Increased Expression of P-Glycoprotein and Doxorubicin Chemoresistance of Metastatic Breast Cancer Is Regulated by miR-298
2012; Elsevier BV; Volume: 180; Issue: 6 Linguagem: Inglês
10.1016/j.ajpath.2012.02.024
ISSN1525-2191
AutoresLili Bao, Sidhartha Hazari, Smriti Mehra, Deepak Kaushal, Krzysztof Moroz, Srikanta Dash,
Tópico(s)RNA Research and Splicing
ResumoMicroRNAs (miRNAs) are short, noncoding RNA molecules that regulate the expression of a number of genes involved in cancer; therefore, they offer great diagnostic and therapeutic targets. We have developed doxorubicin-resistant and -sensitive metastatic human breast cancer cell lines (MDA-MB-231) to study the chemoresistant mechanisms regulated by miRNAs. We found that doxorubicin localized exclusively to the cytoplasm and was unable to reach the nuclei of resistant tumor cells because of the increased nuclear expression of MDR1/P-glycoprotein (P-gp). An miRNA array between doxorubicin-sensitive and -resistant breast cancer cells showed that reduced expression of miR-298 in doxorubicin-resistant human breast cancer cells was associated with increased expression of P-gp. In a transient transfection experiment, miR-298 directly bound to the MDR1 3′ untranslated region and regulated the expression of firefly luciferase reporter in a dose-dependent manner. Overexpression of miR-298 down-regulated P-gp expression, increasing nuclear accumulation of doxorubicin and cytotoxicity in doxorubicin-resistant breast cancer cells. Furthermore, down-regulation of miR-298 increased P-gp expression and induced doxorubicin resistance in sensitive breast cancer cells. In summary, these results suggest that miR-298 directly modulates P-gp expression and is associated with the chemoresistant mechanisms of metastatic human breast cancer. Therefore, miR-298 has diagnostic and therapeutic potential for predicting doxorubicin chemoresistance in human breast cancer. MicroRNAs (miRNAs) are short, noncoding RNA molecules that regulate the expression of a number of genes involved in cancer; therefore, they offer great diagnostic and therapeutic targets. We have developed doxorubicin-resistant and -sensitive metastatic human breast cancer cell lines (MDA-MB-231) to study the chemoresistant mechanisms regulated by miRNAs. We found that doxorubicin localized exclusively to the cytoplasm and was unable to reach the nuclei of resistant tumor cells because of the increased nuclear expression of MDR1/P-glycoprotein (P-gp). An miRNA array between doxorubicin-sensitive and -resistant breast cancer cells showed that reduced expression of miR-298 in doxorubicin-resistant human breast cancer cells was associated with increased expression of P-gp. In a transient transfection experiment, miR-298 directly bound to the MDR1 3′ untranslated region and regulated the expression of firefly luciferase reporter in a dose-dependent manner. Overexpression of miR-298 down-regulated P-gp expression, increasing nuclear accumulation of doxorubicin and cytotoxicity in doxorubicin-resistant breast cancer cells. Furthermore, down-regulation of miR-298 increased P-gp expression and induced doxorubicin resistance in sensitive breast cancer cells. In summary, these results suggest that miR-298 directly modulates P-gp expression and is associated with the chemoresistant mechanisms of metastatic human breast cancer. Therefore, miR-298 has diagnostic and therapeutic potential for predicting doxorubicin chemoresistance in human breast cancer. A number of chemotherapy regimens have been used to treat metastatic breast cancer in humans. The success of treating breast cancer by chemotherapy is hampered by the development of multidrug resistance (MDR) of cancer cells.1Szakacs G. Paterson J.K. Ludwig J.A. Booth-Genthe C. Gottesman M.M. Targeting multidrug resistance in cancer.Nat Rev Drug Discov. 2006; 5: 219-234Crossref PubMed Scopus (2829) Google Scholar, 2Lehnert M. Chemotherapy resistance in breast cancer.Anticancer Res. 1998; 18: 2225-2226PubMed Google Scholar, 3Gottesman M.M. Fojo T. Bates S.E. 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Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs.Genes Dev. 2003; 17: 3011-3016Crossref PubMed Scopus (2131) Google Scholar, 30Lund E. Guttinger S. Calado A. Dahlberg J.E. Kutay U. Nucelar export of microRNA precursors.Science. 2004; 303: 95-98Crossref PubMed Scopus (2016) Google Scholar The strand containing less stable hydrogen bonding at its 5′ end is the mature miRNA and is integrated into the RNA-induced silencing complex, whereas the other strand is degraded.27Denli A.M. Tops B.B. Plasterk R.H. Ketting R.F. Hannon G.J. Processing of primary microRNAs by the microprocessor complex.Nature. 2004; 432: 231-235Crossref PubMed Scopus (1918) Google Scholar To understand the role of miRNAs in the regulation of MDR of breast cancer cells, we developed doxorubicin-sensitive and -resistant metastatic human breast cancer cells (MDA-MB-231). We showed that high-level expression of P-gp leads to the impaired nuclear translocation of doxorubicin and the doxorubicin chemoresistance of MDA-MB-231. To study the role of miRNA involvement in the doxorubicin chemoresistance mechanism, we performed a miRNA array between the doxorubicin-sensitive and -resistant metastatic breast cancer cells. We found significant up-regulation and down-regulation of miRNAs in the doxorubicin-resistant human breast cancer cells compared with the sensitive cells. We have determined that miR-298 is down-regulated significantly in the doxorubicin-resistant MDA-MB-231 cells compared with the doxorubicin-sensitive MDA-MB-231 cells. Using the miRNA database, we found that human miR-298 targeted to the 3′ untranslated region (UTR) of the human P-gp mRNA. Because the role of miRNA-mediated development of resistance to the chemotherapeutic drug is largely unexplored, our study provides the evidence to suggest that the impaired processing of miR-298 because of low expression of Dicer enzyme is associated with an increased expression of P-gp and contributes to the doxorubicin resistance in breast cancer cells. This interaction may have an important functional consequence in the formation of cancer cell resistance to a variety of chemotherapeutic drugs used in the treatment of breast cancer. The MDA-MB-231 and MCF-7 human breast cancer cell lines were obtained from ATCC (Manassas, VA). These two cell lines were cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), sodium pyruvate, nonessential amino acids, and 1% penicillin and streptomycin (Invitrogen, Grand Island, NY) at 37°C in a humidified atmosphere with 5% CO2 and 95% air. Doxorubicin (Adriamycin) was purchased from Sigma Chemical Co. (St. Louis, MO). A stock solution of doxorubicin (1 mg/mL = 1.8 mmol/L) was prepared in distilled water. MDA-MB-231 cells were continuously cultured in growth medium in the presence of 0.18 μmol/L doxorubicin. After several passages, clones that grew in the presence of doxorubicin were selected as drug-resistant cancer (MDA-MB-231-R) cells. The MDA-MB-231-R cells had been cultured for >6 months in the growth medium supplemented with doxorubicin to assure that they were truly resistant to doxorubicin. With the use of a limiting dilution method, MDA-MB-231 cells showing increased doxorubicin-mediated cytotoxicity were isolated. Clones showing the highest cytotoxicity in the MTT assay were selected as doxorubicin-sensitive MDA-MB-231 (MDA-MB-231-S) cells. Doxorubicin-resistant MCF-7 cells (MCF-7-Dox) were obtained from the laboratory of Debasis Mondal (Tulane University School of Medicine, Tulane, LA) and were cultured in high-glucose DMEM supplemented with 10% FBS, sodium pyruvate, nonessential amino acid, 1% penicillin/streptomycin (Invitrogen), and 1 μmol/L insulin (Human Recombination, zinc solution; Invitrogen). Cell viability after doxorubicin treatment was measured by the MTT assay. The assay uses a tetrazolium compound (3-[4,5-dimethylthiazol-2-yl]−2,5-diphenyl tetrazolium bromide; Sigma-Aldrich, St. Louis, MO), which was reduced to formazan intracellularly by the mitochondrial dehydrogenase enzyme. The conversion of tetrazolium into purple formazan by metabolically active cells indicates the extent of cell viability. The viability of tumor cells was measured by the quantification of formazan dye by the colorimetric method. The MDA-MB-231 or MCF-dox cells were seeded on 24-well plates at a density of 2 × 104 cells/well in DMEM with 10% FBS and allowed to adhere at 37°C overnight. After 24 hours, culture medium was replaced and treated with four different concentrations (0.09, 0.18, 0.36, and 0.72 μmol/L) of doxorubicin. After 48 hours of incubation, cells were stained with 100 μL (5 mg/mL of PBS) of MTT solution along with 900 μL of growth medium added in each well. Cells were incubated at 37°C for 3 hours. Then, the tumor cells were washed in PBS and were solubilized with 1 mL of MTT solubilization buffer (anhydrous isopropanol containing 10% Triton X-100, 0.1N HCl) for 5 minutes. Absorbance of converted dye was measured in a spectrophotometer (Beckman Du 530, Life Science UV/Vis spectrophotometer; Beckman Coulter, Inc, Schaumburg, IL) at 570 nm. The percentage of cell viability was determined by comparison with untreated cells as controls. MDA-MB-231-S and MDA-MB-231-R cells (1 × 105) were seeded into 100-mm plates in DMEM with 10% FBS at 37°C overnight. On the next day, the medium was aspirated and replaced with 10 mL of fresh media containing 0.18 μmol/L doxorubicin. After 24 hours of treatment, cells were washed with PBS and trypsinized with 0.05% Trypsin-EDTA (Invitrogen). The cells were fixed in 70% ethanol in PBS and incubated for 2 hours on ice. The fixed cells were subsequently washed twice with PBS and centrifuged at 2000 rpm for 5 minutes. The cells were resuspended in a commercially available propidium iodide staining solution (BD Pharmingen, San Diego, CA). Cells were incubated in the dark for 30 minutes at room temperature. The cell cycle analysis was performed with the use of 2 × 104 cells by a flow cytometer (BD LSR II; BD Biosciences, San Jose, CA). The percentage of tumor cells present in the G1, S, and G2 phases of the cell cycle was analyzed with the computer software (Modfit LT 3.0; Verity Software House, Topsham, ME). Briefly, the cells were cultured in a 6-well plate and lysed with 200 μL of RIPA lysis buffer. Total protein in the lysate was quantified by Bradford protein assay (Bio-Rad Laboratories Inc., Hercules, CA). Approximately 10 μg of protein from each sample was mixed in 4× SDS-loading buffer. Proteins were separated by NuPAGE 12% gel and then transferred onto a nitrocellulose membrane (GE Healthcare, Buckinghamshire, UK). The membrane was blocked with 5% fat-free milk powder in 50 mmol/L Tris-buffered saline (TBS) pH 7.6 with 0.1% Tween-20 (TBS-Tw20) at room temperature for 1 hour. The membrane was washed three times and incubated overnight at 4°C with either the mouse monoclonal antibody to P-gp at 1:500 dilution (C219; AbCam, Inc., Cambridge, MA) or the rabbit monoclonal anti-β-actin clone (Cell Signaling Technologies, Danvers, MA) at 1:1000 dilution in TBS-Tw20 containing 5% fat-free milk powder. After this step, the membrane was washed three times with TBS-Tw20 and reacted for 1 hour with the secondary antibody, anti-mouse or anti-rabbit IgG (Cell Signaling Technologies) that was conjugated with HRP at a dilution of 1:2000. The bound antibodies were detected with the use of ECL Plus Western Blotting Detection system (GE Healthcare), and the chemiluminiscent signals were detected with the use of high-performance chemiluminescence film (GE Healthcare). The MDA-MB-231-S and MDA-MB-231-R cells were cultured overnight on the chamber slides. The next day, slides were fixed with chilled acetone and treated with 1 mL of blocking reagent (Background Sniper; Biocare Medical, Concord, CA) for 10 minutes at room temperature. The slides were then incubated with a primary mouse monoclonal antibody C219 at 1:250 dilution (AbCam, Inc.) at room temperature for 1 hour, followed by a secondary reagent MACH 4 mouse probe (Biocare Medical) for 10 minutes, then a tertiary reagent Mach 4 HRP polymer (Biocare Medical) for 20 minutes. At the final step, the slides were counterstained with hematoxylin and bluing. miRNA was extracted from MDA-MB-231-S and MDA-MB-231-R cells with the use of the Purelink miRNA Isolation Kit (Invitrogen) according to the manufacturer's instructions. To compare the profiles of miRNA expression in these samples, we used the miRCURY LNA miRNA Array, 6th Generation - Human, Mouse & Rat (Exiqon Inc., Woburn, MA). This array platform allows a simultaneous screening of the expression of all known human, mouse, and rat miRNA molecules known to date. Samples extracted from the control (S) cells were labeled with Cy3, and those from the experimental (R) cells were labeled with cyanine 5, using the mercury LNA miRNA labeling kit (Exiqon Inc.). Cyanine-labeled samples were then mixed together in equivalent concentrations and cohybridized to the array overnight at 55°C in a rotary chamber. Slides were scanned on a dual confocal Axon GenePix 4000B scanner (Molecular Devices, Sunnyvale, CA) with the use of GenePix version 6.2 software, and raw data were extracted. A stringent set of criteria was applied to remove background and highly variable data from consideration. The remaining data were log2 transformed and normalized, using Locally Weighted Scatter-plot Smoothing in Spotfire S+, to remove intensity-specific bias. miRNAs were considered to be differentially expressed if they exhibited a twofold perturbation in expression magnitude. Expression miRNA in the doxorubicin-sensitive and -resistant breast cancer cells was examined by Northern blot analysis with the use of a miRNA Northern Blot Assay Kit (Signosis BioSignal Capture, Sunnyvale, CA). Briefly, total RNA was extracted from MDA-MB-231-S and MDA-MB-231-R cells with the use of the Purelink total RNA purification system (Invitrogen). Cellular RNA (5 μg) mixed with 3 μL of RNA loading buffer was heated at 70°C for 5 minutes and chilled on ice. RNA samples in a 10-μL volume were loaded onto a 15% urea-polyacrylamide gel at 60 V in 0.5× TBE buffer until bromophenol blue dye reached approximately 3 cm away from the bottom of the gel. RNAs were transferred to a nylon membrane (Signosis BioSignal Capture) with the use of XCell SureLock Electrophoresis (Invitrogen), immobilized with Stratagene UV cross-linker, and dried at 42°C for 15 minutes. The membrane was transferred into a hybridization tube, soaked with distilled H2O, and incubated with 4 mL of prewarmed (42°C) hybridization buffer then rotated for 30 minutes at 42°C. The membrane was replaced with 4 mL of fresh hybridization buffer along with 10 mL of biotin-labeled miRNA probe and rotated at 42°C overnight. The membrane was removed to an empty container and rinsed with 10 mL of 1× detection wash buffer and then blocked with 15 mL of blocking buffer for 30 minutes at room temperature with moderate shaking. Then the membrane was incubated with 1 mL of the 1× blocking buffer containing 15 mL of streptavidin-HRP conjugate for 45 minutes at room temperature. Then the blocking buffer was removed, and the membrane was washed three times at room temperature with 15 mL of 1× detection washing buffer for 10 minutes each wash. The membrane was incubated with 2 mL of Tris-Buffer pH 7.4 with 200 μL of substrate A and B at room temperature for 5 minutes. After this step, excess substrate was removed by gently applying pressure over the top sheet with the use of a paper towel. The membrane was exposed with Amersham Hyperfilm ECL (GE Healthcare). Membranes were stripped and reprobed to assess other miRNAs. The 3′ UTR of P-gp gene (ABCB1) corresponding to 4262 to 4872 nt (610 bp; Accession no. NM-000927) was cloned into pMirTarget vector by SgfI and MluI restriction sites called pMirTarget-MDR1-3′ UTR (SC208086; Origene Technology, Inc., Rockville, MD). Two different control plasmids with deletion of miR-298 and miR-1253 binding sites were prepared by overlapping PCR and cloned into pMirTarget vector with the use of the unique AscI (GGCGCGCC) and MluI (ACGCGT) restriction sites (Origene Technology, Inc.). The 571-bp DNA fragment with 25-nt deletion in the miR-298 binding site was amplified by overlapping PCR of Fragment 1 (F1) and Fragment 2 (F2). The F1 (411 bp) was PCR amplified with the use of the sense primer (P1), 5′-TCGGGCGCGCCACTCTGACTGTATGAGATGTT-3′, and antisense primer (P2), 5′-AAAGAAAACTTTTTTAAAATTGAGAGAAGATATA-3′, and wild-type MDR1/3′ UTR plasmid as a template. The F2 (160 bp) was amplified from MDR1/3′ UTR plasmid with the use of sense primer (P3), 5′-TATATCTTCTCTCAATTTTAAAAAAGTTTTCTTT-3′, and antisense primer (P4), 5′-GGCACGCGTGAATCAGCAGGATCAAGTCCAAGAAGAATG-3′. AscI site was added to the outer sense primer, and MluI site was added to the outer antisense primer. The PCR-amplified DNA was digested with AscI and MluI restriction endonucleases and cloned into the downstream of the firefly luciferase reporter gene in the AscI/MluI cloning sites of pMirTarget vector. The sequence of the recombinant clones was confirmed by DNA sequencing and was called pMirTarget-MDR1-3′ UTR with deletion of miR-298. The PCR amplification cycles were 94°C for 5 minutes, then 33 cycles at 94°C for 30 seconds, 55°C for 30 second, 72°C for 1 minute, followed by 72°C for 10 minutes. Likewise, the 572-bp DNA fragment with 24-nt deletion in the miR-1253 binding site was amplified by overlapping PCR of Fragment 3 (F3) and Fragment 4 (F4). Briefly, the first 381-bp F3 was amplified with the use of sense primer (P1) and antisense primer (P5), 5′-CTACAATATTCCAATTGGGATAAGATGACTCCAG-3′, using the wild-type MDR1/3′ UTR plasmid as a template. The second 191-bp F4 was amplified with the use of the sense primer (P6), 5′-CTGGAGTCATCTTGTCCCAATTGGAATATTGTAG-3′, and antisense primer (P4) with the use of the wild-type MDR1/3′ UTR plasmid as a template. AscI site was added to the outer sense primer (P1), and Mlu I site was added to the outer antisense primer (P4) so that the recombinant DNA fragment can be cloned into pMirTarget vector with the use of the unique restriction sites. The PCR amplification cycles are the same as above. The sequence of the recombinant clones was confirmed by DNA sequencing and is called pMirTarget-MDR1-3′ UTR with deletion of miR-1253. The MDA-MB-231-R or MCF-7-Dox cells were seeded in 24-well plates and were transfected with 25 ng of the firefly luciferase MDR1-3′ UTR-reporter vector (PS100062; Origene Technology, Inc.) along with the miR-298, miR-1253 mimic (Ambion Pre-miR miRNA Precursor; Ambion by Life Technologies, Carlsbad, CA), or inhibitor (Ambion Anti-miR miRNA Inhibitor; Ambion by Life Technologies) with the use of LipofectAMINE 2000 reagent according to the manufacturer's protocol (Invitrogen). After transfection for 24 hours, cells were lysed with a 1× Passive Lysis Buffer, and the activity of luciferase was assayed with the firefly luciferase reporter assay system (Promega, Valencia, CA) according to the manufacturer's instructions. The values were normalized by total protein of cell lysate. The efficiency of lipofectamine transfection to breast cancer cells was determined by fluorescence microscopy with the use of Cy3-labeled RNA oligonucleotides. Doxorubicin accumulation was assayed by a method described previously.31Bao L. Haque A. Jackson K. Hazari S. Moroz K. Jetley R. Dash S. Increased expression of P-glycoprotein is associated with doxorubicin chemoresistance in 4T1 metastatic breast cancer model.Am J Pathol. 2011; 178: 838-852Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar Briefly, MDA-MB-231-R cells or MDA-MB-231-S cells were transfected with the miRNAs mimics or miR-298 inhibitor with the use of lipofectamine. After 24 hours, transfected cells were incubated overnight with doxorubicin (0.72 μmol/L). Then cells were washed thrice with PBS, fixed with methanol, and observed under a fluorescence microscope (1 × 70; Olympus, Tokyo, Japan). The antitumor effect of doxorubicin on the MDA-MB-231-R and MDA-MB-231-S breast cancer cells was investigated by MTT assay. A significant difference in the doxorubicin-mediated cytotoxicity between the drug-resistant (MDA-MB-231-R) and drug-sensitive (MDA-MB-231-S) breast cancer cell line was observed. The doxorubicin at a concentration of 0.36 μmol/L leads to a 20% growth arrest in the MDA-MB-231-S cells, whereas only 2% to 4% growth arrest was seen with the use of the MDA-MB-231-R cells (Figure 1A). The cellular cytotoxicity of doxorubicin (0.36 μmol/L) with the use of the two cell lines was also measured by MTT assay for 24, 48, and 72 hours, which shows a time-dependent increase in cytotoxicity (Figure 1B). The ability of doxorubicin to induce cell cycle arrest and apoptosis between the MDA-MB-231-R and MDA-MB-231-S cell lines was measured by flow cytometry (Figure 1C). These results indicate that doxorubicin treatment leads to G2/M growth arrest in the majority of cells (89.59%) and that only 10% of cells stay in the S phase of cell cycle in MDA-MB-231-S, whereas only 16% G2/M growth arrest was seen in MDA-MB-231-R. With the use of the TUNEL assay in the 4T1 breast cancer model, we have shown that doxorubicin treatment induced apoptotic cell death because of DNA break in most of the drug-sensitive breast cancer cells,31Bao L. Haque A. Jackson K. Hazari S. Moroz K. Jetley R. Dash S. Increased expression of P-glycoprotein is associated with doxorubicin chemoresistance in 4T1 metastatic breast cancer model.Am J Pathol. 2011; 178: 838-852Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar whereas in the resistant breast cancer cell line no TUNEL-positive cells were present. To understand the mechanism of doxorubicin resistance, the cellular uptake of doxorubicin between the MDA-MB-231-S and MDA-MB-231-R cells was compared by flow cytometry. MDA-MB-231-S and MDA-MB-231-R cells were treated with doxorubicin (0.18 μmol/L). After 24 hours, the intracellular doxorubicin fluorescence was measured by flow cytometry (Figure 2A). The results indicate that a shift in the fluorescence peak because of intracellular doxorubicin between the MDA-MB-231-S and MDA-MB-231-R cel
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