Matrine induced G 0 / G 1 arrest and apoptosis in human acute T-cell lymphoblastic leukemia ( TALL ) cells

Matrine, a natural product extracted from the root of Sophora flavescens, is a promising alternative drug in different types of cancer. Here, we aimed to investigate the therapeutic effects and underlying molecular mechanisms of matrine on human acute lymphoblastic leukemia (ALL) cell line, CCRF-CEM. Cell viability and IC50 values were determined by WST-1 cell cytotoxicity assay. Cell cycle distribution and apoptosis rates were analyzed by flow cytometry. Expression patterns of 44 selected miRNAs and 44 RNAs were analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) using the Applied Biosystems 7500 Fast Real-Time PCR System. Matrine inhibited cell viability and induced apoptosis of CCRF-CEM cells in a dose-dependent manner. Cell cycle analysis demonstrated that matrine-treated CCRF-CEM cells significantly accumulated in the G0/G1 phase compared with the untreated control cells. hsa-miR-376b-3p (-37.09 fold, p = 0.008) and hsa-miR106b-3p (-16.67 fold, p = 0.028) expressions were decreased, whereas IL6 (95.47 fold, p = 0.000011) and CDKN1A (140.03 fold, p = 0.000159) expressions were increased after matrine treatment. Our results suggest that the downregulation of hsa-miR-106b-3p leads to the upregulation of target p21 gene, CDKN1A, and plays a critical role in the cell cycle progression by arresting matrine-treated cells in the G0/G1 phase.


INTRODUCTION
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive form of leukemia characterized by an increased proliferation of lymphoblasts.T-ALL constitutes 10-15% of childhood ALLs and 25% of adult ALLs.An effective chemotherapy combination for T-ALL has not been developed yet, and firstline therapy fails in 25% of pediatric and more than 50% of adult cases [1,2].Recent studies have demonstrated that new treatments targeting specific genes or molecular pathways in cancer patients are more efficient and less toxic.Thus, targeted treatment and the determination of related oncogenes have a priority in terms of patient survival and occurrence of toxicity [2].Therefore, new treatment options should be developed for these cases by determining new molecular targets.
T-ALL is characterized by different gene and chromosome aberrations, including chromosomal translocations in the T-cell receptor (TCR) genes, abnormal expression of oncogenes, deletions, somatic gene mutations, as well as by alterations of signaling pathways and dysregulation of microR-NAs (miRNAs) [2].miRNAs are small noncoding RNAs about 22 nucleotides long which downregulate gene expression at the post-transcriptional level; they can inhibit translation or induce mRNA degradation by binding to the 3'-UTR region of the targeted mRNA.miRNAs regulate different cellular processes, including cell proliferation, transformation, and death; in addition, they control cell development and metabolism [3].Consequently, miRNAs play an important role in the regulation of many diseases such as viral infections, cardiovascular diseases, pulmonary artery hypertension, and cancer [4].Furthermore, cell cycle regulators have a crucial role in the development and progression of cancer and are closely linked to cell death and proliferation [5].
The mechanisms of cell death are evolutionarily conserved processes important for vital functions in all multicellular organisms, and are classified into three types: apoptosis, necrosis, and autophagy [6][7][8].Apoptosis and autophagy are the most important for maintaining tissue homeostasis, and their dysregulation can lead to cancer development.The two cell death processes can be regulated by the same or different signaling pathways.Understanding these pathways will help develop new strategies for inhibiting the proliferation of cancer cells.
The expression of miR-106 has been studied in renal cell carcinoma (RCC) [18], osteosarcoma [19], and gastric [20], lung [21], and colorectal [22] cancers.Moreover, the diagnostic and prognostic significance of miR-106 has also been explored in several cancer types.It was reported that miR-106 is one of four miRNAs whose serum level is closely associated with the overall survival of gastric cancer patients who received adjuvant chemotherapy [20].In another study, serum levels of miR-106a were higher in RCC patients compared to controls, but a decrease in miR-106a levels in the patients was observed one month after surgery [18].High-mobility group AT-hook-2 (HMGA2), E1A binding protein p300 (EP300), DLC1 Rho GTPase activating protein (DLC1), and RE-1 silencing transcription factor (REST) have been reported as target genes of miR-106 [19,[22][23][24].
CDKI 1 (also known as p21 and CDKN1A) is a cell cycle inhibitor protein that controls cell cycle arrest, apoptosis and DNA repair, and it can be regulated by miRNAs, RNA-binding proteins, phosphorylation and ubiquitination [25].Numerous studies have demonstrated that dysregulation of miR-17, miR-20, and miR-93 leads to aberrant p21 expression, which causes enhanced cell proliferation and inhibits progression through the G 0 /G 1 and G 1 /S phases [12,[25][26][27][28].
Matrine (dodecahydro-3a, 7a-diaza-benzo[de]anthracen-8-one; molecular formula: C 15 H 24 N 2 O) is an alkaloid compound obtained from Sophora flavescens plant, that acts as a κ-opioid and μ-opioid receptor agonist.Although in vivo and in vitro studies have shown that matrine has strong antitumor effects, such as inhibiting cell proliferation and inducing apoptosis, the molecular mechanisms by which matrine exerts its effects have not been fully explained [29][30][31][32][33].In this study, we aimed to investigate the effects and underlying molecular mechanisms of matrine on human T-ALL cell line, CCRF-CEM.

Chemicals and materials
Matrine (molecular formula: C 15 H 24 N 2 O; molecular weight: 248.36) was purchased from Sigma-Aldrich (Germany), and 50 mg/mL stock solution was prepared with ultrapure water.Fetal bovine serum (FBS), trypsin, L-glutamine, and RPMI-1640 medium were purchased from Biological Industrie s (USA).Total RNA and miRNA were isolated using RNeasy Mini Kit (Qiagen, Germany).The primers for mRNA and miRNA were obtained from Stellarray and GeneCopoeia (USA).The mRNAs and miRNAs analyzed in this study are listed in Table 1 and 2. Human T-ALL cell line CCRF-CEM was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in RPMI-1640 medium supplemented with 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin and L-glutamine.Untreated CCRF-CEM line was used as a control group, while CCRF-CEM cell lines treated with different concentrations of matrine were defined as matrine-treated groups.

Cytotoxicity assay
WST-1 assay (Roche Applied Science, Mannheim, Germany) was used to determine the cytotoxic effects of matrine at different concentrations.CCRF-CEM cell suspension was placed into a 96-well plate at a density of 1 × 10 4 cells in 100 µl per well.The cells were treated with matrine at a concentration of 0.5, 0.75, 1, 1.5, 2, 2.5, or 3 mg/ml for 72 hours.At the end of the experiment, 10 μl WST1 solution was added to each well and the plate was incubated for 1 hour.The absorbance of each well was measured spectrophotometrically at 450 nm with an enzyme linked immunosorbent assay (ELISA) reader (Thermo Fisher Scientific, Massachusetts, USA).All experiments were performed in triplicate.

Apoptosis analysis
Apoptosis analysis was performed using BD Annexin V/ propidium iodide (PI) apoptosis detection kit (BD Biosciences, USA) and ApoDirect in situ DNA fragmentation assay kit (Biovision, USA).
For the Annexin V/PI staining, the cells were seeded in 6-well plates and treated with matrine at a concentration of 2.4 mg/ml for 48 hours.After the incubation period, the cells were collected and washed twice with cold PBS and then resuspended in binding buffer to a concentration of 1 × 10 6 cells/ml.An aliquot of 100 μl of the resulting solution (1 × 10 5 cells) was transferred to a 5 ml culture tube, after which 5 μl of Annexin V and 5 μl of PI were added to each tube.The cells were incubated for 15 minutes at room temperature in the dark and then, within 30 minutes, analyzed by flow cytometry.
ApoDirect in situ DNA fragmentation assay is based on the principle that terminal deoxynucleotidyl transferase (TdT) catalyzes the addition of FITC-labeled deoxyuridine triphosphates (FITC-dUTP) to the 3'-hydroxyl ends of double-and single-stranded DNA.For fixation of 0.5, 0.75, 1.0, 1.5, 2.0, 2.5, and 3.0 mg/ml matrine-treated CCRF-CEM cells and untreated control CCRF-CEM cells, they were resuspended in 1% paraformaldehyde on ice for 15 minutes.The cells were washed twice with PBS and treated with 70% alcohol while on ice, then kept at −20°C until analysis.For staining, the cells were washed with buffer containing 0.05% sodium azide, then resuspended with staining solution and incubated for 60 minutes at 37°C.After washing, the cells were incubated with PI/RNase A solution at room temperature for 30 minutes.The analysis was performed with BD Accuri C6 Flow Cytometer (BD Biosciences).

Cell cycle analysis
Cell cycle analysis was performed in untreated and matrine-treated CCRF-CEM cells (2.4 mg/ml of matrine for 48 hours) using BD Cycletest assay according to the manufacturer' s instructions (BD Biosciences).Briefly, the cells were centrifuged at 300 × g for 5 minutes at room temperature and the supernatant was discarded.The pellet was resuspended in 1 mL of buffer solution by vortexing.Cell concentration was adjusted with buffer solution to 1 × 10 6 cells/µL.For staining, the supernatant was removed and each tube was incubated at room temperature for 10 minutes with the addition of 250 μL solution-A (trypsin buffer).The tubes were vortexed gently after the addition of 200 μL of solution-B (trypsin inhibitor and RNase buffer) and incubated for 10 minutes at room temperature.Another 200 μL of cold (2-8°C) solution-C (PI lectin) was added to each vial and carefully vortexed.The cell suspension was incubated in the refrigerator for 10 minutes in the dark and analyzed with a flow cytometer within 30 minutes.

mRNA expression analysis
Total RNA was isolated from untreated control CCRF-CEM cells and matrine-treated CCRF-CEM cells (2.5 mg/ml of matrine for 48 hours) using the MagNA Pure LC RNA Isolation Kit (Roche Applied Science, Germany) and was then reverse transcribed into cDNA using the Transcriptor First Strand cDNA Synthesis Kit (Roche Applied Science).According to the manufacturer' s instructions, 1 μg total RNA was mixed with 50 pmol anchored oligo (dT) 18 primer and incubated at 65°C for 10 minutes.Reactions were carried out for 30 minutes at 55°C for the reverse transcription step and 5 minutes at 85°C for the inactivation of transcriptor reverse transcriptase.All cDNA samples were 1: 4 diluted with RNase-free water and used as a template in real-time polymerase chain reaction (real-time PCR).
The mRNA expression levels of genes associated with apoptosis and cell cycle were detected by quantitative reverse transcription PCR (qRT-PCR) method using SYBR Green all-in-one qRT-PCR master mix (Biomatic, USA/ Canada) and ABI-7500 instrument (Applied Biosystems, USA).For each reaction, 5 μL of ×2 all-in-one qRT-PCR master mix, 0.2 μL of ×50 ROX Dye, 5 μM of each primer, and 1 μL of diluted cDNA were mixed.Conditions for the qRT-PCR reactions were as follows: denaturation step, 95°C for 10 minutes; amplification step, 40 cycles at 95°C for 15 seconds, 60°C for 1 minute; and melting curve step, 95°C for 15 seconds, 60°C for 1 minute, 95°C for 30 seconds, and 60°C for 15 seconds.The relative expression of the genes was calculated using the 2 −ΔΔCt method as described previously [34].

miRNA expression analysis
An miRNA isolation kit (Genoid, Hungary) was used to purify miRNA and an all-in-one first strand cDNA synthesis kit (Genoid) was used to obtain cDNA from untreated control CCRF-CEM cells and matrine-treated CCRF-CEM cells (2.5 mg/ml matrine for 48 hours).All qRT-PCR analyses were performed using the ABI 7500 Fast Real-Time PCR System (Applied Biosystems).The expression levels of miRNAs were assessed by relative quantification, and the fold expression changes were determined by the 2 −ΔΔCT method [34].

Statistical analysis
Matrine IC 50 concentrations were calculated with the GraphPad Prism Software version 5.01 (GraphPad Software, Inc., La Jolla, CA, USA).The average of relative expression level, determined by the 2 −ΔΔCT method for each mRNA and miRNA, was compared between untreated control CCRF-CEM cells and matrine-treated groups using Student' s t-test.A p < 0.05 was accepted as statistically significant.

RESULTS
To analyze the cytotoxic effects of matrine, CCRF-CEM cells were treated with different concentrations of matrine (0.5, 0.75, 1, 1.5, 2, 2.5, and 3 mg/ml).After incubation for 24, 48, and 72 hours, the viability was evaluated by WST cytotoxicity assay.Our results indicate that matrine at concentrations of 0.5-2.0 mg/ml does not affect the viability of CCRF-CEM cells.However, the higher doses of matrine decreased the cell viability, and inhibitor concentration (IC 50 ) values were 2.898, 2.400, and 2.390 mg/ml at 24, 48, and 72 hours, respectively (Figure 1).
To evaluate the effects of matrine treatment on apoptosis, CCRF-CEM cells were treated with matrine at the IC 50 dose (2.898, 2.400, and 2.390 mg/ml) for 72 hours, then analyzed using Annexin V-PI staining and DNA fragmentation assay.The apoptotic cell ratios of untreated and matrine-treated CCRF-CEM cells were 0.8% and 5.4%, respectively (Figure 2).The increase in matrine-induced apoptosis in CCRF-CEM cells was more prominent with 2.4 mg/ml dose of matrine.
The cell cycle distribution of untreated and 2.4 mg/ml matrine-treated CCRF-CEM cells was determined by BD Cycletest assay.The distributions of cells in the G 0 /G 1 , S, and G 2 /M phases were 61.0%versus 85.5%, 25.9% versus 6.6%, and 14.2% versus 8.3% in untreated control CCRF-CEM cells and the matrine-treated cells, respectively (Figure 4).These results showed that matrine significantly arrested the CCRF-CEM cells in the G 0 /G 1 phase, resulting in the majority of matrinetreated cells remained at that restriction point.

DISCUSSION
Natural, non-toxic and chemopreventive agents, including matrine, are promising new therapies for the treatment of cancer.The antitumor activities of matrine have been reported in esophageal cancer [29], myeloid leukemia [30], gastric cancer [31], melanoma [32], hepatocellular carcinoma [33], and lung [35], breast [36], prostate [37], pancreatic [38], and colon cancers [39].Up until now, the anticancer effects of matrine have not been investigated in human T-ALL cells.Our results indicated that matrine triggers cell cycle arrest and apoptosis in CCRF-CEM cells in a dose-dependent manner.Moreover, our study is the first to investigate potential miRNAs and target genes involved in the mechanism of matrine-induced cell death in T-ALL.
Studies have shown that miRNAs regulate various cellular processes, including cell division, proliferation and apoptosis [40].miRNAs are usually located in genomic regions involved in cancers and at chromosome fragile sites [41], and aberrant expression levels of miRNAs have been associated with different cancer types in humans [41][42][43][44][45].
Apoptosis is programmed cell death that occurs in both normal physiological and pathologic conditions.The disruption of this delicate balance can lead to the development of cancer.Autophagy is closely associated with eukaryotic cell death and apoptosis.In some cases, autophagy and apoptosis are controlled by the same proteins.Apoptotic signals can regulate autophagy, and autophagy can control apoptosis as well as other death mechanisms.The molecular interactions between autophagy and cell death are complex and, depending on physiological conditions, autophagy may promote or inhibit cell death [46].
Cell cycle checkpoints are controlled by specific miRNAs.Recent studies reported that the dysregulation of miRNAs such as the members of miR-15b, miR-17, and miR-34 precursor families, as well as miR-106, miR-221 and miR-222 disrupts the progression through all cell cycle checkpoints [52].In this study, we showed that the downregulation of hsa-miR-106b-3p in matrine-treated CCRF-CEM cells led to cell cycle arrest in the G 0 /G 1 phase by increasing the level of p21.Our findings are in agreement with those of Ivanovska et al. [12], who reported that the inhibition of miR-106 leads to the accumulation of tumor cells in G 1 phase, also by increasing p21 levels.Moreover, the authors found that the upregulation of miR-106b inhibits the expression of p21 and causes G 2 /M arrest in doxorubicin-treated breast cancer cells [12].Another study similarly showed that the overexpression of miR-106 suppressed the activation of p21 and led to G 2 /M    arrest in radiation-treated prostate cancer cells [53].In gastric cancer cells, the E2F1 gene was identified as the main target of miR-106b-25, which expression is regulated at the posttranscriptional level.It was reported that the dysregulation of E2F1 impairs transforming growth factor beta (TGFb) pathway and indirectly affects the expression of p21 and Bcl-2-like protein 11 (Bim).Due to the suppression of miR-106, the upregulation of E2F1 activates the TGFb tumor suppressor pathway and blocks cell cycle transition at the G 1 /S stage in gastric cancer cells [28].Furthermore, An et al. [54] investigated the therapeutic effects and related molecular mechanisms of matrine on A549 cell line and reported that dose-dependent overexpression of miR-126 inhibits vascular endothelial growth factor gene expression and induces G 0 /G 1 arrest in NSCLC cells [54].Although molecular mechanisms of miR-106b-mediated cell cycle arrest have been investigated in several studies, it remains unclear what causes arrest at the checkpoint.Moreover, the discrepancies between different studies might be due to the differences in selected cell lines and/or treatments.

CONCLUSION
In summary, our findings revealed that matrine suppresses cell growth and has an apoptotic effect in CCRF-CEM human T-ALL cell line.miR-106 affected cell cycle progression by blocking the transition of matrine-treated CCRF-CEM cells from G 0 to G 1 phase.Further in vivo studies are necessary to determine the functional role of matrine in T-ALL patients.

FIGURE 2 .
FIGURE 2. Apoptosis in untreated control CCRF-CEM cells and matrine-treated CCRF-CEM cells was assessed by Annexin V/propidium iodide staining.The apoptotic cell ratio of matrine-treated CCRF-CEM cells was increased significantly at 2.4 mg/ml.

FIGURE 4 .
FIGURE 4. Distribution of G 0 /G 1 , S, and G 2 /M phases in untreated control CCRF-CEM cells and matrine-treated CCRF-CEM cells.The matrine-treated cells were arrested at the G 0 /G 1 phase.

FIGURE 3 .
FIGURE 3. DNA fragmentation ratios of untreated control CCRF-CEM cells and matrine-treated CCRF-CEM cells.DNA fragmentation increased in the matrine-treated cells in a dose-dependent manner.

TABLE 1 .
The effects of matrine treatment on gene expression profile of CCRF-CEM cells

TABLE 2 .
The effects of matrine treatment on miRNA expression in CCRF-CEM cells