Abstract
There is abundant epidemiological evidence that heavy alcohol intake contributes to hepatocellular carcinoma (HCC) development. Previous reports indicated that connexin 32 (Cx32), which is a major hepatocyte gap junction protein, is down-regulated in chronic liver disease and has a protective role in hepatocarcinogenesis. However, functions of Cx32 in alcohol-related hepatocarcinogenesis have not been clarified. To evaluate them, 9-week-old Cx32 dominant negative transgenic (Tg) rats and their wild-type (Wt) littermates were given 1 % or 5 % ethanol (EtOH) or water ad libitum, for 16 weeks after an intraperitoneal injection of diethylnitrosamine (200 mg/kg). EtOH significantly increased the incidence and multiplicity of HCC and total tumors in a dose-dependent manner in Tg rats, but not in Wt rats. Although the number and area of glutathione S-transferase placental form (GST-P) positive foci were not significantly different between the groups, EtOH increased the Ki-67 labeling indices in GST-P positive foci only in Tg rats. EtOH up-regulated phosphorylated Erk1/2 with decrease of the Erk1/2 inhibitor, dual specificity protein phosphatase 1 (Dusp1) in whole livers of Tg and Wt rats. Immunofluorescence staining and quantitative RT-PCR revealed that EtOH significantly increased nucleolar localization of phosphorylated Erk1/2 and contrastingly reduced Dusp1 protein and mRNA expression in GST-P positive foci and HCC of Tg rats as compared to those of Wt rats. These findings suggest that Cx32 dysfunction like in chronic liver disease promoted EtOH-associated hepatocarcinogenesis through dysregulation of Erk-Dusp1 signaling.
Highlights
Hepatocellular carcinoma (HCC) is the second most common cause of cancer-related mortality
The expression of connexin 32 (Cx32) and Cx26, which are hepatocyte gap junction protein, were diffusely decreased in Tg rats as compared to those in Wt rats (Supplementary Figure 2A), and the expression of Cx32 and Cx26 on the cell membrane in Wt rats were not altered by EtOH intake (Figure 1B)
The dual specificity protein phosphatase 1 (Dusp1) expression level was inversely correlated with phosphorylated Erk 1/2 (pErk) expression in nucleoli in both HCC and glutathione S-transferase placental form (GST-P) positive foci of Tg rats that received EtOH (Figure 5B). These results suggest that EtOH treatment and Cx32 dysfunction decreased Dusp1 expression which led to Erk activation in GST-P positive foci
Summary
Hepatocellular carcinoma (HCC) is the second most common cause of cancer-related mortality. Alcohol is one of the most important risk factor for the cause of HCC especially in developed countries, and the incidence of alcoholrelated HCC has recently tended to increase in Japan [2]. Lifelong exposure to 3% EtOH did not induce HCC www.impactjournals.com/oncotarget in rodents [12] whereas 10% EtOH ingestion for 18 months induced HCC development [13]. These results suggest that EtOH is an established carcinogen, which is consistent with the IARC evaluation of EtOH. The detailed molecular mechanisms by which EtOH contributes to hepatocarcinogenesis have not been fully elucidated to date
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