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Ceramide synthase 6 modulates TRAIL sensitivity and nuclear translocation of active caspase-3 in colon cancer cells.

White-Gilbertson S, Mullen T, Senkal C, Lu P, Ogretmen B, Obeid L, Voelkel-Johnson C - Oncogene (2009)

Bottom Line: RNAi against CerS6 resulted in a specific and significant decrease of the C(16)-ceramide species, which was sufficient to inhibit TRAIL-induced apoptosis.In cells with decreased levels of CerS6, caspase-3 was activated but failed to translocate into the nucleus.These results suggest that modulation of CerS6 expression may constitute a new therapeutic strategy to alter apoptotic susceptibility.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Medical University of South Carolina, Charleston, SC 29403, USA.

ABSTRACT
We have previously shown that the death receptor ligand TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) induces an increase of intracellular C(16)-ceramide in sensitive SW480 but not in resistant SW620 cells. Resistance in SW620 cells was overcome by exogenous ceramide, leading us to propose that defective ceramide signaling contributes to TRAIL resistance. In this study we found that the increase in C(16)-ceramide in SW480 cells was inhibited by fumonisin B1, an inhibitor of ceramide synthases (CerS). Protein analysis revealed that TRAIL-resistant SW620 cells expressed lower levels of ceramide synthase 6 (CerS6, also known as longevity assurance homologue 6), which prompted us to investigate the effect of CerS6 modulation on TRAIL phenotype. RNAi against CerS6 resulted in a specific and significant decrease of the C(16)-ceramide species, which was sufficient to inhibit TRAIL-induced apoptosis. In cells with decreased levels of CerS6, caspase-3 was activated but failed to translocate into the nucleus. CerS6 localized primarily to the perinuclear region, suggesting this enzyme may be important in regulation of nuclear permeability. Moderate elevation in CerS6 expression was sufficient to reverse TRAIL resistance in SW620 cells. These results suggest that modulation of CerS6 expression may constitute a new therapeutic strategy to alter apoptotic susceptibility.

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Overexpression of CerS6 in SW620 cells results in sensitization to TRAIL. (A) SW620 cells were transiently transfected with pIRES-GFP as a negative control or pCerS6-IRES-GFP. Cells were visualized at a magnification of 200X 24 hours after treatment with 100 ng/ml TRAIL was initiated. Representative images from one of three experiments are shown. (B/C) SW620 mass clones stably transfected with pIRES-GFP or pCerS6-IRES-GFP were treated with 100 ng/ml TRAIL for 4 hours and examined for morphological evidence of apoptosis (B) or PARP cleavage by western blot analysis (C). A representative experiment is shown. Similar results have been obtained at least three times.
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Figure 9: Overexpression of CerS6 in SW620 cells results in sensitization to TRAIL. (A) SW620 cells were transiently transfected with pIRES-GFP as a negative control or pCerS6-IRES-GFP. Cells were visualized at a magnification of 200X 24 hours after treatment with 100 ng/ml TRAIL was initiated. Representative images from one of three experiments are shown. (B/C) SW620 mass clones stably transfected with pIRES-GFP or pCerS6-IRES-GFP were treated with 100 ng/ml TRAIL for 4 hours and examined for morphological evidence of apoptosis (B) or PARP cleavage by western blot analysis (C). A representative experiment is shown. Similar results have been obtained at least three times.

Mentions: The difference in TRAIL sensitivity between SW480 and SW620 cells does not appear to be mediated by significant alterations to levels of pro- or anti-apoptotic proteins with the possible exception of XIAP (Huerta et al., 2007; Ndozangue-Touriguine et al., 2008). However, in our cells we did not observe elevated expression of anti-apoptotic proteins such as cFLIP, Bcl-2 or XIAP that would explain the TRAIL resistant phenotype of SW620 cells (suppl. Fig. 6). Having observed that the downregulation of CerS6 generates a TRAIL-resistant phenotype in TRAIL sensitive SW480 cells, we investigated whether SW620 cells, which are TRAIL resistant and have reduced CerS6 expression, could be sensitized by increasing levels of CerS6. We generated a plasmid encoding CerS6 and EGFP separated by an IRES sequence, which allows the expression of two proteins from one mRNA, so that GFP serves as an indicator for transfected cells. SW620 cells were transfected with a control plasmid (pIRES2-EGFP) or pCerS6-IRES2-EGFP. Untreated and TRAIL treated cells were then analyzed morphologically. CerS6 over-expression alone did not induce morphological changes associated with apoptosis (Figure 9A). However, when cells transfected with pCerS6-IRES2-EGFP were treated with TRAIL, GFP positive cells appeared to be undergoing apoptosis (Figure 9A, arrows). Some GFP-negative cells also appeared apoptotic, indicating a possible bystander effect that may be mediated by a high local ceramide concentration.


Ceramide synthase 6 modulates TRAIL sensitivity and nuclear translocation of active caspase-3 in colon cancer cells.

White-Gilbertson S, Mullen T, Senkal C, Lu P, Ogretmen B, Obeid L, Voelkel-Johnson C - Oncogene (2009)

Overexpression of CerS6 in SW620 cells results in sensitization to TRAIL. (A) SW620 cells were transiently transfected with pIRES-GFP as a negative control or pCerS6-IRES-GFP. Cells were visualized at a magnification of 200X 24 hours after treatment with 100 ng/ml TRAIL was initiated. Representative images from one of three experiments are shown. (B/C) SW620 mass clones stably transfected with pIRES-GFP or pCerS6-IRES-GFP were treated with 100 ng/ml TRAIL for 4 hours and examined for morphological evidence of apoptosis (B) or PARP cleavage by western blot analysis (C). A representative experiment is shown. Similar results have been obtained at least three times.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2648838&req=5

Figure 9: Overexpression of CerS6 in SW620 cells results in sensitization to TRAIL. (A) SW620 cells were transiently transfected with pIRES-GFP as a negative control or pCerS6-IRES-GFP. Cells were visualized at a magnification of 200X 24 hours after treatment with 100 ng/ml TRAIL was initiated. Representative images from one of three experiments are shown. (B/C) SW620 mass clones stably transfected with pIRES-GFP or pCerS6-IRES-GFP were treated with 100 ng/ml TRAIL for 4 hours and examined for morphological evidence of apoptosis (B) or PARP cleavage by western blot analysis (C). A representative experiment is shown. Similar results have been obtained at least three times.
Mentions: The difference in TRAIL sensitivity between SW480 and SW620 cells does not appear to be mediated by significant alterations to levels of pro- or anti-apoptotic proteins with the possible exception of XIAP (Huerta et al., 2007; Ndozangue-Touriguine et al., 2008). However, in our cells we did not observe elevated expression of anti-apoptotic proteins such as cFLIP, Bcl-2 or XIAP that would explain the TRAIL resistant phenotype of SW620 cells (suppl. Fig. 6). Having observed that the downregulation of CerS6 generates a TRAIL-resistant phenotype in TRAIL sensitive SW480 cells, we investigated whether SW620 cells, which are TRAIL resistant and have reduced CerS6 expression, could be sensitized by increasing levels of CerS6. We generated a plasmid encoding CerS6 and EGFP separated by an IRES sequence, which allows the expression of two proteins from one mRNA, so that GFP serves as an indicator for transfected cells. SW620 cells were transfected with a control plasmid (pIRES2-EGFP) or pCerS6-IRES2-EGFP. Untreated and TRAIL treated cells were then analyzed morphologically. CerS6 over-expression alone did not induce morphological changes associated with apoptosis (Figure 9A). However, when cells transfected with pCerS6-IRES2-EGFP were treated with TRAIL, GFP positive cells appeared to be undergoing apoptosis (Figure 9A, arrows). Some GFP-negative cells also appeared apoptotic, indicating a possible bystander effect that may be mediated by a high local ceramide concentration.

Bottom Line: RNAi against CerS6 resulted in a specific and significant decrease of the C(16)-ceramide species, which was sufficient to inhibit TRAIL-induced apoptosis.In cells with decreased levels of CerS6, caspase-3 was activated but failed to translocate into the nucleus.These results suggest that modulation of CerS6 expression may constitute a new therapeutic strategy to alter apoptotic susceptibility.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Medical University of South Carolina, Charleston, SC 29403, USA.

ABSTRACT
We have previously shown that the death receptor ligand TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) induces an increase of intracellular C(16)-ceramide in sensitive SW480 but not in resistant SW620 cells. Resistance in SW620 cells was overcome by exogenous ceramide, leading us to propose that defective ceramide signaling contributes to TRAIL resistance. In this study we found that the increase in C(16)-ceramide in SW480 cells was inhibited by fumonisin B1, an inhibitor of ceramide synthases (CerS). Protein analysis revealed that TRAIL-resistant SW620 cells expressed lower levels of ceramide synthase 6 (CerS6, also known as longevity assurance homologue 6), which prompted us to investigate the effect of CerS6 modulation on TRAIL phenotype. RNAi against CerS6 resulted in a specific and significant decrease of the C(16)-ceramide species, which was sufficient to inhibit TRAIL-induced apoptosis. In cells with decreased levels of CerS6, caspase-3 was activated but failed to translocate into the nucleus. CerS6 localized primarily to the perinuclear region, suggesting this enzyme may be important in regulation of nuclear permeability. Moderate elevation in CerS6 expression was sufficient to reverse TRAIL resistance in SW620 cells. These results suggest that modulation of CerS6 expression may constitute a new therapeutic strategy to alter apoptotic susceptibility.

Show MeSH
Related in: MedlinePlus