Abstract
BackgroundMatrix 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.MethodsWe 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.ResultsIn 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.ConclusionsOur 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.
Highlights
Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of malignant CD5+ B lymphocytes in the peripheral blood and their progressive infiltration of lymphoid tissues [1,2]
We previously reported that the mechanism by which arsenic trioxide (ATO) induces chronic lymphocytic leukemia (CLL) cell death is via c-jun N-terminal kinase activation and PI3K/Akt downregulation and this was observed in all samples tested, regardless of their prognostic markers [9]
We have studied whether Matrix metalloproteinase-9 (MMP-9) is modulated by fludarabine or ATO treatment and whether it is involved in the CLL cell response to these compounds
Summary
Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of malignant CD5+ B lymphocytes in the peripheral blood and their progressive infiltration of lymphoid tissues [1,2]. Because CLL is a heterogeneous disease, patients carrying specific molecular markers such as del17p13, unmutated IgVH and/or high expression of ZAP-70 or CD38, do not respond well to these treatments [4], making it crucial to continue searching for new compounds useful in these cases. In this regard, arsenic trioxide (ATO), an efficient therapy in acute promyelocytic leukemia [6,7], has been shown to induce apoptosis in all CLL cases including those with unfavorable prognosis [8].
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