Anticancer effect of caudatin in diethylnitrosamine‑induced hepatocarcinogenesis in rats

Abstract

An overwhelming endoplasmic reticulum stress (ERS) and the following unfolded protein response (UPR) can induce hepatic inflammation, fibrosis and hepatocellular carcinoma (HCC). Caudatin, one of the species of C‑21 steroidal glycosides mainly isolated from the roots of Cynanchum bungei Decne, exhibits potent anticancer activities in vivo. However, the effect of caudatin on HCC remains unclear. In the present study, a diethylnitrosamine (DEN)‑induced HCC model was established. Nodules and tumors in rat livers were monitored by T2‑/T1‑weighted‑magnetic resonance imaging (MRI) using a 1.5 T scanner. Caudatin reduced the number and size of nodules and alleviated the inflammatory foci in the liver. In addition, the hepatic pro‑inflammatory levels of interleukin (IL) 6, monocyte chemoattractant protein 1 and IL‑1β were decreased in caudatin‑treated rats. The DEN‑induced surge in malondialdehyde, aspartate aminotransferase, alanine transaminase and TBIL were alleviated following caudatin treatment. The expression of ERS chaperones glucose‑regulated protein, 94 kDa, glucose‑regulated protein, 78 kDa and protein disulfide‑isomerase A4 and the proliferation marker Ki‑67 in liver nodules were all downregulated by caudatin as demonstrated by immunohistochemistry, reverse transcription‑quantitative PCR and western blot analysis. Caudatin reduced the cytoprotective ERS sensor activating transcription factor 6‑mediated signal transduction and inhibited the PKR‑like endoplasmic reticulum kinase/eukaryotic initiation factor 2α/activating transcription factor 4 pathway. However, the effect of caudatin on inositol requiring enzyme 1 signaling was negligible. In conclusion, restoration of the dysregulated UPR program was involved in the antitumor efficacy of caudatin without inducing cumulative hepatotoxicity.

Keywords: caudatin; hepatocellular carcinoma; endoplasmic reticulum stress; glucose-regulated protein; 78 kda; diethylnitrosamine.

Figure 1.

In vivo MRI images of liver tumors in rats. Representative images of tumors from each group. T2- and DWI-weighted MR images of the liver, as well as T1CE MRI following injection of Magnevist (n=6/group). Tumors in T1CE MRI are indicated with red asterisks. MRI, magnetic resonance imaging; DWI, diffusion-weighted imaging; T1CE, tail vein and contrast enhanced.

Figure 2.

H&E staining and immunohistochemical analysis of Ki-67 in liver sections. Hemorrhage (black arrow), hepatic sinus dilation (red arrow), tumor cells (black asterisk) and necrosis are evident in the DEN group. Fibrosis (red asterisk) is observed in the caudatin-treated group. Hepatocytes stained positive with the proliferation marker (Ki-67) were counted under a microscope at ×100 magnification. Graph shows quantitation of Ki-67 immunohistochemistry. **P<0.01 vs. control; ^##P<0.01 vs. DEN group (n=6/group). The Ki-67 intensity is reduced in caudatin-treated groups compared with the DEN group as shown in the immunohistochemical analysis (scale bar=100 µm). H&E, hematoxylin and eosin staining; DEN, diethylnitrosamine.

Figure 3.

Effects of caudatin administration on hepatic pro-inflammatory cytokines. IL-6, TNF-α, MCP-1 and IL-1β levels of liver tissues were examined by ELISA kits (n=6/group). **P<0.01 vs. control; ^#P<0.05, ^##P<0.01 vs. DEN group. IL, interleukin; TNF, tumor necrosis factor; MCP-1, monocyte chemoattractant protein 1; DEN, diethylnitrosamine.

Figure 4.

Effect of caudatin on the expression of chaperones in the DEN-induced HCC model. (A) Reverse transcription-quantitative PCR of GRP78, GRP94 and PDIA4 following the caudatin (50 mg/kg) treatment. **P<0.01 vs. control; ^#P<0.05, ^##P<0.01 vs. DEN group. (B) Western blot analysis of chaperone proteins. Results are representative of three independent experiments. (C) GRP78 immunostaining in the liver. Arrows indicate tumors (scale bar=100 µm). DEN, diethylnitrosamine; HCC, hepatocellular carcinoma; GRP78, glucose-regulated protein, 78 kDa; GRP94, glucose-regulated protein, 94 kDa; PDIA4, protein disulfide-isomerase A4.

Figure 5.

Effect of caudatin on the expression of IRE1α and ATF6 and activation of the PERK/eIF2α/ATF4 pathway in the DEN-induced HCC model. (A) Representative western blot analysis of ERS protein expression. (B) Reverse transcription-quantitative PCR of ERDJ4, ATF4, GADD34 following caudatin (50 mg/kg) treatment. **P<0.01 vs. control, ^##P<0.01 vs. DEN group. IRE1, inositol requiring enzyme 1; ATF, activating transcription factor; PERK, PKR-like endoplasmic reticulum kinase; eIF2α, eukaryotic initiation factor 2α; DEN, diethylnitrosamine; HCC, hepatocellular carcinoma; ERDJ4, endoplasmic reticulum DnaJ homolog 4; GADD34, growth arrest and DNA damage-inducible protein; p-, phosphorylated.