Liver tissues were then frozen in OCT compound (Tissue-Tek; Sakura Finetek, Torrance, Crenolanib buy CA) and cryosectioned (10-��m thick sections) using a cryomicrotome (Leica CM3050 Cryostat; Leica, Wetzlar, Germany). Samples mounted on gelatin-coated slides were visualized under a fluorescence microscope (Leica). For immunoblotting of MMP13, frozen liver tissues were homogenized in ice-cold lysis buffer (10 mmol/l Tris�CCl, 150 mmol/l NaCl, 1 mmol/l ethylenediaminetetraacetic acid, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate, pH 8.0). After resolving proteins by sodium dodecyl sulfate�Cpolyacrylamide gel electrophoresis and transferring to membranes, blots were probed with rabbit monoclonal anti-MMP13 (1:500 dilution; Abcam, Cambridge, MA) or ��-actin antibodies (1:1,000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), and immunoreactive proteins were visualized using a goat anti-rabbit alkaline phosphatase-conjugated secondary antibody.
Histological analysis of liver tissue. Type I collagen content in liver tissue was evaluated using picrosirius red staining. Extracted liver tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5-��m sections. Sections were then deparaffinized, hydrated, and stained with picrosirius red as described by the manufacturer (Sigma, St Louis, MO). The images of stained liver specimens were captured by light microscopy. Measurement of liver damage. The serum levels of AST were measured as a reliable indicator of liver damage. Blood samples were obtained from normal control groups and various groups of pMMP13-treated mice before sacrificing.
AST activities in serum were determined by a UV-kinetic method using commercial kits (YD Diagnostics, Yongin, Korea). Statistics. Statistical analyses were performed using ANOVAs with post-hoc Student�CNewman�CKeuls tests. SigmaStat software (version 3.5; Systat Software, Richmond, CA) was used for all analyses, and a P value <0.05 was considered statistically significant. Acknowledgments This work was supported by the research grants from the Korean Health Technology R&D project (A090945), Ministry for Health, Welfare, and Family Affairs, and from National Research Laboratory (ROA-2006-000-10290-0), South Korea.
DISCLOSURE/CONFLICT OF INTEREST The authors declare no conflict of interest. The authors do not have any financial interest related to the development of PTK/ZK.
The ability of cancer cells to metastasize remains the greatest challenge to the management of malignant disease. The liver is a major site of metastasis for some of the most prevalent human malignancies, particularly carcinomas of the gastrointestinal tract. At present, surgical resection is the only Anacetrapib curative option for liver metastases but its success rate is partial, producing a 25�C30% 5-year survival rate for cancers such as colorectal carcinoma.