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The role of reactive oxygen species and autophagy in safingol-induced cell death.

Ling LU, Tan KB, Lin H, Chiu GN - Cell Death Dis (2011)

Bottom Line: Our data also suggested that autophagy was a cell repair mechanism, as suppression of autophagy by 3-methyladenine or bafilomycin A1 significantly augmented cell death on 2-5 μM safingol treatment.Finally, glucose uptake was shown to be inhibited by safingol treatment, which was associated with an increase in p-AMPK expression.Taken together, our data suggested that ROS was the mediator of safingol-induced cancer cell death, and autophagy is likely to be a mechanism triggered to repair damages from ROS generation on safingol treatment.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacy, Faculty of Science, National University of Singapore, Singapore, Singapore.

ABSTRACT
Safingol is a sphingolipid with promising anticancer potential, which is currently in phase I clinical trial. Yet, the underlying mechanisms of its action remain largely unknown. We reported here that safingol-induced primarily accidental necrotic cell death in MDA-MB-231 and HT-29 cells, as shown by the increase in the percentage of cells stained positive for 7-aminoactinomycin D, collapse of mitochondria membrane potential and depletion of intracellular ATP. Importantly, safingol treatment produced time- and concentration-dependent reactive oxygen species (ROS) generation. Autophagy was triggered following safingol treatment, as reflected by the formation of autophagosomes, acidic vacuoles, increased light chain 3-II and Atg biomarkers expression. Interestingly, scavenging ROS with N-acetyl-L-cysteine could prevent the autophagic features and reverse safingol-induced necrosis. Our data also suggested that autophagy was a cell repair mechanism, as suppression of autophagy by 3-methyladenine or bafilomycin A1 significantly augmented cell death on 2-5 μM safingol treatment. In addition, Bcl-xL and Bax might be involved in the regulation of safingol-induced autophagy. Finally, glucose uptake was shown to be inhibited by safingol treatment, which was associated with an increase in p-AMPK expression. Taken together, our data suggested that ROS was the mediator of safingol-induced cancer cell death, and autophagy is likely to be a mechanism triggered to repair damages from ROS generation on safingol treatment.

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ROS trigger autophagy induction in MDA-MB-231 and HT-29 cells. (a) Detection of AVO in safingol-treated cells in the presence of NAC. Cells were treated with safingol±10 m NAC for 24 h before stained with 1 μg/ml acridine orange for 15 min. Cells were examined by fluorescence microscopy. Representative images of cells from three independent experiments were shown. Bar=50 μm. (b) Downregulation of LC3-II in cells in the presence of NAC. Cells were treated with safingol±10 m NAC for 48 h. Protein lysates were assayed by western blotting and β-actin was used as the loading control. All blots shown are representative of three independent experiments
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fig4: ROS trigger autophagy induction in MDA-MB-231 and HT-29 cells. (a) Detection of AVO in safingol-treated cells in the presence of NAC. Cells were treated with safingol±10 m NAC for 24 h before stained with 1 μg/ml acridine orange for 15 min. Cells were examined by fluorescence microscopy. Representative images of cells from three independent experiments were shown. Bar=50 μm. (b) Downregulation of LC3-II in cells in the presence of NAC. Cells were treated with safingol±10 m NAC for 48 h. Protein lysates were assayed by western blotting and β-actin was used as the loading control. All blots shown are representative of three independent experiments

Mentions: On the basis of the results presented in Figure 2, we postulated that autophagy would be triggered in the cancer cells on safingol treatment, as ROS could induce cellular damages.2 As demonstrated through the ultrastructural morphology using transmission electron microscopy, safingol-treated MDA-MB-231 and HT-29 cells displayed characteristic autophagosomes, as well as cytoplasmic vacuoles (Figure 3a). Concentration-dependent formation of acidic vesicular organelles (AVOs) could be observed in safingol-treated cells when stained with the lysosomo-tropic agent, acridine orange,28 and formation of AVO was markedly suppressed in both cell lines in the presence of 1 m 3-methyladenine (3-MA), the most commonly used autophagy inhibitor29 (Figure 3b). Induction of autophagy is further supported by the conversion of light chain 3 (LC3)-I to LC3-II in a concentration- and time-dependent manner on safingol treatment in the two cell lines (Figures 3c and d). A trend of increasing expression of the Atg proteins (Atg 5, 7 and 12) in MDA-MB-231 and HT-29 cells over the course of 48 h was also observed on safingol treatment (Figure 3d). Importantly, in the presence of the ROS scavenger, N-acetyl--cysteine (NAC), marked reduction in the formation of AVO and in the conversion of LC3-I to LC3-II could be demonstrated in MDA-MB-231 and HT-29 cells (Figures 4a and b). Taken together, our results have demonstrated that safingol is capable of inducing autophagy, which are in line with two recent reports,17, 18 and that autophagy is induced as a result of ROS generation on safingol treatment.


The role of reactive oxygen species and autophagy in safingol-induced cell death.

Ling LU, Tan KB, Lin H, Chiu GN - Cell Death Dis (2011)

ROS trigger autophagy induction in MDA-MB-231 and HT-29 cells. (a) Detection of AVO in safingol-treated cells in the presence of NAC. Cells were treated with safingol±10 m NAC for 24 h before stained with 1 μg/ml acridine orange for 15 min. Cells were examined by fluorescence microscopy. Representative images of cells from three independent experiments were shown. Bar=50 μm. (b) Downregulation of LC3-II in cells in the presence of NAC. Cells were treated with safingol±10 m NAC for 48 h. Protein lysates were assayed by western blotting and β-actin was used as the loading control. All blots shown are representative of three independent experiments
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3101809&req=5

fig4: ROS trigger autophagy induction in MDA-MB-231 and HT-29 cells. (a) Detection of AVO in safingol-treated cells in the presence of NAC. Cells were treated with safingol±10 m NAC for 24 h before stained with 1 μg/ml acridine orange for 15 min. Cells were examined by fluorescence microscopy. Representative images of cells from three independent experiments were shown. Bar=50 μm. (b) Downregulation of LC3-II in cells in the presence of NAC. Cells were treated with safingol±10 m NAC for 48 h. Protein lysates were assayed by western blotting and β-actin was used as the loading control. All blots shown are representative of three independent experiments
Mentions: On the basis of the results presented in Figure 2, we postulated that autophagy would be triggered in the cancer cells on safingol treatment, as ROS could induce cellular damages.2 As demonstrated through the ultrastructural morphology using transmission electron microscopy, safingol-treated MDA-MB-231 and HT-29 cells displayed characteristic autophagosomes, as well as cytoplasmic vacuoles (Figure 3a). Concentration-dependent formation of acidic vesicular organelles (AVOs) could be observed in safingol-treated cells when stained with the lysosomo-tropic agent, acridine orange,28 and formation of AVO was markedly suppressed in both cell lines in the presence of 1 m 3-methyladenine (3-MA), the most commonly used autophagy inhibitor29 (Figure 3b). Induction of autophagy is further supported by the conversion of light chain 3 (LC3)-I to LC3-II in a concentration- and time-dependent manner on safingol treatment in the two cell lines (Figures 3c and d). A trend of increasing expression of the Atg proteins (Atg 5, 7 and 12) in MDA-MB-231 and HT-29 cells over the course of 48 h was also observed on safingol treatment (Figure 3d). Importantly, in the presence of the ROS scavenger, N-acetyl--cysteine (NAC), marked reduction in the formation of AVO and in the conversion of LC3-I to LC3-II could be demonstrated in MDA-MB-231 and HT-29 cells (Figures 4a and b). Taken together, our results have demonstrated that safingol is capable of inducing autophagy, which are in line with two recent reports,17, 18 and that autophagy is induced as a result of ROS generation on safingol treatment.

Bottom Line: Our data also suggested that autophagy was a cell repair mechanism, as suppression of autophagy by 3-methyladenine or bafilomycin A1 significantly augmented cell death on 2-5 μM safingol treatment.Finally, glucose uptake was shown to be inhibited by safingol treatment, which was associated with an increase in p-AMPK expression.Taken together, our data suggested that ROS was the mediator of safingol-induced cancer cell death, and autophagy is likely to be a mechanism triggered to repair damages from ROS generation on safingol treatment.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacy, Faculty of Science, National University of Singapore, Singapore, Singapore.

ABSTRACT
Safingol is a sphingolipid with promising anticancer potential, which is currently in phase I clinical trial. Yet, the underlying mechanisms of its action remain largely unknown. We reported here that safingol-induced primarily accidental necrotic cell death in MDA-MB-231 and HT-29 cells, as shown by the increase in the percentage of cells stained positive for 7-aminoactinomycin D, collapse of mitochondria membrane potential and depletion of intracellular ATP. Importantly, safingol treatment produced time- and concentration-dependent reactive oxygen species (ROS) generation. Autophagy was triggered following safingol treatment, as reflected by the formation of autophagosomes, acidic vacuoles, increased light chain 3-II and Atg biomarkers expression. Interestingly, scavenging ROS with N-acetyl-L-cysteine could prevent the autophagic features and reverse safingol-induced necrosis. Our data also suggested that autophagy was a cell repair mechanism, as suppression of autophagy by 3-methyladenine or bafilomycin A1 significantly augmented cell death on 2-5 μM safingol treatment. In addition, Bcl-xL and Bax might be involved in the regulation of safingol-induced autophagy. Finally, glucose uptake was shown to be inhibited by safingol treatment, which was associated with an increase in p-AMPK expression. Taken together, our data suggested that ROS was the mediator of safingol-induced cancer cell death, and autophagy is likely to be a mechanism triggered to repair damages from ROS generation on safingol treatment.

Show MeSH
Related in: MedlinePlus