|Cancer||Cell death||Cell cycle||Cytoskeleton||Exo/endocytosis||Differentiation||Division||Organelles||Signalling||Stem cells||Trafficking|
Effect of nitric oxide on the daunorubicin efflux mechanism in K562 cells
Juliana Costa Curta, Ana Carolina Rabello de Moraes, Marley Aparecida Licínio, Aline Costa and Maria Cláudia Santos‑Silva1
Departamento de Anlises Clnicas, Universidade Federal de Santa Catarina, Campus Trindade, CEP 88040900, Florianpolis, SC, Brazil
NO (nitric oxide) donating drugs have been investigated for their important role in the sensitization of neoplastic cells to chemotherapy drugs. The goal of this work was to investigate the involvement of NO in the resistance of K562 cells to DNR (daunorubicin). Only simultaneous addition of DNR and SNAP (S-nitroso-N-acetyl-dl-penicillamine) caused significant cell death by apoptosis. Combination of the compounds decreased Bcl-2 and survivin, and increased Bax and active-caspase 3 expression. Fluorescence microscope and cytometric analysis showed that DNR and SNAP together caused DNR intracellular accumulation in K562 cells. RT–PCR (reverse transcription–PCR) analysis showed that DNR and SNAP, alone or in association, produced significant decreases in lrp expression. abcc1 gene expression was unaffected by the presence of SNAP, but when treated with DNR there was a small reduction that was intensified by DNR and SNAP in combination. The transport mechanism involved in the resistance to DNR in K562 cells involves ABCC1 and LRP (lung resistance protein) resistance proteins. DNR and SNAP inhibition of the expression of these proteins occurs by distinct mechanisms, and this disrupts the K562 resistance to DNR.
Key words: ABCC1, apoptosis, danorubicin, lung resistance protein (LRP), nitric oxide, multidrug resistance (MDR)
Abbreviations: AML, acute myeloid leukaemia, DAPI, 4′,6-diamidino-2-phenylindole, DNR, daunorubicin, GAPDH, glyceraldehyde-3-phosphate dehydrogenase, HRP, horseradish peroxidase, LRP, lung resistance protein, MDR, multidrug resistance, MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide, NO, oxide nitric, RT–PCR, reverse transcription–PCR, SNAP, S-nitroso-N-acetyl-dl-penicillamine
1To whom correspondence should be addressed (email email@example.com).
The anthracycline DNR (daunorubicin) is a powerful cytotoxic agent widely used in the treatment of AML (acute myeloid leukaemia). DNR molecular mechanisms of action include DNA intercalation, membrane binding and lipid peroxidation (Michelutti et al., 2006). Interaction of such drug and/or their active metabolites with chromatin components leads to the formation of two major types of DNA damage that may contribute to the mechanism of action of DNR: protein-associated DNA breakage, due to the blockage of topoisomerases I and II, and covalent DNA adducts (Laurent and Jaffrezou, 2001; Ciesielska et al., 2005). The formation of DNA–anthracycline complexes can significantly modify the ability of helicases to split DNA into single strands in an ATP-dependent fashion, thereby hindering the process of strand separation and limiting replication (Duan et al., 2007). However, it has become increasingly evident that the therapeutic effects of DNR derive not only from their cytotoxicity, hence from the dose, but also from the MDR (multidrug resistance) phenotype (Merlin et al., 2000; Michelutti et al., 2006). Intracellular DNR level may be reduced by extrusion of the molecules from the cell by efflux-mediating proteins, such as P-gp (P-glycoprotein; ABCB1, MDR1) and MDR-associated protein (ABCC1 or MRP1). The cytotoxicity of DNR can be prevented by mechanisms that redistribute the drug from its target into subcellular compartments. A putative drug resistance protein that may contribute to the intracellular transport and/or sequestration of drugs is major vault protein/LRP (lung resistance protein; Den Boer et al., 1999).
In some anthracycline-selected cell lines, the subcellular distribution is strikingly different between parental and drug-resistant cell lines. Whereas most parental cell lines accumulate anthracyclines predominately in the nucleus, certain drug-resistant cells sequester anthracyclines in cytoplasmic vesicules. As the cytotoxic targets are nuclear, non-nuclear redistribution could reduce sensitivity (Hurwitz et al., 1997; Merlin et<1?show=[ts]?> al., 2000). However, how and why such an event induces cell death remains unclear, and thus the exploration of the process of apoptosis is needed to reassess the mechanisms that allow myeloid leukaemia cells to respond to DNR (Laurent and Jaffrezou, 2001).
NO (nitric oxide) is a diffusible multifunctional transcellular messenger involved in numerous physiologic and pathologic conditions (Choi et al., 2002). Numerous studies have analysed the role of NO in apoptosis, but controversy exists because NO can be pro- or anti-apoptotic (Wink and Mitchell, 1998; Kolb, 2000; Blaise et al., 2005). NO can be cytotoxic to human cell lines from patients diagnosed with leukaemia and lymphoma (Filep et al., 1996), which raises the possibility of using NO donors in the chemotherapy of haematologic disorders (Tsumori et al., 2002). It has also been suggested that reduced synthesis of NO might be implicated in the beginning of MDR phenotype and that the restoration of NO production could reverse this phenotype (Riganti et al., 2005). If NO levels are increased by NO synthase inducers other than doxorubicin or by NO donors, nitration of the ABCB1 and ABCC3 proteins, and the doxorubicin accumulation and toxicity will be increased, reversing the MDR phenotype (De Boo et al., 2009). The synergy between NO and cytotoxic drugs and its effect on cancer cell death may be due to the accumulation of drugs, as seen with cisplatin and arsenic in human lung cancer cells (Jeannin et al., 2008). The present study investigates the involvement of NO in the molecular mechanisms implicated in the resistance to DNR in human K562 AML cells.
2. Materials and methods
Tissue culture media, serum and antibiotics were purchased from Gibco. SNAP (S-nitroso-N-acetyl-dl-penicillamine) was synthesized according to the Field et al. (1978) method. DNR (Meizler Biopharma S/A). MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide], DAPI (4′,6-diamidino-2-phenylindole), ethidium bromide, acridine orange and all other reagents were purchased from Sigma–Aldrich. All solutions were freshly prepared in culture medium. Mouse polyclonal anti-Bcl-2 antibodies were purchased from Invitrogen™, goat polyclonal anti-survivin, rabbit polyclonal anti-β-actin and anti-mouse HRP (horseradish peroxidase)-conjugated antibodies were purchased from Santa Cruz Biotechnology, rabbit polyclonal anti-caspase 3, anti-Bax and anti-β-actin were purchased from Cell Signaling Technology®, anti-goat, anti-mouse and anti-rabbit IgG HRP-conjugated antibodies were purchased from Millipore. Nitrocellulose membranes, reagent for SDS-electrophoresis and chemiluminescence assay systems were purchased from Amersham Biosciences. All RT–PCR (reverse transcription–PCR) reagents were purchased from Invitrogen.
2.2. Cell culture
K562 cells lines were cultured in RPMI 1640 supplemented with 10% fetal calf serum, 100 units/ml penicillin, 100 μg/ml streptomycin and 10 mM Hepes, pH 7.4 at 37°C in a 5% CO2 humidified air atmosphere.
2.3. Treatments and viability assay (MTT assay)
DNR (1–100 μM) and/or SNAP (0.1–1 mM) were added to cells (3.5×106 cells/well) to a maximum volume of 20 μl. Cells were incubated usually for a further 24 h. Cell viability was assessed by the MTT assay (Van de Loosdrecht et al., 1991).
2.4. Immunoblotting analysis
Cells lysates were denatured in sample buffer (50 mM Tris/HCl, pH 6.8, 1% SDS, 5% 2-mercaptoethanol, 10% glycerol and 0.001% Bromophenol Blue) and heated in a boiling water bath for 5 min. Samples (50 μg total protein) were resolved by SDS/12% PAGE and proteins were transferred to nitrocellulose membranes. Rainbow markers were run in parallel to estimate molecular masses. Membranes were blocked with Tween-TBS (20 mM Tris/HCl, pH 7.5, 500 mM NaCl and 0.1% Tween-20) containing 1% BSA and probed with specific primary antibody: rabbit polyclonal anti-caspase 3 (1:1000, v/v), goat polyclonal anti-survivin (1:1000, v/v), mouse polyclonal anti-Bcl-2 (1:500, v/v), rabbit polyclonal anti-Bax (1:1000, v/v) and rabbit polyclonal anti-β-actin (1:1000, v/v). Membranes were next washed three times with Tween-TBS, followed by 3 h incubation with anti-rabbit IgG antibody (1:2000, v/v), anti-goat IgG antibody (1:7000, v/v) or anti-mouse antibody (1:5000, v/v) HRP-conjugated. Immunoreactive proteins were visualized by chemiluminescence using enhanced chemifluorescence substrate [ECL (enhanced chemiluminescence)]. The bands were quantified by densitometry, using Scion Image Software (Scion Co.). Blots were stripped and reprobed for β-actin as a loading control.
2.5. Morphological assessment of apoptosis
Apoptosis was verified as described by Mcgahon et al (1995). K562 cells were incubated with DNR (100 μM) and/or SNAP (1 mM) for 24 h. After exposure, the cells were centrifuged to concentrate the pellet and remove excess free drug. The pellet was resuspended in 20 μl of a mixture of fluorescence dyes, ethidium bromide (5 μg/ml) and acridine orange (10 μg/ml) (v/v). Slides were examined with a fluorescence microscope (Olympus BX40) using an excitation filter of 484 nm and an emission filter of 501 nm, under a ×40 objective. Images were taken with an Olympus BX40 camera.
2.6. Determination of intracellular DNR levels by fluorescence microscopy
Cellular accumulation and disposition of DNR were visualized as described by Forssen et al. (1996). DNR (100 μM) and/or SNAP (1 mM) were added to K562 cell suspensions (1×106 cells/ml) for 12 h. After incubation, the cells were pelleted as described above, and also to remove excess free drug. The cells were resuspended in PBS and incubated with a DAPI solution (50 nM) for 30 min in the dark before being examined by fluorescence microscopy using an excitation filter of 350 nm and an emission filter of 470 nm to visualize nuclei stained with DAPI, and with an excitation filter of 539 nm and an emission filter of 617 nm to visualize DNR fluorescence.
2.7. Determination of intracellular DNR levels by flow cytometry
The K562 cell lines (1×106 cells/ml) were incubated with SNAP (1 mM) and/or DNR (100 μM). Accumulation of DNR was determined at 6, 12 and 24 h. Cells were washed twice and resuspended in PBS. The samples were kept on ice until intracellular DNR fluorescence was measured by flow cytometry (FACSCalibur™; BD Biosciences). DNR was excited by an argon laser at 488 nm and the orange/red fluorescence signal was collected through a 585/42 bandpass filter set (FL-2 height). Data were analysed using WinMID software. Intracellular DNR content was determined by the mean sample fluorescence intensity/control fluorescence intensity, expressed in arbitrary units.
2.8. Semi-quantitative RT–PCR
Total RNA was isolated with TRIzol™. RNA samples were analysed for RNA integrity by gel electrophoresis. RT used 5 μg of total RNA and up to 10 μl of DEPC [diethyl pyrocarbonate (q.v.)]-treated water (0.1%, v/v). The mixture was heated for 5 min at 70°C and quick chilled in ice for 5 min, followed by 5 μl of first strand buffer, 2 μl of DTT (dithiothreitol) Molecular Grade (0.1 M), 2 μl of random hexamers (100 n/μl), 0.4 μl of dNTP mix (100 mM each), 0.5 μl of RNAseOUT (40 units/μl), and 0.5 μl of SuperScript III Reverse Transcriptase (200 units/μl). The mixture was incubated at 25°C for 5 min, then heated for 60 min at 37°C and the reaction inactivated at 90°C for 5 min. cDNAs were stored at −20°C until the PCR was performed. The expression of abcc1 and lrp gene was assessed by RT–PCR in 50 μl of volume reaction, containing 1 μg cDNA, 5 μl of 10×PCR buffer, 0.4 μl of dNTPmix (100 mM each), 1 μl of sense and antisense primers (10 mM each), 0.4 μl of Taq DNA polymerase (5 units/μl) and 1.5 μl of MgCl2 (50 mM) for GAPDH (glyceraldehyde-3-phosphate dehydrogenase) or 0.75 μl for abcb1 and lrp. GAPDH gene expression served as an endogenous control. Primer sequences, annealing temperatures and thermal cycles were previously described elsewhere (Valera et al., 2004). All RT–PCRs were performed in duplicate. The PCR products were visualized on 2% agarose gel with ethidium bromide staining and analysed densitometrically with NIH ImageJ1.40 software. The intensity of the bands was quantified under UV light and normalized according to those for GAPDH mRNA.
3.1. Cytotoxic effect of SNAP and DNR
Incubation of K562 cells with increasing concentrations of SNAP (Figure 1A) or DNR for 24 h did not induce cell death (Figure 1B). However, when the cells were treated simultaneously for 24 h with DNR (1–100 μM) and SNAP (1 mM) there was increased cytotoxicity compared with DNR alone (Figure 1B).
3.2. Effect of SNAP and DNR on the expression of apoptotic proteins
K562 cells constitutively express anti-apoptotic Bcl-2 and survivin proteins, and treatment with DNR 100 μM or SNAP 1 mM separately did not significantly affect their expression (Figure 2). However, the simultaneous addition reduced the expression of Bcl-2 and survivin to 0.6±0.10 and 0.3±0.15 arbitrary units when compared with the control respectively (P<0.05).
The same Figure shows that the incubation of the cells with DNR 100 μM or SNAP 1 mM left expression of the pro-apoptotic protein, Bax, unchanged. However, compared with the control, 1.3±0.08 arbitrary units increase in its expression occurred when these agents were added simultaneously. Moreover, DNR 100 μM produced a small increase (0.1±0.05 arbitrary units) in active-caspase 3 expression (Figure 2). However, when 100 μM DNR was added with 1 mM SNAP, expression of this protein increased by 0.4±0.10 arbitrary units.
3.3. Morphological changes of K562 cells lines induced by SNAP and DNR
In this assay, cells stained orange or light orange were considered apoptotic, and those stained green were considered viable (Mcgahon et al., 1995; Ribble et al., 2005). The control (untreated cells) and those treated with only DNR or SNAP for 24 h showed cells were viable cells (Figures 3A–3C). Treatment with DNR and SNAP resulted in several cells with nuclear condensation that stained with ethidium bromide orange after 24 h treatment (Figure 3D), indicative of some cell death by apoptosis.
3.4. Effect of SNAP on the distribution and intracellular accumulation of DNR
Fluorescence microscopy of K562 cells in vitro showed significant differences in the distribution of DNR when the cells were treated only with this agent and when given with SNAP 1 mM for 12 h (Figures 4A–4D). Cells treated with only DNR displayed strong fluorescence throughout the cytoplasm, but weak nuclear fluorescence (Figures 4A and 4B). Localized areas of intense fluorescence were also seen in the cytoplasm. Cells treated with SNAP and DNR had considerably greater increase in fluorescent intensity within the whole cell, especially the nucleus (Figures 4C and 4D).
Intracellular DNR concentration was also measured by flow cytometry (Figure 4E), which gave results in accord with the microscopic observations. Cells treated with only DNR had reduced fluorescence intensity, but when treated with DNR and SNAP their fluorescence intensity reached a plateau. The fluorescence intensities of cells treated with DNR in the absence and presence of SNAP were significantly different between 12 and 24 h.
3.5. Effect of SNAP and DNR on the expression of abcc1 and lrp genes
The control group in Figures 4(A)–4(C) shows that K562 cells constitutively express abcc1 and lrp genes. However, the quantification of their expression assessed by the relation between the expression of abcc1 and lrp genes compared with the GAPDH gene shows that the treatment for 6 h with DNR μM significantly reduced in the expression of both abcc1 and lrp, whereas treatment with SNAP 1 mM alone inhibited the lrp gene (Figure 5b). Comparatively, treatment with DNR and SNAP further inhibited expression of the abcc1 gene to constitutive expression and treatment with DNR (Figure 5a).
AML therapy aims at eradicating neoplastic/leukaemic cells and restoring normal haematopoiesis, achieved through aggressive induction chemotherapy (Harris and Hochhauser, 1992). DNR in association with cytarabine is widely used in this treatment (Fathi and Karp, 2009). However, similar to other cytostatic drugs used in conventional chemotherapy, the use of DNR often leads to drug resistance and toxicity, which limits its therapeutic efficacy (Leszczyniecka et al., 2001).
Several groups have demonstrated that NO interferes with the action of anticancer drugs (Wink et al., 1998; Muir et al., 2002; Evig et al., 2004), and can also inhibit drug efflux, thereby reversing doxorubicin resistance (Riganti et al., 2005). Therefore, in aiming to overcome the resistance of neoplastic cells to conventional anticancer agents, NO donors have been intensively investigated as potential anticancer drugs, which could be used alone or in combination with other cytotoxic agents at subtoxic doses (Huerta et al., 2008; Jeannin et al., 2008; De Boo et al., 2009). We have demonstrated that incubation of K562 cells in increasing concentrations of either DNR or SNAP, an NO donor, did not lead to cell death. However, when the cells were incubated simultaneously with both DNR and SNAP, there was significant cytotoxicity that showed DNR concentration-dependency (Figures 1A and 1B). These results suggest that, at these concentrations, K562 cells become resistant to the cytotoxic effects of DNR and SNAP. However, when used in combination, these drugs overcome K562 resistance mechanisms and cause cell death.
Regulation of apoptosis involves a delicate balance between pro- and anti-apoptotic proteins. Members of the Bcl-2 family regulate the initiation of the mitochondrial apoptotic pathway for controlling the release of cytochrome c into the cytoplasm, which activates caspase 9 and consequently the downstream effector caspases. Bcl-2 protein overexpression confers resistance to death signals, such as those induced by anticancer drugs, and is considered a poor prognostic factor in patients with acute leukaemia (Kusenda, 1998; Ruvolo et al., 2001). Furthermore, high expression of survivin, an anti-apoptosis protein, has also been associated with poor prognosis and decreased survival in patients with many types of cancer, including AML (Adida et al., 2000; Xing et al., 2001). Our K562 cells constitutively express Bcl-2 and survivin, and treatment with DNR (100 μM) or SNAP (1 mM) did not significantly affect their expression. However, treatment with both DNR and SNAP reduced Bcl-2 and survivin expression (Figure 2). Incubation of K562 cells with DNR or SNAP did not alter Bax expression, a pro-apoptotic protein. However, DNR and SNAP together increased by 1.3±0.08 arbitrary units Bax expression. Change in the Bax:Bcl-2 ratio leads to apoptosis (Cory and Adams, 2002). Our findings corroborate this finding, since the simultaneous administration of DNR and SNAP was more cytotoxic, associated with decreased Bcl-2 and survivin expression and an increase in Bax expression (Figure 2). Furthermore, imbalance between pro- and anti-apoptotic proteins leads to a cytochrome c release and, consequently, activation of caspase 3, which is the executor of apoptosis. This imbalance in favour of the pro-apoptotic protein Bax resulted in the subsequent activation of caspase 3 protein (Figure 2). Caspase 3 activation induces apoptosis (Figures 3A–3D) in which only the cells treated with combined DNR and SNAP resulted in nuclear condensation and ethidium bromide staining, i.e. cell death by apoptosis.
The ability of some cells to accumulate and retain drugs may contribute to their effectiveness in producing cell death (Den Boer et al., 1999). Merlin et al. (2000) have suggested an intense association between the pattern of intracellular distribution of DNR and its cytotoxicity. Thus, to understand the cytotoxic mechanism of the DNR and SNAP in association, we investigated NO to see whether it would alter intracellular accumulation and retention of DNR. DNR distribution showed accumulation in isolated regions of the cytoplasm (Figures 4A and 4B). However, when incubated with both DNR and SNAP, greater DNR accumulation occurred, preferentially in the nuclear region (Figures 4C and 4D).
The analysis of DNR accumulation and retention by flow cytometry (Figure 4E) showed that DNR and SNAP together caused drug retention that increased linearly in a time-dependent manner. However, when the cells were treated with DNR alone, the drug did not accumulate inside K562 cells and a progressive decrease in DNR intracellular concentration occurred in a time-dependent manner. These results agree with the fluorescence microscopy findings, suggesting an efflux mechanism in K562 cells that this mechanism transports DNR outwardly. Also, the data suggest that SNAP inhibits the efflux mechanism in K562 cells. It is important to note that the primary molecular target of anthracyclines is topoisomerase II located in the nucleus, and that the increase in drug accumulation in the nucleus directly affects the sensitivity of cells to anthracyclines.
Few studies have correlated the intracellular accumulation of anthracyclines through inhibition of drug efflux, which can increase their cytotoxicity in neoplastic cells. Styczyński et al. (2002) suggested that decreased expression of the resistance protein, ABCB1, increases the cytotoxicity caused by DNR in ALL and AML cells. Furthermore, Michelutti et al. (2006) suggested that the accumulation of anthracyclines also occurs due to decreased expression of other proteins related to drug resistance, such as ABCC1 and LRP. Den Boer et al. (1999), in a study of children with ALL, demonstrated that the intracellular concentration of DNR is inversely proportional to LRP expression, but not to ABCB1 expression. Therefore we investigated the effect of DNR and SNAP on the expression of ABCC1 and LRP genes to better understand the effect of NO on the DNR efflux mechanism, and the subsequent decrease in cellular resistance and induction of apoptosis. K562 constitutively express abcc1 and lrp genes (Figure 5). A significant decrease in lrp gene expression was found when cells were treated with DNR or SNAP alone, or with DNR and SNAP in combination. In contrast, abcc1 expression was unaltered in the presence of SNAP alone; however, treatment with DNR caused a small reduction in expression, which was intensified when both DNR and SNAP were administered.
The ABCC1 protein is located in the plasma membrane of cells and intracytoplasmic membranes of cellular organelles, such as the endoplasmic reticulum and the Golgi complex. ABCB1 acts on active transport of substrates across the cell membrane to the extracellular environment or intracellular compartments, resulting in substrate redistribution inside the cell (Swerts et al., 2006; Lu and Shervington, 2008). ABCC1 expression has been detected in several solid tumours, such as melanoma, breast, ovarian and prostate cancer (Hooijberg et al., 2006), and also detected in haematological tumours, such as ALL (Sauerbrey et al., 2002; Stam et al., 2004). In vitro studies have demonstrated that increased expression of ABCC1 in tumour cells may be responsible for cellular resistance against many anti-neoplastic agents. The functional role of LRP in the MDR phenotype remains unclear, but they could act by transporting drugs away from their subcellular targets by mediating the extrusion of drugs from the nucleus and/or their sequestration in vesicles (Swerts et al., 2006).
The results suggest that the transport mechanism involved in the resistance to DNR in human K562 AML cells involves ABCC1 and LRP resistance proteins, and that DNR and SNAP in combination inhibit the expression of these proteins through distinct mechanisms. Furthermore, this inhibition disrupts the complex system of K562 resistance to DNR.
Juliana Costa Curta and Maria Cláudia Santos-Silva designed the research and performed statistical analysis. Juliana Costa Curta, Ana Carolina Rabello de Moraes, Marley Aparecida Licínio and Aline Costa performed the experimental study. Juliana Costa Curta, Ana Carolina Rabello de Moraes, Marley Aparecida Licínio, Aline Costa and Maria Cláudia Santos-Silva participated in data interpretation. Juliana Costa Curta, Ana Carolina Rabello de Moraes and Maria Cláudia Santos-Silva drafted the paper. All authors read and approved the final paper.
This work is part of the master studies of J.C. Curta on the Pharmacy post-graduation course.
This work was supported by grants and fellowships from Coorderação de Aperfeiçoamerto de Pessoal de Nivel Superior-CAPES (Brazil).
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Received 7 April 2011/9 December 2011; accepted 24 February 2012
Published as Cell Biology International Immediate Publication 24 February 2012, doi:10.1042/CBI20110193
© The Author(s) Journal compilation © 2012 International Federation for Cell Biology
ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)
Figure 2 Effects of SNAP and DNR on expression of Bcl-2, Bax, survivin and active-caspase 3 protein on K562 cell lines