(16) Six weeks old female nu/nu nude mice (Charles River Laboratories) were kept under pathogen-free conditions, fed standard chow, and given free access to sterilized water. were evaluated. The SMI inhibitor MO-I-1100 was selected since it reduced ASPH enzymatic activity by 80% and suppressed HCC cell migration, invasion and anchorage impartial growth. Furthermore, substantial inhibition of HCC tumor growth and progression was observed in both animal models. The mechanism(s) for this antitumor effect was associated with reduced activation of Notch signaling both and studies A subcutaneous (s.c.) tumor model was used to analyze the effects of SMIs of hydroxylase activity, as described previously. (16) Six weeks old female nu/nu nude mice (Charles River Laboratories) were kept under pathogen-free conditions, Thiarabine fed standard chow, and given free access to sterilized water. Mice were anesthetized by isoflurane, and then s.c. Rabbit polyclonal to XCR1 xenografts were established by inoculating 1107 FOCUS cells into the right dorsal flank. Palpable tumors were confirmed on day 3 following inoculation, and mice were randomized into treatment or control groups to receive a SMI (MO-I-1100) or saline, respectively. MO-I-1100 (50 mg/kg) was prepared in saline and administered by intraperitoneal (i.p.) injection. Tumor size was measured using calipers every other day and tumor volumes were calculated as AB20.5 (A, length; B, width). An orthotopic xenograft model was also created by direct intrahepatic inoculation of FOCUS cells, as described previously (16). On day 3 after inoculation, MO-I-1100 (50 mg/kg) or saline was administered to mice by i.p. injection on 5 consecutive days per week for 2 weeks. Thiarabine At 4 weeks after initiation of treatment, mice were sacrificed to assess the antitumor effects of MO-I-1100. All procedures were approved by the Institutional Animal Care and Use Committee of Rhode Island Hospital. Immunohistochemistry (Supplemental Methods) RESULTS ASPH expression in human HCC Levels of ASPH expression were evaluated by IHS using the FB-50 mAb that reacts to the N-terminus of ASPH (13), in human HCC tumors (n=27) of diverse etiologies [HBV (n=8), HCV (n=12), and alcohol cirrhosis (n=7) related], dysplastic nodules (n=9), and adjacent non-neoplastic normal liver (n=36) as shown in Fig. 1A and B. ASPH was expressed in 22 of 27 (81.48%) human HCC tumors derived from 86 TMA cores. No immunoreactivity was observed in normal liver parenchyma. Dysplastic regenerating nodules in cirrhotic liver were also unfavorable for ASPH expression indicating that ASPH upregulation may be closely associated with malignant transformation. Most HCCs had a staining intensity of ASPH expression (bottom panels of Fig. 1A) with a mean semi-quantitative value of 1 1.37 (Fig. 1C). The high frequency of ASPH overexpression in human HCC suggests that Thiarabine it may be an important factor in HCC tumor development and progression. Open in a separate window Fig. 1 Expression of ASPH in HCCASPH expression in human HCC tumors, dysplastic nodules and adjacent uninvolved normal liver. (A) represents IHS of normal liver (upper left), dysplastic nodules (upper right) and human HCCs (bottom two). (B) percent of ASPH positive expression in human HCC tumors (86 TMA cores) at 400. ASPH was highly expressed in tumor tissues compared with normal liver. Unfavorable staining was observed for all components of adjacent uninvolved liver and dysplastic (regenerating) nodules. (C) semiquantatative analysis of staining intensity distribution of ASPH levels in HCC (0, unfavorable; 1+, moderate; 2+, strong; and 3+, very strong immunoreactivity). Development and characterizations of -hydroxylase SMIs Fig. 2C depicted representatives of parent compounds synthesized and examined for inhibition of -hydroxylase activity using a high throughput screening approach. In brief, Fig. 2A described the enzymatic reaction catalyzed by ASPH with the liberation of 14CO2 as the readout. Fig. 2B represented a strong hit with MO-I-1100 which inhibits the -hydroxylase activity of ASPH by approximately 80%. Fig. 2C described the structure of representative parent compounds synthesized and Thiarabine examined for inhibition of -hydroxylase activity. When inhibitors of ASPH enzymatic activity were identified, we performed a functional MTT assay to test their effects on cell viability over a range of.