Barrett's esophagus (BE) is an acquired condition in which squamous epithelium in the distal portion of esophagus is replaced by metaplastic columnar epithelium containing goblet cells (1). BE is recognized as a complication of chronic gastro-esophageal reflux disease (GERD) and is reported at endoscopy in 6-12 % of patients with reflux symptoms (2). BE is considered as a precancerous state in the esophagus and may progress to esophageal adenocarcinoma (EA). Compared with individuals in the general population, patients with BE have a 30 to 125-fold higher risk for developing BA. According to the epidemiological data, the incidence of BA has significantly increased over the last three decades (3).

Since BE carries a significantly increased risk of developing EA, three clinical approaches have been developed for secondary prevention of cancer in patients with pre-existing BE. These include: 1) endoscopic ablation and resection; 2) antireflux surgery and 3) pharmacological treatment (chemoprevention) (4). There is some epidemiological and experimental evidence that the long-term use of proton-pump inhibitors (PPI) or the NSAIDs (aspirin, COX-2 inhibitors) may be useful in the prevention of EA in patients with BE (5, 6). Moreover, there is an evidence that dietary supplementation with antioxidants may be beneficial in the chemoprevention of upper GI malignancy including BE (7).

A growing body of literature suggests that statins may have chemopreventive potential against cancer. Well-designed epidemiological studies indicate that reductions in overall cancer risk among statin users (8). The anticancer effect of statins include inhibition of cell proliferation, induction of apoptosis, suppression of angiogenesis and metastasis (9). However, the role of statins has not been investigated in Barrett's carcinogenesis.

The aim of the study was to analyze 1) the impact of HMG-CoAR inhibitor simvastatin on human BA cell growth and 2) effect of simvastatin on apoptosis related proteins Bax/Bcl-2 and COX-2.


MATERIAL AND METHODS
Cell culture and chemicals

The human cell line derived from esophageal adenocarcinoma (OE-19) was purchased from ECACC- the European Collection of Cell Cultures (Sigma-Aldrich, Germany). Cells were cultured in RPMI 1640 medium at pH~7 containing 10% fetal bovine serum (FBS) and antibiotics at 37°C in water saturated atmosphere of 95% air and 5% CO2.

Simvastatin was purchased from AXXORA DEUTSCHLAND GmbH. Stock solution was prepared with DMSO.

Cells viability test

OE-19 cells were seeded into 96-well culture plate at 6x103 in 200µl of RPMI 1640 medium (BIOCHROM AG, Germany) with 10% FBS (Sigma-Aldrich, Germany). Cells were cultured for 24 h under 5% CO2 and at 37°C. After 24 h of starvation in culture medium without serum, cells were treated with simvastatin in different doses (1µM-30µM).

Cell viability was examined using the MTT assay. After exposure to one of simvastatin concentration, medium was replaced by 3-[4,5-dimethyltiazol-2-YL]-2,5-diphenyl tetrazolium bromide-MTT (Sigma-Aldrich, Germany) solution -50uL (1mg/mL) in phosphate-buffored saline -PBS (BIOCHROM AG, Germany). Cells were incubated for 3 h in 5% CO2 ;37°C. Thereafter, MTT solution was removed and purple farmazone crystals produced in mitochondria, were solubilized in 5 nM HCl solution in isopropanol. The optical density at 550 nm was determinated by spectrophotometric analysis using 96-well multiscanner reader (TECAN-spectra mini). Absorbance was read at 550 nm, and cells number was normalized to untreated control cells.

RNA isolation and RT-PCR

For reverse transcryption PCR cells were scraped from culture dishes and suspended in 250µl of TRIZOL (Total RNA Isolation Reagent; Invitrogen, Germany). Further steps of RNA isolation was performed according to manufactured instruction. Isolated RNA was dissolved in 25µl DEPC treated water.

Complementary DNA (cDNA) was generated by reverse transcription of 5 µg total RNA extracted from OE-19 cells, using Moloney murine leukemia virus reverse transcriptase kit (MMLV-RT) (Sratagene, Germany) and oligo dT primes. The cDNA (1µL) was amplified in 25µl volume samples. For quantification, all reactions were performed on iQ5 Mulitcolor Real-Time Detection System; Bio-Rad (Bio-Rad, Germany) contained iQ SYBR green mix (Bio-Rad, Gemany; according to manufacturer's instruction) and specific gene primers (Table 1). Samples without temples were included as negative control. Gene expression was normalized to the ß-actin. All sample were performed in duplicates.

Table 1

Western blot

OE-19 cells were collected to 1.5 mL tubes, washed twice with PBS, and lysed in 0.4 mL of lysis buffer (0.06 mol/L Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 5% beta-mercaptoethanol, 0.0025% bromophenol blue). DNA was sheared by a needle, the solution heated at 95°C for 5 min and centrifuged at 15 000 g for 2 min. The total protein was loaded on SDS-polyacrylamide gel, run at 40 mA and transferred to nitrocellulose (Protran, Schleicher&Schuell, Germany) by electroblotting. Filters were blocked with 5% non fat milk (Roth, Germany) in TBS/Tween-20 buffer (137 mM NaCl, 20 mM Tris-HCl, pH 7.4, 0.1% Tween-20) before incubation with antibodies against Bax (mouse monoclonal, dilution 1:200;Santa Cruz, US), Bcl-2 (mouse monoclonal, dilution 1:200; Santa Cruz, US) or ß-actin (mouse monoclonal, dilution 1:30 000; Sigma Aldrich, Germany), followed by horseradish peroxidase-conjugated anti-mouse-IgG secondary antibody (dilution 1:30 000; Promega, WI, USA) dissolved in 1% non-fat milk in TBS/Tween-20. Immune complexes were detected by the SuperSignal West Pico Chemiluminescent Kit (Pierce, USA) and exposed to an X-ray film (Kodak, Wiesbaden, Germany).

Statistcs

Each experiment was repeated three times. Results are expressed as means ± SEM, and the effects were compared with untreated control cells on the same plate. One-way ANOVA was used for dose-response curve and paired t tests were used to analyze the effect of bile salts. p value of <0.05 was considered significant.


RESULTS
MTT assay was used to determine the antiproliferative effects. As shown in Fig. 1, simvastatin caused a significant decrease in proliferation. The highest inhibition of cell proliferation was observed at a dose 30 µM of simvastatin.

Fig. 1. Effect of different concentrations of Simvastatin (Sim) on viablility OE-19 cells.

Fig. 2 demonstrates the changes in COX-2 expression at mRNA level. In control OE-19 cells, only low expression of COX-2 was detected. Both doses of simvastatin (1 and 10 µM) had no significant influence on COX-2 mRNA expression. To stimulate the COX-2 expression, TNFa was added to the culture medium at 25 ng/ml. Addition of TNFalpha was associated with a significant upregulation of TNFalpha mRNA. The increase in COX-2 mRNA expression after addition of TNFalpha was attenuated by co-incubation with simvastatin at a concentration of 10 µM. At a lower concentration (1 µM) no inhibitory effect on COX-2 mRNA was observed.

Fig. 2. Influence of Simvastatin (Sim) concentrations on COX-2 expression in OE-19 cells incubated with TNF-alpha.

Fig. 3 shows the effect of different simvastatin concentrations on Bcl-2 mRNA expression. In control OE-19 cells a signal for bcl-2 mRNA was detected. The incubation of OE-19 with simvastatin caused a decrease in bcl-2 mRNa expression. The incubation with TNFalpha had no significant effect on Bcl-2 mRNA as compared to control cells. The co-incubation with simvastatin at a lower dose 1 µM had no significant effect on Bcl-2 mRNA expression. At a higher concentrations of 10 µM simvastatin incubated with TNFalpha lowered the Bcl-2 expression to the level observed in cells incubated with simvastatin alone.

Fig. 3. Effect of different Simvastatin (Sim) concentrations on BCL-2 mRNA expression in OE-19 cells.

Similarly to Bcl-2, Bax was expressed in control OE-19 cell line. The incubation with simvastatin at the higher concentration 10 µM (but not at low dose 1 µM) induced up-regulation in Bax mRNA expression. The incubation with TNFalpha had no significant effect on Bax mRNA expression. However, in OE-19 cells incubated with both TNFa and simvastatin at 10 µM, a significant increase in Bax mRNA expression was observed. Simvastatin at a low concentration of 1 µM had no effect on bax mRNA expression in OE-19 cells incubated with TNFalpha (Fig. 4).

Fig. 4. Effect of different Simvastatin (Sim) concentrations on BAX mRNA expression in OE-19 cells.

Fig. 5 shows representative Western blots demonstrating the effects of simvastatin at concentrations of 1 and 10 µM on protein expression of Bax and Bcl-2. The incubation with simvastatin caused concentration-dependent increase in Bax expression and decrease in Bcl-2 expression.

Fig. 5. Effect of different Simvastatin concentrations on expression of apoptosis regulatory proteins Bcl-2(27 kDa) and Bax(23 kDa) in adenocarcinoma cells OE-19 (1-control; 2-Simvastatin 1µM; 3-Simvastatin 10µM.

DISCUSSION
This study reports for the first time on the potential beneficial effect of statin on the Barrett's carcinogenesis and suggests that this compound could be a useful tool in the chemoprevention of EA in patients with pre-existing Barrett's esophagus.

Statins represent a class of agents that inhibit 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoA reducatse), a rate-limiting enzyme in mevalonate synthesis, leading to inhibition of cholesterol synthesis. Mevalonate is also a precursor of farnesyl and geranylgeranyl moieties, which are essential for the activation of variety of intracellular proteins through farnesylation or geranylgeranylation (prenylation). Several important proteins involved in intracellular signalling such as Ras, Rho, nuclear lamins, transducins, etc. are dependent on prenylation (9). A number of epidemiological studies suggest that statins may have a protective effect against cancerogenesis. Furthermore, in vitro studies indicate that statins inhibit tumor cell growth and induce apoptosis (8).

In the present study the effect of statin (simvastatin) on Barrett's carcinoma cell line was investigated. According to our in vitro results the possible anticancer mechanisms of simvastatin include antiproliferative effect as shown in MTT assay, decrease in COX-2 expression and induction of apoptosis due to increase in Bax expression and decrease in Bcl-2 expression.

A number of studies demonstrated that the stimulation of COX-2 plays a crucial role in the progression of carcinogenesis in Barrett's esophagus (10-12). The main factor responsible for the induction of COX-2 in patients with Barrett's esophagus is a persisting inflammatory reaction in the esophageal mucosa due to acid and bile reflux leading to induction of proinflammatory cytokines, especially TNFa (13). In the present study, the incubation of Barrett's cancer cell line (OE-19) caused a significant up-regulation of inducible COX-2 mRNA expression. The interesting observation of our study was the fact that simvastatin caused a significant down-regulation of COX-2 mRNA expression. This mechanism could be potentially of importance in the prevention of Barrett's carcinogenesis.

Deregulation of apotosis plays a crucial role in the pathogenesis of Barrett's cancer (14,15). Apoptosis and the genes regulating this process have recently become a focus of interest in the study of cancer development and progression. Both Bcl-2 and Bax are transcriptional targets for the tumor supressor protein, p53, which induces cell cycle arrest or apoptosis in response to DNA damage. The progression of carcinogenesis in Barrett's esophagus depends on the balance between pro-apoptotic proteins such as Bax and anti-apoptotic proteins such as Bcl-2 (16). In the present study, the incubation of Barrett's carcinoma cell line with simvastatin caused a significant increase in Bax expression with concomitant decrease in anti-apoptotic Bcl-2. This indicates that statin may induce apoptosis and this mechanism could be of importance in the prevention of Barrett's carcinogenesis. These results are keeping with previous results showing a significant increase in apoptosis in different cancer cell lines incubated with statins (17, 18, 19).

There is an increasing interest to use a combination a low doses of chemopreventive agents that differ in their modes of actions to increase their efficacy and minimize toxicity. Since use of NSAIDs including COX-2 inhibitors may results in gastrointestinal, cardiovascular and renal effects, it would be of importance and interest to investigate the effect of combination of low doses of statin and COX-2 inhibitors in the prevention of adenocarcinoma of esophagus in patients with pre-existing Barrett's esophagus.


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