Increased systemic zonula occludens 1 associated with inflammation and independent biomarker in patients with hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the fifth most common primary liver cancer causing more than half a million deaths annually worldwide [1]. HCC progression is alarming in Asia, where hepatitis B virus infection is endemic [2, 3]. It is a complex disease with a poor prognosis, whose pathogenesis is poorly understood. HCC occurrence and mortality rates are increasing in many regions around the globe, specifying a need for the better cure. The most common risk factor for HCC is cirrhosis because of chronic heavy alcohol consumption [4], chronic hepatitis B and C virus infections [1, 3, 5]. Furthermore, inflammation also a key player involved in HCC development [6]. Diabetes [7], cigarette smoking [8] and long-term use of oral contraceptives also appear to be independent risk factors for HCC [8].

The contribution of inflammation to carcinogenesis has received major attention in hepatocarcinogenesis [9, 10]. Epidemiological evidence also suggests that up to 25% of all cancers are due to chronic low-grade inflammation or infection [10, 11]. Most HCC develops in the cirrhotic liver after prolonged inflammation, supporting the hypothesis that inflammation contributes to cancer development [12]. In this context, many published literature has shown evidence that alcohol abuse and hepatitis viral infections lead to chronic inflammation of the liver and are responsible for the progression of HCC worldwide [1, 3, 10, 11]. Moreover, 90% of HCC patients have a natural history of unresolved inflammation [11]. Hence, such considerations are extremely relevant in the design of new preventive approaches to the reduction of cancer risk. Furthermore, the treatment options for HCC have improved indeed; precise diagnostic or prognostic biomarkers are still lacking for the management of HCC.

Tight junctions (TJ) are not only a barrier but also function as a fence to restrict molecules across different cell types of the body based on the charge and size [13]. Furthermore, TJ of hepatocytes play an essential role in the blood-biliary barrier [14]. Zonula occludens 1 (ZO1) is a first tight junction phosphoprotein identified with the molecular weight of 210–225 kDa. ZO-1 appeared in the submembranous domain of tight junction in epithelia and endothelia [15, 16], binds to actin and the integral tight junction proteins occludin and claudins and junctional adhesion molecule (JAM) [16]. Diminished ZO-1 expression was found in many cancers and may closely associate with patient prognosis [17‐19]. Indeed, the blood concentration of ZO-1 and its correlation with inflammation in HCC remains unknown. The aim of the current study was to analyze blood ZO-1 concentration and its association with an inflammatory marker such as hsCRP in HCC cases and control subjects.

This study was a case control study, conducted in the Departments of Biochemistry and Surgical Gastroenterology, Jawaharlal Institute of Post Graduate Medical Education and Research (JIPMER) from March 2014 to March 2016 after obtaining approval from the Institute Ethics Committee (Human Studies) based on Ethical Guidelines of the Helsinki Declaration of 1975, as revised in 1983. Before recruitment, all the study participants were given written informed consent concerning the background and procedure of this study.

The results of this study demonstrated for the first time in HCC patients that increased blood levels of ZO-1 was correlated significantly with increased hsCRP concentrations observed in the same patient samples. Since ZO-1 is a first tight junction protein identified and its expression was shown to decrease in many cancers, including HCC [20]. In this regard, a very recent report indicates in HCC patients after hepatic resection that the low expression of ZO-1 was significantly associated with poor survival [21]. We found similar observation in HCC patient liver, which shows very faint ZO-1 expression compared to noncancerous liver tissue collected from the same HCC patients. Increased blood ZO-1 concentration observed in HCC further supports the hypothesis that decreased hepatic-intestinal barrier dysfunction with marked ZO-1 and other TJ protein disruptions. This finding is also consistent with a very recent study showing in ischemic reperfusion (I/R) injury model that low ZO-1 expression contributing to TJ disruption and increased gut permeability [22] following intestinal I/R insult. Further, it has been reported in response to proinflammatory cytokines, the expression of ZO-1 was reduced and redistributed away from the TJ upon increased intestinal permeability [22, 23]. TNF α a known proinflammatory cytokine was shown to inhibit the expression of ZO-1 through the mechanism involved in the triggering nuclear factor-κ B (NF-κB) thus, increased intestinal epithelial TJ permeability [22, 24]. Moreover, decreased intestinal TJ integrity, a key pathogenic feature involved in the progression of intestinal inflammation [13, 23]. Thus, we speculate the loss of TJ protein during HCC progression on the background of inflammation [20, 24], consequently released in the circulation, perhaps increased plasma ZO-1 in observed in HCC patients.

A growing body of literature showing evidence that a solid pathological association between chronic low-grade inflammation and carcinogenesis [25]. High sensitive CRP, an acute-phase reactant synthesized in the liver that is regulated by pro-inflammatory cytokines, primarily interleukin (IL) 6 [26]. It has been reported to be associated with a poor prognosis in patients with different types of cancer, which including HCC [26]. In this study, hsCRP was found to elevated in HCC patients when compared to healthy volunteers. In this context, the previous studies showing compelling evidence that systemic inflammation is known to associate with tumor development and poor survival in patients with HCC [3, 11]. In addition, a moderate increase of hsCRP showed to predict recurrence and survival in patients with early-stage HCC [27]. Further, it has been demonstrated that a novel prognostic scoring system, which includes the CRP level, predict overall survival in late-stage HCC following treatment with Sorafenib [28]. Shin et al., found that increased serum CRP concentration indicates poor prognosis in patients with HCC [29]. Also, increased immunoreactivity of CRP is considered a key feature of inflammatory hepatic adenomas with an increased risk of malignant transformation [29]. Furthermore, the ratio of hsCRP and albumin predicted outcomes in patients with HCC [30] and considered a novel inflammation-based prognostic score. We found decreased albumin concentrations in HCC patients compared to healthy control. However, there is no correlation between elevated hsCRP and lowered albumin levels in HCC.

Alpha-fetoprotein (AFP), a plasma glycoprotein synthesized during early fetal life by the liver and considered HCC specific marker [31]. However, in the current study we found only 73% of HCC patients had elevated (> 15 ng/mL) serum AFP level whilst 27% had normal (< 15 ng/mL) AFP level consistent with previous report showing that AFP was not an optimal marker for the early detection of HCC patients on the background of hepatitis C infection [32]. This may indicate lower diagnostic efficacy of AFP in HCC however; it needs to prove with larger sample size. Hepatic function markers such as total bilirubin, ALT, AST, ALP, and GGT are widely used in clinical diagnosis of hepatic dysfunction and damage [33]. Increased concentration found in many acute and chronic liver ailments and also associated with HCC progression [34]. In the current study, the plasma concentration of ALT, AST, ALP, GGT and total and direct bilirubin were significantly (p < 0.0001) elevated in HCC patients when compared to control subjects. Indeed, none of the parameters correlated with increased blood ZO-1 concentration. A previous study has shown evidence that AST, ALT, and GGT were elevated in 90% of diagnosed HCC patients whilst half of the patients also showed increased bilirubin or liver-specific ALP concentrations, indicating these are good pre-diagnostic markers of liver cancer [35].

The current study identified elevated serum ZO-1 concentration in HCC patients for the first time, and this was positively correlated with increased hsCRP levels. Thus, our study showing evidence that inflammation promotes plasma ZO-1 concentration and raises the possibility that it could be used as a potential diagnostic biomarker for HCC progression. Moreover, diminished hepatic expression of ZO-1 found in HCC patients further confirms that targeting ZO-1 possibly provides a rational approach to the management of HCC and may serve as a marker for future molecular phenotyping of HCC.