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Antitumor effects of tyropeptin-boronic acid derivatives: New proteasome inhibitors.

Momose I, Abe H, Watanabe T, Ohba S, Yamazaki K, Dan S, Yamori T, Masuda T, Nomoto A - Cancer Sci. (2014)

Bottom Line: Here we report the antitumor effects of two new tyropeptin-boronic acid derivatives, AS-06 and AS-29.We show that these derivatives also suppress the degradation of the NF-κB inhibitor IκB-α and the nuclear translocation of NF-κB p65 in multiple myeloma cells, resulting in the inhibition of NF-κB activation.Our results indicate that tyropeptin-boronic acid derivatives could be lead therapeutic agents against human multiple myeloma.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbial Chemistry, Numazu, Japan.

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Induction of apoptosis. (a) Detection of apoptotic cells. RPMI8226 cells were incubated with 1 μM inhibitors for 22 h. The cells were stained with an annexin V-FITC and propidium iodide and analyzed using a flow cytometer. (b) Induction of caspase activation. RPMI8226 cells were incubated with 0.1 μM inhibitors and caspase activation was detected by western blotting. (c) Induction of caspase-3 activity. RPMI8226 cells were incubated with inhibitors for 16 h and caspase-3 activity was measured using the caspase3/7-Glo assay. Columns, mean of triplicate determinations; bars, SD. (d) Gene expression analysis. RPMI8226 cells were incubated with 0.01, 0.1 and 1 μM inhibitors for 13 h. Gene expression analyses were performed using the Agilent human whole genome microarray.
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fig03: Induction of apoptosis. (a) Detection of apoptotic cells. RPMI8226 cells were incubated with 1 μM inhibitors for 22 h. The cells were stained with an annexin V-FITC and propidium iodide and analyzed using a flow cytometer. (b) Induction of caspase activation. RPMI8226 cells were incubated with 0.1 μM inhibitors and caspase activation was detected by western blotting. (c) Induction of caspase-3 activity. RPMI8226 cells were incubated with inhibitors for 16 h and caspase-3 activity was measured using the caspase3/7-Glo assay. Columns, mean of triplicate determinations; bars, SD. (d) Gene expression analysis. RPMI8226 cells were incubated with 0.01, 0.1 and 1 μM inhibitors for 13 h. Gene expression analyses were performed using the Agilent human whole genome microarray.

Mentions: We investigated whether tyropeptin-boronic acid derivatives induce apoptosis in RPMI8226 cells. Using annexin V and propidium iodide double staining and a flow cytometer (Fig. 3a), AS-06 and AS-29 significantly increased the number of late-apoptotic cells (annexin V-positive/propidium iodide-positive). We examined the effect of tyropeptin-boronic acid derivatives on the activation of caspases, a family of cysteine proteases playing a central role in apoptosis (Fig. 3b). AS-06 and AS-29 induced the degradation of full-length caspase-8 and full-length caspase-9, and cleaved fragments are detected. In addition, bortezomib was reported to activate ER-resident caspase-12 in multiple myeloma cells.(30) Although bortezomib induced the degradation of full-length caspase-12, neither AS-06 nor AS-29 induced the decrease of full-length caspase-12 (Fig. 3b). Caspase-3, a critical executioner of apoptosis, interacts with caspase-8 and caspase-9. AS-06 and AS-29 also stimulated the degradation of full-length caspase-3 and the appearance of cleaved forms, resulting in the cleavage of poly (ADP-ribose) polymerase (PARP), one of the main cleavage targets of caspase-3. In RPMI8226 cells, we found that AS-06 and AS-29 enhance caspase-3 activity in a dose-dependent manner (Fig. 3c). Furthermore, we performed gene expression analysis to compare the effects of tyropeptin-boronic acid derivatives and bortezomib on global gene transcription in RPMI8226 cells (Fig. 3d). We used whole human genome microarrays covering over 41 000 genes and transcripts. The transcription of 757, 744 and 2707 genes was altered over threefold in response to AS-06, AS-29 and bortezomib, respectively. The hierarchical clustering analysis for a total 2803 genes showed that the global gene expression signatures of AS-06 and AS-29 highly correlated with that of bortezomib. In summary, our data demonstrate that tyropeptin-boronic acid derivatives induce apoptosis through the caspase-8 and caspase-9 cascades, and that these derivatives and bortezomib have a similar effect on genome-wide transcriptional expression.


Antitumor effects of tyropeptin-boronic acid derivatives: New proteasome inhibitors.

Momose I, Abe H, Watanabe T, Ohba S, Yamazaki K, Dan S, Yamori T, Masuda T, Nomoto A - Cancer Sci. (2014)

Induction of apoptosis. (a) Detection of apoptotic cells. RPMI8226 cells were incubated with 1 μM inhibitors for 22 h. The cells were stained with an annexin V-FITC and propidium iodide and analyzed using a flow cytometer. (b) Induction of caspase activation. RPMI8226 cells were incubated with 0.1 μM inhibitors and caspase activation was detected by western blotting. (c) Induction of caspase-3 activity. RPMI8226 cells were incubated with inhibitors for 16 h and caspase-3 activity was measured using the caspase3/7-Glo assay. Columns, mean of triplicate determinations; bars, SD. (d) Gene expression analysis. RPMI8226 cells were incubated with 0.01, 0.1 and 1 μM inhibitors for 13 h. Gene expression analyses were performed using the Agilent human whole genome microarray.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig03: Induction of apoptosis. (a) Detection of apoptotic cells. RPMI8226 cells were incubated with 1 μM inhibitors for 22 h. The cells were stained with an annexin V-FITC and propidium iodide and analyzed using a flow cytometer. (b) Induction of caspase activation. RPMI8226 cells were incubated with 0.1 μM inhibitors and caspase activation was detected by western blotting. (c) Induction of caspase-3 activity. RPMI8226 cells were incubated with inhibitors for 16 h and caspase-3 activity was measured using the caspase3/7-Glo assay. Columns, mean of triplicate determinations; bars, SD. (d) Gene expression analysis. RPMI8226 cells were incubated with 0.01, 0.1 and 1 μM inhibitors for 13 h. Gene expression analyses were performed using the Agilent human whole genome microarray.
Mentions: We investigated whether tyropeptin-boronic acid derivatives induce apoptosis in RPMI8226 cells. Using annexin V and propidium iodide double staining and a flow cytometer (Fig. 3a), AS-06 and AS-29 significantly increased the number of late-apoptotic cells (annexin V-positive/propidium iodide-positive). We examined the effect of tyropeptin-boronic acid derivatives on the activation of caspases, a family of cysteine proteases playing a central role in apoptosis (Fig. 3b). AS-06 and AS-29 induced the degradation of full-length caspase-8 and full-length caspase-9, and cleaved fragments are detected. In addition, bortezomib was reported to activate ER-resident caspase-12 in multiple myeloma cells.(30) Although bortezomib induced the degradation of full-length caspase-12, neither AS-06 nor AS-29 induced the decrease of full-length caspase-12 (Fig. 3b). Caspase-3, a critical executioner of apoptosis, interacts with caspase-8 and caspase-9. AS-06 and AS-29 also stimulated the degradation of full-length caspase-3 and the appearance of cleaved forms, resulting in the cleavage of poly (ADP-ribose) polymerase (PARP), one of the main cleavage targets of caspase-3. In RPMI8226 cells, we found that AS-06 and AS-29 enhance caspase-3 activity in a dose-dependent manner (Fig. 3c). Furthermore, we performed gene expression analysis to compare the effects of tyropeptin-boronic acid derivatives and bortezomib on global gene transcription in RPMI8226 cells (Fig. 3d). We used whole human genome microarrays covering over 41 000 genes and transcripts. The transcription of 757, 744 and 2707 genes was altered over threefold in response to AS-06, AS-29 and bortezomib, respectively. The hierarchical clustering analysis for a total 2803 genes showed that the global gene expression signatures of AS-06 and AS-29 highly correlated with that of bortezomib. In summary, our data demonstrate that tyropeptin-boronic acid derivatives induce apoptosis through the caspase-8 and caspase-9 cascades, and that these derivatives and bortezomib have a similar effect on genome-wide transcriptional expression.

Bottom Line: Here we report the antitumor effects of two new tyropeptin-boronic acid derivatives, AS-06 and AS-29.We show that these derivatives also suppress the degradation of the NF-κB inhibitor IκB-α and the nuclear translocation of NF-κB p65 in multiple myeloma cells, resulting in the inhibition of NF-κB activation.Our results indicate that tyropeptin-boronic acid derivatives could be lead therapeutic agents against human multiple myeloma.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbial Chemistry, Numazu, Japan.

Show MeSH
Related in: MedlinePlus