title: Matrix metalloproteinase-9 is involved in chronic lymphocytic leukemia cell response to fludarabine and arsenic trioxide.
abstract: BACKGROUND
  Matrix metalloproteinase-9 (MMP-9) contributes to chronic lymphocytic leukemia (CLL) pathology by regulating cell migration and preventing spontaneous apoptosis. It is not known if MMP-9 is involved in CLL cell response to chemotherapy and we address this in the present study, using arsenic trioxide (ATO) and fludarabine as examples of cytotoxic drugs.
  
  
  METHODS
  We used primary cells from the peripheral blood of CLL patients and MEC-1 cells stably transfected with an empty vector or a vector containing MMP-9. The effect of ATO and fludarabine was determined by flow cytometry and by the MTT assay. Expression of mRNA was measured by RT-PCR and qPCR. Secreted and cell-bound MMP-9 was analyzed by gelatin zymography and flow cytometry, respectively. Protein expression was analyzed by Western blotting and immunoprecipitation. Statistical analyses were performed using the two-tailed Student's t-test.
  
  
  RESULTS
  In response to ATO or fludarabine, CLL cells transcriptionally upregulated MMP-9, preceding the onset of apoptosis. Upregulated MMP-9 primarily localized to the membrane of early apoptotic cells and blocking apoptosis with Z-VAD prevented MMP-9 upregulation, thus linking MMP-9 to the apoptotic process. Culturing CLL cells on MMP-9 or stromal cells induced drug resistance, which was overcome by anti-MMP-9 antibodies. Accordingly, MMP-9-MEC-1 transfectants showed higher viability upon drug treatment than Mock-MEC-1 cells, and this effect was blocked by silencing MMP-9 with specific siRNAs. Following drug exposure, expression of anti-apoptotic proteins (Mcl-1, Bcl-xL, Bcl-2) and the Mcl-1/Bim, Mcl-1/Noxa, Bcl-2/Bax ratios were higher in MMP-9-cells than in Mock-cells. Similar results were obtained upon culturing primary CLL cells on MMP-9.
  
  
  CONCLUSIONS
  Our study describes for the first time that MMP-9 induces drug resistance by modulating proteins of the Bcl-2 family and upregulating the corresponding anti-apoptotic/pro-apoptotic ratios. This is a novel role for MMP-9 contributing to CLL progression. Targeting MMP-9 in combined therapies may thus improve CLL response to treatment.
authors: Cellular and Molecular Medicine Department, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain.
authors: Irene
authors: I
authors: Amigo-Jiménez
authors: Cellular and Molecular Medicine Department, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain.
authors: Elvira
authors: E
authors: Bailón
authors: Cellular and Molecular Medicine Department, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain.
authors: Estefanía
authors: E
authors: Ugarte-Berzal
authors: Cellular and Molecular Medicine Department, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain.
authors: Noemí
authors: N
authors: Aguilera-Montilla
authors: Hematology Unit, Hospital Universitario Puerta de Hierro, Madrid, Spain.
authors: José A
authors: JA
authors: García-Marco
authors: Cellular and Molecular Medicine Department, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain.
authors: Angeles
authors: A
authors: García-Pardo
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D017209:Apoptosis / Q000187:drug effects; D000077237:Arsenic Trioxide; D001152:Arsenicals / Q000494:pharmacology / Q000627:therapeutic use*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects; D019008:Drug Resistance, Neoplasm / Q000187:drug effects; D005260:Female; D057809:HEK293 Cells; D006801:Humans; D018960:Hyaluronan Receptors / Q000378:metabolism; D039041:Integrin alpha4beta1 / Q000378:metabolism; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000188:drug therapy* / Q000201:enzymology* / Q000473:pathology; D008297:Male; D020780:Matrix Metalloproteinase 9 / Q000235:genetics / Q000378:metabolism*; D008875:Middle Aged; D064549:Myeloid Cell Leukemia Sequence 1 Protein / Q000378:metabolism; D010087:Oxides / Q000494:pharmacology / Q000627:therapeutic use*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics; D016755:Proto-Oncogene Proteins c-jun / Q000235:genetics; D014158:Transcription, Genetic / Q000187:drug effects; D015854:Up-Regulation / Q000187:drug effects; D014740:Vidarabine / Q000031:analogs & derivatives* / Q000494:pharmacology / Q000627:therapeutic use
chemical_list: D001152:Arsenicals; D018960:Hyaluronan Receptors; D039041:Integrin alpha4beta1; C579095:MCL1 protein, human; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D010087:Oxides; D019253:Proto-Oncogene Proteins c-bcl-2; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D020780:Matrix Metalloproteinase 9; D014740:Vidarabine; C024352:fludarabine; D000077237:Arsenic Trioxide