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Pro-apoptotic and pro-autophagic effects of the Aurora kinase A inhibitor alisertib (MLN8237) on human osteosarcoma U-2 OS and MG-63 cells through the activation of mitochondria-mediated pathway and inhibition of p38 MAPK/PI3K/Akt/mTOR signaling pathway.

Niu NK, Wang ZL, Pan ST, Ding HQ, Au GH, He ZX, Zhou ZW, Xiao G, Yang YX, Zhang X, Yang T, Chen XW, Qiu JX, Zhou SF - Drug Des Devel Ther (2015)

Bottom Line: The results showed that ALS had potent growth inhibitory, pro-apoptotic, pro-autophagic, and EMT inhibitory effects on U-2 OS and MG-63 cells.ALS markedly induced mitochondria-mediated apoptosis with a significant increase in the expression of key pro-apoptotic proteins and a decrease in main anti-apoptotic proteins.ALS is a promising anticancer agent in OS treatment and further studies are needed to confirm its efficacy and safety in OS chemotherapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Spinal Surgery, General Hospital of Ningxia Medical University, Yinchuan, People's Republic of China ; Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL, USA ; Department of Orthopedics, General Hospital of Tianjin Medical University, Tianjin, People's Republic of China.

ABSTRACT
Osteosarcoma (OS) is the most common malignant bone tumor occurring mostly in children and adolescents between 10 and 20 years of age with poor response to current therapeutics. Alisertib (ALS, MLN8237) is a selective Aurora kinase A inhibitor that displays anticancer effects on several types of cancer. However, the role of ALS in the treatment of OS remains unknown. This study aimed to investigate the effects of ALS on the cell growth, apoptosis, autophagy, and epithelial to mesenchymal transition (EMT) and the underlying mechanisms in two human OS cell lines U-2 OS and MG-63. The results showed that ALS had potent growth inhibitory, pro-apoptotic, pro-autophagic, and EMT inhibitory effects on U-2 OS and MG-63 cells. ALS remarkably induced G2/M arrest and down-regulated the expression levels of cyclin-dependent kinases 1 and 2 and cyclin B1 in both U-2 OS and MG-63 cells. ALS markedly induced mitochondria-mediated apoptosis with a significant increase in the expression of key pro-apoptotic proteins and a decrease in main anti-apoptotic proteins. Furthermore, ALS promoted autophagic cell death via the inhibition of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and p38 mitogen-activated protein kinase (p38 MAPK) signaling pathways, and activation of 5'-AMP-dependent kinase (AMPK) signaling pathway. Inducers or inhibitors of apoptosis or autophagy simultaneously altered ALS-induced apoptotic and autophagic death in both U-2 OS and MG-63 cells, suggesting a crosstalk between these two primary modes of programmed cell death. Moreover, ALS suppressed EMT-like phenotypes with a marked increase in the expression of E-cadherin but a decrease in N-cadherin in U-2 OS and MG-63 cells. ALS treatment also induced reactive oxygen species (ROS) generation but inhibited the expression levels of sirtuin 1 and nuclear factor-erythroid-2-related factor 2 (Nrf2) in both cell lines. Taken together, these findings show that ALS promotes apoptosis and autophagy but inhibits EMT via PI3K/Akt/mTOR, p38 MAPK, and AMPK signaling pathways with involvement of ROS- and sirtuin 1-associated pathways in U-2 OS and MG-63 cells. ALS is a promising anticancer agent in OS treatment and further studies are needed to confirm its efficacy and safety in OS chemotherapy.

No MeSH data available.


Related in: MedlinePlus

ALS inhibits the proliferation of U-2 OS and MG-63 cells and induces G2/M arrest in both cell lines.Notes: U-2 OS and MG-63 cells were treated with ALS and then subjected to flow cytometric analysis using PI as the DNA stain. (A) Representative DNA fluorescence histograms showing the effect of ALS treatment on cell cycle distribution of U-2 OS and MG-63 cells. Cells were treated with ALS at 0.1 to 5 μM for 24 hours and then analyzed by flow cytometry and (B) representative DNA fluorescence histograms showing the effect of 5 μM ALS treatment on cell cycle distribution over a 72-hour period. The bar graphs show the percentage of U-2 OS and MG-63 cells in G1, S, and G2/M phases. Cells in sub G1 are those undergoing apoptosis. Data are the mean ± SD of three independent experiments.Abbreviations: ALS, alisertib; SD, standard deviation; hr, hour; DMSO, dimethyl sulfoxide; OS, osteosarcoma; PI, propidium iodide; Dip, diploid.
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f2-dddt-9-1555: ALS inhibits the proliferation of U-2 OS and MG-63 cells and induces G2/M arrest in both cell lines.Notes: U-2 OS and MG-63 cells were treated with ALS and then subjected to flow cytometric analysis using PI as the DNA stain. (A) Representative DNA fluorescence histograms showing the effect of ALS treatment on cell cycle distribution of U-2 OS and MG-63 cells. Cells were treated with ALS at 0.1 to 5 μM for 24 hours and then analyzed by flow cytometry and (B) representative DNA fluorescence histograms showing the effect of 5 μM ALS treatment on cell cycle distribution over a 72-hour period. The bar graphs show the percentage of U-2 OS and MG-63 cells in G1, S, and G2/M phases. Cells in sub G1 are those undergoing apoptosis. Data are the mean ± SD of three independent experiments.Abbreviations: ALS, alisertib; SD, standard deviation; hr, hour; DMSO, dimethyl sulfoxide; OS, osteosarcoma; PI, propidium iodide; Dip, diploid.

Mentions: Following the test of cell viability, the effects of ALS on cell cycle distribution are shown in Figure 2. Incubation of cells with ALS induced G2/M phase arrest and decreased the percentage of cell numbers in G1 and S phases in both U-2 OS and MG-63 cells (P<0.001; Figure 2A). When U-2 OS cells were incubated with ALS at 0.1, 1, and 5 μM, the percentage of cells in G2/M phase was 33.9%, 90.9%, and 91.6%, respectively; the percentage of cells in G1 phase was 46.6%, 3.1%, and 3.7%, respectively; and the percentage of cells in S phase was 20.5%, 6.1%, and 4.8%, respectively. Similarly, compared with the control cells (22.4%), the percentage of MG-63 cells in G2/M phase was increased in a concentration-dependent manner after ALS treatment (P<0.001; Figure 2A). When MG-63 cells were treated with ALS at 0.1, 1, and 5 μM, the percentage of cells in G2/M phase was 33.9%, 90.9%, and 91.6%, respectively. The percentage of cells in G1 phase was decreased from 23.7% at basal level to 20.5%, 6.1%, and 4.8%, and the percentage of MG-63 cells in S phase was reduced from 54.7% at basal level to 46.6%, 3.1%, and 3.7%, when cells were treated with ALS at 0.1, 1, and 5 μM for 24 hours, respectively. There was no significant difference observed in the number of cells in G1, S, and G2/M phases in both U-2 OS and MG-63 cells between the non-treated control cells and the 0.05% DMSO treated cells.


Pro-apoptotic and pro-autophagic effects of the Aurora kinase A inhibitor alisertib (MLN8237) on human osteosarcoma U-2 OS and MG-63 cells through the activation of mitochondria-mediated pathway and inhibition of p38 MAPK/PI3K/Akt/mTOR signaling pathway.

Niu NK, Wang ZL, Pan ST, Ding HQ, Au GH, He ZX, Zhou ZW, Xiao G, Yang YX, Zhang X, Yang T, Chen XW, Qiu JX, Zhou SF - Drug Des Devel Ther (2015)

ALS inhibits the proliferation of U-2 OS and MG-63 cells and induces G2/M arrest in both cell lines.Notes: U-2 OS and MG-63 cells were treated with ALS and then subjected to flow cytometric analysis using PI as the DNA stain. (A) Representative DNA fluorescence histograms showing the effect of ALS treatment on cell cycle distribution of U-2 OS and MG-63 cells. Cells were treated with ALS at 0.1 to 5 μM for 24 hours and then analyzed by flow cytometry and (B) representative DNA fluorescence histograms showing the effect of 5 μM ALS treatment on cell cycle distribution over a 72-hour period. The bar graphs show the percentage of U-2 OS and MG-63 cells in G1, S, and G2/M phases. Cells in sub G1 are those undergoing apoptosis. Data are the mean ± SD of three independent experiments.Abbreviations: ALS, alisertib; SD, standard deviation; hr, hour; DMSO, dimethyl sulfoxide; OS, osteosarcoma; PI, propidium iodide; Dip, diploid.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4362906&req=5

f2-dddt-9-1555: ALS inhibits the proliferation of U-2 OS and MG-63 cells and induces G2/M arrest in both cell lines.Notes: U-2 OS and MG-63 cells were treated with ALS and then subjected to flow cytometric analysis using PI as the DNA stain. (A) Representative DNA fluorescence histograms showing the effect of ALS treatment on cell cycle distribution of U-2 OS and MG-63 cells. Cells were treated with ALS at 0.1 to 5 μM for 24 hours and then analyzed by flow cytometry and (B) representative DNA fluorescence histograms showing the effect of 5 μM ALS treatment on cell cycle distribution over a 72-hour period. The bar graphs show the percentage of U-2 OS and MG-63 cells in G1, S, and G2/M phases. Cells in sub G1 are those undergoing apoptosis. Data are the mean ± SD of three independent experiments.Abbreviations: ALS, alisertib; SD, standard deviation; hr, hour; DMSO, dimethyl sulfoxide; OS, osteosarcoma; PI, propidium iodide; Dip, diploid.
Mentions: Following the test of cell viability, the effects of ALS on cell cycle distribution are shown in Figure 2. Incubation of cells with ALS induced G2/M phase arrest and decreased the percentage of cell numbers in G1 and S phases in both U-2 OS and MG-63 cells (P<0.001; Figure 2A). When U-2 OS cells were incubated with ALS at 0.1, 1, and 5 μM, the percentage of cells in G2/M phase was 33.9%, 90.9%, and 91.6%, respectively; the percentage of cells in G1 phase was 46.6%, 3.1%, and 3.7%, respectively; and the percentage of cells in S phase was 20.5%, 6.1%, and 4.8%, respectively. Similarly, compared with the control cells (22.4%), the percentage of MG-63 cells in G2/M phase was increased in a concentration-dependent manner after ALS treatment (P<0.001; Figure 2A). When MG-63 cells were treated with ALS at 0.1, 1, and 5 μM, the percentage of cells in G2/M phase was 33.9%, 90.9%, and 91.6%, respectively. The percentage of cells in G1 phase was decreased from 23.7% at basal level to 20.5%, 6.1%, and 4.8%, and the percentage of MG-63 cells in S phase was reduced from 54.7% at basal level to 46.6%, 3.1%, and 3.7%, when cells were treated with ALS at 0.1, 1, and 5 μM for 24 hours, respectively. There was no significant difference observed in the number of cells in G1, S, and G2/M phases in both U-2 OS and MG-63 cells between the non-treated control cells and the 0.05% DMSO treated cells.

Bottom Line: The results showed that ALS had potent growth inhibitory, pro-apoptotic, pro-autophagic, and EMT inhibitory effects on U-2 OS and MG-63 cells.ALS markedly induced mitochondria-mediated apoptosis with a significant increase in the expression of key pro-apoptotic proteins and a decrease in main anti-apoptotic proteins.ALS is a promising anticancer agent in OS treatment and further studies are needed to confirm its efficacy and safety in OS chemotherapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Spinal Surgery, General Hospital of Ningxia Medical University, Yinchuan, People's Republic of China ; Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL, USA ; Department of Orthopedics, General Hospital of Tianjin Medical University, Tianjin, People's Republic of China.

ABSTRACT
Osteosarcoma (OS) is the most common malignant bone tumor occurring mostly in children and adolescents between 10 and 20 years of age with poor response to current therapeutics. Alisertib (ALS, MLN8237) is a selective Aurora kinase A inhibitor that displays anticancer effects on several types of cancer. However, the role of ALS in the treatment of OS remains unknown. This study aimed to investigate the effects of ALS on the cell growth, apoptosis, autophagy, and epithelial to mesenchymal transition (EMT) and the underlying mechanisms in two human OS cell lines U-2 OS and MG-63. The results showed that ALS had potent growth inhibitory, pro-apoptotic, pro-autophagic, and EMT inhibitory effects on U-2 OS and MG-63 cells. ALS remarkably induced G2/M arrest and down-regulated the expression levels of cyclin-dependent kinases 1 and 2 and cyclin B1 in both U-2 OS and MG-63 cells. ALS markedly induced mitochondria-mediated apoptosis with a significant increase in the expression of key pro-apoptotic proteins and a decrease in main anti-apoptotic proteins. Furthermore, ALS promoted autophagic cell death via the inhibition of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and p38 mitogen-activated protein kinase (p38 MAPK) signaling pathways, and activation of 5'-AMP-dependent kinase (AMPK) signaling pathway. Inducers or inhibitors of apoptosis or autophagy simultaneously altered ALS-induced apoptotic and autophagic death in both U-2 OS and MG-63 cells, suggesting a crosstalk between these two primary modes of programmed cell death. Moreover, ALS suppressed EMT-like phenotypes with a marked increase in the expression of E-cadherin but a decrease in N-cadherin in U-2 OS and MG-63 cells. ALS treatment also induced reactive oxygen species (ROS) generation but inhibited the expression levels of sirtuin 1 and nuclear factor-erythroid-2-related factor 2 (Nrf2) in both cell lines. Taken together, these findings show that ALS promotes apoptosis and autophagy but inhibits EMT via PI3K/Akt/mTOR, p38 MAPK, and AMPK signaling pathways with involvement of ROS- and sirtuin 1-associated pathways in U-2 OS and MG-63 cells. ALS is a promising anticancer agent in OS treatment and further studies are needed to confirm its efficacy and safety in OS chemotherapy.

No MeSH data available.


Related in: MedlinePlus