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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

Analysis of MCF10A cell recovery following proteasome inhibition with MG132 and in the presence of the GSK-3β inhibitor VII. a. Representative images of MCF10A cells in control situation and with different treatments at the end of the assay. Note the difference in cell number in MG132 (24 h) and MG132 (24 h) + GSK-3β inhibitor VII (present during treatment and recovery). Scale bar 30 μm b. Proliferative capacity of MCF10A cells during a week following 24 hours of MG132 1 μM and GSK-3β inhibitor VII 20 μM treatment, independently or combined. Note that cells treated with MG132 do not recover the proliferative capacity while co-treated cells (MG132 + GSK-3β inhibitor VII) restored cell proliferation rate.
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f7: Analysis of MCF10A cell recovery following proteasome inhibition with MG132 and in the presence of the GSK-3β inhibitor VII. a. Representative images of MCF10A cells in control situation and with different treatments at the end of the assay. Note the difference in cell number in MG132 (24 h) and MG132 (24 h) + GSK-3β inhibitor VII (present during treatment and recovery). Scale bar 30 μm b. Proliferative capacity of MCF10A cells during a week following 24 hours of MG132 1 μM and GSK-3β inhibitor VII 20 μM treatment, independently or combined. Note that cells treated with MG132 do not recover the proliferative capacity while co-treated cells (MG132 + GSK-3β inhibitor VII) restored cell proliferation rate.

Mentions: As showed above, GSK-3β inhibition rescued MCF10A cells from G1/S arrest induced by MG132 (see Fig. 2). Importantly, as we and other have previously demonstrated pharmacological inhibition of GSK-3β activity induced early autophagy activation2425. In order to test whether this inhibition in MCF10A cells would mimic MCF7 proliferative behavior following 24 hours of MG132 treatment, we inhibit GSK-3β and performed proliferation assays. Treatment with the GSK-3β inhibitor for 168 hours did not affect cell proliferation rate, while proteasome inhibition deeply impaired cell growth even 168 hours after drug removal (Fig. 7a). However, when MCF10A cells were simultaneously treated with both inhibitors for 24 hours, and cells were allowed to grow in the presence of the GSK-3β VII inhibitor after MG132 elimination, cell proliferation began to recover reaching up to 20% of control cell number 168 hours later (Fig. 7b). Thus, these data provide strong evidence supporting that chronic GSK-3β inhibition in addition to early autophagy activation could be responsible for the proliferation recovery of both breast epithelial cells following proteasome inhibition, a mechanism that could be essential for cell survival.


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)

Analysis of MCF10A cell recovery following proteasome inhibition with MG132 and in the presence of the GSK-3β inhibitor VII. a. Representative images of MCF10A cells in control situation and with different treatments at the end of the assay. Note the difference in cell number in MG132 (24 h) and MG132 (24 h) + GSK-3β inhibitor VII (present during treatment and recovery). Scale bar 30 μm b. Proliferative capacity of MCF10A cells during a week following 24 hours of MG132 1 μM and GSK-3β inhibitor VII 20 μM treatment, independently or combined. Note that cells treated with MG132 do not recover the proliferative capacity while co-treated cells (MG132 + GSK-3β inhibitor VII) restored cell proliferation rate.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f7: Analysis of MCF10A cell recovery following proteasome inhibition with MG132 and in the presence of the GSK-3β inhibitor VII. a. Representative images of MCF10A cells in control situation and with different treatments at the end of the assay. Note the difference in cell number in MG132 (24 h) and MG132 (24 h) + GSK-3β inhibitor VII (present during treatment and recovery). Scale bar 30 μm b. Proliferative capacity of MCF10A cells during a week following 24 hours of MG132 1 μM and GSK-3β inhibitor VII 20 μM treatment, independently or combined. Note that cells treated with MG132 do not recover the proliferative capacity while co-treated cells (MG132 + GSK-3β inhibitor VII) restored cell proliferation rate.
Mentions: As showed above, GSK-3β inhibition rescued MCF10A cells from G1/S arrest induced by MG132 (see Fig. 2). Importantly, as we and other have previously demonstrated pharmacological inhibition of GSK-3β activity induced early autophagy activation2425. In order to test whether this inhibition in MCF10A cells would mimic MCF7 proliferative behavior following 24 hours of MG132 treatment, we inhibit GSK-3β and performed proliferation assays. Treatment with the GSK-3β inhibitor for 168 hours did not affect cell proliferation rate, while proteasome inhibition deeply impaired cell growth even 168 hours after drug removal (Fig. 7a). However, when MCF10A cells were simultaneously treated with both inhibitors for 24 hours, and cells were allowed to grow in the presence of the GSK-3β VII inhibitor after MG132 elimination, cell proliferation began to recover reaching up to 20% of control cell number 168 hours later (Fig. 7b). Thus, these data provide strong evidence supporting that chronic GSK-3β inhibition in addition to early autophagy activation could be responsible for the proliferation recovery of both breast epithelial cells following proteasome inhibition, a mechanism that could be essential for cell survival.

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