Azacitidine

SPARC is down-regulated by DNA methylation and functions as a tumor suppressor in T-cell lymphoma

Abstract
The aim of this study was to assess the functional role of SPARC in T-cell non-Hodgkin’s lymphoma (T-NHL), as well as the underlying molecular mechanisms. Here, we first identified SPARC expression in T-NHL tissues and cell lines through western blot and real-time PCR (RT-PCR). Overall survival of T-NHL patients with different levels of SPARC was assessed by Kaplan-Meier survival curves. Then cell proliferation, apoptosis, migration and invasion of T-NHL cells with either knockdown or overexpression of SPARC were determined by MTT, flow cytometry, transwell migration and invasion assay, respectively. Finally, the molecular mechanism by which SPARC modulated T-NHL cell progression was assessed. We confirmed that SPARC was significantly down-regulated in T-NHL tissues and cell lines. T-NHL patients with high levels of SPARC demonstrated a favorable clinical outcome. SPARC significantly suppressed cell proliferation, migration and invasion, and EMT process, but facilitated cell apoptosis in T-NHL cells. Further, we found that loss of SPARC expression in T-NHL tissues and cell lines, both in mRNA and protein levels, was associated with the aberrant DNA methylation in SPRAC gene, and the disrupted SPARC expression could be rescued after treatment with the demethylating agent 5-Aza-2’-deoxycitydine (5-Aza-Cdr). Additionally, 5-Aza-Cdr reversed SPARC hypermethylation to restore its biological role as a tumor suppressor in T-NHL cells, including inhibiting cell proliferation, invasion and migration, while promoting cell apoptosis. Our data provided evidence that DNA methylation in SPARC gene may play a role in the progression of T-NHL.

Introduction
The lymphoma, a type of blood cancer, in histopathology, is roughly classified as Hodgkin’s lymphoma (HD) and non-Hodgkin’s lymphoma (NHL), and NHL represents the most common malignancy [1]. T-cell lymphoma (T-NHL) accounts for approximately 15% of NHL in the United States [2]. Along with the development of molecular biology, a better understanding of the causes of lymphoma is gained. Abnormal gene expression owing to gene mutations [3], chromosomal translocations[4] may be a significant cause of T-NHL.SPARC (secreted protein acidic and rich in cysteine), also known as osteonectin or BM-40, is a 43-kDa glycoprotein that is expressed in various cells and is implicated in the regulation of cell adhesion, differentiation, proliferation and migration, as well as in processes such as tissue remodeling, wound healing, morphogenesis, and angiogenesis [5, 6]. SPARC is differentially expressed in different tumors, usually mesenchymal, with a significant implication in tumor progression. However, the mechanism through which SPARC modulates tumor progression is complex, and may depend on tumor types and the surrounding microenvironment. Higher levels of SPARC have been found in head and neck cancer, breast cancer, and melanomas, and SPARC can facilitate tumor growth and metastasis [7-9]. SPARC has also been reported to act as a chemoattractant for breast and prostate cancer cells, supporting their prior migration and homing to bone [10]. Conversely, lower levels of SPARC have been observed in other cancers, such as ovarian, colon and pancreatic cancers, and disrupted SPARC expression supported enhanced tumor growth and metastasis [11-13]. Nonetheless, the biological role of SPARC in T-NHL still remains unclear.The purpose of this study was to elucidate the definitive link between SPARC and tumor progression in T-NHL, as well as the underlying molecular mechanisms. Here, we examined mRNA and protein expression of SPARC in T-NHL tissues and cell lines. And, functional experiments were carried out to assess the role of SPARC in T-NHL cells, as well as the underlying molecular mechanisms.

From 2008 to 2011, patients who were diagnosed with T-NHL and received standard chemotherapy after lymph node biopsy at The First Affiliated Hospital of Zhengzhou University were included in this study after obtaining their written informed consent. The biopsy specimens of patients (n=30) were prepared by the Department of Clinical Pathology for paraffin-embedded tumor tissue sections. The control group consisted of 16 samples of lymph node that were obtained from standard operations. This study was approved by the Ethics Committee of the medical faculty at the First Affiliated Hospital of Zhengzhou University. The T-NHL cell lines (SNK-6、Hut-78、Hut-102、Jurkat) and human T cell line H9 were maintained in the Key Clinical Laboratory of Henan Province. Cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated FBS (fetalbovine serum), 50 U/ml penicillin and 50 U/ml streptomycin (Sigma-Aldrich, St. Louis, MO, USA) at 37˚C in a humidified atmosphere containing 5% CO2.Total-RNA from tissues and cultured cells was extracted using the TRIzol reagent following the manufacturer’s instructions. The cDNA synthesis was performed following the protocol of the Takara Reverse Transcription System for real-time PCR [Takara Biotechnology (Dalian) Co., Ltd., China] with 2 μg RNA and reverse transcription performed with random primers. The housekeeping gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was employed as an internal control. Primer sequences are: SPARC forward, 5′-GTGGGCAAAGGGAAGTAACA-3′; and reverse, 5′-GGGAGGGTGAAGAAAAGGAG-3′; GAPDH forward, 5′-CACTGGCGTCTTCACCACCATG-3′, and reverse,5′-GCTTCACCACCTTCTTGATGTCA-3′.

Real-time PCR analysis was carried out on a LightCycler real-time PCR instrument using SYBR Green I kit (Tiangen Biotech Co., Ltd., Beijing, China) according to the manufacturer’s instructions. Each reaction was performed in triplicate. Methylation-specific polymerase chain reaction (MSP) and DNA sequencing Methylation status of SPARC gene was assessed using MSP as described previously [14]. Bisulfite-treated DNA (1 mg) was amplified using primers specific for either the methylated or the unmethylated DNA. Primer sequences of unmethylated reactions were 5′-TTTTTTAGATTGTTTGGAGAGTG-3′ (forward)and 5′-AACTAACAACATAAACAAAAATATC-3′ (reverse); primer sequences of methylated reactions were 5′-GAGAGCGCGTTTTGTTTGTC-3′ (forward) and 5′-AACGACGTAAACGAAAATATCG-3′ (reverse). PCR product (8 ml) was loaded onto a 2% agarose gel and visualized using ethidium bromide staining.Western blot was performed as previously described [15]. Total proteins were extracted by lysing cells in buffer (50 mM Tris pH 7.4, 150 mM NaCl, 0.5% NP-40,50 mM NaF, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 25 mg/ml leupeptin and 25 mg/ml aprotinin). The lysates were cleared by centrifugation and the supernatants were collected. Proteins were extracted using the protein extraction kit following the manufacturer’s instructions. Protein concentration was determined using protein assay reagent (Bio-Rad, Hercules, CA, USA). Equal amounts of protein were separated on SDS-PAGE, transferred to PVDF membranes, incubated with antibodies against SPARC, E-cadherin, ZO-1, Vimentin, N-cadherin, and GAPDH, followed by incubation with the secondary antibodies. The membrane was then washed three times and visualized with diaminobenzidine. Quantification of the proteins was detected with the ECL system (Pierce Biotechnology Inc., Rockford, IL, USA). Each value represents the mean of triple experiments, and is presented as the relative density of protein bands normalized to GAPDH.

MTT assay was carried out as previously described [15]. Hut-78 and Jurkat cells were seeded in a 96-well plate at a concentration of 2.5×104/ml (100 μl/well). Then, 20 μl/well of 5 mg/ml MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) was added at 1, 2, 3, 4, 5 and 6 days and were then incubated for another 4 h. The supernatant was removed and the product converted from MTT was dissolved by adding 150 μl/well dimethylsulfoxide (DMSO). An enzyme-linked immunosorbent assay reader was used to examine the absorbance of each well at 570 nm.Annexin V-FITC apoptosis detection kit (BD Biosciences, San Jose, CA, USA) was used to detect apoptosis of Hut-78 and Jurkat cells, as described in our another paper [15]. Briefly, after culturing for 48 h, each group of cells was harvested, washed twice with pre-chilled PBS and resuspended in binding buffer (HEPES-NaOH 10 mM pH 7.4, 144 mM NaCl and 25 mM CaCl2) at a concentration of 1×106 cells/ml. One hundred microliters of this solution (1×105 cells) was mixed with 5 μl of Annexin V-FITC and 5 μl of PI (BD Biosciences) following the manufacturer’s instructions. The mixed solution was incubated in the dark at room temperature (25˚C) for 15 min. Four hundred microliters of 1×dilution buffer were added to each tube and cell apoptosis analysis was performed by flow cytometry (BD FACSCalibur) within 1 h. At least 10,000 events were recorded and represented as dot plots.Transwell migration and invasion assay were performed as reported elsewhere [16]. For the migration assay, exponentially growing cells were seeded into the upper chamber (BD Bioscience) in culture medium with 1% FBS, whereas for the invasion assay, the upper chamber was pre-coated with Matrigel (BD Bioscience) prior to adding the cells, and then they were all incubated for 24 h at 37°C. The lower chamber was added with 600 μL RPMI 1640 containing 10% FBS. Then, the cells on the upper surface of the filters would be scraped off with swabs. After fixed the membrane with 4% paraformaldehyde and stained with crystal violet, the cells were taken photos under microscope. At least 5 microscopic fields were counted for each transwell filter.The SPSS13.0 software package (SPSS, Inc., Chicago, IL, USA) was used for all statistical analyses, and results are expressed as mean ± SEM. The comparison between two groups was evaluated by Student’s t test; the comparison between multiple groups was performed using one-way analysis of variance (ANOVA), followed by the Tukey’s test. Results were considered statistically significant at P<0.05. Results To examine the expression of SPARC in T-NHL, tumor tissues (n=30) and normal lymph node tissues (n=16) were first employed and analyzed by western blot and real-time PCR (RT-PCR). SPARC protein was significantly down-regulated in tumor tissues compared with normal lymph node tissues, both in protein (Fig.1A) and mRNA levels (Fig.1B). Similarly, in agreement with the data derived from T-NHL samples, the expression of SPARC was significantly decreased in all T-NHL cell lines(SNK-6、Hut-78、Hut-102、Jurkat) except in human T cell line H9 (Fig. 1C). In addition, Kaplan-Meier survival curves suggested that T-NHL patients with high levels of SPARC demonstrated a favorable clinical outcome (Fig.1D). Hut 78(cutaneous T-cell lymphoma) and Jurkat (adult T-cell leukemia/lymphoma) cells were selected as representatives for following experiments.To determine the effect of SPARC on the progression of T-NHL, SPARC was overexpressed or silenced in Jurkat (Fig.2A and B) and Hut-78 (Fig.2C and D) cells, and Western blot and RT-PCR were performed to assess transfection efficiency. The effect of SPARC on T-NHL cell proliferation was determined by MTT assay. As shown in Fig. 2E, cell proliferation was significantly promoted in SPARC siRNA-transfected Jurkat cells, but was remarkably inhibited in cells overexpressing SPARC. Similarly, same effects were observed in Hut-78 cells (Fig. 2F). Next, flow cytometry was performed to evaluate the effect of SPARC on cell apoptosis in Jurkat and Hut-78 cells. Conversely, silencing SPARC dramatically reduced cell apoptosis, while apoptosis was notably elevated when SPARC was overexpressed, both in Jurkat (Fig.2G) and Hut-78 cells (Fig.2H). These results suggested that SPARC inhibited cell proliferation and promoted cell apoptosis in T-NHL.SPARC inhibits invasion, migration and epithelial-mesenchymal transition (EMT) process of Jurkat and Hut-78 cellsFig 2 illustrates that SPARC inhibited proliferation and promoted apoptosis in Jurkat and Hut-78 cells, indicating the participation of SPARC in the progression of T-NHL. To further understand the role of SPARC in the metastasis of T-NHL, transwell migration and invasion assays were employed in Jurkat and Hut-78 cells with SPARC overexpression or silence, respectively. Overexpression of SPARC significantly inhibited migratory (Figure 3A) and invasive (Figure 3B) abilities of Jurkat and Hut-78 cells, whereas loss of SPARC in Jurkat and Hut-78 cells significantly compromised both migration (Figure 3A) and invasion (Figure 3B). In consideration of epithelial-mesenchymal transition (EMT) process along with tumor metastasis, we examined the expression of epithelial marker E-cadherin, ZO-1 and mesenchymal marker Vimentin, N-cadherin using western blot in Jurkat and Hut-78 cells. Obviously, down-regulated E-cadherin and ZO-1, concomitant with up-regulated Vimentin and N-cadherin were observed in SPARC siRNA- transfected cells, but SPARC overexpression resulted in opposite results (Fig. 3C). Aberrant methylation and expression in SPARC gene in T-NHL tissues and cell lines Expression of SPARC was examined using RT-PCR and Western blot, while methylation of SPARC gene was determined by MSP assay. As shown in Fig. 4A, loss of SPARC expression was observed in T-NHL tissues, while aberrant methylation was found in them (Fig. 4B). Likewise, concordance between down-regulated SPARC expression (Fig. 4C) and aberrant methylation of SPARC (Fig. 4D) was observed in Jurkat and Hut-78 cells. To confirm that methylation of SPARC gene was responsible for disrupted SPARC expression, Jurkat and Hut-78 cells were treated with the demethylating agent 5-Aza-2’-deoxycytidine (5-Aza-Cdr). 5-Aza-Cdr obviously restored SPARC expression, both in mRNA and protein levels (Fig. 4E).5-Aza-Cdr inhibits proliferation, invasion and migration, but promotes apoptosis of Jurkat and Hut-78 cellsWe then examined the changes of cell proliferation, apoptosis, invasion and migration of Jurkat and Hut-78 cells after 5-Aza-Cdr treatment. Cell proliferation was significantly decreased following 5-Aza-Cdr treatment, both in Jurkat and Hut-78 cells (Fig. 4F). In contrast, cell apoptosis was dramatically elevated after treatment with 5-Aza-Cdr (Fig. 4G). Further, cell migration (Fig. 5A) and invasion (Fig. 5B) were also notably decreased in Jurkat and Hut-78 cells after 5-Aza-Cdr treatment, along with up-regulated E-cadherin and ZO-1 (epithelial biomarkers) as well as down-regulated Vimentin and N-cadherin (mesenchymal biomarkers) (Fig. 5C). Discussion Tumor progression includes tumor cell proliferation, migration and invasion. Thus, inhibiting the proliferative and metastatic behavior of tumor cells is a key problem to ameliorate the clinical outcome of cancer patients [17, 18]. Despite SPARC has been reported to be differentially expressed in different tumors and exert effects on tumor progression [19], little evidence was available with respect to its role in T-NHL progression.In this study, we found that SPARC was prominently down-regulated in T-NHL tissues and cell lines. High levels of SPARC were also associated with a favorable prognosis of T-NHL patients. In addition to inhibiting the proliferation of T-NHL cells, we also found that SPARC significantly inhibited migration and invasion in T-NHL cells, but facilitated apoptosis. Further, SPARC increased the expression of E-cadherin and ZO-1, typical epithelial biomarkers, whereas decreased the expression of Vimentin and N-cadherin, typical mesenchymal biomarkers, suggesting that SPARC restrained the EMT process in T-NHL cells. Our results revealed that SPARC may serve as a tumor suppressor gene in the progression of T-NHL. The present study also uncovered the molecular mechanism by which SPARC expression was reduced in T-NHL cells. Recent studies have shown that several tumor suppressor genes are methylated in T-NHL [20, 21]. Aberrant DNA methylation of tumor suppressor genes has attracted more and more attention in cancer research. DNA methylation is a biochemical modification that primarily affects cytosines when they are part of the symmetrical CpG dinucleotide, which is central to the aberrant epigenetics of cancer [22]. As described in several reports, cancer cells often have a gain of methylation at the promoters of selected CpG islands, resulting in the silencing of hundreds of genes per cancer cell including tumor suppressor genes [23]. Though much progress has been achieved in methylation-related studies, few reports are available related to SPARC inactivation due to hypermethylation in T-NHL. Here, our results showed that the significant down-regulation of SPARC in T-NHL tissues and cell lines was resulted from the aberrant methylation of SPARC gene. Actually, earlier reports from several groups have suggested methylation in SPARC promoter region resulting in disrupted SPARC expression in multiple tumors, such as colon, gastric, ovarian and endometrial carcinomas [5, 24-26]. In this study, the relationship between loss of SPARC expression and methylation of SPARC gene in T-NHL cell lines was confirmed by the excellent uniformity among mRNA expression by RT-PCR, protein expression by western-blotting and DNA methylation in SPARC gene by MSP, largely accounting for the down-regulation of SPARC expression. We also found that the loss of SPARC expression in T-NHL cell lines could be reversed following treatment with the demethylating agent 5-Aza-Cdr, both in mRNA and protein levels, similar to the results previously described in ovarian, colorectal and laryngeal carcinoma cells [24, 27, 28]. As an epigenetic mechanism of gene silencing, DNA hypermethylation can be reversed by DNA methylation inhibitors [29]. 5-Aza-Cdr is a nucleoside anti-metabolite agent and a potent inhibitor of DNA methyltransferase 1 activity, which has been shown antineoplastic activity in patients with leukemia and non-small cell lung cancer [30, 31]. Furthermore, its potential to enhance chemotherapy sensitivity in cancer cells has been reported [32, 33]. In our findings, 5-Aza-Cdr obviously inhibited cell proliferation, invasion and migration, but promoted cell apoptosis, reversing SPARC hypermethylation to restore its biological effect as a tumor suppressor in T-NHL cells. The antineoplastic action of 5-Aza-Cdr in T-NHL cells could also be due to the action of this epigenetic agent on other tumor suppressor genes. In summary, we demonstrate a significant down-regulation of SPARC in T-NHL samples and cell lines owing to its DNA methylation, and low level of SPARC is associated with a poor prognosis of T-NHL patients. Furthermore, our results show that SPARC functions as a tumor suppressor gene in T-NHL. We also provided evidence that DNA methylation in SPARC gene may play a role in the Azacitidine progression of T-NHL.