ASK1, apoptosis signal-regulating kinase 1; DAMP, damage-associated molecular pattern; DEN, diethylnitrosamine; DSB, double-strand break; GFP, green fluorescent protein; γ-H2A.X, phosphorylated histone H2A.X; HCC, hepatocellular carcinoma; IFN-γ,

interferon-γ; IL, interleukin; LPS, lipopolysaccharide; NF-κB, nuclear factor kappa B; NHEJ, nonhomologous end joining; p38 MAPK, p38 mitogen-activated protein kinase; PAMP, pathogen-associated molecular pattern; PCNA, proliferating cell nuclear antigen; ROS, reactive oxygen species; KU-60019 mw TLR4, Toll-like receptor 4; TLR4mut, TLR4 mutant; TUNEL, terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling; WT, wild-type. To explore the role of TLR4 in liver tumorigenesis, TLR4mut or WT mice at age 15 days were subjected to DEN-induced HCC, a well-established chemical carcinogenesis protocol.11 HCC developed in all of newborn WT or TLR4mut male mice injected with DEN (25

mg/kg) within 6 months (Fig. 1A). However, only 20% of female WT littermates developed with HCC as described18 (data not shown). Notably, TLR4mut www.selleckchem.com/products/GDC-0980-RG7422.html mice developed two-fold more visible tumor nodules than WT mice (37.76 ± 5.83 versus 18.09 ± 1.69, P < 0.01) (Fig. 1B). Also, an earlier onset of liver tumors was observed in the TLR4mut mice than WT mice: 60% of WT male mice developed HCC at the end of 5th month after DEN treatment but all of TLR4mut male mice developed HCC (data not show). Moreover, the number of HCC tumor nodules was also found increased in TLR4mut mice than their WT littermates (Fig. 1B).

Most tumor nodules were basophilic HCC (Fig. 1A), and two-fold more tumor area (percentage) was detected in the TLR4mut mice compared with WT mice (39.35 ± 6.42 versus 16.85 ± 3.42; P < 0.01) (Fig. 1B). Consistently, TLR4mut mice displayed a persistent hepatic injury than the WT mice as indicated by increase in the serum alanine aminotransferase (data not shown). Therefore, TLR4mut 上海皓元医药股份有限公司 mice with HCC exhibited significantly shorter mean survival times than WT mice (Fig. 1C). Programmed cell death, including apoptosis and autophagy-associated cell death, is a crucial defense mechanism against tumorigenesis. Compared with WT mice, TLR4mut mice exhibited a remarkable decrease in apoptosis as marked by TUNEL staining (11.12 ± 0.84 versus 4.35 ± 0.45 [P < 0.01], 9.93 ± 0.18 versus 3.84 ± 0.29 [P < 0.01], and 10.35 ± 0.84 versus 4.30 ± 0.62 [P < 0.001], at month 1, 3, and 6 after DEN injection, respectively) (Fig. 1D) as well as a decrease in apoptotic cell death marked by cleaved caspase-3 (Supporting Fig. 1A,B). Moreover, TLR4mut livers displayed a persistent increase in proliferation biomarker proliferating cell nuclear antigen (PCNA) as indicated by immunohistochemistry staining (Fig. 1E) and immune blotting (Supporting Fig. 1A,C).