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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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Apoptosis is enhanced by combination of bozepinib and IFNα. MCF-7, HCT-116, and RKO cell lines were mock-treated or treated with 5 µM bozepinib, 500 IU/mL IFNα, or the bozepinib/IFNα combination for 48 hours. Treated cells were then trypsinized and analyzed by flow cytometry using an Annexin V-fluorescein isothiocyanate detection kit. (A) Data are expressed as the mean ± standard error of the mean of three independent experiments. *P<0.05 (t-test). (B) Representative images from flow cytometry analysis.Abbreviations: FITC, fluorescein isothiocyanate; IFNα, interferon-alpha; M, mock treated cells.
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f2-dddt-7-1301: Apoptosis is enhanced by combination of bozepinib and IFNα. MCF-7, HCT-116, and RKO cell lines were mock-treated or treated with 5 µM bozepinib, 500 IU/mL IFNα, or the bozepinib/IFNα combination for 48 hours. Treated cells were then trypsinized and analyzed by flow cytometry using an Annexin V-fluorescein isothiocyanate detection kit. (A) Data are expressed as the mean ± standard error of the mean of three independent experiments. *P<0.05 (t-test). (B) Representative images from flow cytometry analysis.Abbreviations: FITC, fluorescein isothiocyanate; IFNα, interferon-alpha; M, mock treated cells.

Mentions: In order to determine if the effectiveness of the bozepinib/IFNα combination is due in part to an improvement in the apoptosis phenomenon, we treated MCF-7, HCT-116, and RKO cell lines with bozepinib alone or in combination with 500 IU/mL IFNα. As shown in Figure 2, the apoptosis induced by bozepinib at 48 hours was significantly increased when IFNα was added in all the cell lines analyzed (Figure 2).


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)

Apoptosis is enhanced by combination of bozepinib and IFNα. MCF-7, HCT-116, and RKO cell lines were mock-treated or treated with 5 µM bozepinib, 500 IU/mL IFNα, or the bozepinib/IFNα combination for 48 hours. Treated cells were then trypsinized and analyzed by flow cytometry using an Annexin V-fluorescein isothiocyanate detection kit. (A) Data are expressed as the mean ± standard error of the mean of three independent experiments. *P<0.05 (t-test). (B) Representative images from flow cytometry analysis.Abbreviations: FITC, fluorescein isothiocyanate; IFNα, interferon-alpha; M, mock treated cells.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3815003&req=5

f2-dddt-7-1301: Apoptosis is enhanced by combination of bozepinib and IFNα. MCF-7, HCT-116, and RKO cell lines were mock-treated or treated with 5 µM bozepinib, 500 IU/mL IFNα, or the bozepinib/IFNα combination for 48 hours. Treated cells were then trypsinized and analyzed by flow cytometry using an Annexin V-fluorescein isothiocyanate detection kit. (A) Data are expressed as the mean ± standard error of the mean of three independent experiments. *P<0.05 (t-test). (B) Representative images from flow cytometry analysis.Abbreviations: FITC, fluorescein isothiocyanate; IFNα, interferon-alpha; M, mock treated cells.
Mentions: In order to determine if the effectiveness of the bozepinib/IFNα combination is due in part to an improvement in the apoptosis phenomenon, we treated MCF-7, HCT-116, and RKO cell lines with bozepinib alone or in combination with 500 IU/mL IFNα. As shown in Figure 2, the apoptosis induced by bozepinib at 48 hours was significantly increased when IFNα was added in all the cell lines analyzed (Figure 2).

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