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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

Effects of a series of inducers and inhibitors on the apoptosis and autophagy induced by ALS in U-2 OS and MG-63 cells.Notes: The effects of the compounds on the basal and ALS-induced apoptosis (A) and autophagy (B) of U-2 OS and MG-63 cells; and (C) the bar graphs showing the levels of total apoptosis and autophagy of U-2 OS and MG-63 cells. The apoptosis was determined using the double stain (7-AAD plus annexin V:PE) assay with flow cytometry. The autophagy was determined using the Cyto-ID® green stain with flow cytometry. The flow cytometer collected 10,000 events for apoptosis and autophagy analysis. 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; WM, wortmannin; OS, osteosarcoma; 7-AAD, 7-aminoactinomycin D; PE, phycoerythrin; Q1, debris.
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f10-dddt-9-1555: Effects of a series of inducers and inhibitors on the apoptosis and autophagy induced by ALS in U-2 OS and MG-63 cells.Notes: The effects of the compounds on the basal and ALS-induced apoptosis (A) and autophagy (B) of U-2 OS and MG-63 cells; and (C) the bar graphs showing the levels of total apoptosis and autophagy of U-2 OS and MG-63 cells. The apoptosis was determined using the double stain (7-AAD plus annexin V:PE) assay with flow cytometry. The autophagy was determined using the Cyto-ID® green stain with flow cytometry. The flow cytometer collected 10,000 events for apoptosis and autophagy analysis. 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; WM, wortmannin; OS, osteosarcoma; 7-AAD, 7-aminoactinomycin D; PE, phycoerythrin; Q1, debris.

Mentions: To further explore the mechanisms and potential crosslink between ALS-induced apoptosis and autophagy, we employed several chemical regulators of apoptosis and autophagy in both cell lines. Incubation of U-2 OS and MG-63 cells with 25 nM of FK866 did not affect the basal apoptosis and autophagy compared to the control cells (Figure 9). However, compared to the control cells, when U-2 OS and MG-63 cells were treated with ALS at 5 μM, the total percentage of apoptotic and autophagic cells were both increased (P<0.05, 0.01, or 0.001; Figure 9A and B). Treatment with FK866 did not significantly enhance ALS-induced apoptosis and autophagy in both cell lines. Furthermore, we evaluated the effect of SB202190 (a selective p38 inhibitor and autophagy inducer), WM (a PI3K inhibitor and autophagy blocker), chloroquine (an autophagy inhibitor), bafilomycin A1 (an autophagy inhibitor), and Z-VAD(OMe)-FMK (a pan-caspase inhibitor) on basal and ALS-induced apoptosis in U-2 OS and MG-63 cells (Figure 10A). It was obvious that ALS significantly induced apoptosis compared with the controls in both U-2 OS and MG-63 cells. While only 100 μM bafilomycin A1 significantly reduced ALS-induced apoptosis by 54.6% in U-2 OS cells. Next, we evaluated the effect of autophagy inducers or inhibitors on basal and ALS-induced autophagy in U-2 OS and MG-63 cells. In U-2 OS cells, treatment with 20 μM SB202190 or 10 μM chloroquine for 24 hours induced a 3.7- and 10.0-fold increase in basal autophagy compared to the control cells, respectively (P<0.05 or 0.001; Figure 10B). Bafilomycin A1 at 100 μM, WM at 10 μM, and Z-VAD(OMe)-FMK at 20 μM did not change basal autophagy in U-2 OS cells. Co-incubation of U-2 OS cells with 20 μM SB202190 plus ALS enhanced ALS-induced autophagy by 11.5% (P<0.01; Figure 10B). While 10 μM chloroquine, 100 μM bafilomycin A1, 10 μM WM, and 20 μM Z-VAD(OMe)-FMK reduced ALS-induced autophagy in U-2 OS cells (24.0%, 99.3%, 58.9%, and 41.8%, respectively, P<0.05 or 0.001; Figure 10B). In MG-63 cells, a similar inducing effect of 20 μM SB202190 and 100 μM bafilomycin A1 on basal autophagy was observed. Ten micromolar chloroquine, 10 μM WM, and 20 μM Z-VAD(OMe)-FMK did not affect the basal autophagy. Notably, WM reduced ALS-induced autophagy by 87.4% in MG-63 cells (P<0.001; Figure 10B). Z-VAD(OMe)-FMK at 20 μM did not significantly change ALS-induced autophagy. However, 20 μM SB202190, 100 μM bafilomycin A1, and 10 μM chloroquine increased ALS-induced autophagy (1.6-, 1.4-, and 1.4-fold, respectively, P<0.05 or 0.001; Figure 10B).


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)

Effects of a series of inducers and inhibitors on the apoptosis and autophagy induced by ALS in U-2 OS and MG-63 cells.Notes: The effects of the compounds on the basal and ALS-induced apoptosis (A) and autophagy (B) of U-2 OS and MG-63 cells; and (C) the bar graphs showing the levels of total apoptosis and autophagy of U-2 OS and MG-63 cells. The apoptosis was determined using the double stain (7-AAD plus annexin V:PE) assay with flow cytometry. The autophagy was determined using the Cyto-ID® green stain with flow cytometry. The flow cytometer collected 10,000 events for apoptosis and autophagy analysis. 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; WM, wortmannin; OS, osteosarcoma; 7-AAD, 7-aminoactinomycin D; PE, phycoerythrin; Q1, debris.
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Related In: Results  -  Collection

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f10-dddt-9-1555: Effects of a series of inducers and inhibitors on the apoptosis and autophagy induced by ALS in U-2 OS and MG-63 cells.Notes: The effects of the compounds on the basal and ALS-induced apoptosis (A) and autophagy (B) of U-2 OS and MG-63 cells; and (C) the bar graphs showing the levels of total apoptosis and autophagy of U-2 OS and MG-63 cells. The apoptosis was determined using the double stain (7-AAD plus annexin V:PE) assay with flow cytometry. The autophagy was determined using the Cyto-ID® green stain with flow cytometry. The flow cytometer collected 10,000 events for apoptosis and autophagy analysis. 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; WM, wortmannin; OS, osteosarcoma; 7-AAD, 7-aminoactinomycin D; PE, phycoerythrin; Q1, debris.
Mentions: To further explore the mechanisms and potential crosslink between ALS-induced apoptosis and autophagy, we employed several chemical regulators of apoptosis and autophagy in both cell lines. Incubation of U-2 OS and MG-63 cells with 25 nM of FK866 did not affect the basal apoptosis and autophagy compared to the control cells (Figure 9). However, compared to the control cells, when U-2 OS and MG-63 cells were treated with ALS at 5 μM, the total percentage of apoptotic and autophagic cells were both increased (P<0.05, 0.01, or 0.001; Figure 9A and B). Treatment with FK866 did not significantly enhance ALS-induced apoptosis and autophagy in both cell lines. Furthermore, we evaluated the effect of SB202190 (a selective p38 inhibitor and autophagy inducer), WM (a PI3K inhibitor and autophagy blocker), chloroquine (an autophagy inhibitor), bafilomycin A1 (an autophagy inhibitor), and Z-VAD(OMe)-FMK (a pan-caspase inhibitor) on basal and ALS-induced apoptosis in U-2 OS and MG-63 cells (Figure 10A). It was obvious that ALS significantly induced apoptosis compared with the controls in both U-2 OS and MG-63 cells. While only 100 μM bafilomycin A1 significantly reduced ALS-induced apoptosis by 54.6% in U-2 OS cells. Next, we evaluated the effect of autophagy inducers or inhibitors on basal and ALS-induced autophagy in U-2 OS and MG-63 cells. In U-2 OS cells, treatment with 20 μM SB202190 or 10 μM chloroquine for 24 hours induced a 3.7- and 10.0-fold increase in basal autophagy compared to the control cells, respectively (P<0.05 or 0.001; Figure 10B). Bafilomycin A1 at 100 μM, WM at 10 μM, and Z-VAD(OMe)-FMK at 20 μM did not change basal autophagy in U-2 OS cells. Co-incubation of U-2 OS cells with 20 μM SB202190 plus ALS enhanced ALS-induced autophagy by 11.5% (P<0.01; Figure 10B). While 10 μM chloroquine, 100 μM bafilomycin A1, 10 μM WM, and 20 μM Z-VAD(OMe)-FMK reduced ALS-induced autophagy in U-2 OS cells (24.0%, 99.3%, 58.9%, and 41.8%, respectively, P<0.05 or 0.001; Figure 10B). In MG-63 cells, a similar inducing effect of 20 μM SB202190 and 100 μM bafilomycin A1 on basal autophagy was observed. Ten micromolar chloroquine, 10 μM WM, and 20 μM Z-VAD(OMe)-FMK did not affect the basal autophagy. Notably, WM reduced ALS-induced autophagy by 87.4% in MG-63 cells (P<0.001; Figure 10B). Z-VAD(OMe)-FMK at 20 μM did not significantly change ALS-induced autophagy. However, 20 μM SB202190, 100 μM bafilomycin A1, and 10 μM chloroquine increased ALS-induced autophagy (1.6-, 1.4-, and 1.4-fold, respectively, P<0.05 or 0.001; Figure 10B).

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