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Matrix metalloproteinase-9 is involved in chronic lymphocytic leukemia cell response to fludarabine and arsenic trioxide.

Amigo-Jiménez I, Bailón E, Ugarte-Berzal E, Aguilera-Montilla N, García-Marco JA, García-Pardo A - PLoS ONE (2014)

Bottom Line: 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.Similar results were obtained upon culturing primary CLL cells on MMP-9.Targeting MMP-9 in combined therapies may thus improve CLL response to treatment.

View Article: PubMed Central - PubMed

Affiliation: Cellular and Molecular Medicine Department, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain.

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.

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Fludarabine transcriptionally upregulates MMP-9 and induces its localization to the CLL cell membrane.(A,B) 10–15×106 CLL in RPMI/0.1% FBS cells from two different patients were treated with 3 or 5 µM fludarabine (Fluda) for 48 h and MMP-9 mRNA expression was analyzed by RT-PCR (A) and qPCR (B). Normalized average values (fold change) are shown. (C) 1.5×105 CLL cells from two different patients were incubated with or without 3 µM fludarabine for 48 h and MMP-9 surface expression was analyzed by flow cytometry. White areas, control/untreated cells; grey areas, fludarabine treated cells. Arrows indicate specific fluorescence (SF) values for each cell population. Normalized average values are also shown.
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pone-0099993-g005: Fludarabine transcriptionally upregulates MMP-9 and induces its localization to the CLL cell membrane.(A,B) 10–15×106 CLL in RPMI/0.1% FBS cells from two different patients were treated with 3 or 5 µM fludarabine (Fluda) for 48 h and MMP-9 mRNA expression was analyzed by RT-PCR (A) and qPCR (B). Normalized average values (fold change) are shown. (C) 1.5×105 CLL cells from two different patients were incubated with or without 3 µM fludarabine for 48 h and MMP-9 surface expression was analyzed by flow cytometry. White areas, control/untreated cells; grey areas, fludarabine treated cells. Arrows indicate specific fluorescence (SF) values for each cell population. Normalized average values are also shown.

Mentions: To determine whether MMP-9 modulation was a particular feature of ATO exposure or a more general response to drug-induced apoptosis, we studied the effect of fludarabine, a front-line treatment for CLL, on MMP-9. CLL cells were incubated with or without 3 or 5 µM fludarabine for 48 h and MMP-9 mRNA analyzed by RT-PCR. Figure 5A shows that fludarabine increased MMP-9 transcription in a dose-dependent manner, compared to control cells. These results were confirmed by qPCR, which showed an increase on MMP-9 mRNA of 4.9-fold and 17.5-fold, respectively, for 3 and 5 µM fludarabine (Figure 5B). Parallel flow cytometric analyses indicated that the average percentage of apoptotic cells at this time was 45.2% and 48%, respectively, for 3 and 5 µM fludarabine (not shown). As observed in the case of ATO, MMP-9 expression at the cell surface was enhanced (15.5% to 26.6% positive cells) upon fludarabine treatment (Figure 5C). These results indicated that MMP-9 upregulation in correlation with CLL cell apoptosis was not restricted to ATO action.


Matrix metalloproteinase-9 is involved in chronic lymphocytic leukemia cell response to fludarabine and arsenic trioxide.

Amigo-Jiménez I, Bailón E, Ugarte-Berzal E, Aguilera-Montilla N, García-Marco JA, García-Pardo A - PLoS ONE (2014)

Fludarabine transcriptionally upregulates MMP-9 and induces its localization to the CLL cell membrane.(A,B) 10–15×106 CLL in RPMI/0.1% FBS cells from two different patients were treated with 3 or 5 µM fludarabine (Fluda) for 48 h and MMP-9 mRNA expression was analyzed by RT-PCR (A) and qPCR (B). Normalized average values (fold change) are shown. (C) 1.5×105 CLL cells from two different patients were incubated with or without 3 µM fludarabine for 48 h and MMP-9 surface expression was analyzed by flow cytometry. White areas, control/untreated cells; grey areas, fludarabine treated cells. Arrows indicate specific fluorescence (SF) values for each cell population. Normalized average values are also shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4067296&req=5

pone-0099993-g005: Fludarabine transcriptionally upregulates MMP-9 and induces its localization to the CLL cell membrane.(A,B) 10–15×106 CLL in RPMI/0.1% FBS cells from two different patients were treated with 3 or 5 µM fludarabine (Fluda) for 48 h and MMP-9 mRNA expression was analyzed by RT-PCR (A) and qPCR (B). Normalized average values (fold change) are shown. (C) 1.5×105 CLL cells from two different patients were incubated with or without 3 µM fludarabine for 48 h and MMP-9 surface expression was analyzed by flow cytometry. White areas, control/untreated cells; grey areas, fludarabine treated cells. Arrows indicate specific fluorescence (SF) values for each cell population. Normalized average values are also shown.
Mentions: To determine whether MMP-9 modulation was a particular feature of ATO exposure or a more general response to drug-induced apoptosis, we studied the effect of fludarabine, a front-line treatment for CLL, on MMP-9. CLL cells were incubated with or without 3 or 5 µM fludarabine for 48 h and MMP-9 mRNA analyzed by RT-PCR. Figure 5A shows that fludarabine increased MMP-9 transcription in a dose-dependent manner, compared to control cells. These results were confirmed by qPCR, which showed an increase on MMP-9 mRNA of 4.9-fold and 17.5-fold, respectively, for 3 and 5 µM fludarabine (Figure 5B). Parallel flow cytometric analyses indicated that the average percentage of apoptotic cells at this time was 45.2% and 48%, respectively, for 3 and 5 µM fludarabine (not shown). As observed in the case of ATO, MMP-9 expression at the cell surface was enhanced (15.5% to 26.6% positive cells) upon fludarabine treatment (Figure 5C). These results indicated that MMP-9 upregulation in correlation with CLL cell apoptosis was not restricted to ATO action.

Bottom Line: 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.Similar results were obtained upon culturing primary CLL cells on MMP-9.Targeting MMP-9 in combined therapies may thus improve CLL response to treatment.

View Article: PubMed Central - PubMed

Affiliation: Cellular and Molecular Medicine Department, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain.

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.

Show MeSH
Related in: MedlinePlus