5B). The other two major phosphorylated MAPKs (phosphorylated stress-activated protein kinase/c-Jun N-terminal kinase [p-SAPK/JNK] and p38 MAPK) only increased insignificantly after heat treatment (Supporting Fig.4). Phosphorylation levels returned to baseline at day 12 after heat treatment. Notably, expression of heat shock protein www.selleckchem.com/products/PLX-4032.html (HSP)27, 70, and 90 was significantly increased at day 5 post–heat treatment temperature dependently and also reverted to baseline levels at day 12 (Supporting Fig. 5). Liver specimens from 64 HCC patients, 20 patients with cirrhosis, and
30 subjects with CHC without cirrhosis were examined for Shc expression (Fig. 5C). Shc staining was absent in healthy liver, but dramatically increased in HCC tissue, whereas samples with CHC without cirrhosis showed an intermediate expression (P < 0.0005 for HCV cirrhosis versus HCC and for HCV without cirrhosis versus HCV Palbociclib manufacturer cirrhosis; Fig. 5D). Next, we formed two groups with high and lower Shc-LIs (≥65% or <65%; n = 54 and n = 30, respectively) in patients with advanced fibrosis (without and with HCC). When comparing both
groups by Kaplan-Meier’s analysis, OS rate of patients with Shc-LI ≥65% was significantly lower than with Shc-LI <65% (P = 0.0316; Fig. 5E). When Shc-LI (%) in these patients was compared with hematological parameters associated with hepatocarcinogenesis (alpha-fetaprotein [AFP]-L3%, AFP, and protein induced by vitamin K absence/antagonist-II [PIVKA-II]) and liver function (alanine aminotransferase, aspartate
aminotransferase, total bilirubin, alkaline phosphate, gamma-glutamyl transpeptidase, ALB, and platelet count) in all samples (Supporting Table 3), a strong correlation was only found with AFP-L3 (%) (r = 0.5312; P < 0.0001). However, no strong correlation between Shc-LI and the other parameters was observed. Expression of both phosphorylated Shc-variants, p46- and p52-Shc, was assessed by semiquantitative western blotting in homogenized lysates of human liver specimens (Fig. 5F). Although p52-Shc was strongly expressed in both cirrhosis and HCC specimens (p = 0.0374 and p = 0.0054, respectively), p46-Shc was detected only in HCC, whereas no p66-Shc could be medchemexpress detected in any samples. In all HCC samples, phosphorylated p46-Shc expression was much stronger than phosphorylated p52-Shc expression (P = 0.0313). Five days after heat treatment (50˚C), HEPG2 cells were exposed to the Erk1/2 inhibitor, U0126, whereas HEPG2 cells kept at 37˚C served as controls. Notably, effective Erk1/2 inhibition, as evidenced by complete suppression of Erk1/2 phosphorylation (Fig. 6A), blunted enhanced proliferation (Fig. 6B) and essentially normalized (or reduced) all parameters related to EMT, except for significantly reduced, but still elevated, CK19 and COL1A1 (Figs. 4 and 6C,D).