Background
Esophageal cancer (EC) is the eighth most common cancer worldwide, which arises from the inner lining of the esophagus [
1,
2]. To date, the frequently used therapy for the treatment of EC is chemotherapy in combination with other therapeutic strategies. However, the prognosis of patients with EC remains poor and the 5-year survival rate is less than 20% [
3]. This mainly results from the resistance to the commonly used drugs owing to the abuse of antibiotics [
4]. There are limited salvage options for patients with refractory EC [
5] and targeted therapies are not yet available. Therefore, there is an urgent need for understanding the mechanism of drug-resistance to guide the design of novel approaches for the treatment of EC.
The family of paraoxonase (PON) has three members, PON1, PON2 and PON3, that are located adjacent to each other on chromosome 7 in humans [
6]. They share high levels of homology [
7]. The expression level and specific activities of PON genes were found to be negatively correlated with several inflammatory disorders, such as cardiovascular diseases, type-2 diabetes, and inflammatory bowel disease [
8,
9]. Moreover, PON3 expression is remarkably up-regulated in a variety of human cells, including cancer cells [
10,
11]. Recent study suggested that PON3 promotes cell proliferation and metastasis by regulating PI3K/Akt in oral squamous cell carcinoma [
12]. Despite the extensive studies of PON3 in cancer cells, the roles of PON3 in EC are rarely evaluated, especially the involvement in drug resistance. In this study, we investigated the roles of PON3 in EC cells and found that PON3 is related in various biological processes in EC cells, which will give us hints for a clinical therapy of EC.
Methods
Cell lines and culture
The eight K30, K450, K180, K150, TE-1, K510, K140 and K410 cell lines come from our laboratory. All cell lines were cultured in RPMI1640 (Biological Industries, Israel) +10% fetal bovine serum (Invitrogen, USA) and 1% glutamine at 37 °C in 5% CO2.
Bisulfite sequencing PCR (BSP) analysis
Genomic DNA was isolated by a standard phenol/chloroform purification method, verified by electrophoresis on an agarose gel, and treated by an ammonium bisulfite-based bisulfite conversion method. Then the PCR fragments from the converted DNA were sequenced and analyzed. Raw sequence data files were processed, and the area ratio (%) of C over C + T of the primary CpG dinucleotide was calculated as the % of methylation and plotted [
13].
Transient transfection assays and reagents
siRNA and scrambled (negative control, NC) sequences as well as a riboFECT CP transfection kit were supplied by Guangzhou RiboBio, China. A GFP-tagged PON3 overexpression construct (pReciever-M98) was purchased from Genecopia, Guangzhou, China (Catalog No.: EX-E0804-M98-5). Transfections of the above mentioned ribonucleic acid reagents and reporter plasmids were performed according to the manufacturer’s instructions.
Chemoresistance profiling (IC50 determination)
All of the chemotherapeutic drugs used in this study were of clinical grade. To perform thiazolyl blue tetrazolium blue (MTT)-based cell proliferation assays, experimental groups of cells in the logarithmic phase of growth were seeded in triplicate in 96-well plates at a cell density of 0.5 × 104/well and treated with fourfold serially diluted drugs for 72 h. Then 10 μl (5 mg/ml) of MTT salt (Sigma) was added to the corresponding wells. The cells were incubated at 37 °C for another 4 h, and the reaction was stopped by lysing the cells with 150 μl of DMSO for 5 min. The optical density was measured at 570 nm. A group that received no drug treatment was used as a reference for calculating the relative cell survival rate.
RNA analysis
Total RNA was isolated from cells during the logarithmic phase using TRIzol (Tiangen Biotech). For mRNA analysis, a cDNA primed by an oligo-dT was constructed using a PrimeScript RT reagent kit (Tiangen Biotech). The PON3 mRNA level was quantified using duplex-qRT-PCR analysis, wherein TaqMan probes with a different fluorescence profile were used to detect β-actin (provided by Shing Gene, Shanghai, China) in a FTC-3000P PCR instrument (Funglyn Biotech). Using the 2−ΔΔCt method, target gene expression levels were normalized to the β-actin expression level before the relative levels of the target genes were compared.
Western blot protein analysis
Cells were lysed with lysis buffer (60 mM Tris–HCl [pH 6.8], 2% SDS, 20% glycerol, 0.25% bromophenol blue, and 1.25% 2-mercaptoethanol) and heated at 95 °C for 10 min before electrophoresis/Western blot analysis. The primary anti-PON3 (17422-1-AP) antibodies and anti-GAPDH (60004-1-lg) antibodies were purchased from Proteintech (San Ying Biotechnology, China) and were recognized with anti-rabbit IgG peroxidase-conjugated antibody (30000-0-AP) (San Ying Biotechnology, China), followed by an enhanced chemiluminescence reaction (Thermo Fisher Scientific, Waltham, MA, USA). Relative levels of proteins were quantified using densitometry with a Gel-Pro Analyzer (Media Cybernetics, Rockville, MD, USA). The target bands over the GAPDH band were densitometrically quantified, as indicated under each band (Additional file
1).
Wound-healing assays
For cell motility assays, cells stably expressing si-PON3, GFP-PON3 and the corresponding NC were seeded in 24-well plates and cultured to near confluence. After 6 h of culture in RPMI1640 without FBS, a linear wound was carefully made using a sterile 10 µl pipette tip across the confluent cell monolayer, and the cell debris was removed by washing with phosphate-buffered saline. The cells were incubated in RPMI1640 plus 10% FBS, and the wounded monolayers were then photographed at 0, 8, 12 and 20 h after wounding.
In vitro invasion assays
Cell invasion assays were performed in a 24-well plate with 8 mm pore size chamber inserts (Corning, USA). For invasion assays, 1 × 103 cells stably expressing si-PON3, GFP-PON3 or NC were placed into the upper chamber in each well with the matrigel-coated membrane, which was diluted in serum-free culture medium. In the assay, cells were suspended in 100 µl of RPMI1640 without FBS when they were seeded into the upper chamber. In the lower chamber, 500 µl of RPMI1640 supplemented with 10% FBS was added. After incubation for 36 h at 37 °C and 5% CO2, the membrane inserts were removed from the plate, and non-invading cells were removed with cotton swab from the upper surface of the membrane. Cells that moved to the bottom surface of the chamber were stained with 0.1% crystal violet for 30 min. The cells were then imaged and counted in at least 5 random fields using a CKX41 inverted microscope (Olympus, Japan). The assays were conducted in three independent times.
Signaling pathway analysis
The reporter construct encodes the firefly luciferase reporter gene under the control of a basal promoter element (TATA box) joined to tandem repeats of a specific transcriptional response element. The cells were transfected in triplicate with each firefly luciferase reporter construct in combination with the Renilla luciferase-based control construct using the riboFECT CP transfection reagent, and both the luciferase activities were measured in the cell extracts 24 h after transfection. The luciferase activities (luciferase unit) of the pathway reporter relative to those of the negative control in the transfected cells were calculated as a measurement of the pathway activity.
In vivo studies
Animal experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Male BALB/c nude mice between 3 and 4 weeks old were used for this study [
14]. K510 cells were embedded in BD Matrigel Matrix (Becton, USA) and subcutaneously injected into two sites on the back of each mouse as follows: 1.0 × 10
7 cells/site for K510 into 2 sites/mouse, with 6 mice. Ten days after cell injection, all of the tumors were intratumorally injected with 2 nM NC/si-PON3 every 2 days. Ten days later, after four cell injections, three mice intraperitoneally received DDP (75 μg/mouse) once every other day. The remaining three mice in each group received PBS as a mock treatment control. The mice were euthanized on day 30 after four drug injections, and their tumors were weighed and imaged. Tumor weight was described as the mean ± S.D. The expression levels of PON3 and Ki67 proteins were measured using immunochemical analysis on 5 μm sections of formalin-fixed, paraffin-embedded tumor xenografts in nude mice. The antigens were retrieved by pre-treating the de-waxed sections in a microwave oven at 750 Watts for 5 min in citrate buffer (pH 6) processed with a Super Sensitive Link-Labeled Detection System (Biogenex, Menarini, Florence, Italy), and the slides were developed using 3-amino-9-ethylcarbazole (Dako, Milan, Italy) as a chromogenic substrate. After the slides were counterstained with Mayer’s hematoxylin (Invitrogen), they were mounted in an aqueous mounting medium (Glycergel, Dako). Images were captured using a Leica DM 4000B microscope (Wetzlar, Germany), the relative level of each protein was calculated using Leica software (Wetzlar, Germany), and the percentage of the mock over the chemotherapeutically treated tumors was calculated and plotted.
Statistical analysis
All of the results are represented as the mean ± standard deviation (SD) of three independent experiments. Two-tailed Student’s t-test, one-way analysis of variance or Mann–Whitney U test was used to calculate statistical significance. All of the statistical analyses were performed with Microsoft Excel 2010 (Microsoft, Redmond, WA). A p-value of less than 0.05 was designated statistically significant.
Discussion
Accumulating evidences have been shown that DNA methylation play important roles in drug resistance of cancers, which prevents the effective treatment of cancers [
15]. Altered DNA methylation patterns can influence the expression of genes [
16]. Recent study on the profiling of gene-specific methylation levels in EC provides a useful approach for investigating the individual hypermethylated gene in EC [
17]. Despite extensive studies revealed that methylation is a modulator of cancer, the understanding of DNA methylation on the effect of EC remains limited. In our study, we identified that the promoter region of PON3 is hypermethylated in drug resistant EC cell lines. The hypermethylation of PON3 in return down-regulates its expression in drug resistant EC cells. Furthermore, we showed that the PON3 level is negatively correlated with the drug-resistance of EC cells, and thus suppresses the EC drug resistance. In vivo experiments also found that PON3 inhibits tumor growth in nude mice. All these findings made us to propose that PON3 might be a tumor suppressor, considering its high methylation in the promoter region and low expression level in multiple cancers.
Paraoxonase 3 (PON3) belongs to the paraoxonase family that helps in preventing oxidative stress and anti-inflammatory [
18]. This gene also involves in other diseases including cancer [
19,
20]. PON3 gene has a high expression level in cancer tissues of the lung, liver and colon [
11]. Previous studies also showed that PON3 is hypermethylated in colorectal cancer [
9] and chordomas [
21]. Notably, the genome-wide DNA methylation analysis identified that several genes, including PON3, are aberrantly methylated in the high-grade non-muscle invasive bladder cancer [
22]. These findings suggest that epigenetic modifications are usually associated with the development and/or progression of different type of tumors [
13,
23]. In accordance with previous studies, we identified that the promoter region of PON3 is hypermethylated in EC cancer. The hypermethylation of PON3 may serve as a marker of poor prognosis in human EC. Furthermore, the expression of PON3 negatively correlates with drug resistance in EC cells, and thus appears to act a biomarker for the drug resistance of EC cells. The study may provide a new potential therapeutic target in the treatment of EC. However, the detailed mechanism for the PON3-regulated drug resistance in EC cells remains to be clarified.
Authors’ contributions
DBH, YW, SLH and YYP made substantial contributions to conception and design, acquisition of data, and analysis and interpretation of data, and were involved in drafting and critically revising the manuscript for important intellectual content. DBH, YW and YYP was principally responsible for drafting the manuscript and for several cycles of revision of the manuscript. YFH, GW, WW, XHH, YBS, GDS, MC, GB and XF made substantial contributions to analysis and interpretation of data and were involved in critically revising the manuscript for important intellectual content. BJS, LL made substantial contributions to conception and design, and analysis and interpretation of data and was involved in drafting and critically revising the manuscript for important intellectual content. All authors read and approved the final manuscript.
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