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The synergistic effect of everolimus and chloroquine on endothelial cell number reduction is paralleled by increased apoptosis and reduced autophagy occurrence.

Grimaldi A, Balestrieri ML, D'Onofrio N, Di Domenico G, Nocera C, Lamberti M, Tonini G, Zoccoli A, Santini D, Caraglia M, Pantano F - PLoS ONE (2013)

Bottom Line: In the present study, we evaluated the effects of everolimus (Afinitor, Novartis), a rapamycin analogue, alone or in combination with chloroquine, a 4-alkylamino substituted quinoline family member, one of the autophagy inhibitors, on EPCs biological functions.Moreover, we have studied the mechanisms of cell death induced by the two agents alone or in combination on EPCs and we have found that the synergistic effect of combination on EPC growth inhibition was paralleled by increased apoptosis induction and reduced autophagy.These effects occurred together with biochemical features that are typical of reduced autophagic death such as increased co-immunoprecipitation between Beclin 1 and Bcl-2.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Biophysics and General Pathology, Second University of Naples, Naples, Italy.

ABSTRACT
Endothelial Progenitor Cells (EPCs), a minor subpopulation of the mononuclear cell fraction in peripheral blood, play a critical role in cancer development as they contribute to angiogenesis-mediated pathological neovascularization. In response to tumor cytokines, including VEGF, EPCs mobilize from the bone marrow into the peripheral circulation and move to the tumor bed where they incorporate into sprouting neovessels. In the present study, we evaluated the effects of everolimus (Afinitor, Novartis), a rapamycin analogue, alone or in combination with chloroquine, a 4-alkylamino substituted quinoline family member, one of the autophagy inhibitors, on EPCs biological functions. We found that either everolimus or chloroquine induce growth inhibition on EPCs in a dose-dependent manner after 72 h from the beginning of incubation. The combined administration of the two drugs to EPC was synergistic in inducing growth inhibition; in details, the maximal pharmacological synergism between everolimus and chloroquine in inducing growth inhibition on EPCs cells was recorded when chloroquine was administered 24 h before everolimus. Moreover, we have studied the mechanisms of cell death induced by the two agents alone or in combination on EPCs and we have found that the synergistic effect of combination on EPC growth inhibition was paralleled by increased apoptosis induction and reduced autophagy. These effects occurred together with biochemical features that are typical of reduced autophagic death such as increased co-immunoprecipitation between Beclin 1 and Bcl-2. Chloroquine antagonized the inhibition of the activity of Akt→4EBP1 axis mediated by everolimus and at the same time it blocked the feed-back activation of Erk-1/2 induced by RAD in EPCs. These data suggest a new strategy in order to block angiogenesis in tumours in which this process plays a key role in both the sustainment and spreading of cancer cells.

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FACS analysis of samples of PBMNC derived from human healthy donors (see “Materials and Methods”) labelled with mouse monoclonal antibodies conjugated with fluorophores (CD34-FITC and KDR-PE).Quantitative fluorescence analysis was performed with a FACS-CANTO instrument (BD Biosciences). Cells positive for both CD34-FITC and KDR-PE were considered as EPC. Isolated PBMCs incubated with CLC (20, 40 and 80 µM) (Fig.1 A) or RAD (12.5, 25 and 50 µM) (Fig.1 B) for 72 h in complete media at 37 C. Control cells were also cultured in complete media for 72 h at 37 C. Fig. 1 C shows the CD34+/KDR+ cells after treatment with CLC (20, 40 and 80 µM (p<0.05)) or RAD (12.5, 25 and 50 µM) (p<0.01) expressed as % of control. The figure is representative of three different experiments that always gave similar results.
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pone-0079658-g001: FACS analysis of samples of PBMNC derived from human healthy donors (see “Materials and Methods”) labelled with mouse monoclonal antibodies conjugated with fluorophores (CD34-FITC and KDR-PE).Quantitative fluorescence analysis was performed with a FACS-CANTO instrument (BD Biosciences). Cells positive for both CD34-FITC and KDR-PE were considered as EPC. Isolated PBMCs incubated with CLC (20, 40 and 80 µM) (Fig.1 A) or RAD (12.5, 25 and 50 µM) (Fig.1 B) for 72 h in complete media at 37 C. Control cells were also cultured in complete media for 72 h at 37 C. Fig. 1 C shows the CD34+/KDR+ cells after treatment with CLC (20, 40 and 80 µM (p<0.05)) or RAD (12.5, 25 and 50 µM) (p<0.01) expressed as % of control. The figure is representative of three different experiments that always gave similar results.

Mentions: RAD and CLC reduced EPCs number in a dose-dependent manner after 72 h from the beginning of incubation (Figure 1). Number of CD34+/KDR+ cells was significantly reduced after treatment with 12.5 µM, 25 µM and 50 µM RAD (p<0.01) (Figure 1B), while reduction following CLC treatment was less pronounced (p<0.05) (Figure 1A). Figure 1C shows the % of CD34+/KDR+cells after treatment with CLC (20, 40 and 80 µM) and RAD (12.5, 25 and 50 µM), as percentage of the untreated EPCs.


The synergistic effect of everolimus and chloroquine on endothelial cell number reduction is paralleled by increased apoptosis and reduced autophagy occurrence.

Grimaldi A, Balestrieri ML, D'Onofrio N, Di Domenico G, Nocera C, Lamberti M, Tonini G, Zoccoli A, Santini D, Caraglia M, Pantano F - PLoS ONE (2013)

FACS analysis of samples of PBMNC derived from human healthy donors (see “Materials and Methods”) labelled with mouse monoclonal antibodies conjugated with fluorophores (CD34-FITC and KDR-PE).Quantitative fluorescence analysis was performed with a FACS-CANTO instrument (BD Biosciences). Cells positive for both CD34-FITC and KDR-PE were considered as EPC. Isolated PBMCs incubated with CLC (20, 40 and 80 µM) (Fig.1 A) or RAD (12.5, 25 and 50 µM) (Fig.1 B) for 72 h in complete media at 37 C. Control cells were also cultured in complete media for 72 h at 37 C. Fig. 1 C shows the CD34+/KDR+ cells after treatment with CLC (20, 40 and 80 µM (p<0.05)) or RAD (12.5, 25 and 50 µM) (p<0.01) expressed as % of control. The figure is representative of three different experiments that always gave similar results.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3823580&req=5

pone-0079658-g001: FACS analysis of samples of PBMNC derived from human healthy donors (see “Materials and Methods”) labelled with mouse monoclonal antibodies conjugated with fluorophores (CD34-FITC and KDR-PE).Quantitative fluorescence analysis was performed with a FACS-CANTO instrument (BD Biosciences). Cells positive for both CD34-FITC and KDR-PE were considered as EPC. Isolated PBMCs incubated with CLC (20, 40 and 80 µM) (Fig.1 A) or RAD (12.5, 25 and 50 µM) (Fig.1 B) for 72 h in complete media at 37 C. Control cells were also cultured in complete media for 72 h at 37 C. Fig. 1 C shows the CD34+/KDR+ cells after treatment with CLC (20, 40 and 80 µM (p<0.05)) or RAD (12.5, 25 and 50 µM) (p<0.01) expressed as % of control. The figure is representative of three different experiments that always gave similar results.
Mentions: RAD and CLC reduced EPCs number in a dose-dependent manner after 72 h from the beginning of incubation (Figure 1). Number of CD34+/KDR+ cells was significantly reduced after treatment with 12.5 µM, 25 µM and 50 µM RAD (p<0.01) (Figure 1B), while reduction following CLC treatment was less pronounced (p<0.05) (Figure 1A). Figure 1C shows the % of CD34+/KDR+cells after treatment with CLC (20, 40 and 80 µM) and RAD (12.5, 25 and 50 µM), as percentage of the untreated EPCs.

Bottom Line: In the present study, we evaluated the effects of everolimus (Afinitor, Novartis), a rapamycin analogue, alone or in combination with chloroquine, a 4-alkylamino substituted quinoline family member, one of the autophagy inhibitors, on EPCs biological functions.Moreover, we have studied the mechanisms of cell death induced by the two agents alone or in combination on EPCs and we have found that the synergistic effect of combination on EPC growth inhibition was paralleled by increased apoptosis induction and reduced autophagy.These effects occurred together with biochemical features that are typical of reduced autophagic death such as increased co-immunoprecipitation between Beclin 1 and Bcl-2.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Biophysics and General Pathology, Second University of Naples, Naples, Italy.

ABSTRACT
Endothelial Progenitor Cells (EPCs), a minor subpopulation of the mononuclear cell fraction in peripheral blood, play a critical role in cancer development as they contribute to angiogenesis-mediated pathological neovascularization. In response to tumor cytokines, including VEGF, EPCs mobilize from the bone marrow into the peripheral circulation and move to the tumor bed where they incorporate into sprouting neovessels. In the present study, we evaluated the effects of everolimus (Afinitor, Novartis), a rapamycin analogue, alone or in combination with chloroquine, a 4-alkylamino substituted quinoline family member, one of the autophagy inhibitors, on EPCs biological functions. We found that either everolimus or chloroquine induce growth inhibition on EPCs in a dose-dependent manner after 72 h from the beginning of incubation. The combined administration of the two drugs to EPC was synergistic in inducing growth inhibition; in details, the maximal pharmacological synergism between everolimus and chloroquine in inducing growth inhibition on EPCs cells was recorded when chloroquine was administered 24 h before everolimus. Moreover, we have studied the mechanisms of cell death induced by the two agents alone or in combination on EPCs and we have found that the synergistic effect of combination on EPC growth inhibition was paralleled by increased apoptosis induction and reduced autophagy. These effects occurred together with biochemical features that are typical of reduced autophagic death such as increased co-immunoprecipitation between Beclin 1 and Bcl-2. Chloroquine antagonized the inhibition of the activity of Akt→4EBP1 axis mediated by everolimus and at the same time it blocked the feed-back activation of Erk-1/2 induced by RAD in EPCs. These data suggest a new strategy in order to block angiogenesis in tumours in which this process plays a key role in both the sustainment and spreading of cancer cells.

Show MeSH
Related in: MedlinePlus