Limits...
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.

Show MeSH

Related in: MedlinePlus

ATO transcriptionally upregulates MMP-9 in CLL cells.(A) 1.5×105 CLL cells in RPMI/0.1%FBS were incubated with or without the indicated concentrations of ATO. After 24 h, cells were analyzed by flow cytometry using FITC-Annexin V and PI. (B) 10–15×106 CLL cells were treated with 3 µM ATO for the indicated times and MMP-9 mRNA expression was analyzed by RT-PCR, using GAPDH mRNA as internal control. Normalized average values (fold change) are shown. (C) The same mRNA samples were also analyzed by qPCR using TBP as internal control and normalized average values (fold change) are shown. (D) 10–15×106 CLL cells were cultured with or without 3 µM ATO for 20 h. Cells were then treated or not (Control, Ctl) with 5 µM actinomycin D and mRNA expression was analyzed at the indicated times. Values represent the average MMP-9/GAPDH ratio from the two samples after normalizing control values to 100. Values for GAPDH mRNA are also shown. *P≤0.05; **P≤0.01; ***P≤0.001.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4067296&req=5

pone-0099993-g001: ATO transcriptionally upregulates MMP-9 in CLL cells.(A) 1.5×105 CLL cells in RPMI/0.1%FBS were incubated with or without the indicated concentrations of ATO. After 24 h, cells were analyzed by flow cytometry using FITC-Annexin V and PI. (B) 10–15×106 CLL cells were treated with 3 µM ATO for the indicated times and MMP-9 mRNA expression was analyzed by RT-PCR, using GAPDH mRNA as internal control. Normalized average values (fold change) are shown. (C) The same mRNA samples were also analyzed by qPCR using TBP as internal control and normalized average values (fold change) are shown. (D) 10–15×106 CLL cells were cultured with or without 3 µM ATO for 20 h. Cells were then treated or not (Control, Ctl) with 5 µM actinomycin D and mRNA expression was analyzed at the indicated times. Values represent the average MMP-9/GAPDH ratio from the two samples after normalizing control values to 100. Values for GAPDH mRNA are also shown. *P≤0.05; **P≤0.01; ***P≤0.001.

Mentions: To first establish the best conditions to study ATO action, CLL cells (1.5×106/ml) were cultured for 24 h with or without various concentrations of ATO and apoptosis measured by flow cytometry, using FITC-Annexin V and PI. Figure 1A shows that the percentage of apoptotic cells (Annexin V+PI−) increased in a dose-dependent manner, reaching average values of 46.2% at 3 µM ATO (Figure 1A). 16.6% of the remaining cells were viable (Annexin V−PI−) and 37% were necrotic cells (Annexin V+PI+). As the 46.2% level of apoptosis seemed appropriate for biochemical studies, we chose 3 µM ATO concentration for subsequent experiments, except when indicated.


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)

ATO transcriptionally upregulates MMP-9 in CLL cells.(A) 1.5×105 CLL cells in RPMI/0.1%FBS were incubated with or without the indicated concentrations of ATO. After 24 h, cells were analyzed by flow cytometry using FITC-Annexin V and PI. (B) 10–15×106 CLL cells were treated with 3 µM ATO for the indicated times and MMP-9 mRNA expression was analyzed by RT-PCR, using GAPDH mRNA as internal control. Normalized average values (fold change) are shown. (C) The same mRNA samples were also analyzed by qPCR using TBP as internal control and normalized average values (fold change) are shown. (D) 10–15×106 CLL cells were cultured with or without 3 µM ATO for 20 h. Cells were then treated or not (Control, Ctl) with 5 µM actinomycin D and mRNA expression was analyzed at the indicated times. Values represent the average MMP-9/GAPDH ratio from the two samples after normalizing control values to 100. Values for GAPDH mRNA are also shown. *P≤0.05; **P≤0.01; ***P≤0.001.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4067296&req=5

pone-0099993-g001: ATO transcriptionally upregulates MMP-9 in CLL cells.(A) 1.5×105 CLL cells in RPMI/0.1%FBS were incubated with or without the indicated concentrations of ATO. After 24 h, cells were analyzed by flow cytometry using FITC-Annexin V and PI. (B) 10–15×106 CLL cells were treated with 3 µM ATO for the indicated times and MMP-9 mRNA expression was analyzed by RT-PCR, using GAPDH mRNA as internal control. Normalized average values (fold change) are shown. (C) The same mRNA samples were also analyzed by qPCR using TBP as internal control and normalized average values (fold change) are shown. (D) 10–15×106 CLL cells were cultured with or without 3 µM ATO for 20 h. Cells were then treated or not (Control, Ctl) with 5 µM actinomycin D and mRNA expression was analyzed at the indicated times. Values represent the average MMP-9/GAPDH ratio from the two samples after normalizing control values to 100. Values for GAPDH mRNA are also shown. *P≤0.05; **P≤0.01; ***P≤0.001.
Mentions: To first establish the best conditions to study ATO action, CLL cells (1.5×106/ml) were cultured for 24 h with or without various concentrations of ATO and apoptosis measured by flow cytometry, using FITC-Annexin V and PI. Figure 1A shows that the percentage of apoptotic cells (Annexin V+PI−) increased in a dose-dependent manner, reaching average values of 46.2% at 3 µM ATO (Figure 1A). 16.6% of the remaining cells were viable (Annexin V−PI−) and 37% were necrotic cells (Annexin V+PI+). As the 46.2% level of apoptosis seemed appropriate for biochemical studies, we chose 3 µM ATO concentration for subsequent experiments, except when indicated.

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