Measurement of Serum and Hepatic Eicosanoids by Liquid Chromatography Tandem-Mass Spectrometry (LC-MS/MS) in a Mouse Model of Hepatocellular Carcinoma (HCC) with Delivery of c-Met and Activated β-Catenin by Hepatocyte Hydrodynamic Injection

Abstract

BACKGROUND Most forms of cancer, including hepatocellular carcinoma (HCC), are associated with varying degrees of chronic inflammation. The association between the expression of eicosanoids, which are bioactive lipid mediators of inflammation, and HCC remains unknown. The aim of this study was to measure serum and hepatic eicosanoids in a mouse model of HCC with the delivery of c-Met and activated b-catenin by hepatocyte hydrodynamic injection. MATERIAL AND METHODS The HCC mouse model, and normal control mice, were used in this study with co-delivery of human c-Met combined with activated β-catenin into hepatocytes through hydrodynamic injection. Liquid chromatography tandem-mass spectrometry (LC-MS/MS) analysis was used to measure serum and hepatic eicosanoid levels. RESULTS The combined activation of c-Met and β-catenin was induced in the HCC mouse model. LC-MS/MS showed that a total of 13 eicosanoids in serum and 12 eicosanoids in liver tissue were significantly increased in the HCC mice, when compared with control mice. CONCLUSIONS In a mouse model of HCC, co-activation of the c-Met and β-catenin signaling pathway resulted in increased levels of serum and hepatic eicosanoids.

Figure 1

Exogenous c-Met and ΔN90-β-catenin were successfully delivered three days after hydrodynamic injection in mice. (A) Protein expression of c-Met and ΔN90-β-catenin in liver lysates three days following hydrodynamic injection into the mice tail veins. (B) mRNA level of exogenous c-Met and ΔN90-β-catenin by quantitative real-time polymerase chain reaction (qRT-PCR) analysis. (C) Photomicrograph of the immunohistochemistry (brown staining) for c-Met and β-catenin protein expression in mouse liver tissue sections.

Figure 2

Co-activation of c-Met and ΔN90-β-catenin resulted in liver tumor development in mice. (A) Gross images and hematoxylin staining of tissue sections of mice livers at eight weeks post-injection. (B) Photomicrographs of liver sections were examined by light microscopy following immunohistochemistry with antibody staining for c-Met and β-catenin protein. (C) Liver weight and liver/body weight ratio were compared at eight weeks post-injection. (D) Survival curve following hydrodynamic transfection in mice.

Figure 3

Statistical analysis to compare the control group of mice(CN), with the experimental group of mice with c-Met and β-catenin hepatocellular carcinoma (HCC). (A, B) Orthogonal Projections to Latent Structures Discriminant Analysis (OPLS-DA) score plots and validation plots obtained from permutation test (n=20) for serum samples. (C, D) OPLS-DA score plots and validation plots obtained from permutation test (n=20) for liver tissue samples.

Figure 4

Eicosanoid changes in serum and liver tissue induced by overexpression of C-Met and β-catenin. (A) All detected serum eicosanoid level changes are expressed as a heat map. (B) All detected liver eicosanoid level changes are expressed as a heat map. Fold changes are derived from the fold difference compared with normalized means for each eicosanoid. Green squares indicate a reduction of up to two-fold; black squares indicate no significant fold changes; red squares indicate an increase of up to two-fold. (C) Concentrations of significantly changed eicosanoids detected in mouse serum. (D) Concentrations of significantly changed eicosanoids detected in mouse liver tissue.