Knockdown of MRP4 by lentivirus-mediated siRNA improves sensitivity to adriamycin in adriamycin-resistant acute myeloid leukemia cells

Chemotherapy remains the standard treatment for acute myeloid leukemia; however, the emergence of drug resistance is a major hurdle in the successful treatment of leukemia. The expression of multidrug resistance-associated protein 4 (MRP4) induces resistance in the adriamycin-resistant acute myeloid leukemia cell line, K562/ADR. The aim of this study was to investigate whether knockdown of MRP4 by lentivirus-mediated siRNA could improve the sensitivity of K562/ADR cells to adriamycin. Five lentivirus-mediated short hairpin RNAs (lv-shRNAs-MRP4) were designed to trigger the gene silencing RNA interference (RNAi) pathway. The efficiency of lentivirus-mediated siRNA infection into K562/ADR cells was determined using fluorescence microscopy to observe lentivirus-mediated GFP expression. MRP4 expression in infected K562/ADR cells was evaluated by real-time PCR and Western blot analysis. The MTS assay was used to measure cell viability and flow cytometry was used to measure apoptosis. The transfection efficiency of K562/ADR cells was over 80 percent. The gene silencing efficacy of lv-shRNA1-MRP4 was superior to the other constructs. Infection of K562/ADR cells with lv-shRNA1-MRP4 led to strong inhibition of MRP4 mRNA and protein expression. Combined treatment with lv-shRNA1-MRP4 and adriamycin decreased cell growth and increased apoptosis compared to treatment with lv-shRNA1-MRP4 or adriamycin alone. These data indicate that in K562/ADR cells MRP4 is involved in drug resistance mechanisms and that lentivirus-mediated knockdown of MRP4 may enhance sensitivity to adriamycin.

Acute myeloid leukemia (AML) is a clonal disorder, characterized by the accumulation of an immature cell population in the bone marrow and/or peripheral blood. Despite improvements over the last 30 years in the combined use of various cytarabine (Ara-C) and anthracycline agents, the overall prognosis for AML remains poor. A major issue in the treatment of AML is multidrug resistance (MDR) [1]. Therefore, reversal of multi-drug resistance in leukemia cells is important to effectively treat AML patients [2,3].
Multidrug resistance-associated protein (MRP) is an ATP-binding cassette (ABC) transporter protein that is responsible for the pump resistance in various cancer cells. Over-expression of ABC transporters is commonly seen in MDR. MRP4 is a member of the C subfamily of ABC transporters. The first functional properties of MRP4 were described in a human T-lymphoid cell line in 1999. MRP4 is noteworthy for a particularly broad substrate specificity, including antiviral, antibiotic, cardiovascular and cytotoxic agents and it is a new therapeutic target for the modulation of various pathophysiological signaling processes [4,5]. Studies have shown that high levels of MRP4 expression, but not of any other MRP gene, were significantly associated with poor outcome in neuroblastoma [6]. MRP4 was also identified as a marker for immature stem cells in acute leukemia patients [7]. Moreover, the expression level of MRP4 in 6-mercaptopurine resistant CEM-MP5 leukemia cells was approximately 4-fold higher compared with that in parental CEM cells [8]. However, the importance of MRP4 in adriamycin-resistant acute myeloid leukemia cell lines is unknown. Our previous experiments have shown that MRP4 is over-expressed in K562/ADR cells relative to parental K562 cells ( Figure 1). Thus, we determined whether knockdown of MRP4 using RNAi could reverse resistance and sensitize K562/ADR cells to adriamycin. As with any cancer therapy, effective delivery of the therapeutic agent to the proper target is a rate limiting factor and this holds true for the RNA interference (RNAi) pathway [9]. RNAi strategies that have been employed in cancer gene silencing include delivery of double stranded RNA complexes (siRNA), the use of double stranded DNA vectors encoding short hairpin-containing RNA sequences (shRNA), or the use of viral vectors containing doublestranded DNA encoding shRNA sequences. Viral vectors are particularly attractive because they can induce long-term knockdown of gene transcripts in vivo. Thus, the use of RNAi in a stable viral vector system, such as the lentivirus, is a possible strategy for stable gene knockdown [10][11][12]. Studies have demonstrated that RNAi is a valid approach to counter cellular drug resistance mediated by ABC transporters [2] and that RNAi has therapeutic potential in leukemia cell lines [13].
Our results suggest that MRP4 is a candidate molecule for mediating adriamycin resistance in acute myeloid leukemia cells. In addition, lentiviral-mediated siRNA targeting MRP4 provides an excellent approach for delivering siRNA into K562/ADR cells and this approach may be used for gene therapy of AML to produce an anti-proliferative effect and apoptosis when combined with adriamycin.

Drugs and cell culture
Adriamycin (Sigma, St.Louis, USA) was dissolved in distilled water and stored at -30°C until use. Adriamycinselected human erythroid blast crisis chronic myeloid leukemia (K562/ADR) cells were obtained from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences. Parental K562 cells were provided by the Central Laboratory of the First Affiliated Hospital of Lanzhou University (Lanzhou, China). Cells were routinely maintained in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS), 2 mmol/L L-glutamine, and 1% antibiotics (100 IU/mL penicillin, 100 µg/mL streptomycin) under a humidified atmosphere of 5% CO 2 and air at 37°C. K562/ADR cells were incubated in adriamycin-free medium for over 1 week before experimentation. The IC 50 value of K562/ADR was (60.33 ± 10.68) µg/mL.

Construction of lentivirus-mediated short hairpin RNA (lv-shRNA)
The pGCL-GFP lentiviral vector containing a CMV driven GFP reporter and a U6 promoter upstream of cloning restriction sites (Hpa I and Xho I), which allow the introduction of oligonucleotides encoding shRNAs, has been previously described [14]. Sense and antisense oligonucleotides were designed for 5 targeting sequences ( Table 1). The oligonucleotides were annealed and ligated into the Hpa I and Xho I sites of pGCL-GFP. Each shRNA consists of a T, a 21-nt sense sequence, a short spacer (TTCAAGAGA), the antisense sequence, 6 Ts (a stop signal for RNA polymerase III), and an Xho I site [15]. pGCL-GFP-shRNA-MRP4 constructs were then propagated and purified using a QIAfilter Plasmid Maxi Kit (Qiagen, Germany). Correct insertions of shRNA cassettes were confirmed by restriction mapping and direct DNA sequencing. The new engineered vectors were called lv-shRNA-MRP4; the negative control was named lv-shRNA-NC. The ability of the five lv-shRNA-MRP4 vectors to knock down MRP4 was investigated using

Fluorescence microscopy
To determine the infection efficiency of K562/ADR cells with lv-shRNA-MRP4 and lv-shRNA-NC, cells expressing GFP protein were observed using fluorescence microscopy (Olympus, Tokyo, Japan) 5 d after transfection and images were captured. Successfully infected K562/ADR cells were collected for subsequent experiments.

MTS assay
Cell viability experiments were performed using the MTS

Flow cytometric apoptosis assay
The percentage of apoptotic cells was determined on a FACSCalibur flow cytometer (BD, New Iersey, USA). Annexin V-FITC (fluorescein isothiocyanate)/PI (propidium iodide) analysis was performed according to the manufacturer's protocol (Annexin VAPC Apoptosis Detection Kit, eBioscience, USA). Approximately 1×10 5 cells/well were plated in triplicate in 6-well tissue culture plates. The same experimental groups used in the MTS assay were used in the flow cytometry assay. Cells were washed with PBS and resuspended in 100 µL Annexin V/PI incubation buffer and incubated for 15 min at room temperature in the dark. Binding buffer (400 µL) was then added to each sample and flow cytometry was performed.

Statistical analysis
All experiments were performed at least 3 times and statistical analysis was performed using the SPSS13.0 package (SPSS, Chicago, USA). Data are expressed as the mean ± SD. An ANOVA test was used for the comparison of more than 2 groups. P values of less than 0.05 (P < 0.05) were considered to be statistically significant.

Lentivirus-mediated high-efficiency infection of K562/ADR cells for knockdown of MRP4
The infection efficiency of K562/ADR cells was over 80 percent ( Figure 2). Infected K562/ADR cells were analyzed by real-time PCR and Western blot analysis. Cell viability and apoptosis were determined by the MTS assay and flow cytometry, respectively.

Decrease of MRP4 mRNA levels in human colon cancer RKO cells
We tested the efficiency and specificity of the 5 lv-shRNA-MRP4 vectors in RKO cells. Previous studies have shown that RKO cells express a high level of MRP4 (data not shown). Subconfluent RKO cells were infected with either lv-shRNA-NC or with each of the lv-shRNA-MRP4 vectors. Five days post-infection, infected RKO cells and control cells were collected and MRP4 expression was determined by real-time PCR. As shown in Figure 3, infection of all 5 lv-shRNAs-MRP4 vectors resulted in a considerable decrease in the levels of MRP4 mRNA compared with that of control and lv-shRNA-NC cells (P<0.05), while the expression was similar between the control and lv-shRNA-NC cells (P>0.05). The gene silencing induced by lv-shRNA1-MRP4 was greater (>80%) than that of the other lv-shRNA-MRP4 vectors. Therefore, lv-shRNA1-MRP4 was used for the subsequent experiments in K562/ADR cells.

Inhibition of MRP4 mRNA and protein expression in K562/ADR cells
To determine the effect of lv-shRNA1-MRP4 on the expression of MRP4 in K562/ADR cells, mRNA and protein levels were analyzed. K562/ADR cells were infected with either lv-shRNA-NC or lv-shRNA1-MRP4. Five days postinfection, cells were collected and MRP4 mRNA levels were detected by real-time PCR. Seven and 8 d postinfection, cells were collected and protein levels were detected by Western blot analysis. Lv-shRNA1-MRP4infected cells displayed a remarkable reduction in MRP4 mRNA levels (Figure 4(a)). The level of MRP4 mRNA in lv-shRNA1-MRP4 cells (0.115±0.005) was lower than that of lv-shRNA-NC cells (1.001±0.051) and control cells

Inhibition of MRP4 increases cell death in K562/ ADR cells
MTS cell viability assays were performed to determine whether knockdown of MRP4 increased K562/ADR cell death. Lv-shRNA1-MRP4, lv-shRNA-NC and control cells were assayed 24 and 48 h after an IC 50 dose of adriamycin ( Figure 5). The combination of lv-shRNA1-MRP4 and adriamycin had a significantly greater repressive effect on cell viability than each of the other treatments (P < 0.05). That is, 48 h after infection, the survival of lv-shRNA1-MRP4, lv-shRNA-NC and control cells exposed to adriamycin was 47.7%, 76.3% and 79.1% (normalized to control cells), respectively. Lv-shRNA1-MRP4 or lv-shRNA-NC alone did not significantly inhibit cell proliferation compared to control cells (P > 0.05).

Inhibition of MRP4 sensitizes K562/ADR cells to adriamycin-induced apoptosis
To explore whether loss of MRP4 resulted in increased adriamycin-induced apoptosis, K562/ADR cells were infected with lv-shRNA-NC or lv-shRNA1-MRP4. All cells (including control cells) were harvested for the assay 24 h after exposure to an IC 50 dose of adriamycin, as described in Materials and methods. Annexin-V and PI staining were used to detect apoptotic cells, including cells in early-stage apoptosis (AV + /PI -) and late-stage apoptosis (AV + /PI + ). As shown in Figure 6, the combination of lv-shRNA1-MRP4 infection and adriamycin exposure resulted in a significant increase in apoptosis as compared to each treatment alone (P<0.05). A significant increase in the number of apoptotic cells was observed in control, lv-shRNA-NC and lv-shRNA1-MRP4 cells treated with adriamycin (11.37%, 10.74% and 17.9%, respectively) as compared to control cells (8.03%), lv-shRNA-NC (7.94%) and lv-shRNA1-MRP4 (8.23%) cells without adriamycin treatment (P<0.05), while apoptosis was similar in control, lv-shRNA-NC and lv-shRNA1-MRP4 cells (P>0.05).

Discussion
Leukemia is one of the most common diseases in hematology. It shows frequent recurrence and a poor outcome despite combined chemotherapy because of the multi-drug resistance of leukemia cells [20][21][22]. Improving sensitivity of leukemia cells to chemotherapy is crucial for curing leukemia.
Adriamycin is a standard anthracycline anticancer agent for treating AML patients and is classified as a topoisomerase II inhibitor that induces cancer cells to undergo apoptosis by causing double-strand DNA breaks. Therefore, resistance to adriamycin in leukemia cells is a major obstacle to achieving treatment success. Adriamycin resistance may often result from over-expression of P-glycoprotein (P-gp), which is encoded by the multidrug resistance 1 gene (MDR-1). However, increased expression of MDR-1 does not always contribute to clinical resistance, at least in leukemia patients. The multifaceted nature of cellular resistance led to the discovery of the MRP genes. The MRP family is composed of 9-related ABC transporters that are able to transport structurally diverse lipophilic anions and that function as drug efflux pumps. In cultured cell lines, expression of various MRPs confers resistance to natural product anticancer drugs [23]. Studies have shown that MRPs may also contribute to anthracycline resistance in AML [3]. Furthermore, as yet unidentified MRP homologues that act as transporter proteins may play a role in MDR in AML. The presence of these nine transporters, and the finding that the lack of one transporter can be compensated for by the other transporters, may account for the somewhat disappointing results in some clinical studies using only P-gp inhibitors. MRP4 exports organic anions, including endogenous and exogenous substances, and is involved in MDR [4]. Several studies have shown that MRP4 is over-expressed in neuroblastoma and in the CEM-MP5 leukemia cell line and MRP4 is also a marker for immature stem cells in leukemia patients. In addition, increased levels of MRP5 mRNA were observed in SBC-3/ ADR, MCF7/ADR and K562/ADR cell lines as compared with the respective parental cells, and MRP5 is a candidate gene for adriamycin resistance in addition to MDR1 [24]. Amino acid sequence analysis has revealed that the nine transporters are composed of two nucleotide binding domains (NBDs) and at least two membrane spanning domains. MRP4, similar to MRP5, contains only two membrane spanning domains and is the most recently characterized transporter [25]. These data indicate that MRP4 may also be involved in resistance to adriamycin chemotherapy. Thus, we used K562/ADR cells as a model of drug-resistant leukemia because of the elevated expression of MRP4 as compared to the parental K562 cells. This observation suggests that MRP4 may be responsible for the adriamycin resistance in adriamycin-resistant acute myeloid leukemia cells.
SiRNA has become a powerful tool in molecular biology research [26]. Double stranded RNA (dsRNA) is processed into 21 to 28 nucleotide siRNAs, which are then incorporated into a complex that binds homologous target RNAs. The target RNAs then ultimately undergo cleavage leading to gene silencing. The sequence specific manner with which siRNAs bind mRNA has opened the door to the possibility of using this technique in a model of cancer therapeutics [27][28][29]. While direct use of siRNA is simple and results in functional gene silencing, its effect is transient. DNA-based RNAi has the potential of being stably introduced when used in a gene therapy context, allowing in principle, a single treatment of viral vector-delivered shRNA genes. Lentivirus vector-mediated gene transfer has some advantages for gene therapy: high efficiency of gene transfer into a wide variety of cells, including both dividing and nondividing cells, long-term infection due to gene integration into the chromosome of host cells, no toxicity and no immune response [30][31][32][33][34][35]. Moreover, the RNAi pathway is functional in human myeloid leukemia cell lines, providing a means to overcome the resistance to chemotherapeutic agents and ultimately to augment the efficacy of chemotherapy in myeloid leukemia [13]. Therefore, we used lentivirus-mediated siRNA to study the functions of MRP4 in K562/ADR cells.
In the present study, we initially constructed 5 recombinant lv-shRNA-MRP4 plasmids. The lv-shRNA1-MRP4 plasmid was selected for subsequent experiments over the other four constructs because of its superior gene silencing efficacy; it could efficiently inhibit MRP4 mRNA and protein expression in K562/ADR cells. Then, we determined cell viability using the MTS assay. The viability of K562/ ADR cells was significantly reduced after combined lv-shRNA1-MRP4 infection and adriamycin treatment. To explore the mechanism of this reduced cell viability, flow cytometric analysis of apoptosis was performed. Knockdown of MRP4 by lv-shRNA1-MRP4 sensitized K562/ ADR cells to adriamycin-induced apoptosis. Notably, a similar level of apoptosis was observed between the control, lv-shRNA-NC and lv-shRNA1-MRP4 cells, suggesting that silencing MRP4 alone did not induce apoptosis or reduce K562/ADR cell viability. These data indicate that reduced cell viability was due to apoptosis induced by adriamycin and that the combination of adriamycin treatment with MRP4 depletion by lv-shRNA1-MRP4 may be a potential approach to AML therapy. MRP4 is a membrane-embedded transporter molecule, which can act as an energy-dependent xenobiotic efflux pump [36], the transport activity of which results in decreased intracellular concentrations of anticancer drugs, including adriamycin. Thus, the specific inhibition of MRP4 by lentivirus-mediated siRNA results in the re-sensitization of K562/ADR cells to treatment with adriamycin.
It is known that glutathione (GSH) is involved in a number of biochemical processes and its levels in various cellular compartments are tightly controlled. The two primary mechanisms by which cells regulate intracellular GSH levels are by altering the rate of its biosynthesis and the rate of GSH export from cells. There is growing evidence that some of the MRPs are involved in GSH export and are major contributors to this process in cell models. Previous studies have demonstrated that drug-resistant cancer cells exhibit an elevated level of GSH compared to the drugsensitive parental cells [37] and increased levels of a cisplatin-GSH conjugate have been detected in various cisplatin-resistant cell lines [38]. These data indicate that GSH may be associated with MDR in drug-resistant cancer cells. Supporting evidence has shown that GSH-conjugates are one of the main substrates for MRP4 [1]. It is not known whether the levels of cellular GSH are high or whether GSH is required for the transport of adriamycin in K562/ADR cells. In addition to its role as an efflux pump, MRP4 may contribute to adriamycin resistance in K562/ADR cells in other ways. Thus, further experiments are required to explore the function of MRP4 in these cells.
One potential pitfall of the present study was only focusing on the K562/ADR cell line, which is commonly used as an adriamycin-resistant leukemia model. Further observations with other cell lines would strengthen the significance of our study. Another potential problem is that endogenously expressed shRNA may induce a toxic effect [39]. Therefore, future studies will need to focus on the development of in vivo models.
In conclusion, we show for the first time that MRP4, in addition to MDR1, is likely to be involved in adriamycin resistance in K562/ADR cells. We successfully knocked down MRP4 in K562/ADR cells using lentivirus-mediated siRNA, which resulted in increased adriamycin-induced apoptosis and decreased cell viability, raising the possibility that an appropriate combination of adriamycin application with MRP4 depletion may be a promising approach for the treatment of AML.