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Simultaneous Targeting of Bladder Tumor Growth, Survival, and Epithelial-to-Mesenchymal Transition with a Novel Therapeutic Combination of Acetazolamide (AZ) and Sulforaphane (SFN)

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Abstract

Background

Current chemotherapies for advanced stage metastatic bladder cancer often result in severe side effects, and most patients become drug resistant over time. Thus, there is a need for more effective therapies with minimal side effects.

Objective

The acid/base balance in tumor cells is essential for tumor cell functioning. We reasoned that simultaneous targeting of pH homeostasis and survival pathways would improve therapeutic efficacy. We evaluated the effectiveness of targeting pH homeostasis with the carbonic anhydrase inhibitor acetazolamide (AZ) in combination with the survival pathway targeting isothiocyanate sulforaphane (SFN) on the HTB-9 and RT112(H) human bladder tumor cell lines.

Materials and Methods

We assessed viability, proliferation, and survival in vitro and effect on xenografts in vivo.

Results

Combination AZ + SFN treatment induced dose-dependent suppression of growth, produced a potent anti-proliferative and anti-clonogenic effect, and induced apoptosis through caspase-3 and PARP activation. The anti-proliferative effect was corroborated by significant reductions in Ki-67, pHH3, cyclin D1, and sustained induction of the cell cycle inhibitors, p21 and p27. Both active p-Akt (Ser473) and p-S6 were significantly downregulated in the AZ + SFN combination treated cells with a concomitant inhibition of Akt kinase activity. The inhibitory effects of the AZ + SFN combination treatment showed similar efficacy as the dual PI3K/mTOR pathway inhibitor NVP-BEZ235, albeit at an expected higher dose. In terms of the effect on the metastatic potential of these bladder cancers, we found downregulated expression of carbonic anhydrase 9 (CA9) concomitant with reductions in both E-cadherin, N-cadherin, and vimentin proteins mitigating the epithelial-to-mesenchymal transition (EMT), suggesting negation of this program.

Conclusion

We suggest that reductions in these components could be linked with downregulation of the survival mediated Akt pathway and suggested an active role of the Akt pathway in bladder cancer. Altogether, our in vitro and pre-clinical model data support the potential use of an AZ + SFN combination for the treatment of bladder cancer.

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Acknowledgments

We gratefully thank Dr. Darius Bagli, the Hospital for Sick Children, Toronto, ON, Canada, for the HTB-9 cell line and antibodies, Dr. DA Sens, University of North Dakota, ND, USA, for the UROtsa cell line. We further extend our thanks to Dr. Janet Rossant, the Hospital for Sick Children, Toronto, ON, Canada, for providing several antibodies.

Compliance with Ethical Standards

ᅟ

Funding

None.

Conflict of Interest

The authors Islam SS, Mokhtari RB, Akbari P, Hatina J, Yeger H, and Farhat WA declare that they have no conflict of interest. No funding source disclosed. All animal use was handled with the guidelines of the CCAC and Lab Animal Services, at the Hospital for Sick Children, Toronto, Canada; the Laboratory Animal Safety committee, Research Institute, SickKids, approved the protocols for animal use.

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Authors and Affiliations

  1. Developmental and Stem Cell Biology, Research Institute, The Hospital for Sick Children, Toronto, ON, Canada

    S S Islam, R B Mokhtari, P Akbari, H Yeger & W A Farhat

  2. Faculty of Medicine, Charles University in Prague, Karlovaska, Czech Republic

    J Hatina

  3. Cancer Biology and Experimental Therapeutics Section, Division of Molecular Oncology, King Faisal Specialist Hospital and Research Centre, MBC# 03-66, PO Box 3354, Riyadh, 11211, Saudi Arabia

    S S Islam

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  1. S S Islam
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Correspondence to S S Islam.

Electronic Supplementary Material

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Supplementary Table 1

Frequency and absolute number of tumors in xenograft. Table represents total number of cells (1×106) implanted in each mouse (n = 5/group) and total number of tumors formed in each treatment group. (DOCX 17.2 kb)

Supplementary Table 2

In vivo xenograft tumor weight (gm) comparison within the group (Cont. vs AZ; AZ vs SFN and SFN vs AZ + SFN) in the drug treated mice. 1 × 106 cells were implanted in each mouse (n = 5/group) (Tumors were weighted on the day of 16 at experiment termination). (DOC 56 kb)

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Islam, S.S., Mokhtari, R.B., Akbari, P. et al. Simultaneous Targeting of Bladder Tumor Growth, Survival, and Epithelial-to-Mesenchymal Transition with a Novel Therapeutic Combination of Acetazolamide (AZ) and Sulforaphane (SFN). Targ Oncol 11, 209–227 (2016). https://doi.org/10.1007/s11523-015-0386-5

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