PRDX1 Influences The Occurrence and Progression of Liver Cancer by Inhibiting Mitochondrial Apoptosis Pathway

Abstract

Objective: The aim of this study is to elucidate the role of PRDX1 in hepatocellular carcinoma using hepatoma cells.

Materials and methods: In this experimental study, we elucidated role of PRDX1, using hepatoma cell lines.

Results: PRDX1 was upregulated in different types of cancers, including lung adenocarcinoma, breast cancer and liver cancer reported by several studies. nevertheless, mechanism of inducing liver cell death by PRDX1 remains largely unknown. Here, we showed that PRDX1 expression is enhanced in different cell lines. Here, we used western blot, quantitative real time polymerase chain reaction (qRT-PCR) and different biochemical assays to explore the role of PRDX1. We observed that overexpression of PRDX1 significantly enhanced proliferation of hepatoma cell lines, while knock-down of this gene showed significant inhibitory effects. We found that knock-down of PRDX1 activated cleaved caspase-3, caspase-9 proteins and Poly [ADP-ribose] polymerase 1 (PARP-1), which further executed apoptotic process, leading to cell death. We found that PRDX1 knock-down significantly produced mitochondrial fragmentation. We showed that silencing PRDX1 led to the loss of B-cell lymphoma 2 (Bcl-2) and activated Bcl-2-like protein 11 (Bim) which further induced Bax activation. Bax further released cytochrome c from mitochondria and induced apoptotic proteins, suggesting a significant role of PRDX1 knock-down in apoptosis. Finally, we showed that knock-down of PRDX1 significantly activated expression of Dynein-related protein 1 (Drp1), fission 1 (Fis1) and dynamin-2 (Dyn2) suggesting a crucial role of PRDX1 in mitochondrial fragmentation and apoptosis conditions. This study highlighted an important role of PRDX1 in regulating proliferation of hepatoma cells and thus future studies are required to validate its effect on hepatcoytes.

Conclusion: We propose that future works on PRDX1 inhibitors may act as a therapeutic candidate for treatment of liver cancer.

Keywords: Hepatocellular Carcinoma; PRDX1; Peroxiredoxins; liver cancer.

Fig 1

Expression of peroxiredoxin 1 (PRDX1) in different cell lines of liver cancers. A. Box diagram of PRDX1 expression, with significant difference between cancerous and paracancerous tissues of liver cancer patients. B. PRDX1 mRNA expression was significantly higher in cancerous cells. C. Western blot results showed an increased expression of PRDX1 protein in cancerous cells, with the highest expression level observed in hepatoma (HepG2) cells. D. Quantifications of protein levels. The experiment was repeated three times. Data represent mean ± SEM. *; P<0.05, **; P<0.01, and ***; P<0.001.

Fig 2

PRDX1 knock-down induced cell death via apoptosis in HCC cells. A. Significant increase of PRDX1 mRNA expression level when overexpressed in hepatoma cells. B. Western blot showed that PRDX1 overexpression enhanced expression level of PRDX1, while knock-down of this gene substantially reduced the expression. C. Quantification of the protein levels. D. CCK-8 assay showed a significant reduction in the IC₅₀ value of hepatoma cells. E. Clonal formation assay demonstrated effect of PRDX1 expression on the proliferation of hepatoma cells. Knock-down PRDX1 showed a significant inhibitory effect on cells proliferation. F. Quantification of the number of colonies. G. Western results showed the effect of knock-down of PRDX1 protein on caspase proteins in hepatoma cells. H. Quantification of the protein levels. The experiment was repeated three times. Data represent mean ± SEM. **; P<0.01, and ***; P<0.001.

Fig 3

PRDX1 knock-down reduced mitochondrial transmembrane potential in HCC cells. A. Western blot results depicted that PRDX1 knock-down substantially reduced expression of Bcl-2 and increased Bax expression. The lower bottom showed quantifications of protein levels. B. Mitotracker red assay was used to see the effect of si-PRDX1 expression on mitochondrial morphology of hepatoma cells. PRDX1 knock-down induced significantly increased filamentous mitochondrial morphology, assessed by fluorescent intensity. Representative graphs show quantifications. The experiment was repeated three times. Data represent mean ± SEM. **; P<0.01, and ***; P<0.001.

Fig 4

Effect of PRDX1 knock-down on apoptosis and mitochondrial fission machinery of HCC cells. A. Western results showed that knock-down of PRDX1 significantly enhanced expression of Drp1, Fis1 and Dyn2 in hepatoma cells. B. Images and graphs showed that overexpression of PRDX1 decreased apoptotic proteins (Bim, cytochrome C and Apaf-1) expression level, while knock-down of this gene significantly enhanced expression of caspase proteins. The right panel shows quantifications of protein levels. The experiment was repeated three times. Data represent mean ± SEM. **; P<0.01, and ***; P<0.001.