Physique 11A shows that Mcl-1 was significantly upregulated, both in the absence or presence of ATO, in cells cultured on MMP-9 compared to cells cultured on BSA

Physique 11A shows that Mcl-1 was significantly upregulated, both in the absence or presence of ATO, in cells cultured on MMP-9 compared to cells cultured on BSA. 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. Comparable results were obtained upon culturing main 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. Introduction Chronic lymphocytic leukemia (CLL) is usually characterized by the accumulation of malignant CD5+ B lymphocytes in the peripheral blood Fluralaner and their progressive infiltration of lymphoid tissues [1], [2]. Frontline therapies for CLL consist in the administration of the purine analogue fludarabine, alone or in combination with other drugs such as anti-CD20 monoclonal antibodies or kinase inhibitors [3]C[5]. Because CLL is usually 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 Fluralaner [8]. We previously reported that this mechanism by which ATO induces CLL cell death is usually 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]. ATO may thus constitute an efficient option/complementary treatment for CLL. As with CD127 most tumors, CLL cell response to therapy is usually influenced by the microenvironment, whose cellular and molecular components provide survival signals that favor drug resistance [10], [11]. A consistent component of CLL niches is usually matrix metalloproteinase-9 (MMP-9) [12], which is also produced by CLL cells and upregulated by several stimuli [13]C[15]. Endogenous or/and exogenous MMP-9 binds to CLL cells via specific docking receptors and regulates cell migration [16]. Surface-bound MMP-9 also prevents CLL cell spontaneous apoptosis by a non-catalytic mechanism, consisting in Lyn/STAT3 activation and Mcl-1 upregulation [17], thus contributing to CLL progression. It is not known if MMP-9 affects CLL cell response to chemotherapy. This is important to elucidate since MMP-9, as other MMPs, may play dual functions in apoptosis, either facilitating or antagonizing drug Fluralaner action [18], [19]. To approach this issue, we have analyzed whether MMP-9 is usually modulated by fludarabine or ATO treatment and whether it is involved in the CLL cell response to these compounds. Using main CLL cells and a CLL-derived cell collection stably expressing MMP-9 [20], we show that MMP-9 contributes to chemoresistance by preventing downregulation of anti-apoptotic proteins. Materials and Methods Patients, cells and cell culture Approval was obtained from the CSIC Bioethics Review Table for these studies. All patients signed an informed consent before blood was drawn. B-lymphocytes were purified from your 20 CLL samples listed in Table 1 as reported [9], [17], using Ficoll-Paque.