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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.

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Effects of butyrate, doxorubicin or their combination on caspase 3 and 7 activation and AIF release in myeloma cells. (A, B) Effects of butyrate, doxorubicin and their combination on caspase 3 and 7 activation. (A) NCI H929, RPMI 8226 and U266 cells were treated with butyrate (SB; 300 μ for NCI H929 and 600 μ for RPMI 8226 and U266), doxorubicin (Dox; 40 n) or with their combination. After 24 h treatments, fold change in caspase 3 and 7 activity relative to untreated cells was assessed by caspase 3 and 7 glo kit (Promega Inc.). TRAIL (50 ng ml−1, Peprotech Inc., Rocky Hill, NJ, USA) was used as a positive control. Each data point in the bar graph is mean±s.e.m. of three independent experiments performed in triplicate. (B) Caspase 3 cleavage was assessed after 16, 24 or 36 h by subjecting 30 μg of whole-cell lysates (WCL) of RPMI 8226 cells to immunoblot analysis with a caspase 3-specific antibody. TRAIL-treated sample was used as a positive control and β-Actin as a loading control. (C, D) Effects of caspase 3 inhibitor DEVD-CHO on butyrate- and doxorubicin-induced apoptosis of myeloma cells. RPMI 8226 cells (1 × 106) were pretreated with either vehicle (DMSO) or 1 μ of cell permeable caspase 3-specific inhibitor DEVD-CHO (Biomol Inc.) for 2 h. Then the cells were left untreated or treated with TRAIL (50 ng ml−1) or butyrate (600 μ) plus doxorubicin (40 n). Caspase 3 and 7 activity was determined as in Figure 3A, and percentage of cells undergoing apoptosis was determined 48 h post-treatment by TUNEL assay as in Figure 2. Scatter plot shown is one of two independent experiments with similar results, in which 10 000 events were collected (top panel). Each bar on the graph is mean±s.e.m. of two independent experiments, and P-values of significantly different treatments are provided. (E) Butyrate plus doxorubicin combination results in nuclear translocation of AIF in RPMI 8226 and NCI H929 cells. RPMI 8226 or NCI H929 cells were left untreated or treated with indicated concentrations of butyrate, doxorubicin or their combination for 48 h. The localisation of AIF was assessed by indirect immunofluorescence staining with an AIF antibody followed by Alexa Flour-488-conjugated secondary antibody (Green staining). Nuclei of the cells were stained with DAPI (blue). Merged images were produced by superimposing both images. Results shown are representative of three independent experiments with similar results.
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fig2: Effects of butyrate, doxorubicin or their combination on caspase 3 and 7 activation and AIF release in myeloma cells. (A, B) Effects of butyrate, doxorubicin and their combination on caspase 3 and 7 activation. (A) NCI H929, RPMI 8226 and U266 cells were treated with butyrate (SB; 300 μ for NCI H929 and 600 μ for RPMI 8226 and U266), doxorubicin (Dox; 40 n) or with their combination. After 24 h treatments, fold change in caspase 3 and 7 activity relative to untreated cells was assessed by caspase 3 and 7 glo kit (Promega Inc.). TRAIL (50 ng ml−1, Peprotech Inc., Rocky Hill, NJ, USA) was used as a positive control. Each data point in the bar graph is mean±s.e.m. of three independent experiments performed in triplicate. (B) Caspase 3 cleavage was assessed after 16, 24 or 36 h by subjecting 30 μg of whole-cell lysates (WCL) of RPMI 8226 cells to immunoblot analysis with a caspase 3-specific antibody. TRAIL-treated sample was used as a positive control and β-Actin as a loading control. (C, D) Effects of caspase 3 inhibitor DEVD-CHO on butyrate- and doxorubicin-induced apoptosis of myeloma cells. RPMI 8226 cells (1 × 106) were pretreated with either vehicle (DMSO) or 1 μ of cell permeable caspase 3-specific inhibitor DEVD-CHO (Biomol Inc.) for 2 h. Then the cells were left untreated or treated with TRAIL (50 ng ml−1) or butyrate (600 μ) plus doxorubicin (40 n). Caspase 3 and 7 activity was determined as in Figure 3A, and percentage of cells undergoing apoptosis was determined 48 h post-treatment by TUNEL assay as in Figure 2. Scatter plot shown is one of two independent experiments with similar results, in which 10 000 events were collected (top panel). Each bar on the graph is mean±s.e.m. of two independent experiments, and P-values of significantly different treatments are provided. (E) Butyrate plus doxorubicin combination results in nuclear translocation of AIF in RPMI 8226 and NCI H929 cells. RPMI 8226 or NCI H929 cells were left untreated or treated with indicated concentrations of butyrate, doxorubicin or their combination for 48 h. The localisation of AIF was assessed by indirect immunofluorescence staining with an AIF antibody followed by Alexa Flour-488-conjugated secondary antibody (Green staining). Nuclei of the cells were stained with DAPI (blue). Merged images were produced by superimposing both images. Results shown are representative of three independent experiments with similar results.

Mentions: To gain a better understanding of the mechanism of apoptosis potentiated by butyrate and doxorubicin, effects of these agents alone or in combination on caspase-dependent and -independent apoptosis were investigated. Butyrate and doxorubicin treatments had no marked effect on the activity of caspase 3 and 7 in NCI H929, RPMI 8226 and U266 cell lines at 24 h (Figure 2A). Under the same conditions TRAIL, a potent inducer of apoptosis, markedly increased the activity of caspase 3 and 7 in sensitive cell lines (NCI H929 and RPMI 8226), suggesting the absence of an intrinsic block in caspase activation pathways in these cell lines. Lack of caspase 3 activation was confirmed by caspase 3 cleavage assay at 16, 24 and 36 h (Figure 2B). The role of caspase 3 in apoptosis potentiated by butyrate and doxorubicin was further tested by pretreating RPMI 8226 cells with DEVD-CHO, a cell permeable caspase 3-specific peptide inhibitor. DEVD-CHO inhibited the activity of caspase 3 and 7 (Figure 2C). Consistent with its inhibition of caspase activity, DEVD-CHO significantly reduced the apoptosis induced by TRAIL from 41.4 to 19.5% (P=0.0065); however, it had no apparent effect on apoptosis potentiated by the butyrate and doxorubicin combination (Figures 2C and D). Influence of caspases other than caspase 3 and 7 on butyrate and doxorubicin combination potentiated apoptosis was tested using z-VAD-FMK, a pan-caspase inhibitor. Compared with vehicle-treated cells, ∼12% decrease in apoptosis was observed in z-VAD-FMK-treated cells (Supplementary Figure 3). Together, these results suggest the involvement of caspases other than 3 and 7 in the potentiation of apoptosis by HDACi and doxorubicin.


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)

Effects of butyrate, doxorubicin or their combination on caspase 3 and 7 activation and AIF release in myeloma cells. (A, B) Effects of butyrate, doxorubicin and their combination on caspase 3 and 7 activation. (A) NCI H929, RPMI 8226 and U266 cells were treated with butyrate (SB; 300 μ for NCI H929 and 600 μ for RPMI 8226 and U266), doxorubicin (Dox; 40 n) or with their combination. After 24 h treatments, fold change in caspase 3 and 7 activity relative to untreated cells was assessed by caspase 3 and 7 glo kit (Promega Inc.). TRAIL (50 ng ml−1, Peprotech Inc., Rocky Hill, NJ, USA) was used as a positive control. Each data point in the bar graph is mean±s.e.m. of three independent experiments performed in triplicate. (B) Caspase 3 cleavage was assessed after 16, 24 or 36 h by subjecting 30 μg of whole-cell lysates (WCL) of RPMI 8226 cells to immunoblot analysis with a caspase 3-specific antibody. TRAIL-treated sample was used as a positive control and β-Actin as a loading control. (C, D) Effects of caspase 3 inhibitor DEVD-CHO on butyrate- and doxorubicin-induced apoptosis of myeloma cells. RPMI 8226 cells (1 × 106) were pretreated with either vehicle (DMSO) or 1 μ of cell permeable caspase 3-specific inhibitor DEVD-CHO (Biomol Inc.) for 2 h. Then the cells were left untreated or treated with TRAIL (50 ng ml−1) or butyrate (600 μ) plus doxorubicin (40 n). Caspase 3 and 7 activity was determined as in Figure 3A, and percentage of cells undergoing apoptosis was determined 48 h post-treatment by TUNEL assay as in Figure 2. Scatter plot shown is one of two independent experiments with similar results, in which 10 000 events were collected (top panel). Each bar on the graph is mean±s.e.m. of two independent experiments, and P-values of significantly different treatments are provided. (E) Butyrate plus doxorubicin combination results in nuclear translocation of AIF in RPMI 8226 and NCI H929 cells. RPMI 8226 or NCI H929 cells were left untreated or treated with indicated concentrations of butyrate, doxorubicin or their combination for 48 h. The localisation of AIF was assessed by indirect immunofluorescence staining with an AIF antibody followed by Alexa Flour-488-conjugated secondary antibody (Green staining). Nuclei of the cells were stained with DAPI (blue). Merged images were produced by superimposing both images. Results shown are representative of three independent experiments with similar results.
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fig2: Effects of butyrate, doxorubicin or their combination on caspase 3 and 7 activation and AIF release in myeloma cells. (A, B) Effects of butyrate, doxorubicin and their combination on caspase 3 and 7 activation. (A) NCI H929, RPMI 8226 and U266 cells were treated with butyrate (SB; 300 μ for NCI H929 and 600 μ for RPMI 8226 and U266), doxorubicin (Dox; 40 n) or with their combination. After 24 h treatments, fold change in caspase 3 and 7 activity relative to untreated cells was assessed by caspase 3 and 7 glo kit (Promega Inc.). TRAIL (50 ng ml−1, Peprotech Inc., Rocky Hill, NJ, USA) was used as a positive control. Each data point in the bar graph is mean±s.e.m. of three independent experiments performed in triplicate. (B) Caspase 3 cleavage was assessed after 16, 24 or 36 h by subjecting 30 μg of whole-cell lysates (WCL) of RPMI 8226 cells to immunoblot analysis with a caspase 3-specific antibody. TRAIL-treated sample was used as a positive control and β-Actin as a loading control. (C, D) Effects of caspase 3 inhibitor DEVD-CHO on butyrate- and doxorubicin-induced apoptosis of myeloma cells. RPMI 8226 cells (1 × 106) were pretreated with either vehicle (DMSO) or 1 μ of cell permeable caspase 3-specific inhibitor DEVD-CHO (Biomol Inc.) for 2 h. Then the cells were left untreated or treated with TRAIL (50 ng ml−1) or butyrate (600 μ) plus doxorubicin (40 n). Caspase 3 and 7 activity was determined as in Figure 3A, and percentage of cells undergoing apoptosis was determined 48 h post-treatment by TUNEL assay as in Figure 2. Scatter plot shown is one of two independent experiments with similar results, in which 10 000 events were collected (top panel). Each bar on the graph is mean±s.e.m. of two independent experiments, and P-values of significantly different treatments are provided. (E) Butyrate plus doxorubicin combination results in nuclear translocation of AIF in RPMI 8226 and NCI H929 cells. RPMI 8226 or NCI H929 cells were left untreated or treated with indicated concentrations of butyrate, doxorubicin or their combination for 48 h. The localisation of AIF was assessed by indirect immunofluorescence staining with an AIF antibody followed by Alexa Flour-488-conjugated secondary antibody (Green staining). Nuclei of the cells were stained with DAPI (blue). Merged images were produced by superimposing both images. Results shown are representative of three independent experiments with similar results.
Mentions: To gain a better understanding of the mechanism of apoptosis potentiated by butyrate and doxorubicin, effects of these agents alone or in combination on caspase-dependent and -independent apoptosis were investigated. Butyrate and doxorubicin treatments had no marked effect on the activity of caspase 3 and 7 in NCI H929, RPMI 8226 and U266 cell lines at 24 h (Figure 2A). Under the same conditions TRAIL, a potent inducer of apoptosis, markedly increased the activity of caspase 3 and 7 in sensitive cell lines (NCI H929 and RPMI 8226), suggesting the absence of an intrinsic block in caspase activation pathways in these cell lines. Lack of caspase 3 activation was confirmed by caspase 3 cleavage assay at 16, 24 and 36 h (Figure 2B). The role of caspase 3 in apoptosis potentiated by butyrate and doxorubicin was further tested by pretreating RPMI 8226 cells with DEVD-CHO, a cell permeable caspase 3-specific peptide inhibitor. DEVD-CHO inhibited the activity of caspase 3 and 7 (Figure 2C). Consistent with its inhibition of caspase activity, DEVD-CHO significantly reduced the apoptosis induced by TRAIL from 41.4 to 19.5% (P=0.0065); however, it had no apparent effect on apoptosis potentiated by the butyrate and doxorubicin combination (Figures 2C and D). Influence of caspases other than caspase 3 and 7 on butyrate and doxorubicin combination potentiated apoptosis was tested using z-VAD-FMK, a pan-caspase inhibitor. Compared with vehicle-treated cells, ∼12% decrease in apoptosis was observed in z-VAD-FMK-treated cells (Supplementary Figure 3). Together, these results suggest the involvement of caspases other than 3 and 7 in the potentiation of apoptosis by HDACi and doxorubicin.

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