Expression of Plasminogen Activator Inhibitor-2 is Negatively Associated with Invasive Potential in Hepatocellular Carcinoma Cells△

Ye Jin, Li Zhou, Ke-min Jin, and Bao-cai Xing

1Clinical Research Laboratory, 2Department of General Surgery, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100730, China 3Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Department of Hepatobiliary Surgery I, Peking University Cancer Hospital, Beijing 100142, China

HEPATOCELLULAR carcinoma (HCC), a life-thr- eatening malignancy, has high incidence, especially in endemic areas of hepatitis.1-3Besides, prognosis of HCC remains poor because most patients had no chance to receive curative therapy.3,4The extensive invasion and metastasis might account at least partially for the poor prognosis. Therefore, identification of molecular and genetic alterations associated with invasive phenotypes of HCC has attracted much interest.5-10

It has been proven that components of urokinase plasminogen activator system play different roles in invasion/metastasis of cancer cells.11In that system, uroki- nase-type plasminogen activator (uPA) promotes invasive phenotypes mainly by involving degradation of basement membrane and extracellular matrix.11A specific inhibitor of uPA, plasminogen activator inhibitor (PAI)-2, functions as a protease inhibitor.12PAI-2 is differentially expressed and has prognostic significance in some types of cancers.13-16PAI-2 expression and significance in HCC have been preliminarily studied.17,18However, the association between PAI-2 expression and invasive potential of HCC cells has not been elucidated. The present study was designed to explore this topic using three HCC cell lines with different metastatic potentials.

MATERIALS AND METHODS

Cell lines and cell culture

Two HCC cell lines, MHCC97-H with high metastatic potential and MHCC97-L with low metastatic potential,19were established and kindly provided by Liver Cancer Institute of Zhongshan Hospital Affiliated to Fudan University (Shanghai, China). The non-metastatic HCC cell line, SMMC-7721,20was purchased from Shanghai Institutes for Biological Sciences. The cells were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C in 5% CO2.

Matrigel invasion assay

Matrigel invasion assay was performed in vitro using 24-well millicell inserts (Costar Inc., Cambridge, MA, USA) with polycarbonate filters (pore size, 8 μm). The upper side of the filter was coated with 50 μg/mL matrigel (BD Biosciences, San Jose, CA, USA). The chambers were incubated at 37°C in 5% CO2for 30 minutes to promote coagulation of matrigel. The cells were harvested and re-suspended in a serum-free medium. Then 1×104cells in 100 μL of the serum-free medium were placed in the upper chamber, while the lower chamber was filled with 500 μL medium containing 20% FBS. The cells were cultured for 24 hours. The cells on the upper surface of the filters were removed using cotton swabs, and those on the lower surface were fixed with ice-cooled methanol and stained with hematoxylin eosin. The cell numbers in five fields (up, down, middle, left, and right of each chamber, ×200) were counted, and average values were calculated. Four chambers containing the same cell lines were observed, and the experiments were repeated thrice.

Western blot

The cells were digested with 0.25% trypsin and lyzed with RIPA buffer (Pierce, Rockford, IL, USA). The quantity of protein in the lysate was determined with the bicinchoninic acid protein assay kit (Pierce). Total proteins (60 μg) were separated using sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred for 2 hours to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). After blocked for 1 hour with 5% non-fat dry milk in 0.05% TBS/Tween (TBS-T), the membrane was incubated overnight with primary antibodies (sc-25745 for PAI-2 and sc-81178 for β-actin, Santa Cruz Biotechnology, Dallas, TX, USA) at 4°C. After washed with TBS-T for 4 times, the membrane was incubated with horse radish peroxidase- conjugated secondary antibodies for 1.5 hours at room temperature. Protein bands were appeared using an ECL kit (Millipore). Beta-actin was used as the internal control.

Statistical analysis

Statistical analysis was performed with SPSS 11.5 software package. The comparison of measurement data was conducted with Student t-test for independent samples. The Pearson's correlation analysis was used to detect correlation between variables. Statistical significance was defined as P ＜0.05.

RESULTS

Invasive potentials of the three HCC cell lines

Matrigel invasion assay revealed that the number of invaded cells in MHCC97-H (Fig. 1A) was significantly higher than that in MHCC97-L (P=0.017, Fig. 1B) and SMMC-7721 (P=0.001, Fig. 1C). The number of invaded cells in MHCC97-L was also signifiantly higher than that in SMMC-7721 (P=0.005). The numbers of invaded cell in the three cell lines were displayed in a histogram (Fig. 1D).

PAI-2 expression in the three HCC cell lines

Figure 1. Matrigel invasion results of three hepatocellular carcinoma (HCC) cell lines.A. the HCC cell line with high metastatic potential, MHCC97-H (HE ×200)； B. the HCC cell line with low metastatic potential,MHCC97-L (HE ×200)； C. the non-metastatic HCC cell line, SMMC-7721 (HE ×200)； D. the numbers of invaded cells in the three cell lines.*P＜0.05 compared with SMMC-7721； #P＜0.05 compared with MHCC97-L.

According to the result of Western blot, PAI-2 expression in MHCC97-H was significantly lower than that in MHCC97-L (P＜0.001) and SMMC-7721 (P=0.001) (Fig. 2A). PAI-2 expression in MHCC97-L was also significantly lower than that in SMMC-7721 (P=0.001, Fig. 2A). The protein bands representing PAI-2 expression were shown in Figure 2B.

The Pearson's correlation analysis revealed a negative association between the number of invaded cells and PAI-2 expression (r=-0.892, P=0.001).

Figure 2. Expression of plasminogen activator inhibitor (PAI)-2 in three HCC cell lines. A. Expression of PAI-2 relative to β-actin； B. Protein bands of Western blot detecting PAI-2 expression. *P=0.001 compared with SMMC-7721； #P＜0.001 compared with MHCC97-L.

DISCUSSION

In general, PAI-2, a classical endogenous inhibitor of uPA, inhibits uPA activity via a serpin “suicide” trapping mechanism.12It has been demonstrated to be involved in many physiological and pathological conditions, such as pregnancy, inflammation, and malignant tumors.12In particular, it is expressed and plays different clinicopathological roles in numerous kinds of cancer.13-16In HCC, limited publications focused on expression and implication of PAI-2, based on studies with tumor tissues.17,18The present study was designed to investigate the relationship between PAI-2 expression and invasive potential in HCC, on the basis of in vitro experiments using three HCC cell lines with different metastatic potentials, namely MHCC97- H, MHCC97-L, and SMMC-7721. Matrigel invasion assay detected a statistically significant decrease in the number of invaded cells from MHCC97-H to SMMC-7721. Western blot revealed an obvious trend of increase in PAI-2 protein expression from MHCC97-H to SMMC-7721. More importantly,the Pearson's correlation analysis confirmed the negative association between invaded cell number and PAI-2 expression (r=-0.892, P=0.001). The findings of the present study support the hypothesis that PAI-2 might serve as a negative regulator of the invasive proclivity of HCC cells. It has been indicated that PAI-2 inhibits cell invasion in gastric, breast, and cervical cancers through some important molecules and pathways, such as nuclear factor-κB and RhoA.21,22However, high PAI-2 was linked to favorable or poor prognosis in different malignancies.13-16Therefore, the biological role of PAI-2 in cancer might be tissue/cell type-specific. The present study provides a clue for the invasion-inhibitory role of PAI-2 in HCC. These preliminary findings need to be validated and deepened by further in-depth studies in the future.

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