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Bozepinib, a novel small antitumor agent, induces PKR-mediated apoptosis and synergizes with IFNα triggering apoptosis, autophagy and senescence.

Marchal JA, Carrasco E, Ramirez A, Jiménez G, Olmedo C, Peran M, Agil A, Conejo-García A, Cruz-López O, Campos JM, García MÁ - Drug Des Devel Ther (2013)

Bottom Line: In addition, the efficacy of bozepinib was improved when combined with the interferon-alpha (IFNα) cytokine, which enhanced bozepinib-induced apoptosis with involvement of protein kinase PKR.Curiously, this population showed β-galactosidase activity and a percentage of cells arrested in S phase, that was more evident in cells treated with the bozepinib/IFNα combination than in cells treated with bozepinib or IFNα alone.Considering the resistance of some cancer cells to conventional chemotherapy, combinations enhancing the diversity of the cell death outcome might succeed in delivering more effective and less toxic chemotherapy.

View Article: PubMed Central - PubMed

Affiliation: Biopathology and Regenerative Medicine Institute, Centre for Biomedical Research, Spain ; Department of Human Anatomy and Embryology, Faculty of Medicine, University of Granada, Granada, Spain.

ABSTRACT
Bozepinib [(RS)-2,6-dichloro-9-[1-(p-nitrobenzenesulfonyl)-1,2,3,5-tetrahydro-4,1-benzoxazepin-3-yl]-9H-purine] is a potent antitumor compound that is able to induce apoptosis in breast cancer cells. In the present study, we show that bozepinib also has antitumor activity in colon cancer cells, showing 50% inhibitory concentration (IC50) values lower than those described for breast cancer cells and suggesting great potential of this synthetic drug in the treatment of cancer. We identified that the double-stranded RNA-dependent protein kinase (PKR) is a target of bozepinib, being upregulated and activated by the drug. However, p53 was not affected by bozepinib, and was not necessary for induction of apoptosis in either breast or colon cancer cells. In addition, the efficacy of bozepinib was improved when combined with the interferon-alpha (IFNα) cytokine, which enhanced bozepinib-induced apoptosis with involvement of protein kinase PKR. Moreover, we report here, for the first time, that in combined therapy, IFNα induces a clear process of autophagosome formation, and prior treatment with chloroquine, an autophagy inhibitor, is able to significantly reduce IFNα/bozepinib-induced cell death. Finally, we observed that a minor population of caspase 3-deficient MCF-7 cells persisted during long-term treatment with lower doses of bozepinib and the bozepinib/IFNα combination. Curiously, this population showed β-galactosidase activity and a percentage of cells arrested in S phase, that was more evident in cells treated with the bozepinib/IFNα combination than in cells treated with bozepinib or IFNα alone. Considering the resistance of some cancer cells to conventional chemotherapy, combinations enhancing the diversity of the cell death outcome might succeed in delivering more effective and less toxic chemotherapy.

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Bozepinib induced LC3-autophagosome formation that was strongly enhanced when combined with IFNα. (A) MCF-7 cells were mock-treated or treated with 5 µM bozepinib, 500 IU/mL human IFNα, or a combination of bozepinib/IFNα for 48 hours. Total proteins were extracted for immunoblot analysis using anti-LC3 and anti-β-actin antibodies. (B) MCF-7 cells were plated on cover slips supported in six-well plates and transfected with 5 μg of GFP-LC3 or GFP-control plasmids as described in the Materials and methods section. After 24 hours, the cells were treated with 5 µM bozepinib, 500 IU/mL human IFNα, or a combination of bozepinib/IFNα for 48 hours. Cells were fixed and visualized using a Radiance 2000 confocal microscope. (C) MCF-7 and HCT-116 cells were mock-treated or treated with 5 µM bozepinib, 500 IU/mL human IFNα, or a combination of bozepinib/IFNα for 48 hours. Cells were fixed and prepared for visualization by transmission electron microscopy as described in the Materials and methods section. Transmission electron microscopy images show that the treated cells included typical autophagolysosomes (arrows) containing organelles and lamellar structures. (D) MCF-7 cells were treated with 20 μM of chloroquine or 25 μM of Z-VAD inhibitors 2 hours before 5 μM bozepinib, 500 IU/mL IFNα, or a combination of bozepinib/IFNα. After 48 hours, the cells were treated with a Cell Counting Kit-8, measured at 450 nm optical density and represented as described in the Materials and methods section. Total proteins were extracted for immunoblot analysis using anti-LC3 and anti-β-actin antibodies. *P<0.05 (t-test). Western blot signals were quantified using Image J software, and relative β-actin-normalized values were assigned in reference to nontreated cells (value 1).Abbreviations: CQ, chloroquine; IFNα, interferon-alpha; M, mock treated cells.
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f4-dddt-7-1301: Bozepinib induced LC3-autophagosome formation that was strongly enhanced when combined with IFNα. (A) MCF-7 cells were mock-treated or treated with 5 µM bozepinib, 500 IU/mL human IFNα, or a combination of bozepinib/IFNα for 48 hours. Total proteins were extracted for immunoblot analysis using anti-LC3 and anti-β-actin antibodies. (B) MCF-7 cells were plated on cover slips supported in six-well plates and transfected with 5 μg of GFP-LC3 or GFP-control plasmids as described in the Materials and methods section. After 24 hours, the cells were treated with 5 µM bozepinib, 500 IU/mL human IFNα, or a combination of bozepinib/IFNα for 48 hours. Cells were fixed and visualized using a Radiance 2000 confocal microscope. (C) MCF-7 and HCT-116 cells were mock-treated or treated with 5 µM bozepinib, 500 IU/mL human IFNα, or a combination of bozepinib/IFNα for 48 hours. Cells were fixed and prepared for visualization by transmission electron microscopy as described in the Materials and methods section. Transmission electron microscopy images show that the treated cells included typical autophagolysosomes (arrows) containing organelles and lamellar structures. (D) MCF-7 cells were treated with 20 μM of chloroquine or 25 μM of Z-VAD inhibitors 2 hours before 5 μM bozepinib, 500 IU/mL IFNα, or a combination of bozepinib/IFNα. After 48 hours, the cells were treated with a Cell Counting Kit-8, measured at 450 nm optical density and represented as described in the Materials and methods section. Total proteins were extracted for immunoblot analysis using anti-LC3 and anti-β-actin antibodies. *P<0.05 (t-test). Western blot signals were quantified using Image J software, and relative β-actin-normalized values were assigned in reference to nontreated cells (value 1).Abbreviations: CQ, chloroquine; IFNα, interferon-alpha; M, mock treated cells.

Mentions: We analyzed the ability of bozepinib to induce autophagy as well as regulation of this process by IFNα in the MCF-7 cell line, which is deficient in caspase 3 activation.28 Despite the low level of endogenous LC3 protein, LC3-II levels were weakly detected 48 hours after treatment with bozepinib and were more evident when IFNα was added (Figure 4A). Confocal microscopy was used to analyze the redistribution of LC3 protein into the autophagosomes of MCF-7 cells transfected with the pCMV-GFP-LC3 vector and the pCMV-GFP control vector, and the cells were then mock-treated or treated with bozepinib, IFNα, or the bozepinib/IFNα combination. As shown in Figure 4B, 48 hours post-treatment, the mock-treated cells displayed diffuse staining. However, a speckled fluorescent staining pattern was detected in almost all cells analyzed after treatment with bozepinib/IFNα, indicating redistribution of LC3 to autophagosomes. The speckled fluorescent stain was less pronounced after treatment with bozepinib alone or IFNα alone, and was detected in less than half of the cells analyzed (Figure 4B). Cells expressing the control vector pCMV-GFP displayed diffuse staining, even in the presence of treatment with bozepinib/IFNα (Figure 4B).


Bozepinib, a novel small antitumor agent, induces PKR-mediated apoptosis and synergizes with IFNα triggering apoptosis, autophagy and senescence.

Marchal JA, Carrasco E, Ramirez A, Jiménez G, Olmedo C, Peran M, Agil A, Conejo-García A, Cruz-López O, Campos JM, García MÁ - Drug Des Devel Ther (2013)

Bozepinib induced LC3-autophagosome formation that was strongly enhanced when combined with IFNα. (A) MCF-7 cells were mock-treated or treated with 5 µM bozepinib, 500 IU/mL human IFNα, or a combination of bozepinib/IFNα for 48 hours. Total proteins were extracted for immunoblot analysis using anti-LC3 and anti-β-actin antibodies. (B) MCF-7 cells were plated on cover slips supported in six-well plates and transfected with 5 μg of GFP-LC3 or GFP-control plasmids as described in the Materials and methods section. After 24 hours, the cells were treated with 5 µM bozepinib, 500 IU/mL human IFNα, or a combination of bozepinib/IFNα for 48 hours. Cells were fixed and visualized using a Radiance 2000 confocal microscope. (C) MCF-7 and HCT-116 cells were mock-treated or treated with 5 µM bozepinib, 500 IU/mL human IFNα, or a combination of bozepinib/IFNα for 48 hours. Cells were fixed and prepared for visualization by transmission electron microscopy as described in the Materials and methods section. Transmission electron microscopy images show that the treated cells included typical autophagolysosomes (arrows) containing organelles and lamellar structures. (D) MCF-7 cells were treated with 20 μM of chloroquine or 25 μM of Z-VAD inhibitors 2 hours before 5 μM bozepinib, 500 IU/mL IFNα, or a combination of bozepinib/IFNα. After 48 hours, the cells were treated with a Cell Counting Kit-8, measured at 450 nm optical density and represented as described in the Materials and methods section. Total proteins were extracted for immunoblot analysis using anti-LC3 and anti-β-actin antibodies. *P<0.05 (t-test). Western blot signals were quantified using Image J software, and relative β-actin-normalized values were assigned in reference to nontreated cells (value 1).Abbreviations: CQ, chloroquine; IFNα, interferon-alpha; M, mock treated cells.
© Copyright Policy
Related In: Results  -  Collection

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f4-dddt-7-1301: Bozepinib induced LC3-autophagosome formation that was strongly enhanced when combined with IFNα. (A) MCF-7 cells were mock-treated or treated with 5 µM bozepinib, 500 IU/mL human IFNα, or a combination of bozepinib/IFNα for 48 hours. Total proteins were extracted for immunoblot analysis using anti-LC3 and anti-β-actin antibodies. (B) MCF-7 cells were plated on cover slips supported in six-well plates and transfected with 5 μg of GFP-LC3 or GFP-control plasmids as described in the Materials and methods section. After 24 hours, the cells were treated with 5 µM bozepinib, 500 IU/mL human IFNα, or a combination of bozepinib/IFNα for 48 hours. Cells were fixed and visualized using a Radiance 2000 confocal microscope. (C) MCF-7 and HCT-116 cells were mock-treated or treated with 5 µM bozepinib, 500 IU/mL human IFNα, or a combination of bozepinib/IFNα for 48 hours. Cells were fixed and prepared for visualization by transmission electron microscopy as described in the Materials and methods section. Transmission electron microscopy images show that the treated cells included typical autophagolysosomes (arrows) containing organelles and lamellar structures. (D) MCF-7 cells were treated with 20 μM of chloroquine or 25 μM of Z-VAD inhibitors 2 hours before 5 μM bozepinib, 500 IU/mL IFNα, or a combination of bozepinib/IFNα. After 48 hours, the cells were treated with a Cell Counting Kit-8, measured at 450 nm optical density and represented as described in the Materials and methods section. Total proteins were extracted for immunoblot analysis using anti-LC3 and anti-β-actin antibodies. *P<0.05 (t-test). Western blot signals were quantified using Image J software, and relative β-actin-normalized values were assigned in reference to nontreated cells (value 1).Abbreviations: CQ, chloroquine; IFNα, interferon-alpha; M, mock treated cells.
Mentions: We analyzed the ability of bozepinib to induce autophagy as well as regulation of this process by IFNα in the MCF-7 cell line, which is deficient in caspase 3 activation.28 Despite the low level of endogenous LC3 protein, LC3-II levels were weakly detected 48 hours after treatment with bozepinib and were more evident when IFNα was added (Figure 4A). Confocal microscopy was used to analyze the redistribution of LC3 protein into the autophagosomes of MCF-7 cells transfected with the pCMV-GFP-LC3 vector and the pCMV-GFP control vector, and the cells were then mock-treated or treated with bozepinib, IFNα, or the bozepinib/IFNα combination. As shown in Figure 4B, 48 hours post-treatment, the mock-treated cells displayed diffuse staining. However, a speckled fluorescent staining pattern was detected in almost all cells analyzed after treatment with bozepinib/IFNα, indicating redistribution of LC3 to autophagosomes. The speckled fluorescent stain was less pronounced after treatment with bozepinib alone or IFNα alone, and was detected in less than half of the cells analyzed (Figure 4B). Cells expressing the control vector pCMV-GFP displayed diffuse staining, even in the presence of treatment with bozepinib/IFNα (Figure 4B).

Bottom Line: In addition, the efficacy of bozepinib was improved when combined with the interferon-alpha (IFNα) cytokine, which enhanced bozepinib-induced apoptosis with involvement of protein kinase PKR.Curiously, this population showed β-galactosidase activity and a percentage of cells arrested in S phase, that was more evident in cells treated with the bozepinib/IFNα combination than in cells treated with bozepinib or IFNα alone.Considering the resistance of some cancer cells to conventional chemotherapy, combinations enhancing the diversity of the cell death outcome might succeed in delivering more effective and less toxic chemotherapy.

View Article: PubMed Central - PubMed

Affiliation: Biopathology and Regenerative Medicine Institute, Centre for Biomedical Research, Spain ; Department of Human Anatomy and Embryology, Faculty of Medicine, University of Granada, Granada, Spain.

ABSTRACT
Bozepinib [(RS)-2,6-dichloro-9-[1-(p-nitrobenzenesulfonyl)-1,2,3,5-tetrahydro-4,1-benzoxazepin-3-yl]-9H-purine] is a potent antitumor compound that is able to induce apoptosis in breast cancer cells. In the present study, we show that bozepinib also has antitumor activity in colon cancer cells, showing 50% inhibitory concentration (IC50) values lower than those described for breast cancer cells and suggesting great potential of this synthetic drug in the treatment of cancer. We identified that the double-stranded RNA-dependent protein kinase (PKR) is a target of bozepinib, being upregulated and activated by the drug. However, p53 was not affected by bozepinib, and was not necessary for induction of apoptosis in either breast or colon cancer cells. In addition, the efficacy of bozepinib was improved when combined with the interferon-alpha (IFNα) cytokine, which enhanced bozepinib-induced apoptosis with involvement of protein kinase PKR. Moreover, we report here, for the first time, that in combined therapy, IFNα induces a clear process of autophagosome formation, and prior treatment with chloroquine, an autophagy inhibitor, is able to significantly reduce IFNα/bozepinib-induced cell death. Finally, we observed that a minor population of caspase 3-deficient MCF-7 cells persisted during long-term treatment with lower doses of bozepinib and the bozepinib/IFNα combination. Curiously, this population showed β-galactosidase activity and a percentage of cells arrested in S phase, that was more evident in cells treated with the bozepinib/IFNα combination than in cells treated with bozepinib or IFNα alone. Considering the resistance of some cancer cells to conventional chemotherapy, combinations enhancing the diversity of the cell death outcome might succeed in delivering more effective and less toxic chemotherapy.

Show MeSH
Related in: MedlinePlus