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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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Evaluation of pathways involved in the regulation of proliferation and survival.A) EPCs were treated with RAD and/or CLC alone or in combination. Thereafter, both the activity and the expression of the different proteins were evaluated after blotting with specific antibodies, as described in “Materials and Methods”. Expression of the house-keeping protein γ-tubulin was used as loading control. Untreated cells, 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. The figure is representative of three different experiments that always gave similar results. B) Representation of the intensities of the bands associated to the different proteins normalized for the expression of the total proteins. The intensities of the bands were expressed as arbitrary units when compared to those of the untreated cells. The values are the mean of three independent experiments. SDs, standard deviations.
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pone-0079658-g007: Evaluation of pathways involved in the regulation of proliferation and survival.A) EPCs were treated with RAD and/or CLC alone or in combination. Thereafter, both the activity and the expression of the different proteins were evaluated after blotting with specific antibodies, as described in “Materials and Methods”. Expression of the house-keeping protein γ-tubulin was used as loading control. Untreated cells, 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. The figure is representative of three different experiments that always gave similar results. B) Representation of the intensities of the bands associated to the different proteins normalized for the expression of the total proteins. The intensities of the bands were expressed as arbitrary units when compared to those of the untreated cells. The values are the mean of three independent experiments. SDs, standard deviations.

Mentions: We next investigated the molecular mechanisms of autophagy studying the interaction between two molecules involved in this process: Beclin-1 and Bcl-2. It is known that Bcl-2, interacting with Beclin-1, inhibits Beclin-1-dependent autophagy [39]. When these proteins were co-immunoprecipitated, we found that combined treatment increased Beclin-1/Bcl-2 complex formation if compared to the two drugs alone (p<0.01) (Figure 6). The expression of total Beclin-1 and Bcl-2 remained unchanged in both the single and sequential treatments. On the other hand, a significant increase of Beclin-1/Bcl-2 complex was recorded in EPCs treated with the two drugs in combination if compared to drugs alone or to untreated EPCs. Another key component that regulates the balance between cell growth and autophagy in response to cellular physiological conditions and environmental stress is mammalian target of rapamycin (mTOR), the target of RAD. The activation of mTOR leads to the phosphorylation of its two major downstream components, p70S6K and eIF4E-binding protein 1 (4EBP1). 4EBP1 is believed to primarily control cap-dependent translation by binding and inactivating eIF4E, an initiation factor, which binds to the mRNA cap structure, thereby mediating the initiation of translation. Phosphorylation of 4EBP1 results in the release of eIF4E and subsequent activation of the eIF4G scaffolding protein. Both PI3 kinase/AKT pathway and mTOR kinase regulate 4EBP1 activity [40]. As shown in Figure 7, we found that the combination between CLC/RAD and treatment with CLC increased 4EBP1 phosphorylation if compared to the RAD (p<0.01) while 4EBP1 phosphorylation was significantly reduced also by RAD alone (p<0.01). Therefore, the antagonism induced by CLC on RAD-mediated autophagy was paralleled by an immobilization of the pro-autophagic protein Beclin 1 mediated by Bcl2 and by a decrease of the phosphorylation of 4EBP1, a target of the anti-autophagic signalling protein mTOR.


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)

Evaluation of pathways involved in the regulation of proliferation and survival.A) EPCs were treated with RAD and/or CLC alone or in combination. Thereafter, both the activity and the expression of the different proteins were evaluated after blotting with specific antibodies, as described in “Materials and Methods”. Expression of the house-keeping protein γ-tubulin was used as loading control. Untreated cells, 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. The figure is representative of three different experiments that always gave similar results. B) Representation of the intensities of the bands associated to the different proteins normalized for the expression of the total proteins. The intensities of the bands were expressed as arbitrary units when compared to those of the untreated cells. The values are the mean of three independent experiments. SDs, standard deviations.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3823580&req=5

pone-0079658-g007: Evaluation of pathways involved in the regulation of proliferation and survival.A) EPCs were treated with RAD and/or CLC alone or in combination. Thereafter, both the activity and the expression of the different proteins were evaluated after blotting with specific antibodies, as described in “Materials and Methods”. Expression of the house-keeping protein γ-tubulin was used as loading control. Untreated cells, 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. The figure is representative of three different experiments that always gave similar results. B) Representation of the intensities of the bands associated to the different proteins normalized for the expression of the total proteins. The intensities of the bands were expressed as arbitrary units when compared to those of the untreated cells. The values are the mean of three independent experiments. SDs, standard deviations.
Mentions: We next investigated the molecular mechanisms of autophagy studying the interaction between two molecules involved in this process: Beclin-1 and Bcl-2. It is known that Bcl-2, interacting with Beclin-1, inhibits Beclin-1-dependent autophagy [39]. When these proteins were co-immunoprecipitated, we found that combined treatment increased Beclin-1/Bcl-2 complex formation if compared to the two drugs alone (p<0.01) (Figure 6). The expression of total Beclin-1 and Bcl-2 remained unchanged in both the single and sequential treatments. On the other hand, a significant increase of Beclin-1/Bcl-2 complex was recorded in EPCs treated with the two drugs in combination if compared to drugs alone or to untreated EPCs. Another key component that regulates the balance between cell growth and autophagy in response to cellular physiological conditions and environmental stress is mammalian target of rapamycin (mTOR), the target of RAD. The activation of mTOR leads to the phosphorylation of its two major downstream components, p70S6K and eIF4E-binding protein 1 (4EBP1). 4EBP1 is believed to primarily control cap-dependent translation by binding and inactivating eIF4E, an initiation factor, which binds to the mRNA cap structure, thereby mediating the initiation of translation. Phosphorylation of 4EBP1 results in the release of eIF4E and subsequent activation of the eIF4G scaffolding protein. Both PI3 kinase/AKT pathway and mTOR kinase regulate 4EBP1 activity [40]. As shown in Figure 7, we found that the combination between CLC/RAD and treatment with CLC increased 4EBP1 phosphorylation if compared to the RAD (p<0.01) while 4EBP1 phosphorylation was significantly reduced also by RAD alone (p<0.01). Therefore, the antagonism induced by CLC on RAD-mediated autophagy was paralleled by an immobilization of the pro-autophagic protein Beclin 1 mediated by Bcl2 and by a decrease of the phosphorylation of 4EBP1, a target of the anti-autophagic signalling protein mTOR.

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