Limits...
Potentiation of apoptosis by histone deacetylase inhibitors and doxorubicin combination: cytoplasmic cathepsin B as a mediator of apoptosis in multiple myeloma.

Cheriyath V, Kuhns MA, Kalaycio ME, Borden EC - Br. J. Cancer (2011)

Bottom Line: Consistent with this, butyrate and doxorubicin combination significantly increased the activity of cytoplasmic cathepsin B.Finally, ex vivo, clinically relevant concentrations of butyrate or SAHA (suberoylanilide hydroxamic acid, vorinostat, an HDACi in clinical testing) in combination with doxorubicin significantly (P<0.0001) reduced the survival of primary myeloma cells.Our results support a molecular model of lysosomal-mitochondrial crosstalk in HDACi- and doxorubicin-potentiated apoptosis through the activation of cathepsin B.

View Article: PubMed Central - PubMed

Affiliation: Translational Hematology and Oncology Research, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH 44195, USA. cheriyv@ccf.org

ABSTRACT

Background: Although inhibitors of histone deacetylase inhibitors (HDACis) in combination with genotoxins potentiate apoptosis, the role of proteases other than caspases in this process remained elusive. Therefore, we examined the potentiation of apoptosis and related mechanisms of HDACis and doxorubicin combination in a panel of myeloma cell lines and in 25 primary myelomas.

Results: At IC(50) concentrations, sodium butyrate (an HDACi) or doxorubicin alone caused little apoptosis. However, their combination potentiated apoptosis and synergistically reduced the viability of myeloma cells independent of p53 and caspase 3-7 activation. Potentiated apoptosis correlated with nuclear translocation of apoptosis-inducing factor, suggesting the induction of caspase 3- and 7-independent pathways. Consistent with this, butyrate and doxorubicin combination significantly increased the activity of cytoplasmic cathepsin B. Inhibition of cathepsin B either with a small-molecule inhibitor or downregulation with a siRNA reversed butyrate- and doxorubicin-potentiated apoptosis. Finally, ex vivo, clinically relevant concentrations of butyrate or SAHA (suberoylanilide hydroxamic acid, vorinostat, an HDACi in clinical testing) in combination with doxorubicin significantly (P<0.0001) reduced the survival of primary myeloma cells.

Conclusions: Cathepsin B has a prominent function in mediating apoptosis potentiated by HDACi and doxorubicin combinations in myeloma. Our results support a molecular model of lysosomal-mitochondrial crosstalk in HDACi- and doxorubicin-potentiated apoptosis through the activation of cathepsin B.

Show MeSH

Related in: MedlinePlus

Combinations of butyrate and doxorubicin significantly increased the activity of cytoplasmic cathepsin B. (A) Optimisation of cytoplasmic cathepsin B extraction by permeabilisation of plasma membrane with digitonin. RPMI 8226 cells plasma membranes were permeabilised with increasing concentrations of digitonin for 10 min in ice. Permeabilisation of plasma membranes was monitored by assessing LDH activity (left Y axis) and permeabilisation of lysosomes was monitored by cathepsin B activity (right Y axis). Each point on the graph is mean±s.e.m. of two independent experiments. (B) Effects of butyrate and doxorubicin combination on the activity of cytoplasmic cathepsin B in myeloma cells. RPMI 8226 cells were treated with butyrate (SB, 600 μ), doxorubicin (Dox, 40 n) or their combination. Cells were harvested at indicated time periods, permeabilised with 50 μg ml−1 digitonin, and the activity of cathepsin B was measured using enzyme assay kits (Biovision Inc.). Cathepsin B activity was normalised to LDH activity; *P<0.05, **P<0.001 and ***P<0.0001. (C, D) Effects of butyrate and doxorubicin combination on the activity of calpain and cathepsin D in RPMI 8226 cells. Relative increase in total calpain (C) and cathepsin D (D) activities were calculated by normalising to untreated samples. Each data point on the graph is mean±s.e.m. of two independent experiments performed in triplicate.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3065279&req=5

fig3: Combinations of butyrate and doxorubicin significantly increased the activity of cytoplasmic cathepsin B. (A) Optimisation of cytoplasmic cathepsin B extraction by permeabilisation of plasma membrane with digitonin. RPMI 8226 cells plasma membranes were permeabilised with increasing concentrations of digitonin for 10 min in ice. Permeabilisation of plasma membranes was monitored by assessing LDH activity (left Y axis) and permeabilisation of lysosomes was monitored by cathepsin B activity (right Y axis). Each point on the graph is mean±s.e.m. of two independent experiments. (B) Effects of butyrate and doxorubicin combination on the activity of cytoplasmic cathepsin B in myeloma cells. RPMI 8226 cells were treated with butyrate (SB, 600 μ), doxorubicin (Dox, 40 n) or their combination. Cells were harvested at indicated time periods, permeabilised with 50 μg ml−1 digitonin, and the activity of cathepsin B was measured using enzyme assay kits (Biovision Inc.). Cathepsin B activity was normalised to LDH activity; *P<0.05, **P<0.001 and ***P<0.0001. (C, D) Effects of butyrate and doxorubicin combination on the activity of calpain and cathepsin D in RPMI 8226 cells. Relative increase in total calpain (C) and cathepsin D (D) activities were calculated by normalising to untreated samples. Each data point on the graph is mean±s.e.m. of two independent experiments performed in triplicate.

Mentions: On the basis of the role of cathepsin B in mediating AIF activation and apoptosis induced by agents that act on DNA (Bidere et al, 2003; Broker et al, 2004; Biswas et al, 2005), we postulated that increased activity of cathepsin B in the cytoplasm may be a critical mediator of HDACi and doxorubicin combination induced apoptosis. To test this hypothesis, activity of cathepsin B in cytoplasmic extracts of untreated and treated RPMI 8226 cells was determined. Permeabilisation of cells with digitonin was monitored by measuring the activity of cytoplasmic enzyme LDH (Foghsgaard et al, 2002). Incubation of cells in cytoplasmic extraction buffer with 50 μg of digitonin resulted in the maximal release of LDH with minimal increase in the activity of cathepsin B, indicating the permeabilisation of the plasma membrane, but not lysosomes (Figure 3A). In kinetic studies compared with untreated and single agents, the combination of butyrate and doxorubicin significantly increased the cytoplasmic activity of cathepsin B at 16 h in RPMI 8226 cells (Figure 3B). An increase in the activity of cytoplasmic cathepsin B was also observed in NCI H929 cells treated with butyrate and doxorubicin combination (data not shown).


Potentiation of apoptosis by histone deacetylase inhibitors and doxorubicin combination: cytoplasmic cathepsin B as a mediator of apoptosis in multiple myeloma.

Cheriyath V, Kuhns MA, Kalaycio ME, Borden EC - Br. J. Cancer (2011)

Combinations of butyrate and doxorubicin significantly increased the activity of cytoplasmic cathepsin B. (A) Optimisation of cytoplasmic cathepsin B extraction by permeabilisation of plasma membrane with digitonin. RPMI 8226 cells plasma membranes were permeabilised with increasing concentrations of digitonin for 10 min in ice. Permeabilisation of plasma membranes was monitored by assessing LDH activity (left Y axis) and permeabilisation of lysosomes was monitored by cathepsin B activity (right Y axis). Each point on the graph is mean±s.e.m. of two independent experiments. (B) Effects of butyrate and doxorubicin combination on the activity of cytoplasmic cathepsin B in myeloma cells. RPMI 8226 cells were treated with butyrate (SB, 600 μ), doxorubicin (Dox, 40 n) or their combination. Cells were harvested at indicated time periods, permeabilised with 50 μg ml−1 digitonin, and the activity of cathepsin B was measured using enzyme assay kits (Biovision Inc.). Cathepsin B activity was normalised to LDH activity; *P<0.05, **P<0.001 and ***P<0.0001. (C, D) Effects of butyrate and doxorubicin combination on the activity of calpain and cathepsin D in RPMI 8226 cells. Relative increase in total calpain (C) and cathepsin D (D) activities were calculated by normalising to untreated samples. Each data point on the graph is mean±s.e.m. of two independent experiments performed in triplicate.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3065279&req=5

fig3: Combinations of butyrate and doxorubicin significantly increased the activity of cytoplasmic cathepsin B. (A) Optimisation of cytoplasmic cathepsin B extraction by permeabilisation of plasma membrane with digitonin. RPMI 8226 cells plasma membranes were permeabilised with increasing concentrations of digitonin for 10 min in ice. Permeabilisation of plasma membranes was monitored by assessing LDH activity (left Y axis) and permeabilisation of lysosomes was monitored by cathepsin B activity (right Y axis). Each point on the graph is mean±s.e.m. of two independent experiments. (B) Effects of butyrate and doxorubicin combination on the activity of cytoplasmic cathepsin B in myeloma cells. RPMI 8226 cells were treated with butyrate (SB, 600 μ), doxorubicin (Dox, 40 n) or their combination. Cells were harvested at indicated time periods, permeabilised with 50 μg ml−1 digitonin, and the activity of cathepsin B was measured using enzyme assay kits (Biovision Inc.). Cathepsin B activity was normalised to LDH activity; *P<0.05, **P<0.001 and ***P<0.0001. (C, D) Effects of butyrate and doxorubicin combination on the activity of calpain and cathepsin D in RPMI 8226 cells. Relative increase in total calpain (C) and cathepsin D (D) activities were calculated by normalising to untreated samples. Each data point on the graph is mean±s.e.m. of two independent experiments performed in triplicate.
Mentions: On the basis of the role of cathepsin B in mediating AIF activation and apoptosis induced by agents that act on DNA (Bidere et al, 2003; Broker et al, 2004; Biswas et al, 2005), we postulated that increased activity of cathepsin B in the cytoplasm may be a critical mediator of HDACi and doxorubicin combination induced apoptosis. To test this hypothesis, activity of cathepsin B in cytoplasmic extracts of untreated and treated RPMI 8226 cells was determined. Permeabilisation of cells with digitonin was monitored by measuring the activity of cytoplasmic enzyme LDH (Foghsgaard et al, 2002). Incubation of cells in cytoplasmic extraction buffer with 50 μg of digitonin resulted in the maximal release of LDH with minimal increase in the activity of cathepsin B, indicating the permeabilisation of the plasma membrane, but not lysosomes (Figure 3A). In kinetic studies compared with untreated and single agents, the combination of butyrate and doxorubicin significantly increased the cytoplasmic activity of cathepsin B at 16 h in RPMI 8226 cells (Figure 3B). An increase in the activity of cytoplasmic cathepsin B was also observed in NCI H929 cells treated with butyrate and doxorubicin combination (data not shown).

Bottom Line: Consistent with this, butyrate and doxorubicin combination significantly increased the activity of cytoplasmic cathepsin B.Finally, ex vivo, clinically relevant concentrations of butyrate or SAHA (suberoylanilide hydroxamic acid, vorinostat, an HDACi in clinical testing) in combination with doxorubicin significantly (P<0.0001) reduced the survival of primary myeloma cells.Our results support a molecular model of lysosomal-mitochondrial crosstalk in HDACi- and doxorubicin-potentiated apoptosis through the activation of cathepsin B.

View Article: PubMed Central - PubMed

Affiliation: Translational Hematology and Oncology Research, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH 44195, USA. cheriyv@ccf.org

ABSTRACT

Background: Although inhibitors of histone deacetylase inhibitors (HDACis) in combination with genotoxins potentiate apoptosis, the role of proteases other than caspases in this process remained elusive. Therefore, we examined the potentiation of apoptosis and related mechanisms of HDACis and doxorubicin combination in a panel of myeloma cell lines and in 25 primary myelomas.

Results: At IC(50) concentrations, sodium butyrate (an HDACi) or doxorubicin alone caused little apoptosis. However, their combination potentiated apoptosis and synergistically reduced the viability of myeloma cells independent of p53 and caspase 3-7 activation. Potentiated apoptosis correlated with nuclear translocation of apoptosis-inducing factor, suggesting the induction of caspase 3- and 7-independent pathways. Consistent with this, butyrate and doxorubicin combination significantly increased the activity of cytoplasmic cathepsin B. Inhibition of cathepsin B either with a small-molecule inhibitor or downregulation with a siRNA reversed butyrate- and doxorubicin-potentiated apoptosis. Finally, ex vivo, clinically relevant concentrations of butyrate or SAHA (suberoylanilide hydroxamic acid, vorinostat, an HDACi in clinical testing) in combination with doxorubicin significantly (P<0.0001) reduced the survival of primary myeloma cells.

Conclusions: Cathepsin B has a prominent function in mediating apoptosis potentiated by HDACi and doxorubicin combinations in myeloma. Our results support a molecular model of lysosomal-mitochondrial crosstalk in HDACi- and doxorubicin-potentiated apoptosis through the activation of cathepsin B.

Show MeSH
Related in: MedlinePlus