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Chorein addiction in VPS13A overexpressing rhabdomyosarcoma cells.

Honisch S, Yu W, Liu G, Alesutan I, Towhid ST, Tsapara A, Schleicher S, Handgretinger R, Stournaras C, Lang F - Oncotarget (2015)

Bottom Line: Chorein encoded by VPS13A (vacuolar protein sorting-associated protein 13A) is defective in chorea-acanthocytosis.Chorein silencing significantly reduced the ratio of phosphorylated (and thus activated) to total phosphoinositide 3 kinase (PI-3K), pointing to inactivation of this crucial pro-survival signaling molecule.In cells with high chorein expression levels chorein silencing promotes apoptotic cell death, an effect paralleled by down-regulation of PI-3K activity and BCL-2/Bax expression ratio.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Tübingen, Tübingen, Germany.

ABSTRACT
Chorein encoded by VPS13A (vacuolar protein sorting-associated protein 13A) is defective in chorea-acanthocytosis. Chorein fosters neuronal cell survival, cortical actin polymerization and cell stiffness. In view of its anti-apoptotic effect in neurons, we explored whether chorein is expressed in cancer cells and influences cancer cell survival. RT-PCR was employed to determine transcript levels, specific siRNA to silence chorein, FACS analysis to follow apoptosis and Western blotting to quantify protein abundance. Chorein transcripts were detected in various cancer cell types. The mRNA coding for chorein and chorein protein were most abundant in drug resistant, poorly differentiated human rhabdomyosarcoma cells. Chorein silencing significantly reduced the ratio of phosphorylated (and thus activated) to total phosphoinositide 3 kinase (PI-3K), pointing to inactivation of this crucial pro-survival signaling molecule. Moreover, chorein silencing diminished transcript levels and protein expression of anti-apoptotic BCL-2 and enhanced transcript levels of pro-apoptotic Bax. Silencing of chorein in rhabdomyosarcoma cells was followed by mitochondrial depolarization, caspase 3 activation and stimulation of early and late apoptosis. In conclusion, chorein is expressed in various cancer cells. In cells with high chorein expression levels chorein silencing promotes apoptotic cell death, an effect paralleled by down-regulation of PI-3K activity and BCL-2/Bax expression ratio.

No MeSH data available.


Related in: MedlinePlus

Chorein sensitive Caspase-3 activity and mitochondrial depolarization in ZF rhabdomyosarcoma cellsA. Cells were stained with conjugated inhibitor of active Caspase-3 (FITC-DEVD-FMK) and measured by FACS. Shown are arithmetic means (left) ± SE (n=4) and representative original histograms (right) demonstrating caspase activity in ZF cells transfected with control siRNA (siNeg) or siRNA for chorein (siVPS13A). * significant difference (p<0.05; unpaired t-test). B. Arithmetic means (left) ± SEM (n=4) and representative original histograms (right) of mitochondrial depolarization measured by FACS in ZF cells transfected with negative control siRNA (siNeg) or siRNA for chorein (siVPS13A).*** indicates significant difference (p<0.001, unpaired t-test).
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Figure 4: Chorein sensitive Caspase-3 activity and mitochondrial depolarization in ZF rhabdomyosarcoma cellsA. Cells were stained with conjugated inhibitor of active Caspase-3 (FITC-DEVD-FMK) and measured by FACS. Shown are arithmetic means (left) ± SE (n=4) and representative original histograms (right) demonstrating caspase activity in ZF cells transfected with control siRNA (siNeg) or siRNA for chorein (siVPS13A). * significant difference (p<0.05; unpaired t-test). B. Arithmetic means (left) ± SEM (n=4) and representative original histograms (right) of mitochondrial depolarization measured by FACS in ZF cells transfected with negative control siRNA (siNeg) or siRNA for chorein (siVPS13A).*** indicates significant difference (p<0.001, unpaired t-test).

Mentions: Caspase 3 activity was determined to elucidate whether chorein influenced apoptotic signaling. As illustrated in Fig. 4A, chorein silencing significantly enhanced caspase-3 activity in ZF rhabdomyosarcoma cells, an observation pointing to triggering of apoptosis. In line with this observation, chorein silencing of ZF rhabdomyosarcoma cells was followed by significant mitochondrial depolarization (Fig. 4B). Mitochondrial depolarization reached statistical significance already 24 h after chorein silencing and persisted throughout 72 h (Suppl. Fig. 2C).


Chorein addiction in VPS13A overexpressing rhabdomyosarcoma cells.

Honisch S, Yu W, Liu G, Alesutan I, Towhid ST, Tsapara A, Schleicher S, Handgretinger R, Stournaras C, Lang F - Oncotarget (2015)

Chorein sensitive Caspase-3 activity and mitochondrial depolarization in ZF rhabdomyosarcoma cellsA. Cells were stained with conjugated inhibitor of active Caspase-3 (FITC-DEVD-FMK) and measured by FACS. Shown are arithmetic means (left) ± SE (n=4) and representative original histograms (right) demonstrating caspase activity in ZF cells transfected with control siRNA (siNeg) or siRNA for chorein (siVPS13A). * significant difference (p<0.05; unpaired t-test). B. Arithmetic means (left) ± SEM (n=4) and representative original histograms (right) of mitochondrial depolarization measured by FACS in ZF cells transfected with negative control siRNA (siNeg) or siRNA for chorein (siVPS13A).*** indicates significant difference (p<0.001, unpaired t-test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496357&req=5

Figure 4: Chorein sensitive Caspase-3 activity and mitochondrial depolarization in ZF rhabdomyosarcoma cellsA. Cells were stained with conjugated inhibitor of active Caspase-3 (FITC-DEVD-FMK) and measured by FACS. Shown are arithmetic means (left) ± SE (n=4) and representative original histograms (right) demonstrating caspase activity in ZF cells transfected with control siRNA (siNeg) or siRNA for chorein (siVPS13A). * significant difference (p<0.05; unpaired t-test). B. Arithmetic means (left) ± SEM (n=4) and representative original histograms (right) of mitochondrial depolarization measured by FACS in ZF cells transfected with negative control siRNA (siNeg) or siRNA for chorein (siVPS13A).*** indicates significant difference (p<0.001, unpaired t-test).
Mentions: Caspase 3 activity was determined to elucidate whether chorein influenced apoptotic signaling. As illustrated in Fig. 4A, chorein silencing significantly enhanced caspase-3 activity in ZF rhabdomyosarcoma cells, an observation pointing to triggering of apoptosis. In line with this observation, chorein silencing of ZF rhabdomyosarcoma cells was followed by significant mitochondrial depolarization (Fig. 4B). Mitochondrial depolarization reached statistical significance already 24 h after chorein silencing and persisted throughout 72 h (Suppl. Fig. 2C).

Bottom Line: Chorein encoded by VPS13A (vacuolar protein sorting-associated protein 13A) is defective in chorea-acanthocytosis.Chorein silencing significantly reduced the ratio of phosphorylated (and thus activated) to total phosphoinositide 3 kinase (PI-3K), pointing to inactivation of this crucial pro-survival signaling molecule.In cells with high chorein expression levels chorein silencing promotes apoptotic cell death, an effect paralleled by down-regulation of PI-3K activity and BCL-2/Bax expression ratio.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Tübingen, Tübingen, Germany.

ABSTRACT
Chorein encoded by VPS13A (vacuolar protein sorting-associated protein 13A) is defective in chorea-acanthocytosis. Chorein fosters neuronal cell survival, cortical actin polymerization and cell stiffness. In view of its anti-apoptotic effect in neurons, we explored whether chorein is expressed in cancer cells and influences cancer cell survival. RT-PCR was employed to determine transcript levels, specific siRNA to silence chorein, FACS analysis to follow apoptosis and Western blotting to quantify protein abundance. Chorein transcripts were detected in various cancer cell types. The mRNA coding for chorein and chorein protein were most abundant in drug resistant, poorly differentiated human rhabdomyosarcoma cells. Chorein silencing significantly reduced the ratio of phosphorylated (and thus activated) to total phosphoinositide 3 kinase (PI-3K), pointing to inactivation of this crucial pro-survival signaling molecule. Moreover, chorein silencing diminished transcript levels and protein expression of anti-apoptotic BCL-2 and enhanced transcript levels of pro-apoptotic Bax. Silencing of chorein in rhabdomyosarcoma cells was followed by mitochondrial depolarization, caspase 3 activation and stimulation of early and late apoptosis. In conclusion, chorein is expressed in various cancer cells. In cells with high chorein expression levels chorein silencing promotes apoptotic cell death, an effect paralleled by down-regulation of PI-3K activity and BCL-2/Bax expression ratio.

No MeSH data available.


Related in: MedlinePlus