AIM: To evaluate the clinical significance of oxidative stress markers in patients with hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC).

METHODS: Sixty-four consecutive patients who were admitted to Kagoshima University Medical and Dental Hospital were enrolled in this retrospective study. All patients had chronic liver disease (CLD) due to infection with HCV. Thirty patients with HCV-related HCC, 34 with HCV-related CLD without HCC (non-HCC), and 20 healthy volunteers (HVs) were enrolled. Possible associations between serum manganese superoxide dismutase (MnSOD) and thioredoxin (TRX) levels and clinical parameters or patient prognosis were analyzed over a mean follow-up period of 31.7 mo.

RESULTS: The serum MnSOD levels were significantly higher in patients with HCV-related HCC than in patients without HCC (P = 0.03) or HVs (P < 0.001). Similarly, serum TRX levels were also significantly higher in patients with HCV-related HCC than in patients without HCC (P = 0.04) or HVs (P < 0.01). However, serum levels of MnSOD and TRX were not correlated in patients with HCC. Among patients with HCC, the overall survival rate (OSR) was lower in patients with MnSOD levels ≥ 110 ng/mL than in patients with levels < 110 ng/mL (P = 0.01), and the OSR tended to be lower in patients with TRX levels < 80 ng/mL (P = 0.05). In addition, patient prognosis with HCC was poorest with serum MnSOD levels ≥ 110 ng/mL and serum TRX levels < 80 ng/mL. Furthermore, a multivariate analysis using a Cox proportional hazard model and serum levels of five factors (MnSOD, prothrombin time, serum albumin, serum α-fetoprotein (AFP), and serum des-γ-carboxy prothrombin) revealed that MnSOD levels ≥ 110 ng/mL (risk ratio: 4.12, 95% confidential interval: 1.22-13.88, P = 0.02) and AFP levels ≥ 40 ng/mL (risk ratio: 6.75; 95% confidential interval: 1.70-26.85, P < 0.01) were independent risk factors associated with a poor patient prognosis.

CONCLUSION: Serum MnSOD and TRX levels are potential clinical biomarkers that predict patient prognosis in HCV-related HCC.

As a significant cause of global cancer morbidity and mortality, hepatocellular carcinoma (HCC) is the fifth- and seventh-most frequently diagnosed cancer worldwide in men and women, respectively, and is the second- and sixth-most frequent cause of cancer deaths in men and women, respectively[1]. HCC is most frequently caused by persistent infection with hepatitis C or B virus. Early HCC diagnosis and better treatments have helped to improve the prognosis for patients with HCC. Also, interferon (IFN)-based treatments not only eliminate hepatitis C virus (HCV) infection, but also prevent HCC in patients with chronic hepatitis C (CHC)[2]. However, IFN-based therapies do not always effectively eliminate HCV infection or prevent HCC. Thus, biomarkers that are indicative of HCC pathological condition would have many clinical benefits, including aiding in the selection of the most appropriate treatment for a patient’s disease.

Oxidative stress results from an imbalance in the production of reactive oxygen species (ROS) and the antioxidative defenses that maintain a cellular redox state. ROS include superoxide anions, hydrogen peroxide, hydroxyl radicals and nitric oxide, all of which are indispensable elements in many biochemical processes[3]. ROS are mainly derived from Kupffer and inflammatory cells in the liver[4], and upon exposure to other cells are thought to induce apoptosis, necrosis, inflammation, immune responses, fibrosis and tissue regeneration[5]. In liver disease, there is an overproduction of ROS from endogenous sources such as the mitochondria, peroxisomes, and activated inflammatory cells. In particular, ROS of mitochondrial origin were recently reported to be elevated in patients with alcoholic liver disease, non-alcoholic steatohepatitis (NASH)[6,7] and HCV-related chronic liver disease (CLD)[8]. Conversely, cells are protected from oxidative stress by intracellular antioxidants such as glutathione (GSH) and thioredoxin (TRX) and by various antioxidant enzymes such as superoxide dismutase (SOD), GSH peroxidase, catalase, and heme oxygenase-1[9-11]. Collectively, the relative expression levels of these molecules may serve as biomarkers for various liver diseases, including HCV-related HCC.

Manganese SOD (MnSOD) is an antioxidant enzyme that catalyzes the dismutation of the highly reactive superoxide anion to O₂ and to the less reactive species H₂O₂. We have previously demonstrated that MnSOD expression was induced in primary cultured hepatocytes that were loaded with hydrogen peroxide in vitro and that serum MnSOD levels can be used to distinguish between NASH and simple steatosis in patients with nonalcoholic fatty liver disease[7]. However, the clinical significance of serum MnSOD levels in HCV-related CLD has not been fully investigated.

TRX was originally discovered in Escherichia coli as a proton donor for ribonucleotide reductase[12]. Subsequently, the human TRX gene was cloned as an adult T-cell leukemia-derived factor and was originally described as an interleukin-2 receptor inducer present in the cell culture supernatant of human T-lymphotropic virus type-1 -transformed cells[13]. TRX expression is induced by various oxidative stressors in patients with acquired immunodeficiency syndrome[14], Sjögren’s syndrome[15], rheumatoid arthritis[16], and malignant neoplasms[17,18]. Previous studies have reported that serum TRX is an oxidative stress marker and that serum TRX levels increase in patients with HCV-related CLD during liver fibrosis progression[19]. In addition, serum TRX levels are reported to be elevated in patients with NASH compared to patients with simple steatosis[20]. However, the clinical significance of elevated TRX levels among patients infected with HCV in relation to HCC diagnosis and prognosis has not been elucidated.

In this study, we aimed to clarify the clinical significance of serum levels of MnSOD and TRX in patients with HCV-related CLD, and in particular among patients with HCC.

HCV infection is the most important known contributor to the etiology of HCC. An increasing incidence of HCC has been largely attributed to a rise in HCV infections in the general population during the last 50 to 60 years[26]. During HCV infection, ROS production increases and persists throughout the infection. In addition, ROS are thought to play a major role in the pathogenesis of chronic inflammatory changes in the liver, leading to increased hepatic fibrosis and decreased hepatic function. In this study, we have shown that both serum MnSOD and TRX levels are elevated in patients with HCV-related HCC, with no correlation between these two markers. In addition, serum MnSOD and TRX levels were a useful predictor of overall patient survival. Serum MnSOD and TRX levels are reported to be biomarkers of oxidative stress in several diseases, including liver disease[7,14,17,19,27-29]. There were a small number of enrolled patients in this study and other contributors to liver diseases such as chronic hepatitis B infection should be further evaluated. However, our study has clearly demonstrated the clinical significance of these markers in patients with HCV-related HCC.

Serum MnSOD and TRX levels should both reflect hepatic oxidative stress. The results of the current study showed that both of these markers were increased in the HCC group relative to levels in the non-HCC group and the HV group (Figure 1A and B). However, there was no correlation between these two markers in the HCC group (Figure 1C). MnSOD is primarily localized to the mitochondrial matrix[3] and abnormal mitochondrial morphologies are frequently observed in CHC[8]. Therefore, MnSOD may be an indicator of mitochondrial disorders that are induced by oxidative stress. On the other hand, there are two TRX proteins, cytoplasmic TRX1 and mitochondrial TRX2[30]. TRX1 negatively regulates the apoptosis signal-regulating kinase 1 (ASK1)-c-Jun N-terminal kinase/P38 apoptotic pathway by binding to and inhibiting the kinase activity of ASK1, which plays an important role in ROS-induced cellular responses[31]. TRX2 is an essential regulator of mitochondrial ROS levels that has been associated with mitochondrial outer membrane permeability[32]. In the present study, we examined the serum levels of TRX1, but not TRX2, using a sandwich ELISA. Thus, the MnSOD and TRX proteins that were examined in this study have different origins in the mitochondria and cytoplasm, respectively, which could contribute to the lack of correlation between these two markers.

Several studies have shown that the HCV core protein directly inhibits the electron transport system and modulates apoptosis, transcription, and cell signaling[33]. Abdalla et al[34] reported that expression of not only the HCV core protein but also the HCV NS proteins increases ROS and further showed that the presence of these proteins can increase endogenous expression levels of antioxidant enzymes and prooxidants such as MnSOD. Several reports have shown that serum MnSOD levels in patients with HCV-related CLD[35-37] are associated with various clinical findings, such as fibrosis and hepatic oxidative stress. However, the significance of serum MnSOD levels has not been fully examined in patients with HCC. We previously reported that serum MnSOD levels may be correlated with fibrosis in patients with NAFLD[7]. In addition, serum MnSOD levels decreased in patients with CHC after administration of an interferon-based treatment (data not shown). These results indicate that serum MnSOD levels are likely associated with hepatic fibrosis or oxidative stress in patients with CHC. In the present study, however, MnSOD levels were not associated with platelet counts, which is a simple predictor of hepatic fibrosis in this patient population[38]. Thus, advanced hepatic fibrosis or oxidative stress may be one reason why serum MnSOD levels have diagnostic and prognostic utility with HCC, but other mechanisms should also be considered.

The present study revealed that serum MnSOD levels were significantly higher in the HCC group than in the non-HCC group (Figure 1A). In the HCC group, serum MnSOD levels were negatively correlated with serum Alb and tended to negatively correlate with PT (Table 3); these results showed an association between MnSOD and Child-Pugh stage (Figure 2A). It is known that in humans, MnSOD activity is comparatively higher in the liver compared to other tissues[39]. In addition, although a previous immunohistochemical study showed that MnSOD expression was higher in both cancerous and non-cancerous liver tissues from patients with HCC, this positive immunoreactivity was strongly observed in non-cancerous liver tissues, especially in normal hepatocytes surrounding HCC, regenerative small hepatocytes in the tumor boundary, and mononuclear inflammatory cells in necroinflammatory lesions[40]. Furthermore, ROS are overproduced by Kupffer cells and inflammatory cells in liver disease[5,41]. In the present study, serum MnSOD levels were also positively correlated with the serum tumor markers AFP and DCP (Table 3) and with Child-Pugh stage and CLIP score (Figure 2). These results indicate that increased MnSOD expression reflects hepatocyte oxidative stress and correlates with decreased hepatic function, increased hepatic fibrosis and ROS production by inflammatory cells in liver cirrhosis. These features comprise the main background characteristics leading to HCC and may be associated with the indirect effects of liver cancer progression. These associations may also explain why serum MnSOD levels predicted the overall survival of patients with HCC.

It was previously reported that serum levels of TRX, which is a stress-induced protein, increase relative to the degree of hepatic fibrosis, and that high serum concentrations of TRX may indicate advanced hepatic fibrosis[19,20]. In contrast, it has also been reported that a higher degree of hepatic fibrosis is associated with lower platelet counts[38]. Therefore, the present study may present a conflict, since results indicated that serum TRX level was positively correlated with platelet count. A previous report showed that the survival rate following LPS plus GalN-induced hepatitis was much higher in transgenic mice overexpressing TRX than in wild-type mice, and that thioacetamide-induced hepatic fibrosis was suppressed in TRX transgenic mice compared to wild-type mice[42]. Although it is still unclear why TRX and platelet counts are positively correlated, we speculate that elevated serum TRX in patients with HCC and advanced hepatic fibrosis potentially improves overall survival by suppressing oxidative stress[43]. In addition, patients with HCC, low levels of TRX, and high levels of MnSOD, which may be indicative of excessive oxidative stress without TRX attenuation, have the poorest prognosis. This result supports the hypotheses presented above. In order to better assess these findings, future studies are needed that incorporate sequential observations of serum TRX and MnSOD levels over time in patients with chronic hepatitis, cirrhosis and HCC.

Serum MnSOD and TRX may be useful biomarkers for HCC diagnosis (Figure 1). AFP is also a diagnostic marker for HCC, and the present results indicate that AFP can be used to distinguish between patients with and without HCC (Table 2). However, AFP is not a sufficiently sensitive marker for identification of the majority of patients with small HCCs[44,45], and AFP testing is not currently included in the recommendations for HCC surveillance in the updated HCC guidelines published by the American Association for the Study of Liver Disease[46]. Therefore, clinicians and clinical researchers should consider using MnSOD and TRX as diagnostic biomarkers for early HCC or as additional markers in a HCC surveillance program using ultrasonography or AFP. In addition, it is highly important to know whether these markers decrease in response to HCC therapy and reductions in tumor burden. These markers also may have utility in patients on a transplant waiting list who are treated with neo-adjuvant therapy for tumor downstaging.

Our study demonstrated that elevated serum AFP level is indicative of a poor prognosis for patients with HCC (Table 4), as was previously reported[47]. The CLIP score, which is calculated based on four factors such as the AFP value, was also useful to predict the prognosis of HCC patients in this study as well as in a previous report[48]. Other markers such as the protein survivin have been reported as poor prognostic factors for HCC[49]. Similarly, MnSOD was an independent predictive factor for overall survival in the HCC group (Figure 3A, Table 5). Although TRX was not an independent predictor of overall survival in patients with HCC (Table 4), we speculate that a combination assay using both MnSOD and TRX could be used to predict overall patient survival. It will be important to conduct further prospective evaluations of each individual marker as well as a combination of these markers using a large number of patients.

In conclusion, serum MnSOD and TRX levels increased as HCV-related chronic liver disease progressed, especially among patients with HCC. Although there was no correlation between serum levels of MnSOD and TRX, higher serum MnSOD levels and lower TRX levels in patients with HCC trended towards an indication of poor patient prognosis. These results suggest that serum MnSOD and TRX levels are not only a potential biomarker for HCV-related progressed liver disease, but may also serve as prognostic markers in HCC.

We thank Ms. Yuko Nakamura for technical assistance.