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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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Autophagy evaluation.A) After treatment with CLC and/or RAD alone or in sequence, EPCs were incubated with MDC and analyzed by flow cytometry as described in “Materials and Methods” in order to evaluate the autophagy onset. Untreated cells unexposed to MDC, CTR-; untreated cells exposed to MDC, CTR+; CLC added for 72 h, CLC 72 h; RAD added for 48 h, RAD 48 h; CLC added for 72 h and RAD for the last 48 h, CLC → RAD. B) Representation of autophagy expressed as percentage of mean fluorescence intensity (MFI) derived by MDC stained EPC cells treated with CLC and/or RAD alone or in sequence. CLC added for 72 h, CLC 72 h; RAD added for 48 h, RAD 48 h; CLC added for 72 h and RAD for the last 48 h, CLC → RAD. The experiments were repeated at least three times and always gave similar results. Bars, SDs.
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pone-0079658-g005: Autophagy evaluation.A) After treatment with CLC and/or RAD alone or in sequence, EPCs were incubated with MDC and analyzed by flow cytometry as described in “Materials and Methods” in order to evaluate the autophagy onset. Untreated cells unexposed to MDC, CTR-; untreated cells exposed to MDC, CTR+; CLC added for 72 h, CLC 72 h; RAD added for 48 h, RAD 48 h; CLC added for 72 h and RAD for the last 48 h, CLC → RAD. B) Representation of autophagy expressed as percentage of mean fluorescence intensity (MFI) derived by MDC stained EPC cells treated with CLC and/or RAD alone or in sequence. CLC added for 72 h, CLC 72 h; RAD added for 48 h, RAD 48 h; CLC added for 72 h and RAD for the last 48 h, CLC → RAD. The experiments were repeated at least three times and always gave similar results. Bars, SDs.

Mentions: Moreover, we studied the effects of the two agents alone or in combination on autophagy occurrence, an alternative cell death mechanism that could be involved in the triggering of tumour cell resistance to RAD. Autophagy was evaluated by FACS analysis after staining with autofluorescent molecule associated to early autophagosomes MDC, as described in “Materials and Methods” section. As shown in Figure 5, treatment of EPC with CLC and RAD alone induced a 25 and 36 % increase of MFI, respectively, if compared to untreated cells, while the treatment with the sequence CLC→RAD restored the autophagy levels to those of untreated cells (p<0.01). These findings suggest that when synergistic conditions on both EPCs growth inhibition and apoptosis were recorded autophagy occurrence was almost completely antagonized by the sequential treatment with the two agents.


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)

Autophagy evaluation.A) After treatment with CLC and/or RAD alone or in sequence, EPCs were incubated with MDC and analyzed by flow cytometry as described in “Materials and Methods” in order to evaluate the autophagy onset. Untreated cells unexposed to MDC, CTR-; untreated cells exposed to MDC, CTR+; CLC added for 72 h, CLC 72 h; RAD added for 48 h, RAD 48 h; CLC added for 72 h and RAD for the last 48 h, CLC → RAD. B) Representation of autophagy expressed as percentage of mean fluorescence intensity (MFI) derived by MDC stained EPC cells treated with CLC and/or RAD alone or in sequence. CLC added for 72 h, CLC 72 h; RAD added for 48 h, RAD 48 h; CLC added for 72 h and RAD for the last 48 h, CLC → RAD. The experiments were repeated at least three times and always gave similar results. Bars, SDs.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3823580&req=5

pone-0079658-g005: Autophagy evaluation.A) After treatment with CLC and/or RAD alone or in sequence, EPCs were incubated with MDC and analyzed by flow cytometry as described in “Materials and Methods” in order to evaluate the autophagy onset. Untreated cells unexposed to MDC, CTR-; untreated cells exposed to MDC, CTR+; CLC added for 72 h, CLC 72 h; RAD added for 48 h, RAD 48 h; CLC added for 72 h and RAD for the last 48 h, CLC → RAD. B) Representation of autophagy expressed as percentage of mean fluorescence intensity (MFI) derived by MDC stained EPC cells treated with CLC and/or RAD alone or in sequence. CLC added for 72 h, CLC 72 h; RAD added for 48 h, RAD 48 h; CLC added for 72 h and RAD for the last 48 h, CLC → RAD. The experiments were repeated at least three times and always gave similar results. Bars, SDs.
Mentions: Moreover, we studied the effects of the two agents alone or in combination on autophagy occurrence, an alternative cell death mechanism that could be involved in the triggering of tumour cell resistance to RAD. Autophagy was evaluated by FACS analysis after staining with autofluorescent molecule associated to early autophagosomes MDC, as described in “Materials and Methods” section. As shown in Figure 5, treatment of EPC with CLC and RAD alone induced a 25 and 36 % increase of MFI, respectively, if compared to untreated cells, while the treatment with the sequence CLC→RAD restored the autophagy levels to those of untreated cells (p<0.01). These findings suggest that when synergistic conditions on both EPCs growth inhibition and apoptosis were recorded autophagy occurrence was almost completely antagonized by the sequential treatment with the two agents.

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