Limits...
Sphingosine analogue drug FTY720 targets I2PP2A/SET and mediates lung tumour suppression via activation of PP2A-RIPK1-dependent necroptosis.

Saddoughi SA, Gencer S, Peterson YK, Ward KE, Mukhopadhyay A, Oaks J, Bielawski J, Szulc ZM, Thomas RJ, Selvam SP, Senkal CE, Garrett-Mayer E, De Palma RM, Fedarovich D, Liu A, Habib AA, Stahelin RV, Perrotti D, Ogretmen B - EMBO Mol Med (2012)

Bottom Line: Mechanistically, targeting I2PP2A/SET by FTY720 mediated PP2A/RIPK1-dependent programmed necrosis (necroptosis), but not by apoptosis.The RIPK1 inhibitor necrostatin and knockdown or genetic loss of RIPK1 prevented growth inhibition by FTY720.Expression of WT- or death-domain-deleted (DDD)-RIPK1, but not the kinase-domain-deleted (KDD)-RIPK1, restored FTY720-mediated necroptosis in RIPK1(-/-) MEFs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC, USA.

Show MeSH

Related in: MedlinePlus

FTY720 mediates cell death via PP2A/RIPK1-dependent necroptosisA–C. Effects of FTY720 at various concentrations on growth of MEFs obtained from caspase 3/7−/− (A), Bax/Bak−/− (B) or ATG5−/− (C) mice were measured using MTT assays compared to vehicle-treated controls.D. Effects of Z-VAD or necrostatin on FTY720-mediated cell death were measured in A549 cells compared to vehicle treated controls. Cell death was measured by LDH release in the media.E. Effects of FTY720 (15 µM) on cell death were measured in WT and RIPK1−/− MEF's compared to vehicle-treated controls.F,G. Roles of siRNA-mediated knockdown of RIPK1 compared to Scr-siRNAs-transfected controls, confirmed by Western blotting (F), in FTY720-mediated cell death were measured by detection of LDH release (G). Error bars represent s.d. (*p < 0.05, **p < 0.01). Full blots can be seen in Supporting Information Fig S16.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3569657&req=5

fig08: FTY720 mediates cell death via PP2A/RIPK1-dependent necroptosisA–C. Effects of FTY720 at various concentrations on growth of MEFs obtained from caspase 3/7−/− (A), Bax/Bak−/− (B) or ATG5−/− (C) mice were measured using MTT assays compared to vehicle-treated controls.D. Effects of Z-VAD or necrostatin on FTY720-mediated cell death were measured in A549 cells compared to vehicle treated controls. Cell death was measured by LDH release in the media.E. Effects of FTY720 (15 µM) on cell death were measured in WT and RIPK1−/− MEF's compared to vehicle-treated controls.F,G. Roles of siRNA-mediated knockdown of RIPK1 compared to Scr-siRNAs-transfected controls, confirmed by Western blotting (F), in FTY720-mediated cell death were measured by detection of LDH release (G). Error bars represent s.d. (*p < 0.05, **p < 0.01). Full blots can be seen in Supporting Information Fig S16.

Mentions: To delineate the downstream mechanism by which targeting I2PP2A/SET by FTY720 results in cell death [as confirmed by increased Annexin V/7-aminoactinomycin D (7-AAD) positive A549 populations compared to controls using flow cytometry; Supporting Information Fig S11A], we examined the effects of FTY720 on apoptotis- or autophagy-dependent cell death using MEFs obtained from caspase3/7−/− DKO, Bax/Bak−/− DKO or ATG5−/− KO mice. The data showed that genetic loss of caspases3/7, Bax/Bak, or ATG5 had no protective effect on FTY720-induced cell death in these immortalized MEFs (Fig 8A–C), which express endogenous I2PP2A/SET (Supporting Information Fig S11B). Consistent with these data, treatment with a pan-caspase inhibitor Z-VAD had no effect on A549 cell death in response to FTY720 (Fig 8D). Accordingly, ATG5−/− MEFs were more sensitive to taxol-induced apoptosis (80 nM and 24 h) as measured by increased caspase 3 activity compared to WT MEFs and used as an additional control to confirm their expected response to chemotherapy-induced apoptosis (Supporting Information Fig S11C). Overall, these data suggest that FTY720 induces cell death independently of caspase/Bax/Bak-mediated apoptosis or ATG5-mediated autophagy.


Sphingosine analogue drug FTY720 targets I2PP2A/SET and mediates lung tumour suppression via activation of PP2A-RIPK1-dependent necroptosis.

Saddoughi SA, Gencer S, Peterson YK, Ward KE, Mukhopadhyay A, Oaks J, Bielawski J, Szulc ZM, Thomas RJ, Selvam SP, Senkal CE, Garrett-Mayer E, De Palma RM, Fedarovich D, Liu A, Habib AA, Stahelin RV, Perrotti D, Ogretmen B - EMBO Mol Med (2012)

FTY720 mediates cell death via PP2A/RIPK1-dependent necroptosisA–C. Effects of FTY720 at various concentrations on growth of MEFs obtained from caspase 3/7−/− (A), Bax/Bak−/− (B) or ATG5−/− (C) mice were measured using MTT assays compared to vehicle-treated controls.D. Effects of Z-VAD or necrostatin on FTY720-mediated cell death were measured in A549 cells compared to vehicle treated controls. Cell death was measured by LDH release in the media.E. Effects of FTY720 (15 µM) on cell death were measured in WT and RIPK1−/− MEF's compared to vehicle-treated controls.F,G. Roles of siRNA-mediated knockdown of RIPK1 compared to Scr-siRNAs-transfected controls, confirmed by Western blotting (F), in FTY720-mediated cell death were measured by detection of LDH release (G). Error bars represent s.d. (*p < 0.05, **p < 0.01). Full blots can be seen in Supporting Information Fig S16.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3569657&req=5

fig08: FTY720 mediates cell death via PP2A/RIPK1-dependent necroptosisA–C. Effects of FTY720 at various concentrations on growth of MEFs obtained from caspase 3/7−/− (A), Bax/Bak−/− (B) or ATG5−/− (C) mice were measured using MTT assays compared to vehicle-treated controls.D. Effects of Z-VAD or necrostatin on FTY720-mediated cell death were measured in A549 cells compared to vehicle treated controls. Cell death was measured by LDH release in the media.E. Effects of FTY720 (15 µM) on cell death were measured in WT and RIPK1−/− MEF's compared to vehicle-treated controls.F,G. Roles of siRNA-mediated knockdown of RIPK1 compared to Scr-siRNAs-transfected controls, confirmed by Western blotting (F), in FTY720-mediated cell death were measured by detection of LDH release (G). Error bars represent s.d. (*p < 0.05, **p < 0.01). Full blots can be seen in Supporting Information Fig S16.
Mentions: To delineate the downstream mechanism by which targeting I2PP2A/SET by FTY720 results in cell death [as confirmed by increased Annexin V/7-aminoactinomycin D (7-AAD) positive A549 populations compared to controls using flow cytometry; Supporting Information Fig S11A], we examined the effects of FTY720 on apoptotis- or autophagy-dependent cell death using MEFs obtained from caspase3/7−/− DKO, Bax/Bak−/− DKO or ATG5−/− KO mice. The data showed that genetic loss of caspases3/7, Bax/Bak, or ATG5 had no protective effect on FTY720-induced cell death in these immortalized MEFs (Fig 8A–C), which express endogenous I2PP2A/SET (Supporting Information Fig S11B). Consistent with these data, treatment with a pan-caspase inhibitor Z-VAD had no effect on A549 cell death in response to FTY720 (Fig 8D). Accordingly, ATG5−/− MEFs were more sensitive to taxol-induced apoptosis (80 nM and 24 h) as measured by increased caspase 3 activity compared to WT MEFs and used as an additional control to confirm their expected response to chemotherapy-induced apoptosis (Supporting Information Fig S11C). Overall, these data suggest that FTY720 induces cell death independently of caspase/Bax/Bak-mediated apoptosis or ATG5-mediated autophagy.

Bottom Line: Mechanistically, targeting I2PP2A/SET by FTY720 mediated PP2A/RIPK1-dependent programmed necrosis (necroptosis), but not by apoptosis.The RIPK1 inhibitor necrostatin and knockdown or genetic loss of RIPK1 prevented growth inhibition by FTY720.Expression of WT- or death-domain-deleted (DDD)-RIPK1, but not the kinase-domain-deleted (KDD)-RIPK1, restored FTY720-mediated necroptosis in RIPK1(-/-) MEFs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC, USA.

Show MeSH
Related in: MedlinePlus