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Breast cancer cell line MCF7 escapes from G1/S arrest induced by proteasome inhibition through a GSK-3β dependent mechanism.

Gavilán E, Giráldez S, Sánchez-Aguayo I, Romero F, Ruano D, Daza P - Sci Rep (2015)

Bottom Line: The study was performed in the breast cancer cell line MCF7 compared to the normal mammary cell line MCF10A.We found that the proteasome inhibitor MG132 induced G1/S arrest in MCF10A, but G2/M arrest in MCF7 cells.The effect of MG132 on MCF7 was reproduced on MCF10A cells in the presence of the glycogen synthase kinase 3β (GSK-3β) inhibitor VII.

View Article: PubMed Central - PubMed

Affiliation: 1] Instituto de Biomedicina de Sevilla (IBIS)-Hospital Universitario Virgen del Rocío, Universidad de Sevilla, Sevilla, Spain [2] Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Sevilla, Sevilla, Spain.

ABSTRACT
Targeting the ubiquitin proteasome pathway has emerged as a rational approach in the treatment of human cancers. Autophagy has been described as a cytoprotective mechanism to increase tumor cell survival under stress conditions. Here, we have focused on the role of proteasome inhibition in cell cycle progression and the role of autophagy in the proliferation recovery. The study was performed in the breast cancer cell line MCF7 compared to the normal mammary cell line MCF10A. We found that the proteasome inhibitor MG132 induced G1/S arrest in MCF10A, but G2/M arrest in MCF7 cells. The effect of MG132 on MCF7 was reproduced on MCF10A cells in the presence of the glycogen synthase kinase 3β (GSK-3β) inhibitor VII. Similarly, MCF7 cells overexpressing constitutively active GSK-3β behaved like MCF10A cells. On the other hand, MCF10A cells remained arrested after MG132 removal while MCF7 recovered the proliferative capacity. Importantly, this recovery was abolished in the presence of the autophagy inhibitor 3-methyladenine (3-MA). Thus, our results support the relevance of GSK-3β and autophagy as two targets for controlling cell cycle progression and proliferative capacity in MCF7, highlighting the co-treatment of breast cancer cells with 3-MA to synergize the effect of the proteasome inhibition.

No MeSH data available.


Related in: MedlinePlus

Ultrastructural and biochemical analysis of autophagy resolution during proteasome inhibition recovery. a. TEM analysis of MCF10A (upper panel) and MCF7 cells (lower panel) following 24 hours of proteasome inhibition and 24, 48 and 72 hours after removing MG132. Scale bar 4 μm. b. Representative western blots of specific autophagic markers (LC3 and p62) and phosphorilated GSK-3β protein expression in MCF10A (left) and MCF7 cells (right), following 24 hours of proteasome inhibition and 24, 48 and 72 hours after removing MG132. Gels were run in parallel and under the same experimental conditions.
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f5: Ultrastructural and biochemical analysis of autophagy resolution during proteasome inhibition recovery. a. TEM analysis of MCF10A (upper panel) and MCF7 cells (lower panel) following 24 hours of proteasome inhibition and 24, 48 and 72 hours after removing MG132. Scale bar 4 μm. b. Representative western blots of specific autophagic markers (LC3 and p62) and phosphorilated GSK-3β protein expression in MCF10A (left) and MCF7 cells (right), following 24 hours of proteasome inhibition and 24, 48 and 72 hours after removing MG132. Gels were run in parallel and under the same experimental conditions.

Mentions: On the other hand, MCF10A cells augmented in size and diminished in number after MG132 elimination. Moreover, the yuxtanuclear aggregates were disappearing at the same time that cytosolic vacuoles were appearing throughout the recovery period, showing most of them a highly vacuolated cytoplasm. This was also confirmed by ultrastructural analysis by TEM in ultrathin sections. As shown in Fig. 5a, cytoplasmic vacuoles in MCF10A cells increased in size, in a time dependent manner, filling the entire cytosolic surface and surrounding the cytoplasmic aggregates in some cells. Some cytosolic vacuoles observed in MCF10A cells had the appearance of autophagic vacuoles as judged by the double membrane and the heterogeneous content, supporting immunofluorescence data. In addition, we also observed large empty vacuoles in both cell types which resembled senescent vacuoles. In fact, the proportion of senescent cells, determined by SA-β-gal assay, was significantly higher in MCF10 than in MCF7 cells 48 hours following MG132 removal (P = 0.0003; Supplementary Fig S2).


Breast cancer cell line MCF7 escapes from G1/S arrest induced by proteasome inhibition through a GSK-3β dependent mechanism.

Gavilán E, Giráldez S, Sánchez-Aguayo I, Romero F, Ruano D, Daza P - Sci Rep (2015)

Ultrastructural and biochemical analysis of autophagy resolution during proteasome inhibition recovery. a. TEM analysis of MCF10A (upper panel) and MCF7 cells (lower panel) following 24 hours of proteasome inhibition and 24, 48 and 72 hours after removing MG132. Scale bar 4 μm. b. Representative western blots of specific autophagic markers (LC3 and p62) and phosphorilated GSK-3β protein expression in MCF10A (left) and MCF7 cells (right), following 24 hours of proteasome inhibition and 24, 48 and 72 hours after removing MG132. Gels were run in parallel and under the same experimental conditions.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4419540&req=5

f5: Ultrastructural and biochemical analysis of autophagy resolution during proteasome inhibition recovery. a. TEM analysis of MCF10A (upper panel) and MCF7 cells (lower panel) following 24 hours of proteasome inhibition and 24, 48 and 72 hours after removing MG132. Scale bar 4 μm. b. Representative western blots of specific autophagic markers (LC3 and p62) and phosphorilated GSK-3β protein expression in MCF10A (left) and MCF7 cells (right), following 24 hours of proteasome inhibition and 24, 48 and 72 hours after removing MG132. Gels were run in parallel and under the same experimental conditions.
Mentions: On the other hand, MCF10A cells augmented in size and diminished in number after MG132 elimination. Moreover, the yuxtanuclear aggregates were disappearing at the same time that cytosolic vacuoles were appearing throughout the recovery period, showing most of them a highly vacuolated cytoplasm. This was also confirmed by ultrastructural analysis by TEM in ultrathin sections. As shown in Fig. 5a, cytoplasmic vacuoles in MCF10A cells increased in size, in a time dependent manner, filling the entire cytosolic surface and surrounding the cytoplasmic aggregates in some cells. Some cytosolic vacuoles observed in MCF10A cells had the appearance of autophagic vacuoles as judged by the double membrane and the heterogeneous content, supporting immunofluorescence data. In addition, we also observed large empty vacuoles in both cell types which resembled senescent vacuoles. In fact, the proportion of senescent cells, determined by SA-β-gal assay, was significantly higher in MCF10 than in MCF7 cells 48 hours following MG132 removal (P = 0.0003; Supplementary Fig S2).

Bottom Line: The study was performed in the breast cancer cell line MCF7 compared to the normal mammary cell line MCF10A.We found that the proteasome inhibitor MG132 induced G1/S arrest in MCF10A, but G2/M arrest in MCF7 cells.The effect of MG132 on MCF7 was reproduced on MCF10A cells in the presence of the glycogen synthase kinase 3β (GSK-3β) inhibitor VII.

View Article: PubMed Central - PubMed

Affiliation: 1] Instituto de Biomedicina de Sevilla (IBIS)-Hospital Universitario Virgen del Rocío, Universidad de Sevilla, Sevilla, Spain [2] Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Sevilla, Sevilla, Spain.

ABSTRACT
Targeting the ubiquitin proteasome pathway has emerged as a rational approach in the treatment of human cancers. Autophagy has been described as a cytoprotective mechanism to increase tumor cell survival under stress conditions. Here, we have focused on the role of proteasome inhibition in cell cycle progression and the role of autophagy in the proliferation recovery. The study was performed in the breast cancer cell line MCF7 compared to the normal mammary cell line MCF10A. We found that the proteasome inhibitor MG132 induced G1/S arrest in MCF10A, but G2/M arrest in MCF7 cells. The effect of MG132 on MCF7 was reproduced on MCF10A cells in the presence of the glycogen synthase kinase 3β (GSK-3β) inhibitor VII. Similarly, MCF7 cells overexpressing constitutively active GSK-3β behaved like MCF10A cells. On the other hand, MCF10A cells remained arrested after MG132 removal while MCF7 recovered the proliferative capacity. Importantly, this recovery was abolished in the presence of the autophagy inhibitor 3-methyladenine (3-MA). Thus, our results support the relevance of GSK-3β and autophagy as two targets for controlling cell cycle progression and proliferative capacity in MCF7, highlighting the co-treatment of breast cancer cells with 3-MA to synergize the effect of the proteasome inhibition.

No MeSH data available.


Related in: MedlinePlus