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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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Cell death mechanism induced by safingol treatment in MDA-MB-231 and HT-29 cells. (a) Flow cytometric analysis of MDA-MB-231 and HT-29 cells after 2, 5 and 10 μ safingol treatment for 48 h. In all, 2% paraformaldehyde was used as a positive control to induce necrosis. Cells were harvested and stained with annexin V-FITC/7AAD before analyzed by flow cytometry. Results from treatment with 10 μ safingol for 24 h in the two cell lines are shown in the inset. Each reported value represents the mean±S.E.M. from three independent experiments. (b) Effect of safingol on MMP in HT-29 cells. Cells were treated with 10 μ safingol for 8, 24 and 48 h, stained with JC-1 and subjected to flow cytometry. Representative dot plots from three independent experiments were shown, and numerical values represent means±S.E.M. (n=3). (c) Effect of safingol on intracellular ATP. Cells were treated with 2, 5 and 10 μ safingol for 48 h before measurement of ATP by a luminometric assay. Results shown are means±S.E.M. from three independent experiments. (d) Effect of necrostatin-1 on the viability of safingol-treated cells. Cells were treated with safingol±100 μ necrostatin-1 for 48 h. Viability was assessed using MTT assay. Results shown are means±S.E.M. from three independent experiments. *P<0.05, compared with untreated control
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fig1: Cell death mechanism induced by safingol treatment in MDA-MB-231 and HT-29 cells. (a) Flow cytometric analysis of MDA-MB-231 and HT-29 cells after 2, 5 and 10 μ safingol treatment for 48 h. In all, 2% paraformaldehyde was used as a positive control to induce necrosis. Cells were harvested and stained with annexin V-FITC/7AAD before analyzed by flow cytometry. Results from treatment with 10 μ safingol for 24 h in the two cell lines are shown in the inset. Each reported value represents the mean±S.E.M. from three independent experiments. (b) Effect of safingol on MMP in HT-29 cells. Cells were treated with 10 μ safingol for 8, 24 and 48 h, stained with JC-1 and subjected to flow cytometry. Representative dot plots from three independent experiments were shown, and numerical values represent means±S.E.M. (n=3). (c) Effect of safingol on intracellular ATP. Cells were treated with 2, 5 and 10 μ safingol for 48 h before measurement of ATP by a luminometric assay. Results shown are means±S.E.M. from three independent experiments. (d) Effect of necrostatin-1 on the viability of safingol-treated cells. Cells were treated with safingol±100 μ necrostatin-1 for 48 h. Viability was assessed using MTT assay. Results shown are means±S.E.M. from three independent experiments. *P<0.05, compared with untreated control

Mentions: Previously, safingol was shown to induce cell death in a caspase-independent manner,17, 19, 20, 26 and we could obtain similar results in MDA-MB-231 and HT-29 cells with specific inhibitors to caspases 8 and 9 and pan-caspase inhibitor (data not shown). In order to characterize the mechanism by which safingol exerts its cell killing effect, we performed flow cytometric analysis after staining the cells with annexin V-FITC/7-aminoactinomycin (7AAD). Paraformaldehyde 2% was included as a positive control for inducing necrosis in cells. No significant increase in the percentage of cells stained positive for annexin V-FITC was observed in MDA-MB-231 and HT-29 cells after a 48-h exposure to safingol (Figure 1a). In contrast, concentration-dependent increase in the percentage of cells stained positive for 7AAD was observed (Figure 1a), indicating a loss of cell membrane integrity that is suggestive of necrotic cell death. A shorter treatment time of 24 h with 10 μ safingol yielded similar results (Figure 1 inset), indicating that plasma membrane permeabilization, as reflected by 7AAD positivity, occurred early on as opposed to delayed occurrence that is associated with late-stage apoptosis. As the loss of MMP and depletion of ATP have been described as the hallmarks of necrosis,3, 4 we further performed measurements on these two biochemical events. Remarkable loss of MMP in HT-29 cells was observed after a 48-h exposure to 10 μ safingol (Figure 1b). Significant depletion of cellular ATP was also observed in both cell lines with 10 μ safingol (Figure 1c). Taken together, these data suggest that necrosis, rather than apoptosis, is the main mode through which cancer cell death occurred on safingol treatment. We further examined whether the execution of necrotic cell death is a programmed event that involves RIP1 kinase. Cells were exposed to safingol with or without necrostatin-1, a small molecule inhibitor of RIP1 kinase.27 Our results indicated that necrostatin-1 did not prevent safingol-induced cell death (Figure 1d), thus suggesting an accidental form of necrotic cell death.


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)

Cell death mechanism induced by safingol treatment in MDA-MB-231 and HT-29 cells. (a) Flow cytometric analysis of MDA-MB-231 and HT-29 cells after 2, 5 and 10 μ safingol treatment for 48 h. In all, 2% paraformaldehyde was used as a positive control to induce necrosis. Cells were harvested and stained with annexin V-FITC/7AAD before analyzed by flow cytometry. Results from treatment with 10 μ safingol for 24 h in the two cell lines are shown in the inset. Each reported value represents the mean±S.E.M. from three independent experiments. (b) Effect of safingol on MMP in HT-29 cells. Cells were treated with 10 μ safingol for 8, 24 and 48 h, stained with JC-1 and subjected to flow cytometry. Representative dot plots from three independent experiments were shown, and numerical values represent means±S.E.M. (n=3). (c) Effect of safingol on intracellular ATP. Cells were treated with 2, 5 and 10 μ safingol for 48 h before measurement of ATP by a luminometric assay. Results shown are means±S.E.M. from three independent experiments. (d) Effect of necrostatin-1 on the viability of safingol-treated cells. Cells were treated with safingol±100 μ necrostatin-1 for 48 h. Viability was assessed using MTT assay. Results shown are means±S.E.M. from three independent experiments. *P<0.05, compared with untreated control
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig1: Cell death mechanism induced by safingol treatment in MDA-MB-231 and HT-29 cells. (a) Flow cytometric analysis of MDA-MB-231 and HT-29 cells after 2, 5 and 10 μ safingol treatment for 48 h. In all, 2% paraformaldehyde was used as a positive control to induce necrosis. Cells were harvested and stained with annexin V-FITC/7AAD before analyzed by flow cytometry. Results from treatment with 10 μ safingol for 24 h in the two cell lines are shown in the inset. Each reported value represents the mean±S.E.M. from three independent experiments. (b) Effect of safingol on MMP in HT-29 cells. Cells were treated with 10 μ safingol for 8, 24 and 48 h, stained with JC-1 and subjected to flow cytometry. Representative dot plots from three independent experiments were shown, and numerical values represent means±S.E.M. (n=3). (c) Effect of safingol on intracellular ATP. Cells were treated with 2, 5 and 10 μ safingol for 48 h before measurement of ATP by a luminometric assay. Results shown are means±S.E.M. from three independent experiments. (d) Effect of necrostatin-1 on the viability of safingol-treated cells. Cells were treated with safingol±100 μ necrostatin-1 for 48 h. Viability was assessed using MTT assay. Results shown are means±S.E.M. from three independent experiments. *P<0.05, compared with untreated control
Mentions: Previously, safingol was shown to induce cell death in a caspase-independent manner,17, 19, 20, 26 and we could obtain similar results in MDA-MB-231 and HT-29 cells with specific inhibitors to caspases 8 and 9 and pan-caspase inhibitor (data not shown). In order to characterize the mechanism by which safingol exerts its cell killing effect, we performed flow cytometric analysis after staining the cells with annexin V-FITC/7-aminoactinomycin (7AAD). Paraformaldehyde 2% was included as a positive control for inducing necrosis in cells. No significant increase in the percentage of cells stained positive for annexin V-FITC was observed in MDA-MB-231 and HT-29 cells after a 48-h exposure to safingol (Figure 1a). In contrast, concentration-dependent increase in the percentage of cells stained positive for 7AAD was observed (Figure 1a), indicating a loss of cell membrane integrity that is suggestive of necrotic cell death. A shorter treatment time of 24 h with 10 μ safingol yielded similar results (Figure 1 inset), indicating that plasma membrane permeabilization, as reflected by 7AAD positivity, occurred early on as opposed to delayed occurrence that is associated with late-stage apoptosis. As the loss of MMP and depletion of ATP have been described as the hallmarks of necrosis,3, 4 we further performed measurements on these two biochemical events. Remarkable loss of MMP in HT-29 cells was observed after a 48-h exposure to 10 μ safingol (Figure 1b). Significant depletion of cellular ATP was also observed in both cell lines with 10 μ safingol (Figure 1c). Taken together, these data suggest that necrosis, rather than apoptosis, is the main mode through which cancer cell death occurred on safingol treatment. We further examined whether the execution of necrotic cell death is a programmed event that involves RIP1 kinase. Cells were exposed to safingol with or without necrostatin-1, a small molecule inhibitor of RIP1 kinase.27 Our results indicated that necrostatin-1 did not prevent safingol-induced cell death (Figure 1d), thus suggesting an accidental form of necrotic cell death.

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