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Introduction

The cancer microenvironment has been implicated in playing a critical role(s) in the development of drug resistance (1). Mesenchymal stem cells (MSCs) comprise a cell type found in the cancer microenvironment (2). It has been reported that stromal cells protect tumor cells from apoptosis induced by chemotherapy either through cell-cell interactions with tumor cells or by the local release of soluble factors, such as interleukin-6, which promote survival and tumor growth (3,4). In contrast to solid tumors that invade bone marrow, leukemia originates in the marrow in close proximity to stromal cells that provide growth and survival signals (5). These signals provide the primary drug resistance mechanism in leukemia, also referred to as cell adhesion-mediated drug resistance, and may account for the minimal residual disease in tissue (6,7). We recognized the importance of MSC in drug resistance, and we propose that determining how leukemia cells communicate with the surrounding tissue will pave the way for eliminating residual leukemia cells that are 'hiding' in stromal niches.

Leukemia-stroma interactions and the consequences on tumor survival involve CXCR4/CXCL12 signaling (3). C-X-C motif chemokine 12 (CXCL12), previously called stromal cell-derived factor-1 (SDF-1), is a chemokine that is constitutively secreted by MSC and facilitates the interaction between leukemia cells and stromal cells via its cognate receptor CXCR4 (8). Intracellular CXCR4 levels are significantly elevated in B-CLL (9,10), B-cell acute lymphoblastic leukemia (11), multiple myeloma (12), some AML (13), and CML (3). The CXCR4 antagonist, AMD3100 (Plerixafor), has been in clinical trials to resensitize leukemia cells to cytotoxic drugs in co-cultures with MSC (14). However, blocking the CXCR4 only partially overcomes stromal cell-emediated drug resistance (14–17). Therefore, other interactions between leukemia cells and the tumor microenvironment may provide alternative therapeutic targets.

Arsenic trioxide (As2O3) is a clinically effective agent in the treatment of acute promyelocytic leukemia (APL) in primary cases (18,19), relapsed cases (20), and in ATRA-resistant patients (21). As2O3 has also been shown to induce apoptosis and display antiproliferative effects in Hodgkin's lymphoma, myeloid leukemias that express BCR-ABL tyrosine kinase as well as HL-60 cell types that overexpress Bcl-2, Bcl-X (L), MPR, and MRP proteins (22–25). Low-dose As2O3 induces partial differentiation, while high-dose As2O3 triggers apoptosis (26). Degradation of PML-RARα and PML is the most important role that As2O3 plays in the differentiation of APL cells (27). Mitochondrial pathway is the principle mechanism of As2O3-induced apoptosis (28). Intracellular reactive oxygen species (ROS) also play a role in apoptosis (29). However, the molecular mechanism by which As2O3 modulates apoptosis remains unclear. Identification of the genes regulated by As2O3 will aid in developing our understanding of the mechanisms of As2O3 therapy for the treatment of leukemia.

A downstream target of the TGFβ tumor suppressor pathway, RUNX3, functions as a candidate tumor suppressor (30,31), RUNX3 modulates cellular proliferation and apoptosis through transcriptional regulation of genes, such as the growth regulator p21 and the pro-apoptotic gene Bim (32,33). RUNX3 is an important molecule that links the oncongenic Wnt and the tumor-suppressive TGFβ pathways in intestinal carcinogenesis (34). RUNX3 exhibits dynamic shuttling between the nuclear and cytoplasmic compartments (35), and RUNX3 is found tightly associated with components of the nuclear architecture. The targeting of RUNX3 to the nuclear matrix may have significant biological consequences and may regulate RUNX3 activity (35,36). In our study, we report that RUNX3 plays a critical role in As2O3-induced apoptosis and MSC-mediated drug resistance in leukemia cells.

Materials and methods

Generation of mesenchymal stem cells

Three milliliters of bone marrow cells were obtained from normal donors aged between 20 and 50 years old who had provided written consent under the Ethics Committee of Sun Yat-sen University, which approved the protocol according to the Declaration of Helsinki. To isolate MSC, bone marrow mononuclear cells were isolated via density gradient centrifugation over Percoll (Amersham Pharmacia Biotech, Little Chalfont, UK). Immediately after centrifugation, isolated mononuclear cells were resuspended at 5×103 cells/cm2 in α-modified eagle's medium (αMEM) with high glucose concentration (Gibco, UK), 10% fetal bovine serum (FBS; Lonza, UK), and 1% penicillin-streptomycin (Gibco). Cells were cultured at 37°C in a humidified atmosphere containing 5% carbon dioxide. After 72 h, non-adherent cells were removed. When the cells were 70–80% confluent, adherent cells were trypsinized and expanded for 3–5 weeks. Before use in experiments, the identity of MSC was verified by checking for positivity of CD105, CD106, CD73,CD90,CD144, HLA-class I, and the lack of expression of CD45, CD34, CD133.

Co-culture experiments

For co-culture experiments, mesenchymal stem cells were seeded the day before the experiment onto 6-well plates (Corning Life Sciences) at a concentrationof 1×105 cells/well and incubated at 37°C in 5% CO2. After confirming the confluence of the stromal layer by phase contrast microscopy, K562 or NB4 cells were added onto the MSC layers separately at a ratio of 20:1. For assessment of MSC-derived drug resistance, cells were treated with As2O3 at the indicated time points. Next, K562 or NB4 cells were collected from the top layer leaving the adherent stromal layer intact. Then, K562 and NB4 cells were assayed for cell viability. MSC contamination, assessed by FACS as the fraction of CD19-negative cells, was always <1%. For experiments with AMD3100 and As2O3 treatment, K562 cells were pre-incubated for 4 h with AMD3100 and seeded onto confluent marrow stromal cell layers. After 4 h, As2O3 was added for a further 24 h. Then, the K562 cell layer was vigorously washed off and collected as previously described.

Cell transfection

A short hairpin RNA (shRNAs) plasmid against human RUNX3 and control shRNA were purchased from Joekai Biotechonology LLC (Shanghai, China) and transduced into cells by electroporation. Electroporation was performed with a Gene Pulser Xcell Unit (Bio-Rad Laboratories, Inc., Hercules, CA, USA) under conditions of 250 V, 1200 µF, and exponential wave. Clones were selected with G418 (500 µg/ml). The RUNX3 expression plasmid PCDNA3.1/RUNX3 was constructed using full-length RUNX3 cDNA, as described previously (37). K562 cells were transfected using Lipofectamine LTX from Invitrogen. Empty vectors were transfected as controls.

Cell cycle analysis

K562 cells expressing scrambled or RUNX3-targeting shRNA were incubated with As2O3. After incubation, the cells were harvested and resuspended in 500 µl of cold PBS and cold ethanol (1.5 ml) for 2 h at 4°C, followed by incubation with 0.1% sodium citrate containing propidium iodide (PI) 0.05 mg and 50 µg RNase for 30 min. Finally, the cell cycle analysis was detected by flow cytometry (FC500; Beckman Coulter, Miami, FL, USA), and the proportion of cells within the G0/G1, S, and G2/M phases of the cell cycle were analyzed using the MultiCycle AV DNA Analysis software (Phoenix Flow Systems).

Quantitative reverse transcriptase-polymerase chain reaction

Total RNA was extracted from each sample using the Total RNA Extraction kit (Invitrogen), following the manufacturer's instructions. The concentration of RNA was measured by spectrophotometry. Total RNA was reverse transcribed to cDNA with reverse transcriptase reagents (Takara, Nanjing, China), according to the manufacturer's protocol. Specific primers for RUNX3 and GAPDH genes were designed based on sequence data from the GenBank database. The primers were purchased from Invitrogen Biological Engineering Technology & Services Co., Ltd. For human RUNX3 transcripts, 5′-AGGCATTGCGCAGCTCAGCGGAGTA-3′ was used as a sense primer and 5′-TCTGCTCCGTGCTGCCCTCGCACTG-3′ as an antisense primer. For the human GAPDH transcripts, 5′-TCACAGGGGTTCCCTTCTCTC-3′ was used as a sense primer and 5′-CACTTCTTTGTGCCATCCATGG-3′ as an antisense primer. Conditions for all PCRs were optimised on an iCycler iQ (Bio-Rad Laboratories, Inc.), and the optimum annealing temperature was 60°C.

Immunofluorescence microscopy

Cells were incubated on poly-L-lysine-coated slides for 30 min, washed gently with PBS and fixed in 10% methanol at room temperature. After washing with PBS, the cells were permeabilized with 0.1% Triton in PBS for 60 min at room temperature. The cells were then incubated with the rabbit anti-RUNX3 antibodies (BD Biosciences) overnight at 4°C. After washing with PBS, the slides were incubated with the secondary antibody, Alexa 555-conjugated goat anti-rabbit immunoglobulin G (IgG; Molecular Probes), for 2 h and then washed with PBS. Nuclei were stained with 10 nM DAPI, and cells were then subjected to fluorescence microscopy.

Morphological detection of apoptosis

Morphological evaluation of apoptotic cell death was performed using the Hoechst 33258 kit (KeyGen Biotech), according to the manufacturer's instructions. Cells were cytospun, stained with Hoechst 33258, and observed with light microscopy.

CXCL12 enzyme-linked immunosorbent assay

After culturing mesenchymal stem cells in 24-well plates for 24 h, medium was collected and concentrated by ultrafiltration. CXCL12 levels in the medium were assayed with a Human CXCL12/SDF-1 α immunoassay kit (R&D Systems, Abingdon, UK) according to the manufacturer's instructions. Samples were run in triplicate.

Flow cytometry analysis of CXCR4 expression

Monoclonal antibodies against human CXCR4-PE (BD Pharmingen, DakoCytomation) were used for flow cytometry analysis. PE-conjugated IgG1 and IgG2a control monoclonal antibodies were from Cell Signaling Technology, Inc.

Transmigration assays

For quantitative transmigration assays, K562 cells (5×104 cells) were placed in the upper chamber of a Transwell system (24-well plate, 5 µM-pore filter; Corning Life Sciences) in a total volume of 150 µl DMEM medium. Next, 600 ml of the supernatant was concentrated 10-fold, and placed in the lower chamber of the Transwell. Further control wells assessed the chemotactic activity of recombinant CXCL12 (rCXCL12) at concentrations of 100 ng/ml. Cells were harvested from the lower chamber after 3 h and counted using a hemocytometer.

Annexin V staining

Apoptosis was measured using the Calbiochem assay kit (Calbiochem, San Diego, CA, USA) according to the manufacturer's instructions. K562 cells expressing scrambled or RUNX3-targeting shRNA were incubated with As2O3. After incubation, cells were washed with phosphate-buffered saline (PBS) and stained using phycoerythrin-labeled Annexin V. Numbers of both RFP- and Annexin V-positive cells were determined by flow cytometry. In some cases, the Annexin V-propidium iodide staining kit (Calbiochem) was used instead, according to the manufacturer's protocol.

Western blot analysis

Cells were cultured with the indicated concentrations of As2O3 for the specified times, harvested, washed, and lysed using RIPA buffer containing protease inhibitors. After normalization for total protein content (60 µg/lane), the samples were subjected to 12% SDS-PAGE and then transferred to a PVDF membrane. After blocking with 5% non-fat dry milk and 0.1% Tween-20 in Tris-buffered saline, the membranes were incubated with the following primary antibodies: Mcl-1 (Cell Signaling Technology, Inc.), RUNX3 (BD Pharmingen), p21, Bax, Bcl-2, cleaved caspase-3, cleaved caspase-9, and β-actin (all from Cell Signaling Technology, Inc.). After extensive rinsing with TBST, the membranes were incubated with HRP-conjugated anti-mouse or rabbit secondary antibodies (Cell Signaling Technology, Inc.), and proteins were detected with the use of an enhanced chemiluminescence system (EMD Millipore, Billerica, MA, USA) and captured on a light-sensitive imaging film.

Statistical analysis

All experiments were repeated in triplicate. The data are reported as the mean ± SEM. Statistical analyses were performed using Student's t-test or one-way ANOVA. Probability (P) value of 0.05 was considered to be significant (SPSS v13.0 for windows).

Results

As2O3 induces expression of RUNX3 and translocation of RUNX3 into the nucleus

To determine whether the expression of RUNX3 is induced by As2O3 treatment, western blot analysis was performed to detect the level of total RUNX3 and the RUNX3 target gene, p21. As shown in (Fig. 1A), As2O3 treatment resulted in the elevation of total RUNX3 in K562 and NB4 cells in a dose-dependent manner. Elevation of RUNX3 levels was observed at 3 h post-treatment with 5 µM As2O3 in NB4 cells, and expression levels reached a peak at 9 h post-treatment. An increase in RUNX3 expression was detected at 1 h following treatment with 3 µM As2O3 in K562 cells, and expression levels reached a peak at 4 h post-treatment (Fig. 1B). Similarly, we also observed that the expression of RUNX3 mRNA was enhanced after As2O3 treatment in both K562 and NB4 cell lines. Protein levels of p21 were elevated in a dose- and time-dependent manner and corresponded to the elevation of RUNX3 observed following As2O3 treatment. Immunofluorescence staining revealed that RUNX3 was located mainly in the cytoplasm before As2O3 treatment and accumulated in the nucleus following As2O3 treatment (Fig. 1C). Consistent with the results obtained using NB4 cells, our analysis of K562 cells and primary APL blast cells from three patients revealed that RUNX3 localized in the cytoplasm prior to As2O3 treatment and translocated into the nucleus following As2O3 treatment. It has been previously reported that As2O3 treatment resulted in elevation in cellular ROS stores and production. In our studies, treatment of cells with As2O3 also resulted in the generation of ROS (Fig. 1D). This ROS induction seems to be necessary for As2O3-dependent elevation of RUNX3 because pre-treatment of cells with NAC, a scavenger of ROS, resulted in the inhibition of As2O3-induced expression of RUNX3 (Fig. 1E).

Figure 1 As2O3 induces expression of RUNX3 and translocation of RUNX3 into the nucleus. (A) NB4 or K562 cells were incubated with different concentrations of As2O3 for the indicated periods. Total protein was extracted and analyzed by western blot analysis with antibodies against the indicated antigens. β-actin was used as an internal control. (B) NB4 cells, K562 cells or primary APL blast cells were incubated with 6 or 3 µM As2O3 for 12 h. After fixation and permeabilization, cells were incubated with anti-RUNX3 antibody and Alexa 555-conjugated goat anti-rabbit IgG (red). The nuclei were stained with DAPI (blue), and cells were subjected to fluorescence microscopy. Scale bar, 20 µm. (C) NB4 or K562 cells were pre-incubated with As2O3 (6 or 3 µM) for the indicated time with or without MSC. Cells were then analyzed by flow cytometry for the presence of ROS as described in Materials and methods. Data are expressed as the fold increase in mean fluorescence over untreated samples and represent the mean ± SD of three independent experiments. *P<0.05 compared with K562 incubated with MSC. (D) Pre-treatment of cells with N-acetylcysteine (5 mM, 30 min) attenuated the expression of RUNX3 in NB4 or K562 cells. (e) The expression of RUNX3 was analyzed by western blot analysis in cells treated with As2O3 for the indicated times.

RUNX3 is required for As2O3-induced apoptosis

We next determined the role of RUNX3 in As2O3-induced apoptosis using RNA interference to reduce RUNX3 mRNA. We established two stable clones expressing shRNA specific to RUNX3, designated A2 and B4, and a scramble shRNA-expressing clone, designated SCR. Western blot analysis with anti-RUNX3 antibody revealed that endogenous RUNX3 protein levels were markedly diminished in A2 clones and significantly reduced in the B4 clone compared with SCR (Fig. 2A). Cell growth assays showed that A2 cells exhibited significantly enhanced growth compared with SCR cells. As2O3 inhibited the growth of SCR cells, but this growth inhibition was compromised in the RUNX3 shRNA-transduced cells treated with As2O3 (Fig. 2B). Next, we examined the involvement of RUNX3 in As2O3-induced apoptosis and cell cycle arrest. As shown in (Fig. 3A), the ratio of Annexin V and propidium iodide-positive cells was increased in a time-dependent manner, and 21.6% of the SCR cells were positive on the second day after As2O3 treatment. By contrast, apoptosis was significantly reduced during the observation periods up to day 2 in RUNX3 knockdown clones (Fig. 3A and B). Similar results were obtained from our Hoechst 33258 staining analysis (Fig. 2C and D). Fig. 6H shows that re-expression of RUNX3 enhanced sensitivity of K562 cells to As2O3 in a dose-dependent manner. Transduction of control constructs alone did not alter cellular response to As2O3. The percentages of cells in G1, S, and G2/M phase are illustrated in Fig. 3C and D. In SCR cells, treatment with As2O3 increased the fraction of cells in G2/M progressively from 8.95 to 27.61%. However, suppression of RUNX3 expression by RUNX3 shRNA attenuated As2O3-induced cell cycle arrest. Consistent with these results, p21 expression was suppressed in the A2 and B4 clones after As2O3 treatment. A decrease in the Mcl-1 protein was observed after treatment with As2O3 in SCR clones, though this reduction in Mcl-1 levels was compromised in A2 and B4 clones (Fig. 3E).

Figure 2 Knockdown of RUNX3 suppresses As2O3-induced cell cycle arrest and apoptosis in K562 cells. (A) Total protein was extracted from SCR or RUNX3 shRNA expressing cells and subjected to western blot analysis with antibodies against the indicated antigens. β-actin was used as an internal control. (B) SCR or RUNX3 shRNA expressing cells were incubated with 3 µM As2O3 for the indicated time periods. Cell growth was assessed by the Cell Counting kit-8. The results are presented as the mean ± SE of three independent experiments relative to the results obtained at day 0. *P<0.05 compared with SCR treated with As2O3. (C and D) Morphologic changes were examined by hoechst 33258 staining and fluorescence microscopy. *P<0.05; Scale bar, 50 µm.

Figure 3 (A and B) Annexin V-positive cells were detected by FACS analysis. The results are presented as the mean ± Se of three independent experiments. *P<0.05 compared with SCR. (C and D) Cell cycle arrest was determined by propidium iodide staining and FACS analysis. *P<0.05, **P<0.01 compared with SCR. (e) SCR or RUNX3 shRNA expressing cells were incubated with 3 µM As2O3 for 12 h. Total protein was extracted and analyzed by western blot analysis with antibodies against the indicated antigens. β-actin was used as an internal control.

Figure 6 The expression of transcription factor RUNX3 was modulated via CXCL12/CXCR4 signaling and restore the sensitivity of K562 cells to As2O3. (A) K562 cells were cultured with MSC and treated with AMD3100 (50 µM), As2O3 (5 µM), or a combination of both. The expression level of CXCR4 compared with non-treated cells was analyzed by flow cytometry. *P<0.05 compared with non-treated cells. #P<0.05 compared with cells not treated with AMD3100. $P<0.05 compared with cells treated with As2O3 without AMD3100. (B) Levels of CXCL12 produced by mesenchymal stem cells from patients were detected by ELISA. The results are presented as the mean ± SE of three independent experiments. *P<0.05 compared with non-treated cells. (C) K562 cells were pre-incubated for 4 h with or without AMD3100 before being assayed for chemotaxis toward supernatant collected from MSC culture or toward rCXCL12 in a Transwell system. *P<0.05 compared with non-treated cells. #P<0.05 compared with cells not treated with AMD3100. $P<0.05 compared with cells treated with CXCL12 without AMD3100. (D) K562 cells were treated with As2O3 both in the absence and in the presence of CXCL12 (100 ng/ml) for the indicated times. The expression of RUNX3 was analyzed by western blot analysis. (E) K562 cells were cultured with MSC and treated with AMD3100 (50 µg/ml) or left untreated. Subsequently, cells were treated with As2O3 (3 µM) as indicated. Western blot analysis of total lysates from cells cultured for 10 h was performed. A representative experiment is shown. (F) Total protein was collected from vector control (VCont) and PCDNA3.1/RUNX3 expressing cells and subjected to western blot analysis with antibodies against RUNX3 and β-actin. (G) Total protein was extracted and analyzed by western blot analysis with antibodies against the indicated antigens. β-actin was used as an internal control. (H) Cells were cultured alone or in the presence of MSC. After 24 h, cells were treated with As2O3 at various concentrations, and Annexin V and PI-positive cells were detected by FACS 24 h post-treatment. Average histograms from three individual experiments are shown. *P<0.05 compared with non-treated cells.

Mesenchymal stromal stem cells protect leukemia cells from As2O3-induced apoptosis

To assess whether MSC could protect leukemia cells from As2O3-induced apoptosis, K562 and NB4 cells were exposed to a range of As2O3 concentrations in the presence or absence of MSC. After 24 and 48 h, cells were analyzed by probing for Annexin V and propidium iodide (Fig. 4A). While exposure of NB4 cells to As2O3 for 24 or 48 h in control cultures induced apoptosis in a dose-dependent fashion (6.5±2, 9.2±3.5 and 13.5±3.7% of Annexin V/propidium iodide+ cells for 24 h; 8±2.8, 15.9±4.1 and 38.1±5.4% for 48 h for As2O3 at 3, 6 and 9 µM, respectively), the proportion of apoptotic NB4 cells in the presence of MSC was significantly reduced (6.7±1.8, 7.1±3 and 8.1±2.6% for 24 h; 7.7±3.3, 8.4±3.5 and 16±4.2% for 48 h, with As2O3 at 3, 6 and 9 µM, respectively; P=0.003, 0.015 and 0.024, NB4 vs. NB4 + MSC with 3, 6 and 9 µM As2O3, respectively) (Fig. 4A). As shown in Fig. 4A, the protective effect of MSC was also observed for K562 cells. No significant protection was observed when CML cells were cultured and treated on a monolayer of endothelial cells.

Figure 4 Mesenchymal stromal stem cells reduce As2O3-induced responses. (A) K562 or NB4 cells were cultured alone or in the presence of MSC. After 24 h, cells were treated with As2O3 at various concentrations and apoptosis was quantified after a further 24 or 48 h by Annexin V and propidium iodide staining. *P<0.05 compared with K562 incubated without MSC. (B and C) Cell cycle progression was measured by propidium iodide staining.

We also determined whether MSC affect As2O3-induced cell cycle arrest. As shown in (Fig. 4B and C), As2O3 treatment of K562 cells resulted in a dose-dependent accumulation of cells with 4N and <2N sub-G1 DNA content, which suggests induction of G2/M-phase cell cycle arrest and apoptosis. MSC robustly reduced the proportion of G2/M-phase and sub-G1 cells. MSC did not significantly affect cell cycle arrest induced by As2O3 in NB4 cells. To determine if MSC modulate the level of ROS, K562 and NB4 cells incubated with or without As2O3, either in the presence or absence of MSC, were analyzed for ROS by DCFDA staining. As shown in Fig. 1D, the level of ROS in NB4 cells increased after treatment with 6 µM As2O3 in a time-dependent manner; however, MSC were able to attenuate the quantity of ROS produced following As2O3 treatment.

The intrinsic, mitochondrial pathway is the principal mechanism of As2O3-induced apoptosis. In the mitochondrial apoptotic pathway, the ratio of expression of pro-apoptotic Bax to that of anti-apoptotic Bcl-2 ultimately results in either cell death or survival. We determined the effect of MSC on the Bax/Bcl-2 ratio. Upregulation of Bax expression, as well as downregulation of Bcl-2 expression induced by As2O3, was blocked in the presence of MSC (Fig. 5D).

Figure 5 MSC reduce the expression of RUNX3 induced by As2O3. (A) K562 or NB4 cells were cultured alone or in the presence of MSC. After 24 h, cells were treated with As2O3 at various concentrations. Total protein was extracted 24 h post-treatment and analyzed by western blot analysis with antibodies against the indicated antigens with RUNX3 and p21. β-actin was used as an internal control. (B and C) Nuclear extract from K562 or NB4 cells was prepared at the indicated time points and subjected to western blot analysis. (D) Total protein was extracted and analyzed by western blot analysis with antibodies against the indicated antigens. β-actin was used as an internal control.

MSCs have been found to protect NB4 and K562 cells from As2O3-induced apoptosis, and this protection mechanism is related to the expression of apoptotic proteins. We measured protein levels of cleaved caspase-3 and cleaved caspase-8. Consistent with the previous report, As2O3 treatment induced the activation of caspase-8, whereas this modulation was not noted in the presence of MSC. As2O3-induced death of cells is associated with strong activation of caspase-3, which was significantly inhibited when cells were treated in the presence of MSC (Fig. 5D). Additionally, there were low levels of Mcl-1 protein in NB4 and K562 cells treated with As2O3. However, in the presence of MSC, the decrease in Mcl-1 levels was compromised (Fig. 5D).

MSC reduce the expression of RUNX3 induced by As2O3 via CXCL12/CXCR4 signaling

We have demonstrated that RUNX3 is required for As2O3-induced apoptosis. To determine whether MSC modulate the expression of RUNX3, western blot analysis was used to detect the level of total RUNX3 in K562 and NB4 cells treated with As2O3 in the presence or absence of MSC (Fig. 5A). The presence of MSC reduced RUNX3 protein expression of cells treated with different concentrations of As2O3 compared to the cultures in which the leukemic cells were exposed to As2O3 without MSC. The expression of p21 induced by As2O3 was also inhibited in the presence of MSC. We next studied the subcellular distribution of RUNX3 by performing a western blot analysis on nuclear extracts from NB4 and K562 cells treated with As2O3 for different time intervals in the presence or in the absence of MSC. In K562 cells, the expression of RUNX3 was induced in 2 h, peaking at 4 h. In NB4 cells, RUNX3 expression was induced later and peaked at 9 h. In the presence of MSC, the level of RUNX3 in the nuclear fraction was significantly reduced at the indicated time in NB4 and K562 cells (Fig. 5B and C).

CXCL12/CXCR4 signaling is an important mediator for leukemia-stroma interaction, and this interaction has consequences on tumor survival. Therefore, we determined whether CXCL12/CXCR4 modulates the expression of transcription factor RUNX3 in K562 cells. The expression level of CXCR4 was analyzed in K562 cells by flow cytometry (Fig. 6A). K562 cells showed higher expression of CXCR4 when co-cultured with MSC. Furthermore, this increase in CXCR4 expression was enhanced in the presence of As2O3. The expression of CXCR4 peaked 10 min post-treatment with As2O3 when K562 cells were co-cultured with MSC. Second, the production of CXCL12 by MSC was analyzed in MSC supernatants by western blot analysis (Fig. 6B). To determine whether the CXCL12/CXCR4 interaction had functional activity, we investigated the chemotactic properties of MSC on K562 cells in a transmigration assay. The K562 cells migrated toward a CXCL12 gradient, demonstrating that CXCR4 was fully functional. As shown in Fig. 6C, MSC supernatant exhibited potent chemoattractive activity on K562 cells, as 24±3.2% of K562 cells migrated toward undiluted supernatant compared to 0.95±0.06% of cells that migrated in the presence of media (P=0.013). Blocking CXCR4 led to a significant reduction in chemoattractive activity in response to MSC, as well as the chemokine CXCL12 (P=0.014; Fig. 6C). All of these data indicate the presence of a functional CXCR4/CXCL12 signaling between CML cells and MSC and that this signaling is important for promoting migration.

To assess whether CXCR4/CXCL12 could modulate the expression of RUNX3, the expression of RUNX3 was analyzed in the presence of CXCL12 by western blot analysis. As shown in Fig. 6D, CXCL12 induced a significant reduction of RUNX3 mRNA expression in K562 cells. Remarkably, treatment of K562 cells with the CXCR4 antagonist AMD3100 induced RUNX3 expression (Fig. 6E). Moreover, the expression of RUNX3 was upregulated after treatment with AMD3100 in the presence of MSC, indicating that the inhibition of CXCR4/CXCL12 signaling was rescuing the expression of RUNX3.

Expression of RUNX3 overcomes MSC-mediated As2O3 resistance

Mesenchymal stromal cells have been found to protect leukemia cells from As2O3-induced apoptosis. We tested the hypothesis that the overexpression of RUNX3 could restore the effect of As2O3 on K562 cells. K562 cells were transfected with a RUNX3 expression vector or an empty control vector. The expression of RUNX3 in these cells was confirmed by western blot analysis (Fig. 6F). When K562 cells transfected with RUNX3 were co-cultured with MSC, the Annexin V and PI-positive cells treated with increasing concentrations of As2O3 was significantly increased compared to the cultures in which leukemic cells transfected with empty vector were exposed to As2O3 in the presence of MSC. Similar results were observed from APL primary blast patients (Fig. 6H). In addition, we found that p21 protein expression was significantly increased after exposure to As2O3 in cells transfected with the RUNX3 expression vector in the presence of MSC, but not for cells transfected with control vector (Fig. 6G). The influence of overexpression of RUNX3 on the expression of anti-apoptotic proteins in the presence of the MSC was determined by immunoblotting. Mcl-1 protein upregulation was observed in K562 cells grown with stromal cells. However, cells transfected with RUNX3 downregulated stroma-induced Mcl-1 levels in K562 cells (Fig. 6G).

Discussion

In this study, we examined the involvement of RUNX3 in As2O3-induced cellular apoptosis using the APL-derived, NB4 cell line and the CML-derived, K562 cell line. As2O3-induced expression of RUNX3 and nuclear localization of RUNX3 were both observed in NB4 and K562 cells. The transcriptional activation of RUNX3 following treatment with As2O3 was confirmed by the upregulation of p21, a target gene for RUNX3. It is now known that the generation of ROS is one of the major mechanisms through which As2O3 affects neoplastic cells. Treatment with NAC, a scavenger of ROS, resulted in the inhibition of As2O3-induced expression of RUNX3. Furthermore, knockdown of endogenous RUNX3 by shRNA blocked As2O3-induced apoptosis and cell cycle arrest in the K562 cell lines. Our data strongly suggest that RUNX3 may be a key molecule in a series of cellular responses induced by As2O3 treatment during leukemia therapy.

In this study, we provide evidence that a cell-contact mediated interaction between MSC and leukemia cells effectively protects leukemia cells from As2O3-induced cell death. In agreement with earlier results, we found that the leukemia-stroma interaction and the consequences that this interaction has on tumor survival following exposure to As2O3, involve CXCR4/CXCL12 signaling. However, the precise mechanism by which CXCR4/CXCL12 signaling guard leukemia cells from As2O3-induced apoptosis was unclear. In this study, we identified RUNX3 as a key transcription factor involved in the protection of leukemia cells through an interaction with MSC in As2O3-treated cell lines. We show at the protein level that the presence of CXCL12 or MSC the upregulation of RUNX3 that occurs during treatment of cells with As2O3. A CXCR4 antagonist, AMD3100, which blocks CXCL12/CXCR4 signaling, could efficiently rescue the expression of RUNX3. Regulation of RUNX3 expression appears to be the effect of CXCL12/CXCR4 signaling in K562 cells. This finding suggests that downregulation of RUNX3 expression is involved in the development of drug-resistance signals derived from CML-MSC interactions. Taken together with our observation that active RUNX3 restores sensitivity of chronic myeloid leukemia cells to As2O3, MSC-induced drug-resistance in leukemia cells may be overcome by forced activation of RUNX3 and subsequent inhibition of Mcl-1.

Mcl-1 is an early response gene that functions as a regulator of cell viability (38). Mcl-1 can undergo rapid upregulation, as well as downregulation (39), which allows Mcl-1 to provide an acute protective function from apoptosis induced by various factors, including DNA damage (40), growth factor withdrawal (41,42) and treatment with cytotoxic agents (43). Western blot analysis suggested that Mcl-1 expression decreased in K562/RUNX3 and increased in K562/siRUNX3 compared with respective control cells. However, this result did not necessarily mean that RUNX3 was a transcriptional repressor of Mcl-1. Further study is needed to clarify the regulatory effects of RUNX3 on Mcl-1 expression. Mcl-1 protein upregulation was observed in K562 cells grown in the presence of stromal cells. The overexpression of RUNX3 downregulated endogenous as well as stroma-induced Mcl-1 levels in K562 cells. Those results suggest the induction of RUNX3 is associated with the expression of RUNX3 and bone marrow stromal cells may support K562 cells via induction of Mcl-1.

In summary, we report that RUNX3 plays a critical role in As2O3-induced apoptosis and CXCR4/CXCL12 signaling involved in the leukemia-stroma interaction. CXCR4/CXCL12 signaling downregulates the expression of the transcription factor RUNX3. Reduced levels of RUNX3 in turn compromise the apoptosis induced by As2O3. The overexpression of RUNX3 can effectively antagonize survival and drug-resistance signals derived from CML-MSC interactions.

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References