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Figure 1. Stichoposide D (STD) induces apoptosis of K562 and HL-60 cells through the activation of ceramide synthase 6 (CerS6)A. Structure of STD. B. K562 and HL-60 cells (1 × 105 cells/well) were each incubated with various concentrations (0, 0.3, 0.5, 1.0, 1.5 μM) of STD for 24 h or 6 h. After treatment for 24 h, cell viability was determined by MTT assay (upper panel). After treatment for 6 h, the percentage of apoptotic cells was determined by Annexin V-FITC/PI staining (lower panel). These data represent the mean ± SD of three independent experiments. IC50 of STD in each cell is indicated. *P < 0.05, **P < 0.01, ***P < 0.001 versus control. C–F. K562 and HL-60 cells were transiently transfected by electroporation with no siRNA (shock), nonspecific control (NC) siRNA, CerS6 siRNA-1, or CerS6 siRNA-2 for 48 h. (C) Western blot analysis of protein lysates. (D) Transfected K562 cells were exposed to 1.0 μM STD for 2 h and fixed. After permeabilization, samples were stained with PE-anti-CerS6, ceramide, or Fas antibodies and with Alexa 488-labeled cholera toxin B antibody. The pictures are representative of three separate experiments. (E) Left panel: The culture medium was changed, and K562 and HL-60 cells were treated with or without STD (1.0 μM and 1.5 μM, respectively) for 6 h. The percentage of apoptotic cells was determined by annexin V-FITC/PI staining. Results are the mean ± SD of three independent experiments. *P < 0.05, ***P < 0.001, cells treated with STD alone versus cells transfected with CerS6–1 or CerS6–2 siRNA and treated with STD. Right panel: Cells stained with DiOC6. Reduction of Djm was determined by monitoring DiOC6 uptake using flow cytometry. Low Djm values are expressed as the percentage of cells exhibiting diminished mitochondrial potential. Results are the mean ± SD of three independent experiments. ***P < 0.001 for cells treated with STD alone vs. cells transfected with CerS6–1 siRNA and treated with STD. (F) Western blot for mitochondrial proteins (AIF, Smac/DIABLO, cytochrome oxidase IV, and cytochrome c). Cytochrome oxidase (COX IV) was used as a mitochondrial marker. Western blots (C, F) are each representative of three separate experiments; equal protein loading was ensured by demonstrating uniform β-actin expression. Densitometry results are expressed above the bands.
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Figure 2. Knockdown of Fas can inhibit STD-induced apoptosis in K562 and HL-60 cellsK562 and HL-60 cells were transiently transfected by electroporation with no siRNA (shock), nonspecific control (NC) siRNA, or Fas siRNA for 48 h. A. Western blot analysis of protein lysates. B. Transfected K562 cells were treated with STD for 2 h and fixed. After permeabilization, samples were stained with PE-anti-Fas, CerS6, or ceramide antibodies and Alexa 488-labeled cholera toxin B antibody. The pictures are representative of three separate experiments. C. The culture medium was changed, and cells were treated with or without STD for 6 h. The percentage of apoptotic cells was determined by annexin V-FITC/PI staining. Results are the mean ± SD of three independent experiments. *P < 0.05, ***P < 0.001, cells treated with STD versus cells transfected with Fas siRNA and treated with STD. D. Transfected K562 and HL-60 cells treated with or without STD for 6 h. Cells were stained with DiOC6, and reduction of Δψm was determined by monitoring DiOC6 uptake using flow cytometry. Low Δψm values are expressed as the percentage of cells exhibiting diminished mitochondrial potential. The values obtained from the DiOC6 assays represent the mean ± SD of three independent experiments. ***P < 0.001, cells treated with STD versus cells transfected with Fas siRNA and treated with STD. E. Western blot for mitochondrial proteins (AIF, Smac/DIABLO, cytochrome oxidase IV, and cytochrome c). Cytochrome oxidase IV (COX IV) was used as a mitochondrial marker. Western blots (A, E) are each representative of three separate experiments; equal protein loading was ensured by demonstrating uniform β-actin expression. Densitometry results are expressed above the bands.
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Figure 3. STD induces apoptosis of K562 and HL-60 cells through the activation of p38 kinaseA. K562 and HL-60 cells were treated with STD for the indicated times. Protein lysates were prepared and subjected to Western blot analysis. Equal protein loading was ensured by showing uniform β-actin expression. The blot is representative of three separate experiments. B. K562 and HL-60 cells (1 × 105 cells/well) were pretreated with SB203580 (a p38 kinase inhibitor), SP600125 (a JNK inhibitor), or PD98059 (an ERK inhibitor) before treatment with STD (1.0 μM, 1.5 μM, respectively) for 6 h. After treatment for the indicated times, the percentage of apoptotic cells was determined by Annexin V-FITC/PI staining. Upper panel: mean ± SD of three independent experiments. ***P < 0.001, versus cells treated with STD in the absence of SB203580. Lower panel: representative of three independent experiments. C. In parallel, whole cell lysates from K562 and HL-60 cells incubated with STD for 6 h in the presence or absence of SB203580 were prepared and analyzed by Western blot. In each case, 30 μg of protein was separated by SDS-PAGE, after which blots were probed with the corresponding antibodies. Blots were subsequently stripped and reprobed with antibodies directed against β-actin to ensure equivalent loading and transfer. D. K562 cells were treated with STD for 2 h in the presence or absence of SB203580 and fixed. After permeabilization, samples were stained with PE-anti-Fas, CerS6, ceramide, p-p38, or caspase-8 antibodies and Alexa 488-labeled cholera toxin B antibody. The pictures are representative of three separate experiments. E. K562 and HL-60 cells were pretreated with SB203580 for 1 h before treatment with STD for 6 h. Cells were stained with DiOC6, and reduction of Δψm was determined by monitoring DiOC6 uptake using flow cytometry. Low Δψm values are expressed as the percentage of cells exhibiting diminished mitochondrial potential. The values obtained from the DiOC6 assays represent the mean ± SD of three independent experiments. **P < 0.01, ***P < 0.001 versus cells treated with STD in the absence of SB203580. F. Western blot for mitochondrial proteins (AIF, Smac/DIABLO, cytochrome oxidase IV, and cytochrome c). Cytochrome oxidase IV (COX IV) was used as a mitochondrial marker. Western blots (C, F) are each representative of three separate experiments; equal protein loading was ensured by demonstrating uniform β-actin expression. Densitometry results are expressed above the bands.
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Figure 4. Knockdown of p38 kinase can inhibit STD-induced apoptosis in K562 and HL-60 cellsK562 and HL-60 cells were transiently transfected by electroporation with no siRNA (shock), nonspecific control (NC) siRNA, or p38 siRNA for 48 h. A. Transfected K562 cells were treated with or without STD for 6 h. Protein lysates were prepared and subjected to Western blot analysis. B. The culture medium was changed, and cells were treated with or without STD for 6 h. The percentage of apoptotic cells was determined by annexin V-FITC/PI staining. Left panel: representative of three independent experiments in each cell line. Right panel: mean ± SD of three independent experiments. **P < 0.01 and ***P < 0.001, cells treated with STD versus cells transfected with p38 kinase siRNA and treated with STD. C. The transfected K562 and HL-60 cells were treated with or without STD for 6 h. Cells were stained with DiOC6, and reduction of Δψm was determined by monitoring DiOC6 uptake using flow cytometry. Low Δψm values are expressed as the percentage of cells exhibiting diminished mitochondrial potential. The values obtained from the DiOC6 assays represent the mean ± SD of three independent experiments. **P < 0.01 and ***P < 0.001, cells treated with STD versus cells transfected with p38 kinase siRNA and treated with STD. D. Western blot analysis for mitochondrial proteins (AIF, Smac/DIABLO, cytochrome oxidase IV, and cytochrome c). Cytochrome oxidase IV (COX IV) was used as a mitochondrial marker. Western blots (A, D) are each representative of three separate experiments; equal protein loading was ensured by demonstrating uniform β-actin expression. Densitometry results are expressed above the bands.
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Figure 5. Clustering of Fas and its downstream molecules in lipid rafts during STD-induced apoptosisA. K562 and HL-60 cells were pretreated with MβCD (20 μg/ml) and nystatin (20 μg/ml) for 1 h and cultured in medium containing STD (1.0 μM, 1.5 μM, respectively) for 6 h. After treatment for the indicated times, the percentage of apoptotic cells was determined by Annexin V-FITC/PI staining. Upper panel: representative of three independent experiments. Lower panel: mean ± SD of three independent experiments. **P < 0.01; ***P < 0.001 versus STD-treated cells. B. Whole cell lysates from K562 cells incubated with STD for 6 h in the presence or absence of MβCD were prepared in parallel and subjected to Western blot analysis. C. K562 cells were treated with STD in the presence or absence of MβCD or nystatin for 2 h and fixed. After permeabilization, samples were stained with PE-anti-Fas, CerS6, ceramide, p-p38, or cleaved caspase-8 antibodies and Alexa 488-labeled cholera toxin B antibody. The pictures are representative of three separate experiments. D. Cells were pretreated with MβCD or nystatin for 1 h before treatment with STD for 6 h. Cells were stained with DiOC6, and reduction of Δψm was determined by monitoring DiOC6 uptake using flow cytometry. Low Δψm values are expressed as the percentage of cells exhibiting diminished mitochondrial potential. The values obtained from the DiOC6 assays represent the mean ± SD of three independent experiments. *P < 0.05 and **P < 0.01 versus STD-treated cells. E. Western blot analysis for mitochondrial proteins (AIF, Smac/DIABLO, cytochrome oxidase IV, and cytochrome c). Cytochrome oxidase IV (COX IV) was used as a mitochondrial marker. Western blots (B, E) are each representative of three separate experiments; equal protein loading was ensured by demonstrating uniform β-actin expression. Densitometry results are expressed above the bands.
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Figure 8. Possible molecular mechanisms of STD-induced apoptosis in human leukemia cellsSTD induces apoptosis of human leukemic cells through ceramide generation by activation of CerS6 from Fas clustering and activation of p38 kinase in the lipid rafts.
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