Nimesulide inhibits the proliferation of HepG2 by up-regulation of Smad4.

OBJECTIVES: Hepatocellular carcinoma (HCC) is receiving increased attention. This study was designed to investigate the effect of selective Cyclooxygenase-2 (COX-2) inhibitor, nimesulide, on the expression of Smad4 in human hepatocellular carcinoma HepG2.
MATERIALS AND METHODS: HepG2 cells were incubated in various concentrations of nimesulide (25, 50, 100, 200, 400 μmol/L) to detect the effect of proliferation by MTS. The apoptosis of HepG2 was determined by TUNEL; fluorescence microscope was used to observe the expression of Smad4.
RESULTS: The result showed that nimesulide inhibited the proliferation of HepG2 cell in a concentrations-dependent manner, and promoted the karyopyknosis and fragmentation of HepG2 cell nucleus, induced its apoptosis, the number of fluorescence labeling of Smad4 in Nimesulide group was higher than control group (P<0.05).
CONCLUSIONS: Nimesulide inhibits the proliferation and promotes apoptosis of HepG2 by up-regulation of Smad4 in HepG2.

Introduction

Hepatocellular carcinoma (HCC) commonly occurs in hepatitis B endemic areas, arising almost exclusively under certain established high-risk situations, especially in China. The incidence of (HCC) has increased considerably in recent years, and is known as high as 10-20 cases per 100,000 per year.[1] Current effective treatment for HCC is surgical resection and transplantation. Unfortunately, this is possible in only a minority of patients, with good performance status and Child-PughA, as diagnosis is made at a late stage in most cases. Therefore, more attention was paid on the therapies of HCC.

Cyclooxygenase (COX) is a key enzyme in catalysising arachidonic Acid to produce prostsgandins (prostsgandins, PGs). COX-2, an inducible form, has a close relationship with the tumorigeness and development.[2] Current studies suggest that COX-2 is highly expressed in patients with various liver disease and hepatocellular carcinoma (HCC), suggesting its possible role in chronic liver disease and progression of HCC.[34] Overexpression of COX-2 is well correlated with the differentiation grade, metastasis and prognosis.[5-7] The mechanism is not clear, these effects may related to regulating some cytokines like vascular endothelial growth factor (VEGF).[78] Smads mediated transforming growth factor (TGF-β) signal transduction system has a series of important roles in controlling growth, survival, apoptosis and differentiation of many cells. Depressive effect of TGF-β is considered to be related to the occurrence, development and biological behavior of malignant tumors.[9] SMAD family member 4 (Smad4) plays a central role in TGF-β signaling to the nucleus, and it is known as a key tumor suppressor.[910] In the present study, we investigated the influence of selective inhibitor of COX-2 nimesulide (4-Nitro-2-henoxyme thane-sulfoanilide) on HepG2, in addition, we took the Smad4 as a target to investigate whether nimesulide can inhibit the growth of HCC cells by regulating the expression of Smad4.

Materials and Methods

Materials

HepG2 was obtained from Chinese Academy of Sciences. Dulbecco's modified Eagle's medium (DMEM) is a product of Hyclone company, nimesulide was purchased from Sigma company, MTS and TUNEL were from Promega. Anti-Smad4 (Mothers against decapentaplegic homolog4) and Anti- Smad4/ FITC were purchased from Beijing Biosynthesis Biotechnology Co., LTD, China.

Culturing of Cell Lines

HepG2 cell lines were cultured in DMEM supplemented with 15% fetal bovine serum (FBS, Sijiqiing) and incubated at 37°C in the humidified incubator with 5% CO2 in air.

Cells Proliferation Assay

HepG2 were plated in 96 well dishes (corning com- pany) at a density of 15*103 cells per well, respectively. All cells were suspended in DMEM. After 24 hours, media was renewed and cells exposed for another 24 hours to different concentration of nimesulide (0,25,50,100,200,400 μmol/L) 100μl in the presence of 20μl MTS for 4 hours, Every concentration was performed in triplicate. The optimal density (OD) was read on an enzyme-labeled at 492nm, results were expressed as Mean (SD) ±Standord Deviation (x˒±s). Values were calculated for all groups.

Apoptosis of Cells

Cells were plated on slides of 24 well dishes, after exposed for 48 hours to nimesulide, media was renewed, washed by PBS for 3 minites, repeat PBS wash, fixed cells in freshly prepared 4% methanol-freeformaldehyde solution in PBS (pH 7.4) in a Coplin jar for 25 minutes at 4°C. Washed by PBS for 3 times. Permeabilized cells by 0.2% Triton® X-100 solution in PBS for 5 minutes, washed by PBS for 3 times. Then the examimation were carried out according to the manual of the kit. Every concentration was performed in triplicate.

The Expression of Smad4

To process the cells in the same way with TUNEL, 0.2% Triton® X-100 solution in PBS for 5 minutes, washing by PBS for 3 times, then operated according to the introduction.

Statistical Analysis

To compare means the independent samples T test, a P-value of less than 0.05 was considered statistically significant. All analyses were performed using the statistical software SPSS13.0 for Windows.

Results

Cell Proliferation Assay

The strong inhibitory effect of nimesulide on the proliferation of HepgG2 showed a concentration-dependent fashion [Figure 1 and Table 1]. To compared with control group, the difference has statistical significance when the concentration of nimesulide is 100, 200, 400μmol/L (P<0.05).

Figure 1

Detection of A values after different concentration of nimesulide

Table 1

Inhibition effect of nimesulide on proliferation of HepG2 (x̄ ± s)

Cell Apoptosis Detection

As shown in Figure 2, cell apoptosis in nimesulide group is obvious, karyopyknosis and fragmentation of HepG2 cell nucleus can be seen, while few apoptotic cells in control group.

Figure 2

Apoptotic effect of nimesulide on HepG2

Expression of Smad4

Fluorescence labelling of Smad4 in nimesulide is more than control group [Figure 3].

Figure 3

The effect of nimesulide on expression of Smad4 in HepG2

Discussion

COX is a key enzyme in catalysising arachidonic Acid to produce prostsgandins (PGs). COX-2, an inducible form, has a close relationship with the tumorigeness and development.[2] Studies prompt that COX-2 take an effect in chronic hepatitis to HCC.[34] Over expression of COX-2 in HCC cells and tissues is well related with the cell differentiation, transference and prognosis.[5-7] The aim of the present study was to investigate the effects of nimesulide on HepG2. The results showed that nimesulide can inhibit the proliferation of HepG2 in a conceration-dependent manner. To compared with control group, nimesulide can induce the apoptosis of HepG2.

The mechanism of the inhibitory effect of nimesulide on HepG2 is less clear. Smad4, a member of TGF-β super family signaling, the production of DPC4, has been shown an important role in TGF-β/Smads signaling pathway. Some studies have shown that Smad4 can inhibit growth of the tumor by decreasing the expression of VEGF in cancer cells, inducing apoptosis of cancer cells and the interaction with other extracellular matrix and signaling path way.[11-13] These results suggest that the expression of Smad4 is lower in HCC. Schutte show that the expression of Smad4 is loss in 20% HCC, which lower the inhibitory effect on cell multiplication.[14] Antisense Smad4 cDNA was introduced into rat HCC liver, which can block the progression of liver fibrosis and the development of HCC.[15] Reports may remind us that loss or low expression of Smad4 in HCC cells or issue will lead to interruption of TGF-β/S pathway and inducing HCC. In this study, we have described the expression of Smad4 in nimesulide group and control group. Fluorescent expression of Smad4 in nimesulide group is higher than control group, suggesting that nimesulide could inhibit the proliferation and induce apoptosis of HepG2 by up-regulating the Smad4 in HepG2, however, whether this effect is produced by COX-2 is still not clear. In vivo animal experiments with nimesulide are necessary to further explore this effect.

Conclusions

We conclude that nimesulide inhibits the proliferation and promotes apoptosis of HepG2 by up-regulation of Smad4 in HepG2.