, Japan) and housed in a specific pathogen-free room. Mice

(6 weeks old) were injected subcutaneously in a flask with 5 × 106 viable cancer cells. After tumor volumes reached 100-150 mm3, mice were treated with dimethyl sulfoxide (DMSO), chaetocin (0.25 mg/kg, intraperitoneally [i.p.]), and/or doxorubicin (1 mg/kg, i.p.) once a day. Tumor volumes Selleckchem Everolimus were measured with a caliper and calculated using the equation volume = ab2/2, where “a” is the maximal width and “b” is maximal orthogonal width. All procedures were conducted in accordance with the guidelines of the Laboratory Animal Ethics Committee of Seoul National University. All data were analyzed using Microsoft Excel 2007 or

SPSS (v. 10.0) software and results are expressed as means and 95% confidence intervals or standard deviations. The two-sided Mann-Whitney U test was used to compare luciferase activities, vascular endothelial growth factor (VEGF) concentrations, and HIF-1α-positive cell, CD31-positive vessel, and Transferase-Mediated dUTP Nick-End Labeling (TUNEL)-positive cell numbers. Tumor sizes were compared using analysis of variance (ANOVA) followed by Duncan’s multiple range test. Differences were considered significant for P < 0.05. All statistical tests were two-sided. To examine if chaetocin has anticancer activity against

hepatoma, chaetocin was injected for 2 weeks into mice bearing Meloxicam Hepa 1c1c-7 tumors. Tumor growth was significantly retarded after chaetocin treatment (Fig. 1A). Interestingly, chaetocin did not induce massive cell death or deformation (Fig. 1B, upper), suggesting that cytotoxicity does not primarily underlie the anticancer effect. Moreover, neither histological changes nor apoptosis was observed in the livers of mice treated with chaetocin for 2 weeks (Fig. 1B, lower). Because a hypoxic microenvironment and angiogenesis are critical for tumor growth, we assessed HIF-1α expression and vascular density immunohistochemically. In chaetocin-treated tumors, HIF-1α-positive cells and CD31-positive threadlike vessels were noticeably reduced, but TUNEL-positive apoptotic cells increased (Fig. 1B, upper); the results are summarized in Fig. 1C. Furthermore, VEGF mRNA was down-regulated overall in chaetocin-treated tumors (Fig. 1D).