Limits...
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.

Show MeSH

Related in: MedlinePlus

Apoptosis resistance despite caspase-3 cleavage in SW480 cells stably transfected with CerS6 shRNA. Cells were treated with 100 ng/ml TRAIL for 22 hrs. (A) Quantitation of nuclear condensation by Hoechst stain. Condensed nuclei in three randomly chosen fields/well were counted and apoptotic nuclei expressed as a percentage of total nuclei (n=2). (B) Western blot analysis. *The actin membrane was reprobed with CerS6. The upper band in the CerS6 blot is the actin signal.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2648838&req=5

Figure 6: Apoptosis resistance despite caspase-3 cleavage in SW480 cells stably transfected with CerS6 shRNA. Cells were treated with 100 ng/ml TRAIL for 22 hrs. (A) Quantitation of nuclear condensation by Hoechst stain. Condensed nuclei in three randomly chosen fields/well were counted and apoptotic nuclei expressed as a percentage of total nuclei (n=2). (B) Western blot analysis. *The actin membrane was reprobed with CerS6. The upper band in the CerS6 blot is the actin signal.

Mentions: To pinpoint the lesion in the TRAIL apoptotic pathway resulting from CerS6 downregulation, we analyzed caspase cleavage. Surprisingly, we failed to find significant differences in cleavage of caspases-8, -2, -9, or -3 regardless of CerS6 expression (Figure 5A). A decrease in CerS6 resulted only in inhibition of PARP cleavage. PARP is a known target of caspase-3 and therefore we wished to verify that the cleaved form of caspase-3 detected by Western blot was functionally active. However, downregulation of CerS6 did not result in significant differences in caspase-3/7 activity following TRAIL treatment (Figure 5B). To confirm these results, we generated SW480 cells that stably expressed a CerS6 shRNA against a different target sequence than the siRNA. An shRNA against GFP served as a control in these experiments. Consistent with data obtained with siRNA, shRNA against CerS6 also inhibited nuclear condensation and PARP cleavage but not cleavage of caspase-3 (Fig. 6, supplementary Fig. 3). Similar data were obtained with mass and different individual clones at both 8 and 22 hours after TRAIL treatment (data not shown).


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)

Apoptosis resistance despite caspase-3 cleavage in SW480 cells stably transfected with CerS6 shRNA. Cells were treated with 100 ng/ml TRAIL for 22 hrs. (A) Quantitation of nuclear condensation by Hoechst stain. Condensed nuclei in three randomly chosen fields/well were counted and apoptotic nuclei expressed as a percentage of total nuclei (n=2). (B) Western blot analysis. *The actin membrane was reprobed with CerS6. The upper band in the CerS6 blot is the actin signal.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2648838&req=5

Figure 6: Apoptosis resistance despite caspase-3 cleavage in SW480 cells stably transfected with CerS6 shRNA. Cells were treated with 100 ng/ml TRAIL for 22 hrs. (A) Quantitation of nuclear condensation by Hoechst stain. Condensed nuclei in three randomly chosen fields/well were counted and apoptotic nuclei expressed as a percentage of total nuclei (n=2). (B) Western blot analysis. *The actin membrane was reprobed with CerS6. The upper band in the CerS6 blot is the actin signal.
Mentions: To pinpoint the lesion in the TRAIL apoptotic pathway resulting from CerS6 downregulation, we analyzed caspase cleavage. Surprisingly, we failed to find significant differences in cleavage of caspases-8, -2, -9, or -3 regardless of CerS6 expression (Figure 5A). A decrease in CerS6 resulted only in inhibition of PARP cleavage. PARP is a known target of caspase-3 and therefore we wished to verify that the cleaved form of caspase-3 detected by Western blot was functionally active. However, downregulation of CerS6 did not result in significant differences in caspase-3/7 activity following TRAIL treatment (Figure 5B). To confirm these results, we generated SW480 cells that stably expressed a CerS6 shRNA against a different target sequence than the siRNA. An shRNA against GFP served as a control in these experiments. Consistent with data obtained with siRNA, shRNA against CerS6 also inhibited nuclear condensation and PARP cleavage but not cleavage of caspase-3 (Fig. 6, supplementary Fig. 3). Similar data were obtained with mass and different individual clones at both 8 and 22 hours after TRAIL treatment (data not shown).

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