Butaselen prevents hepatocarcinogenesis and progression through inhibiting thioredoxin reductase activity

Abstract

Hepatocellular carcinoma (HCC) accounts for most of primary liver cancer, of which five-year survival rate remains low and chemoprevention has become a strategy to reduce disease burden of HCC. We aim to explore the in vivo chemopreventive effect of an organoselenium-containing compound butaselen (BS) against hepatocarcinogenesis and its underlying mechanisms. Pre- and sustained BS treatment (9, 18 and 36mg/Kg BS) could dose-dependently inhibit chronic hepatic inflammation, fibrosis, cirrhosis and HCC on murine models with 24 weeks treatment scheme. The thioredoxin reductase (TrxR), NF-κB pathway and pro-inflammatory factors were activated during hepatocarcinogenesis, while their expression were decreased by BS treatment. BS treatment could also significantly reduce tumor volume in H22-bearing models and remarkably slow tumor growth. HCC cell lines HepG2, Bel7402 and Huh7 were time- and dose-dependently inhibited by BS treatment. G2/M arrest and apoptosis were observed in HepG2 cells after BS treatment, which were mediated by TrxR/Ref-1 and NF-κB pathways inhibition. BS generated reactive oxygen species (ROS), which could be reduced by antioxidant N-acetyl-L-cysteine (NAC) and NADPH oxidase inhibitor DPI. NAC could markedly increase HepG2 cells viability. TrxR activity of HepG2 cells treated with BS were significantly decreased in parallel with proliferative inhibition. The TrxR1-knockdown HepG2 cells also exhibited low TrxR1 activity, high ROS level, relatively low proliferation rate and increased resistance to BS treatment. In conclusion, BS can prevent hepatocarcinogenesis through inhibiting chronic inflammation, cirrhosis and tumor progression. The underlying mechanisms may include TrxR activity inhibition, leading to ROS elevation, G2/M arrest and apoptosis.

Keywords: Chemoprevention; Hepatocellular carcinoma (HCC); NF-κB; Reactive oxygen species (ROS); Thioredoxin reductase (TrxR).

Fig. 1

Experimental schedule and preventative effect of butaselen against hepatocarcinogenesis. (A) Chemical structure of butaselen (BS). (B) Diethylnitrosamine (DEN), CCl₄ and 10% ethanol were used to induce model of hepatocellular carcinoma (HCC) for 24 weeks in C57BL/6J mice. Different doses of butaselen (BS) were administered in other three groups. (C) The body weight change and (D) survival proportions of mice in 180 days. (E) Representative hepatocellular damage at week 6. (F) Gross pathology and HE stain of cirrhosis at week 16. (G) Gross and pathological change of HCC at week 24. Data were shown as mean ± std, n ≥ 28 and analyzed with ANOVA or Log-rank test. *, ^#p < 0.05 compared with control or model group. BSL, 9 mg/kg BS; BSM, 18 mg/kg BS; BSH, 36 mg/kg BS.

Fig. 2

Biomarkers and pathological changes during hepatocarcinogenesis. (A) Histogram analysis of necrosis grade of hepatocytes at week 6. (B) Serum ALT and AST levels at week 6. (C) Statistical analysis of hepatic fibrosis grade and (D) Masson stain of mice liver tissue at week 16. Collagen is in blue color. (E) Histogram analysis of hepatocellular dysplasia grade at week 24. Data were shown as mean ± std, n ≥ 6 and analyzed with ANOVA. *, ^#p < 0.05 compared with control or model group. BSL, 9 mg/kg BS; BSM, 18 mg/kg BS; BSH, 36 mg/kg BS.

Fig. 3

The biomarkers change and underlying mechanisms of butaselen (BS) to prevent hepatocarcinogenesis. (A) Histoimmunochemical and immunoblotting analysis of glypican-3 (GPC3) and CD34. Serum level of (B) alpha-fetal protein (AFP), and (C) thioredoxin reductase (TrxR) activity at week 24. (D) Protein expression of TrxR and NF-κB pathway in liver of every group. (E) Gene and (F) protein level of inflammatory factors in mice liver. Data were shown as mean ± std, n ≥ 6 and analyzed with One-way ANOVA. *, ^#p < 0.05 compared with control or model group.

Fig. 4

The in vivo inhibitory effect of butaselen (BS) on hepatocellular carcinoma (HCC). (A) The tumor volume changes and (B) gross pathology of H22 mice models injected with 1 × 10⁶ cells after BS treatments. (C) Tumor volume analysis, (D) Gross pathology and (E) HCC formation rate in control or 180 mg/kg BS treatment mice models implanted with 1 × 10⁴–1 × 10⁶ H22 cells. Data were shown as mean ± std, n = 6, analyzed with One-way ANOVA and Log-rank test. *, ^#p < 0.05 compared with control or BS 180 mg/kg group.

Fig. 5

Proliferative inhibition, G2/M arrest and apoptosis in hepatoma cells induced by BS treatment. (A) Dose- and time-dependent inhibition of BS on Bel7402, HepG2 and Huh7 cells after 24–72 h treatment. (B) Representative figure and histogram analysis of G2/M arrest in HepG2 cells after 20–40 µM BS treatment for 24 h. (C) Annexin V-PI analysis and statistical analysis of early and total apoptosis rate after BS treatment for 24 h on HepG2 cells. Data were shown as mean ± std, n ≥ 3 and analyzed with One-way ANOVA. *^{, #, ※}p < 0.05 compared with control group, 20 μM BS group or 30 μM BS group.

Fig. 6

ROS production mediated by TrxR activity inhibition after butaselen (BS) treatment for 24 h. (A) Representative histogram plot and statistical analysis of ROS stained by DCF-DA and DHE. Pretreatment of antioxidant N-acetyl-L-cysteine (NAC, 5 mM) decreased ROS level induced by BS. (B) The ratio of glutathione (GSH) to glutathione disulfide (GSSG) was markedly reduced after BS treatment. (C) Proliferative inhibition was significantly lower of BS added with 5 mM NAC compared with BS alone. (D) TrxR activity inhibition were generally in parallel with proliferative inhibition of HepG2 cells. Glutathione peroxidase and glutathione reductase activities were to some extent elevated. (E) Significantly lower TrxR1 expression, TrxR activity and higher ROS level after HepG2 cells transfected with LV-TrxR1-shRNA compared with LV-NC-shRNA. (F) The ROS induced by 40 μM BS was inhibited by NADPH oxidase inhibitor DPI. Data were shown as mean ± std, n ≥ 3 and analyzed with One-way ANOVA. *^{, #, ※,★}p < 0.05 compared with control group, 20 μM BS group, 30 μM BS group or 40 μM BS group. DPI, diphenyleneiodonium chloride; MT, mito-tempo.

Fig. 7

The effects of TrxR1 knock down on proliferation, viability and mechanisms mediating G2/M arrest and apoptosis of HepG2 cells (A) Knock-down of TrxR1 inhibited HepG2 cells proliferation and (B) increased resistance to BS treatment. (C) Immunofluorescent analysis of Trx level upon BS treatment. (D) Gene and (E) protein levels of Trx, Ref-1 and molecules mediating G2/M arrest. (F) Downregulation of NF-κB pathway. Level of (G) mRNA and (H) proteins mediating mitochondria-dependent apoptosis. Data were shown as mean ± std, n ≥ 3 and analyzed with One-way ANOVA. *^{, #}p < 0.05 compared with control or LV-NC-shRNA group.

Fig. S4

Statistical analysis of NF-κB pathway and key molecules in mitochondria-dependent apoptosis. Protein expression of (A) pIκB-α and (B) pNF-κB p65. (C) The gene and protein level of Bcl-2/Bax. (D) Protein levels of pro-caspase-3. Data were shown as mean ± std, n ≥ 3 and analyzed with One-way ANOVA. * p < 0.05 compared with control group. ^#p < 0.05 compared with 20 μM BS group. ^※P<0.05 compared with 30 μM BS group.