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

Abstract

Background: Hepatocellular carcinoma (HCC) is a frequent type of primary liver cancer, and its prevalence is increasing worldwide. Indeed, the underlying molecular mechanism is not well understood. Previous studies have shown evidence that tight junction (TJ) components were correlated with carcinogenesis and tumor development. Our aims were to determine the serum levels of tight junction protein Zonula Occludens (ZO)-1 and an inflammatory marker such as high-sensitive C-reactive protein (hs-CRP) in HCC patients compared to healthy volunteers and also to identify the association between ZO-1 and inflammation in HCC.

Methods: Thirty HCC patients and 30 healthy volunteers were recruited in the current study. Clinical data regarding child class, BCLC staging, the number of lesions, tumor size, absence or presence of metastasis, cirrhosis and hepatitis infection were also collected in HCC patients. Plasma ZO-1 and serum hsCRP were analyzed by EIA and ELISA respectively and biochemical parameters by autoanalyser (AU680 Beckman Coulter, USA). Furthermore, hepatic ZO-1 protein expression and tissue localization were examined.

Results: Compared to healthy individuals, the serum levels of bilirubin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyltransferase (GGT) and alkaline phosphatase (ALP) were elevated significantly (P < 0.0001) whilst serum albumin level was significantly (P < 0.0001) decreased in HCC patients. Furthermore, tight junction protein ZO-1 concentration was significantly elevated in HCC patients compared to control subjects (648 ± 183.8 vs. 396.4 ± 135.8 pg/ml, respectively; P < 0.0001). Serum hsCRP level was also significantly increased in HCC patients compared to control subjects (17.25 ± 3.57 vs. 5.54 ± 2.62 mg/L, respectively; P < 0.0001). Moreover, decreased protein expression of ZO-1 was found in liver tissue obtained from HCC patients.

Conclusion: Our findings show for the first time that the systemic concentration of ZO-1 was significantly elevated in HCC patients and is positively correlated with inflammatory markers. Thus, the current 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.

Keywords: Blood biomarker; Hepatic marker enzymes; Inflammation; Liver cancer; Tight junction.

Fig. 1

a Plasma ZO-1 levels in healthy volunteers and HCC patients. Values are expressed as mean ± standard deviation. P < 0.0001, controls Vs HCC. b Plasma ZO-1 levels according to Child-Pugh class in HCC patients. Values are expressed as mean ± standard deviation. ***P = 0.0003, Child-Pugh class A Vs C ***P = 0.0004, Child-Pugh class B Vs C; ns-No significant. c Plasma ZO-1 levels according to BCLC staging system in HCC patients. Values are expressed as mean ± standard deviation. ****P < 0.0001, BCLC staging B Vs D; **P < 0.01, BCLC staging C Vs D; ns-No significant

Fig. 2

a Serum hs-CRP levels in healthy volunteers and HCC patients. Values are expressed as mean ± standard deviation. p < 0.0001, controls Vs HCC. b Serum hs-CRP levels according to Child-Pugh class in HCC patients. Values are expressed as mean ± standard deviation. **P < 0.01, Child-Pugh class A Vs B and A Vs C, respectively. c Serum hs-CRP levels according to BCLC staging system in HCC patients. Values are expressed as mean ± standard deviation. ns-No significant

Fig. 3

Prognostic potential of serum ZO-1 in HCC patients. Serum ZO-1 levels were positively correlated with increased hsCRP in HCC. Correlation coefficient (r) and statistical significance (p value) are indicated. Individual samples are represented as dots (n = 28)

Fig. 4

ROC curve analysis of ZO-1 (a) and hs-CRP (b). ZO-1 at cut off of 472.99 (pg/ml) had a sensitivity of 76.92%, specificity of 80.00% with AUC of 0.8692. hs-CRP at cut off of 9.2 (mg/L) had a sensitivity of 86.67%, specificity of 85.00% with Area Under Curve of 0.8767

Fig. 5

Protein expression of ZO-1 in HCC liver and noncancerous liver tissues (histologically proven control) from HCC patients. Values are expressed as mean ± standard deviation. P < 0.05, controls Vs HCC

Fig. 6

Immunohistochemical localization of ZO-1 in noncancerous liver tissue (histologically proven control) (a) and HCC liver (b) from HCC patients. ZO-1 is not expressed in HCC liver (b), whereas control liver tissue (a) expressed increased ZO-1 particularly in the bile ducts