This article is mentioned in:

Introduction

Aberrant activation of the Wingless-type MMTV integration site family (WNT)/β-catenin pathway has been implicated in the pathogenesis of many malignancies (1–3). The phenomenon has also been observed in hematological malignancies (4,5). In addition, abnormal methylation of the promoters of specific WNT/β-catenin inhibitors has been reported in leukemia (6,7). The WNT/β-catenin signaling pathway plays an important role in the survival, proliferation and differentiation of hematopoietic stem cells. Aberrant activation of WNT/β-catenin signaling is closely associated with the pathogenesis of leukemia (8–10). As a result, WNT/β-catenin signaling may be an important treatment target for leukemia. The secreted frizzled-related protein (SFRP) family and Dickkopf (DKK) family are WNT signaling antagonists; the WNT/β-catenin signaling pathway is regulated tightly by the SFRP and DKK families (11). The functional loss of WNT antagonists contributes to activation of the WNT signaling pathway. Activation of the canonical WNT pathway causes the hypophosphorylation and stabilization of β-catenin. Following translocation into the nucleus, non-phosphorylated β-catenin associates with the T-cell factor family of transcription factors, thereby modulating the expression of target genes such as c-myc, cyclin D, matrix metalloproteinase-7 and bone morphogenetic protein-4 (12–15).

Arsenic trioxide (As2O3) is a traditional Chinese medicine. It has been found to be effective in the treatment of malignant hematopoietic diseases by inducing apoptosis and inhibiting cellular proliferation, and has been used to treat acute promyelocytic leukemia and multiple myeloma with good results (16,17). The anticancer effects of As2O3 are exerted through the induction of apoptosis and differentiation of leukemia cells and reduction of telomerase activity (18,19). However, some studies have found that the metabolism of As2O3 involves detoxification via methylation, which is similar to the methylation processes of oncogenes and tumor suppressor genes (20,21). In addition, As2O3 has been reported to utilize S-adenosyl methionine (SAM) (22), an essential co-factor of DNA methyltransferases, which results in DNA hypomethylation. Therefore, As2O3 may regulate tumor suppressor genes by interfering with DNA methylation patterns. Although As2O3 has been shown to have antileukemic effects, its demethylating and dose-dependent effects on genes associated with other tumors have already been postulated (20,23).

Different molecular mechanisms have been implicated in aberrant activation of the WNT/β-catenin signaling pathway. Abnormal methylation of WNT antagonists is a frequent event in several human malignancies (7,24,25). Previous studies have indicated that methylation of SFRP exists in leukemia (1,2). Our previous studies demonstrated that inhibitory factors of the WNT pathway, such as WNT inhibitory factor, DKK1 and SFRP1, are hypermethylated in leukemia cells and patients with leukemia (26–28). As2O3 has been shown to exert a demethylation effect through the inhibition of DNA methyltransferase, and has a similar demethylation effect to decitabine (29,30). SFRP1 is a member of the SFRP family, and the SFRP1 gene is located on chromosome 8p11.2. As an important inhibitor of the WNT pathway, the SFRP1 gene is involved in the regulation of cell growth and proliferation, and is closely associated with the occurrence of leukemia (31,32). However, it is unclear whether As2O3 affects the SFRP1 gene, WNT pathway or WNT downstream genes in Jurkat cells. In the present study, the methylation status of the SFRP1 gene in Jurkat cells was examined and the effect of As2O3 on SFRP1 and the WNT/β-catenin signaling pathway was investigated.

Materials and methods

Cells

Normal bone-marrow mononuclear cells (BMMNCs) and the leukemia cell lines HL60 (acute myeloid leukemia), K562 (chronic myeloid leukemia) and Jurkat (acute T cell leukemia) were purchased from Shandong University Medical School (Shandong, China) and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 10% non-essential amino acids, 1% penicillin and 0.1% amphotericin B (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The cells were maintained in an incubator at 37°C in an atmosphere with 5% CO2. Sub-culturing of the cells was performed after 80% confluence was reached. Cells in the exponential growth phase were treated with As2O3 (Yida Pharmaceutical Co., Ltd., Harbin, China) at concentrations of 0, 1.25, 2.5 and 5.0 µmol/l for 48 h. Untreated cells were used as the control.

DNA extraction and methylation modification

DNA was extracted from the cells using the Wizard Genomic DNA Purification kit (Promega Corporation, Madison, WI, USA) according to manufacturer's protocol. To ensure the purity of the extracted DNA, the ratio of the optical density of the DNA at 260 nm to that at 280 nm was confirmed to be ~1.8. In addition, the DNA concentration was confirmed to be between 0.1 and 0.9 ng/µl using a Smartspec 3000 spectrophotometer (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Approximately 1 µg extracted DNA was used for bisulfate modification using the EZ DNA Methylation Gold kit (Zymo Research, Seattle, WA, USA) according to the manufacturer's instructions.

Methylation-specific polymerase chain reaction (MSP)

The modified DNA was resuspended in 20 µl TE buffer (10 mM Tris-HCl, and 1 mM EDTA; pH=8.0) and immediately subjected to polymerase chain reaction (PCR) or stored at −20°C. PCR was performed with Zymo Taq PreMix (Zymo Research) using the primers listed in Table I. Approximately 50 ng bisulfite-modified DNA was amplified by MSP using the following reaction conditions: 95°C for 2 min; 95°C for 20 sec, 60°C (methylated) or 62°C (unmethylated) for 30 sec (40 cycles); and extension at 72°C for 7 min. DNA from normal BMMNCs treated with Sss I transmethylase (Zymo Research) was used as a positive control for methylation, and water was used as a negative control. PCR was performed using a PTC-200 cycler (Bio-Rad Laboratories, Inc.). The amplified PCR products (5 µl) were analyzed on 2% agarose gel and visualized under ultra violet illumination. MSP experiments were repeated three times for each sample.

Table I. Primer sequences.

Table I.

Primer sequences.

Genes	Primer sequences
SFRP1	Forward: 5′-TTGAGCATTTGAAAGGTGTGCTA-3′
	Reverse: 5′-ACAGCTACACTACCAGGGAAATCC-3′
SFRP1 (methylated)	Forward: 5′-GCGTTGGGTATTTAGTAGGATTTATTCG-3′
	Reverse: 5′-CGAACCCAACAGATCCCACGA-3′
SFRP1 (unmethylated)	Forward: 5′-GTGTTTGGTATTCAGTAGGATTTATTTG-3′
	Reverse: 5′-CAAACCCAAACAATCCCACAAC-3′
β-catenin	Forward: 5′- ATCCCACTGGCCTCTGATAAAG-3′
	Reverse: 5′-GTACGGCGCTGGGTATCCT-3′
Cyclin Dl	Forward: 5′-CGTGGCCTCTAAGATGAAGGA-3′
	Reverse: 5′-TCGGTGTAGATGCACAGCTTCT-3′
c-myc	Forward: 5′-TGAATCTGCCGCAGCTAGAA-3′
	Reverse: 5′- TCCCCTCGTTGCTCTTGTTC-3′
GAPDH	Forward: 5′-AGAAGGCTGGGGCTCATTTG-3′
	Reverse: 5′-AGGGGCCATCCACAGTCTTC-3′

[i] SFRP1, secreted frizzled-related protein 1.

Reverse transcription-quantitative PCR (RT-qPCR)

Total RNA was extracted from the cells using TRIzol (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Then, DNase I (Thermo Fisher Scientific, Inc.) treatment was used to remove genomic DNA contamination from the total RNA. RT-qPCR was performed using 0.3 µg total RNA following the instructions of the PrimeScript RT reagent kit (#DRR037A; Takara Biotechnology Co., Ltd., Dalian, China). The reverse transcription system included 5X PrimeScript Buffer (2 µl), PrimeScript RT Enzyme Mix I (0.5 µl), oligo dT primer (0.5 µl), random 6 mers (2 µl), total RNA (0.3 µg), and RNase-free dH2O (4.7 µl). The reaction conditions were as follows: Initial denaturation at 95°C for 30 sec, 40 cycles of denaturation at 95°C for 5 sec and annealing at 60°C for 30 sec. The PCR amplification system contained cDNA (2 µl), 10X buffer (2 µl), Mg2+ (1 µl), dNTP (10 mM) (1 µl), probe (1 µl; TaqMan GAPDH detection reagents; Takara Biotechnology Co., Ltd.), Primer-F (1 µl; Table I), Primer-R (1 µl; Table I), ddH2O (10.8 µl) and Taq DNA polymerase (0.2 µl; PrimeScript RT-PCR reagent kit; Takara Biotechnology Co., Ltd.). The reaction system was amplified using an Applied Biosystems 7500 Fast cycler (Thermo Fisher Scientific, Inc.). The reaction conditions were as follows: 95°C for 2 min; 94°C for 20 sec, and 60°C for 20 sec (40 cycles); and 72°C for 30 sec. The experiment was repeated for 3 times. GAPDH was used as internal standard to calculate the relative fold differences using the comparative Cq (2−ΔΔCq) method (33), and fold differences in SFRP1, β-catenin, cyclin Dl and c-myc expression compared with untreated cells were determined.

Western blotting

Cells were trypsinized and precooled radioimmunoprecipitation assay lysis buffer (600 µl; 50 mM Tris-base; 1 mM EDTA; 150 mM NaCl; 0.1% sodium dodecyl sulfate; 1% Triton X-100; 1% sodium deoxycholate; Beyotime Institute of Biotechnology, Haimen, China) was added to the samples Following lysis for 50 min on ice, the mixture was centrifuged at 12,000 × g and 4°C for 5 min. The supernatant was used to determine protein concentration with a bicinchoninic acid protein concentration determination kit [RTP7102; Real-Times (Beijing) Biotechnology Co., Ltd., Beijing, China]. Proteins (20 µg) were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions and transferred to nitrocellulose membranes (Whatman; GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). The membranes were incubated with phosphate-buffered saline containing 0.1% Tween-20 and 5% non-fat dry milk to block nonspecific binding. The membranes were then incubated for 1.5 h with rabbit anti-human SFRP1 (1:1,000; #4690), β-catenin (1:1,000; #8480), cyclin Dl (1:1,000; #2978), c-myc (1:1,000; #5605; all Cell Signaling Technology, Inc., Danvers, MA, USA) and β-actin antibodies (1:1,000; #8227; Abcam, Cambridge, UK), followed by 1 h incubation with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G secondary antibody (1:10,000; #111-005-045; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). Signals were visualized using an enhanced chemiluminescence detection system (ChemiDoc-It System; UVP, Inc., Upland, CA, USA). The intensity of protein fragments was quantified using Basic Quantity One software (v4.5.0; Bio-Rad Laboratories, Inc.). The relative expression of each protein was normalized to β-actin control.

Statistical analysis

Data are expressed as the mean ± standard deviations. Comparisons were made using an independent samples t-test with SPSS software (version 16.0; SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant result.

Results

As2O3 induces demethylation of the CpG islands of SFRP1 in Jurkat cells

To test the effect of different concentrations of As2O3 on the methylation status of DNA, MSP analysis was performed. DNA from normal BMMNCs treated with SssI transmethylase was used as a positive control for methylation, DNA from BMMNCs of healthy controls served as negative control, and water was used as a blank control. The data showed that the SFRP1 gene promoter was completely methylated in Jurkat cells, partially methylated in HL60 and K562 cells, and unmethylated in normal bone marrow mononuclear cells (Fig. 1A). In addition, the methylation-specific bands of the SFRP1 gene were significantly decreased in Jurkat cells treated with 1.25, 2.5 and 5.0 µmol/l As2O3 for 48 h. By contrast, the non-methylation-specific bands of SFRP1 genes were significantly increased in Jurkat cells treated with 1.25, 2.5 and 5.0 µmol/l As2O3 for 48 h. Notably, the attenuation of the hypermethylation of SFRP1 by As2O3 was not dose-dependent in Jurkat cells (Fig. 1B). These results suggest that As2O3 induces the demethylation of CpG islands of SFRP1 in Jurkat cells.

Figure 1. Methylation-specific polymerase chain reaction analysis of (A) SFRP1 gene expression in normal BMMNCs and leukemia cell lines (HL60, K562 and Jurkat). (B) SFRP1 gene expression in Jurkat cells treated with As2O3 at different concentrations (0, 1.25, 2.5 and 5.0 µM) for 48 h. BMMNC, normal bone marrow mononuclear cell; P, positive control; u, unmethylated; m, methylated; SFRP1, secreted frizzled-related protein 1.

As2O3 increases the mRNA expression level of SFRP1 in Jurkat cells

To study the effect of As2O3 on SFRP1, β-catenin, cyclin Dl and c-myc mRNA expression in Jurkat cells, RT-qPCR was employed. As2O3 decreased the expression levels of β-catenin (Fig. 2A) and cyclin Dl (Fig. 2B) in a dose-dependent manner (P<0.05), but had no effect on the expression of c-myc mRNA (P>0.05; Fig. 2C). Notably, SFRP1 mRNA expression in Jurkat cells was increased in a dose-dependent manner by treatment with increasing concentrations of As2O3 for 48 h; the increases resulting from treatment with 2.5 and 5.0 µmol/l were significant (P<0.05; Fig. 2D). These results indicate that As2O3 increases the mRNA expression level of SFRP1 in Jurkat cells.

Figure 2. Expression of (A) β-catenin, (B) cyclin Dl, (C) c-myc and (D) SFRP1 mRNA in Jurkat cells treated with 0, 1.25, 2.5 and 5.0 µM As2O3. Reverse transcription-quantitative polymerase chain reaction was performed to measure mRNA expression. Data are expressed as the mean ± SD (n=3–6). *P<0.05 vs. 0 µM As2O3. SFRP1, secreted frizzled-related protein 1.

As2O3 increases SFRP1 protein expression, but decreases β-catenin, cyclin Dl and c-myc protein expression levels in Jurkat cells

To determine the effect of As2O3 on the protein expression of SFRP1, β-catenin, cyclin Dl and c-myc, western blotting was conducted. Western blots showed that As2O3 increased the protein expression of SFRP1 in a dose-dependent manner (P<0.05; Fig. 3). However, As2O3 decreased the protein expression of β-catenin, cyclin Dl and c-myc in a dose-dependent manner (P<0.05; Fig. 4). These results suggest that As2O3 increases SFRP1 protein expression, but decreases β-catenin, cyclin Dl and c-myc protein expression in Jurkat cells.

Figure 3. Western blot analysis of SFRP1 protein expression in Jurkat cells treated with 0, 1.25, 2.5 and 5.0 µM As2O3. Data are expressed as the mean ± SD (n=3–6). *P<0.05 vs. 0 µM As2O3. SFRP1, secreted frizzled-related protein 1.

Figure 4. Western blot analysis of proteins in Jurkat cells treated with 0, 1.25, 2.5 and 5.0 µM As2O3. (A) Representative western blots and expression levels of (B) β-catenin, (C) cyclin Dl and (D) c-myc proteins. Data are expressed as the mean ± SD (n=3–6). *P<0.05 vs. 0 µM As2O3.

Discussion

The results of the present study indicate that As2O3 induces the demethylation of SFRP1 genes in Jurkat cells, and upregulates SFRP1 gene expression at the mRNA and protein levels. However, the demethylation effect of As2O3 was not found to be completely dose-dependent, which conflicts with previous studies (34,35). This observation may be due to differences in the leukemia cell lines used and genes analyzed. This suggests that the different types of leukemia may have different pathogeneses. The mechanism of action of As2O3 also appears to also differ among leukemia cell lines.

β-catenin is an important component of the WNT pathway, and reflects the activity of the WNT pathway (36). In the present study, following treatment with As2O3 the expression of β-catenin mRNA and protein was significantly reduced, suggesting that As2O3 affects the activity of the WNT pathway. Cyclin Dl, c-myc and B-cell lymphoma-2 (Bcl-2) are downstream genes of the WNT pathway, and play important roles in cell proliferation, differentiation and apoptosis (37). Cyclin D1 positively regulates cell cycle, and facilitates cells to cross G1/S check point to enter S phase. Cyclin D1 is overexpressed in multiple tumors (13). As2O3 arrests some tumor cells at G1 phase and inhibits tumor cell proliferation (38). In the present study, RT-qPCR and western blotting demonstrated that As2O3 treatment reduced cyclin D1 expression in Jurkat cells. Therefore, As2O3 has an anti-leukemia effect by inhibiting the proliferation of Jurkat cells via the down-regulation of cyclin D1 expression. In the present study, the expression of c-myc mRNA was not significantly altered, but the expression of c-myc protein was downregulated. The inconsistency between mRNA and protein expression may be due to the fact that mRNA is affected by a number of molecular regulatory factors, such as by microRNA, or it may be possible that post-translational regulation serves a role. Gene regulation in the cell cycle is complex, and multiple genes may be associated with the same protein (39). The regulatory effect of As2O3 on B-cell lymphoma-2 has been extensively studied (40), and so was not examined in the present study.

To summarize, the results of the present study indicate that As2O3 increases the expression of the WNT suppressor gene SFRP1 by demethylation, and reduces the expression of β-catenin, thereby inhibiting the WNT pathway through downregulation of the expression of the WNT downstream target genes cyclin Dl and c-myc. The demethylation effect of As2O3 in leukemia cell lines suggests the potential of As2O3 as a demethylation drug that could be widely used in the treatment of leukemia. However, the methylation effects of As2O3 on other important genes are worthy of further study.

Acknowledgements

This study was supported by Natural Science Foundation of Shandong Province (grant no. ZR-2012HL06), Shandong Province Science and Technology Plans (grant no. 2014GSF118029) and Shandong Medical and Health Science and Technology Development Plan (grant no. 2014WS0105).

References