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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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Cytotoxic effect of bozepinib and combined bozepinib/IFNα therapy. (A) Chemical structure of bozepinib. (B) MCF-7, HCT-116, and RKO cell lines treated with increasing amounts of bozepinib alone (circle) or in combination with 50 IU/mL IFNα (square) for 6 days as described in the Materials and methods section. Cell lines have been defined in material and methods section. The curve for cell survival is represented as a percentage compared to mock-treated cells. Values shown represent the mean of triplicate determinations calculated from a single experiment. Experiments were repeated at least three times.Abbreviation: IFNα, interferon-alpha.
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f1-dddt-7-1301: Cytotoxic effect of bozepinib and combined bozepinib/IFNα therapy. (A) Chemical structure of bozepinib. (B) MCF-7, HCT-116, and RKO cell lines treated with increasing amounts of bozepinib alone (circle) or in combination with 50 IU/mL IFNα (square) for 6 days as described in the Materials and methods section. Cell lines have been defined in material and methods section. The curve for cell survival is represented as a percentage compared to mock-treated cells. Values shown represent the mean of triplicate determinations calculated from a single experiment. Experiments were repeated at least three times.Abbreviation: IFNα, interferon-alpha.

Mentions: A human breast MCF-7 cell line (ECACC: 86012803) and human colon cancer RKO (ATCC: CRL-2577) and HCT-116 (ECACC: 91091005) cell lines were provided by the Cell Bank of the University of Granada (Granada, Spain). PKR+/+ and PKR−/− mouse embryonic fibroblasts25 and human colon HCT-116 p53+/+ and p53−/− cells26 were kindly provided by M Esteban (National Center of Biotechnology, Madrid, Spain) and B Vogelstein (Johns Hopkins Oncology Center, Baltimore, MD, USA), respectively. PKR was knocked down by RNA interference using shRNA-PKR as described previously.24 The cells were maintained in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, and 1% nonessential amino acid solution. Exponentially growing cells were used for all experiments. Bozepinib (Figure 1A) was synthesized as previously described,4 dissolved in dimethyl sulfoxide, and stored at −20°C. For each experiment, the stock solutions were further diluted in medium to obtain the desired concentrations. Human IFNα2b (Intron A®) was obtained from Schering-Plough (Union, NJ, USA) and mouse IFNα was sourced from Peprotech (Rocky Hill, NJ, USA). Z-VAD-FMK, a pan-caspase inhibitor, was provided by Santa Cruz Biotechnology (Santa Cruz, CA, USA) and chloroquine was obtained from Sigma-Aldrich (St Louis, MO, USA).


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)

Cytotoxic effect of bozepinib and combined bozepinib/IFNα therapy. (A) Chemical structure of bozepinib. (B) MCF-7, HCT-116, and RKO cell lines treated with increasing amounts of bozepinib alone (circle) or in combination with 50 IU/mL IFNα (square) for 6 days as described in the Materials and methods section. Cell lines have been defined in material and methods section. The curve for cell survival is represented as a percentage compared to mock-treated cells. Values shown represent the mean of triplicate determinations calculated from a single experiment. Experiments were repeated at least three times.Abbreviation: IFNα, interferon-alpha.
© Copyright Policy
Related In: Results  -  Collection

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

f1-dddt-7-1301: Cytotoxic effect of bozepinib and combined bozepinib/IFNα therapy. (A) Chemical structure of bozepinib. (B) MCF-7, HCT-116, and RKO cell lines treated with increasing amounts of bozepinib alone (circle) or in combination with 50 IU/mL IFNα (square) for 6 days as described in the Materials and methods section. Cell lines have been defined in material and methods section. The curve for cell survival is represented as a percentage compared to mock-treated cells. Values shown represent the mean of triplicate determinations calculated from a single experiment. Experiments were repeated at least three times.Abbreviation: IFNα, interferon-alpha.
Mentions: A human breast MCF-7 cell line (ECACC: 86012803) and human colon cancer RKO (ATCC: CRL-2577) and HCT-116 (ECACC: 91091005) cell lines were provided by the Cell Bank of the University of Granada (Granada, Spain). PKR+/+ and PKR−/− mouse embryonic fibroblasts25 and human colon HCT-116 p53+/+ and p53−/− cells26 were kindly provided by M Esteban (National Center of Biotechnology, Madrid, Spain) and B Vogelstein (Johns Hopkins Oncology Center, Baltimore, MD, USA), respectively. PKR was knocked down by RNA interference using shRNA-PKR as described previously.24 The cells were maintained in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, and 1% nonessential amino acid solution. Exponentially growing cells were used for all experiments. Bozepinib (Figure 1A) was synthesized as previously described,4 dissolved in dimethyl sulfoxide, and stored at −20°C. For each experiment, the stock solutions were further diluted in medium to obtain the desired concentrations. Human IFNα2b (Intron A®) was obtained from Schering-Plough (Union, NJ, USA) and mouse IFNα was sourced from Peprotech (Rocky Hill, NJ, USA). Z-VAD-FMK, a pan-caspase inhibitor, was provided by Santa Cruz Biotechnology (Santa Cruz, CA, USA) and chloroquine was obtained from Sigma-Aldrich (St Louis, MO, USA).

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