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Knockdown of BAG3 sensitizes bladder cancer cells to treatment with the BH3 mimetic ABT-737

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World Journal of Urology Aims and scope Submit manuscript

Abstract

Purpose

BAG3 is overexpressed in several malignancies and mediates a non-canonical, selective form of (macro)autophagy. By stabilizing pro-survival Bcl-2 proteins in complex with HSP70, BAG3 can also exert an apoptosis-antagonizing function. ABT-737 is a high affinity Bcl-2 inhibitor that fails to target Mcl-1. This failure may confer resistance in various cancers.

Methods

Urothelial cancer cells were treated with the BH3 mimetics ABT-737 and (−)-gossypol, a pan-Bcl-2 inhibitor which inhibits also Mcl-1. To clarify the importance of the core autophagy regulator ATG5 and BAG3 in ABT-737 treatment, cell lines carrying a stable lentiviral knockdown of ATG5 and BAG3 were created. The synergistic effect of ABT-737 and pharmaceutical inhibition of BAG3 with the HSF1 inhibitor KRIBB11 or sorafenib was also evaluated. Total cell death and apoptosis were quantified by FACS analysis of propidium iodide, annexin. Target protein analysis was conducted by Western blotting.

Results

Knockdown of BAG3 significantly downregulated Mcl-1 protein levels and sensitized urothelial cancer cells to apoptotic cell death induced by ABT-737, while inhibition of bulk autophagy through depletion of ATG5 had no discernible effect on cell death. Similar to knockdown of BAG3, pharmacological targeting of the BAG3/Mcl-1 pathway with KRIBB11 was capable to sensitize both cell lines to treatment with ABT-737.

Conclusion

Our results show that BAG3, but not bulk autophagy has a major role in the response of bladder cancer cells to BH3 mimetics. They also suggest that BAG3 is a suitable target for combined therapies aimed at synergistically inducing apoptosis in bladder cancer.

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Abbreviations

CMA:

Chaperone-mediated autophagy

ATG:

Autophagy-related genes

z-VAD:

Z-Val-Ala-DL-Asp(OMe)-fluoromethylketone

STS:

Staurosporine

PI:

Propidium iodide

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Acknowledgments

The authors would like to thank Gabriele Köpf for excellent technical assistance. This study was supported by a grant of the Medical Faculty, Goethe University Frankfurt (Frankfurter Forschungsförderung 2012), to JM.

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Authors and Affiliations

  1. Department of Urology, Goethe University Hospital, Theodor-Stern-Kai 7, 60590, Frankfurt am Main, Germany

    Jens Mani, Roman Blaheta, Georg Bartsch & Axel Haferkamp

  2. Experimental Neurosurgery, Neuroscience Center, Goethe University Hospital, Theodor-Stern-Kai 7, 60590, Frankfurt am Main, Germany

    Patrick Antonietti, Stefanie Rakel & Donat Kögel

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  1. Jens Mani
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Correspondence to Jens Mani.

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Mani, J., Antonietti, P., Rakel, S. et al. Knockdown of BAG3 sensitizes bladder cancer cells to treatment with the BH3 mimetic ABT-737. World J Urol 34, 197–205 (2016). https://doi.org/10.1007/s00345-015-1616-2

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  • DOI: https://doi.org/10.1007/s00345-015-1616-2

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