Abstract
Background/Aim: Altered redox status has been reported to play a significant role in the development of multidrug resistance (MDR) in leukemia cells. Human biliverdin reductase (hBVR) has been recently identified as a major cytoprotectant; however, its role in MDR has not been yet investigated. In the present study we evaluated the possible role of hBVR in MDR development in the human HL60 leukemia cell line. Materials and Methods: The relationship between hBVR and MDR was examined using the drug-sensitive HL60 cells and the drug-resistant HL60 subline, HL60/ADR. A possible chemosensitization by siRNA pre-treatment was also investigated. Results: We observed that hBVR expression, protein, and enzymatic activity were significantly increased in multidrug-resistant HL60 leukemia cells. We also found that knockdown of hBVR reverses multidrug resistance in resistant leukemic HL60 cells by a ROS-dependent mechanism. Conclusion: hBVR plays a pivotal role in the development of multidrug resistance in human HL60 leukemia cells.
Cancer treatment-related multidrug resistance (MDR) occurs when tumor cells develop the ability to protect themselves against multiple classes of chemotherapeutic drugs, which maybe structurally and mechanistically unrelated (1). MDR is common in leukemia, particularly during relapse, whereby dose intensification and combination therapy often result in significant comorbid conditions and organ failure. For these reason, MDR has been extensively studied in the specific type of cancer. One of the most investigated mechanisms underlying this MDR phenotype is the active cellular extrusion of anticancer agents by drug efflux pumps, such as p-glycoprotein (P-gp) and the multidrug resistance-associated protein (MRP) (1). Inhibiting pump proteins as a method to reverse MDR in cancer patients has been widely studied, but the results have generally been disappointing as patients in phase II trials often show continued drug resistance or relapse (1, 2). Several additional mechanisms have also been implicated in the development of MDR. For example, alterations in the apoptotic signaling pathways and activation of cytoprotective and antioxidant enzymes are also considered major contributors (1, 3).
Two antioxidants of particular interest are biliverdin and bilirubin, the end-product of heme metabolism. The reduction of biliverdin to bilirubin is catalyzed by the ubiquitously expressed enzyme human biliverdin reductase (hBVR). Previously, it was demonstrated that hBVR is a multifunctional enzyme involved in various cellular processes, including regulation of gene expression as a dual-specificity kinase, nuclear targeting, and inflammatory response (4). More recently, hBVR has been shown to be a major cytoprotectant, responsible for the maintenance of intracellular redox homeostasis (5, 6). It was also recently reported that exogenous BVR can enhance drug resistance in normal mouse fibroblasts (7).
Although many researchers have focused on the antioxidant defense system in MDR, the role of biliverdin reductase has not been previously investigated. In the presentstudy, we evaluate the possible role of hBVR in the development of MDR in the HL60 human leukemia cell line.
Materials and Methods
Cell culture and reagents. Human HL60 cells (American Type Culture Collection,Manassas, VA, USA) were grown in RPMI 1640 medium (Gibco, NY, USA) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/ml streptomycin at 37°C in 5% CO2. Doxorubicin-resistant cells (HL60/ADR) were developed by stepwise exposure of HL60 to increasing concentrations of the drug, as described previously (8). Doxorubicin and vincristine were obtained commercially from Sigma-Aldrich (St. Louis, MO, USA).
Cell viability assay. Cell viability was measured using the XTT (sodium 3’-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4 methoxy- 6-nitro) benzene sulfonic acid hydrate) cell proliferation assay (Roche, Inc., NJ, USA). The cell viability is based on the ability of the mitochondrial succinate-terazoliumreductase system to convert yellow tetrazolium salt XTT to orange formazan dye. Half-maximal concentration (IC50) values were calculated by log expression using the software of Microsoft Excel. Relative reversal rate=(IC50A – IC50B)/(IC50A − IC50C), where IC50A was IC50 value of resistant cells before RNAi, IC50B was IC50 value of resistant cells after RNAi, IC50C was IC50 value of sensitive HL60 cells (9).
qRT-PCR. Quantitative RT-PCR was performed using real-time PCR with the SYBR Green reporter. Total RNA was extracted from HL60 and HL60/ADR cells using TRIzol reagent (Invitrogen, Carlsbad, USA) according to the manufacturer's protocol. Quantitative RT-PCR was performed using SYBR Green PCR master kit (Invitrogen, Carlsbad, USA). The primers used for this experiment were purchased from BIONEER.
Immunoblot analysis. Cells were solubilized with a lysis buffer containing 50 mM Tris, 1 mM EDTA, 150 mM NaCl, 1% SDS, protease inhibitor cocktail (Roche, Indianapolis, USA), 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM NaF, and 1 mM sodium orthovanadate. Proteins were resolved by SDS-PAGE, transferred onto nitrocellulose membranes, and blocked with TBST in 2.5% skim milk. The membranes were incubated with hBVR antibody (Abcam, Cambridge, USA) at 4°C overnight. Secondary antibodies were added for 40 min at room temperature. The antibody–antigen complexes were detected using the ECL detection system (Pierce, Rockford, USA).
In vitro measurement of biliverdin reductase activity. Biliverdin reductase activity was measured in HL60 and HL60/ADR cells plated in 60-mm plastic dishes and pooled samples of cells using a colorimetric reaction to measure the formation of bilirubin, as described by the manufacturer's protocol (Sigma).
Analysis of apoptosis by annexin V staining. The amount of phosphatidylserine (PS) on cell surfaces was accessed using an Annexin V-FITC apoptosis detection kit (Abcam, Cambridge, USA), according to the manufacturer's protocol.
RNA interference and transfection. The specific siRNA for hBVR and scrambled control siRNA were purchased from BIONEER Co. RNA interference of the hBVR was performed as described (8). Briefly, HL60 and HL60/ADR cells were plated in a 100-mm dish, transfected with 50 nmole of siRNA and Oligofectamine™ reagent in serum-free medium and incubated for 4 h at 37°C in a CO2 incubator. Following incubation, HL60 and HL60/ADR cells were supplied with growth medium containing 10% fetal bovine seerum.
Measurement of intracellular ROS levels. The intracellular levels of ROS were accessed using dichlorodihydrofluoresceindiacetate (DCF-DA) (Sigma). HL60 and HL60/ADR cells were stained with 50 μM of DCF-DA for 30 min and then collected. The fluorescent intensities were measured using a cytometer (Becton Dickinson FACSorter). To access the effect of N-acetylcystein (NAC) (Sigma,), cell were treated with 20 mM of NAC for 24 h.
IC50 values of DOX and VCR in HL60/ADR and HL60 cells.
Statistical analysis. Significance was calculated using one-way analysis of variance (ANOVA), followed by a Dunnett's post-hoc test. For all data sets comparing the mean of only two groups, an unpaired Student's t-test was employed.
Results
hBVR levels are increased in multidrug-resistant HL60 leukemia cells. To determine whether hBVR levels changed during MDR development, we initially examined the hBVR expression levels in HL60 and HL60/ADR leukemia cell lines. The HL60/ADR cell line is approximately 83-fold more resistant to doxorubicin (DOX) compared to the parental HL60 cell line and shows 9.2-fold greater cross-resistance to vincristine (VCR) (Table I). Western blot analysis revealed that the hBVR protein is significantly overexpressed in drug-resistant HL60/ADR cells compared to their parental cell line (Figure 1A). Real-time PCR analysis also showed that there is a significant increase in hBVR mRNA levels in HL60/ADR cells, indicating that transcriptional regulation plays an important role in determining the hBVR expression in HL60/ADR cells (Figure 1B). Consistent with the increased mRNA and protein expression levels, hBVR enzymatic activity was also up-regulated in HL60/ADR cells (Figure 1C). When we examined the DOX effect on hBVR activity in normal HL60 cells, we observed a dose-dependent increase in hBVR enzyme activity in response to DOX treatment. However, the same dosage of DOX did not significantly alter hBVR activity in the HL60/ADR cells (Figure 1C).
Knockdown of hBVR sensitizes drug-resistant HL60 leukemia cells to chemotherapeutics. To evaluate whether hBVR depletion can affect the sensitization of HL60/ADR cells to anticancer agents, hBVR gene silencing was utilized. hBVR-knockdown efficiency was evaluated using western blot analysis, which showed that the levels of hBVR protein decreased dramatically 48 h after transfection of hBVR siRNA (Figure 2). Using an XTT assay, we found that hBVR gene silencing significantly decreased HL60/ADR cell viability, but does not noticeably affect cell viability in drug-sensitive HL60 cells (Figure 3A, B, C). The IC50 concentration of DOX in cells transfected with scrambled control siRNA was 1,497 nM, whereas that in hBVR-knockdown cells was 506 nM (Table II, Figure 3C). This difference in DOX IC50 concentration shows that the inhibition of hBVR sensitizes HL60/ADR cells to being highly susceptible to DOX. We obtained similar results when HL60/ADR cells were treated with VCR (Table II). The relative reversal rates for HL60/ADR cells treated with DOX and VCR were 67.6% and 49.1%, respectively (Table II). To further investigate the apoptotic effects of DOX on hBVR inhibition, Annexin-V-positive cells were measured by flow cytometry. As shown in Figure 3E, knockdown of hBVR in HL60/ADR cells markedly increased the number of Annexin-V positive cells, whereas there was no significant difference among control siRNA transfected cells and the HL60 hBVR knockdown cells (Figure 3D).
hBVR expression is up-regulated in multidrug-resistant HL60 leukemia cells. A: Cell lysates from HL60 and HL60/ADR were subjected to immunoblot analysis using an anti-hBVR antibody. The left panel is a representative blot from 3 independent experiments. Protein levels were quantified by densitometry analysis (right panel). B: Total RNA was extracted from HL60 and HL60/ADR cells, and analyzed by real-time PCR for hBVR mRNA expression. C: After treatment with DOX for the indicated concentrations, the cell extracts were prepared and the reductase activity was measured at pH 8.7. Data are means±SEM from three independent experiments. Statistical significance was determined by one-way ANOVA followed by a Dunnett's post-hoc test or unpaired Student's t-test; *p<0.05, **p<0.01, ***p<0.001, significantly different between HL60 cells and HL60/ADR cells; #p<0.05, significantly different from control cells (NT). NT, Non-treated controls.
Knockdown of hBVR induces ROS-dependent apoptosis in DOX-resistant HL60 leukemia cells. Since hBVR has been reported to possess antioxidant and radical scavenging activities, we investigated whether depletion of hBVR could affect the cellular redox status of MDR cells. Using dichlorofluoresceindiacetate (DCF-DA), a cell-permeable fluorescent dye, we examined changes in intracellular ROS levels in hBVR-depleted HL60 and HL60/ADRcells after DOX treatment. As shown in Figure 4B, knockdown of hBVR in HL60/ADR cells showed a significant increase in ROS generation compared to control scrambled siRNA-transfected cells. However, in sensitive HL60 cells, there was no significant change in ROS status with hBVR depletion (Figure 4A). Increased ROS levels in hBVR-depleted HL60/ADR prompted us to investigate whether N-acetyl-cysteine (NAC), a well-known ROS scavenger, could block DOX-induced apoptosis in these cells. In fact, pre-treatment with NAC significantly reduced DOX-induced apoptotic cell death in hBVR-silenced HL60/ADR cells (Figure 4C), indicating that ROS production is likely the main mediator of apoptosis observed in these cells.
Efficacy of hBVR-specific siRNAs in human HL60 cells. HL60 cells were transfected with 3 different hBVR-specific siRNAs and a scrambled control siRNA (Ctrl) using Oligofectamine. Two days after transfection, relative hBVR protein levels were calculated by western blotting. Relative protein levels were quantified using densitometry analysis (upper panel, β-actin as a loading control). The lower panel is a representative blot from 3 independent experiments. NT, Non-treated controls.
Discussion
The development of MDR is a major obstacle to successful chemotherapy treatment for many types of cancers (1). The mechanism of MDR is believed to differ depending on the type of cancer and the type of anticancer drugs used for treatment. A number of anticancer agents induce oxidative stress by generating ROS and recent studies suggest that this drug-induced ROS formation might be an alternative mechanism for their cytotoxic effects by inducing apoptosis in tumor cells (11-13). However, prolonged treatment with the same drug induces antioxidant enzyme-gene expression, thus increasing the ROS scavenging capacity of cancer cells, leading to drug-resistance (13, 14).
IC50 (nM) values of DOX and VCR on siRNA-transfected HL60 cells.
hBVR is an evolutionarily-conserved, ubiquitously-expressed enzyme that has recently been identified as a potent enzymatic antioxidant responsible for the maintenance of intracellular redox homeostasis through the conversion of biliverdin to bilirubin during heme metabolism (5). Furthermore, it has been reported that as little as 10 nM of bilirubin protected against 10,000-fold higher concentrations of hydrogen peroxide (6). To explain the powerful antioxidant capacity of bilirubin, the hBVR redox cycle hypothesis has been proposed, which suggests that the oxidation of bilirubin into biliverdin, triggered by high ROS levels is quickly recycled back to its reduced form by hBVR (5). This hBVR redox cycle is expected to be particularly effective against lipophilic ROS because of the lipophilic nature of bilirubin. In contrast, the glutathione redox cycling system, another major antioxidant defense system for the detoxification of ROS, is primarily effective against hydrophilic ROS, because of the hydrophilic nature of glutathione (5, 15). Therefore, the hBVR redox cycle is presumed to be complementary to the glutathione redox cycling system and together will scavenge both hydrophilic and lipophilic ROS. In the present study, we have shown that hBVR activity is increased in MDR cells and hBVR gene silencing by siRNA markedly increased ROS levels compared to control siRNA. These data indicate that overexpression of the hBVR ROS-reducing system has a role in the development of multidrug resistance. We also demonstrated that knockdown of hBVR expression can sensitize drug-resistant HL60 cells to anticancer agents and that this depletion of hBVR is likely associated with increased ROS formation since pre-treatment with NAC abolished these sensitizing effects. Taken together, these data suggest that ROS production is likely to be one of the primary signals inducing the development of hBVR-dependent MDR phenotype.
Aside from its role as a reductase, hBVR is also involved in the transcriptional regulation of a number of stress-activated genes. In the nucleus, hBVR being a leucine zipper-like DNA binding protein, it can function as a transcription factor for activator protein-1 (AP-1)-regulated genes, including heme oxygenase-1 (HO-1) (16). The heme degradation pathway provides two powerful enzymatic antioxidants: hemeoxygenase (HO) and hBVR. HO proteinsare heat-shock protein-32 family members, consisting of HO-1, a highly inducible cytoprotective enzyme, and HO-2, aconstitutive isoform of HO. Like hBVR, HO also plays a crucial role in cellular homeostasis and since hBVR can regulate HO expression, both entities are intimately intertwined with cell survival and cellular defense mechanisms (17). Thus, the signaling and transcriptional activity of hBVR may act synergistically with the cyclic generation of bilirubin, helping to further explain the mechanism of hBVR-dependent drug resistance. Further studies should be performed to evaluate the effects of hBVR on stress-related gene expression during the development of drug resistance and the interaction of hBVR with the various stress-activated signaling pathways should be examined in greater detail.
Knockdown of hBVR reverses multidrug resistance in HL60 cells. A: HL60 and HL60/ADR cells were transfected with an siRNA targeting hBVR or a scrambled control siRNA (Ctrl) and then an XTT assay was performed as described in the Materials and Methods. NT, Non-treated controls. B and C: HL60 and HL60/ADR cells were transfected with an siRNA targeting hBVR or a scrambled control siRNA and treated with or without indicated concentration of DOX for 48 h, and then an XTT assay was performed as described in the Materials and Methods. D and E: After transfection as described above, HL60 and HL60/ADR cells were treated with IC50 concentration of DOX for 48 h. Assay for Annexin-V staining was then performed as described in the Materials and Methods. Statistical analyses were determined independently for the 3 subgroups without siRNA, with scrambled control siRNA and with hBVR siRNA. Data are expressed as the mean±SEM from 3 independent experiments. Significance was assessed by ANOVA followed by a Dunnett's post-hoc test or unpaired Student's t-test; *p<0.05, **p<0.01, significantly different between control siRNA- and hBVR siRNA-treated cells. Ctrl, Control.
hBVR mediates multidrug resistance in a ROS-dependent manner. A and B, HL60 and HL60/ADR cells transfected with an siRNA targeting hBVR, or a scrambled control siRNA (Ctrl) were exposed to IC50 concentration of DOX for an additional 48 h. The cells were stained with DCF-DA, fixed, and immediately accessed by fluorescence-activated cell sorting (FACS). Statistical significance was calculated by one-way ANOVA followed by a Dunnett's post-hoc test; *p<0.05, **p<0.01, significantly different from scrambled control siRNA (Ctrl)-transfected cells. C, HL60/ADR cells transfected with an siRNA against hBVR, or the scrambled control siRNA (Ctrl), were cultured in the presence or absence of 20 mM NAC for 24 h. After incubation, cells were treated with DOX for an additional 48 h, and then Annexin-V staining assay was performed as described in Materials and Methods. Data are means±SEM from three independent experiments. Statistical significance was determined by unpaired Student's t-test; **p<0.01, significantly different from NAC-untreated cells. NT, Non-treated controls; Ctrl, control.
In conclusion, we have shown that hBVR is overexpressed in multidrug-resistant HL60 leukemia cells and controls cell ROS levels, limiting drug toxicity. Our observations indicate that hBVR plays a crucial role in the acquisition of the MDR phenotype and manipulation of hBVR status can serve as a novel therapeutic strategy for leukemia treatment.
- Received September 21, 2013.
- Revision received October 10, 2013.
- Accepted October 15, 2013.
- Copyright© 2013 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
References
In this issue
Jump to section
Related Articles
Cited By...
- No citing articles found.