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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 regulates the expression of key pro- and anti-autophagic proteins in U-2 OS and MG-63 cells.Notes: U-2 OS and MG-63 cells were treated with ALS at 0.1, 1, and 5 μM for 24 hours and then subjected to Western blotting assay. (A) Representative blots of the phosphorylated PI3K, AMPK, p38 MAPK, Akt, and mTOR and the total protein levels of PI3K, AMPK, p38 MAPK, Akt, mTOR, PTEN, beclin 1, LC3-I, and LC3-II in U-2 OS and MG-63 cells and (B) the ratio of p-PI3K/PI3K, p-AMPK/AMPK, p-p38 MAPK/p38 MAPK, p-Akt/Akt, p-mTOR/mTOR, and LC3-II/I and the expression levels of PTEN and beclin 1 in U-2 OS and MG-63 cells. β-actin was used as the internal control. Data are the mean ± SD of three independent experiments. *P<0.05; **P<0.01; and ***P<0.001 by one-way ANOVA.Abbreviations: ALS, alisertib; SD, standard deviation; ANOVA, analysis of variance; PI3K, phosphatidylinositol 3-kinase; AMPK, AMP-activated protein kinase; MAPK, mitogen-activated protein kinase; Akt, protein kinase B; mTOR, mammalian target of rapamycin; PTEN, phosphatase and tensin homolog; LC3, microtubule-associated protein 1A/1B-light chain 3; OS, osteosarcoma.
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f8-dddt-9-1555: ALS regulates the expression of key pro- and anti-autophagic proteins in U-2 OS and MG-63 cells.Notes: U-2 OS and MG-63 cells were treated with ALS at 0.1, 1, and 5 μM for 24 hours and then subjected to Western blotting assay. (A) Representative blots of the phosphorylated PI3K, AMPK, p38 MAPK, Akt, and mTOR and the total protein levels of PI3K, AMPK, p38 MAPK, Akt, mTOR, PTEN, beclin 1, LC3-I, and LC3-II in U-2 OS and MG-63 cells and (B) the ratio of p-PI3K/PI3K, p-AMPK/AMPK, p-p38 MAPK/p38 MAPK, p-Akt/Akt, p-mTOR/mTOR, and LC3-II/I and the expression levels of PTEN and beclin 1 in U-2 OS and MG-63 cells. β-actin was used as the internal control. Data are the mean ± SD of three independent experiments. *P<0.05; **P<0.01; and ***P<0.001 by one-way ANOVA.Abbreviations: ALS, alisertib; SD, standard deviation; ANOVA, analysis of variance; PI3K, phosphatidylinositol 3-kinase; AMPK, AMP-activated protein kinase; MAPK, mitogen-activated protein kinase; Akt, protein kinase B; mTOR, mammalian target of rapamycin; PTEN, phosphatase and tensin homolog; LC3, microtubule-associated protein 1A/1B-light chain 3; OS, osteosarcoma.

Mentions: Next, we investigated the mechanisms for the autophagy-inducing effect of ALS in U-2 OS and MG-63 cells. We examined the phosphorylation levels of PI3K at Tyr199, AMPK at Thr172, and p38 MAPK at Thr180/Tyr182 which are the upstream signaling molecules of Akt/mTOR pathway and play an important role in regulating cell proliferation and cell death.34 PI3K catalyzes the formation of phos-phatidylinositol-3, 4, 5-triphosphate via phosphorylation of phosphatidylinositol, phosphatidylinositol-4-phosphate, and phosphatidylinositol-4, 5-bisphosphate.29 Growth factors and hormones trigger this phosphorylation event, which in turn coordinates cell growth, cell cycle, cell migration, and cell survival. Incubation of cells with ALS significantly inhibited the phosphorylation of PI3K at Tyr199 in both cell lines in a concentration-dependent manner (Figure 8A). In MG-63 cells, there was a significant decrease in expression level of total PI3K when treated with 5 μM ALS (Figure 8A). However, ALS did not significantly affect the expression of total PI3K in U-2 OS cells. The ratio of p-PI3K over total PI3K was decreased in both cell lines. In U-2 OS cells, the p-PI3K/PI3K ratio was decreased by 42.3% when treated with 5 μM ALS (P<0.001; Figure 8B). In MG-63 cells, 1 and 5 μM ALS decreased the ratio of p-PI3K/PI3K by 53.7% and 30.8%, respectively (P<0.01 or 0.001; Figure 8B).


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 regulates the expression of key pro- and anti-autophagic proteins in U-2 OS and MG-63 cells.Notes: U-2 OS and MG-63 cells were treated with ALS at 0.1, 1, and 5 μM for 24 hours and then subjected to Western blotting assay. (A) Representative blots of the phosphorylated PI3K, AMPK, p38 MAPK, Akt, and mTOR and the total protein levels of PI3K, AMPK, p38 MAPK, Akt, mTOR, PTEN, beclin 1, LC3-I, and LC3-II in U-2 OS and MG-63 cells and (B) the ratio of p-PI3K/PI3K, p-AMPK/AMPK, p-p38 MAPK/p38 MAPK, p-Akt/Akt, p-mTOR/mTOR, and LC3-II/I and the expression levels of PTEN and beclin 1 in U-2 OS and MG-63 cells. β-actin was used as the internal control. Data are the mean ± SD of three independent experiments. *P<0.05; **P<0.01; and ***P<0.001 by one-way ANOVA.Abbreviations: ALS, alisertib; SD, standard deviation; ANOVA, analysis of variance; PI3K, phosphatidylinositol 3-kinase; AMPK, AMP-activated protein kinase; MAPK, mitogen-activated protein kinase; Akt, protein kinase B; mTOR, mammalian target of rapamycin; PTEN, phosphatase and tensin homolog; LC3, microtubule-associated protein 1A/1B-light chain 3; OS, osteosarcoma.
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Related In: Results  -  Collection

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f8-dddt-9-1555: ALS regulates the expression of key pro- and anti-autophagic proteins in U-2 OS and MG-63 cells.Notes: U-2 OS and MG-63 cells were treated with ALS at 0.1, 1, and 5 μM for 24 hours and then subjected to Western blotting assay. (A) Representative blots of the phosphorylated PI3K, AMPK, p38 MAPK, Akt, and mTOR and the total protein levels of PI3K, AMPK, p38 MAPK, Akt, mTOR, PTEN, beclin 1, LC3-I, and LC3-II in U-2 OS and MG-63 cells and (B) the ratio of p-PI3K/PI3K, p-AMPK/AMPK, p-p38 MAPK/p38 MAPK, p-Akt/Akt, p-mTOR/mTOR, and LC3-II/I and the expression levels of PTEN and beclin 1 in U-2 OS and MG-63 cells. β-actin was used as the internal control. Data are the mean ± SD of three independent experiments. *P<0.05; **P<0.01; and ***P<0.001 by one-way ANOVA.Abbreviations: ALS, alisertib; SD, standard deviation; ANOVA, analysis of variance; PI3K, phosphatidylinositol 3-kinase; AMPK, AMP-activated protein kinase; MAPK, mitogen-activated protein kinase; Akt, protein kinase B; mTOR, mammalian target of rapamycin; PTEN, phosphatase and tensin homolog; LC3, microtubule-associated protein 1A/1B-light chain 3; OS, osteosarcoma.
Mentions: Next, we investigated the mechanisms for the autophagy-inducing effect of ALS in U-2 OS and MG-63 cells. We examined the phosphorylation levels of PI3K at Tyr199, AMPK at Thr172, and p38 MAPK at Thr180/Tyr182 which are the upstream signaling molecules of Akt/mTOR pathway and play an important role in regulating cell proliferation and cell death.34 PI3K catalyzes the formation of phos-phatidylinositol-3, 4, 5-triphosphate via phosphorylation of phosphatidylinositol, phosphatidylinositol-4-phosphate, and phosphatidylinositol-4, 5-bisphosphate.29 Growth factors and hormones trigger this phosphorylation event, which in turn coordinates cell growth, cell cycle, cell migration, and cell survival. Incubation of cells with ALS significantly inhibited the phosphorylation of PI3K at Tyr199 in both cell lines in a concentration-dependent manner (Figure 8A). In MG-63 cells, there was a significant decrease in expression level of total PI3K when treated with 5 μM ALS (Figure 8A). However, ALS did not significantly affect the expression of total PI3K in U-2 OS cells. The ratio of p-PI3K over total PI3K was decreased in both cell lines. In U-2 OS cells, the p-PI3K/PI3K ratio was decreased by 42.3% when treated with 5 μM ALS (P<0.001; Figure 8B). In MG-63 cells, 1 and 5 μM ALS decreased the ratio of p-PI3K/PI3K by 53.7% and 30.8%, respectively (P<0.01 or 0.001; Figure 8B).

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