Yonsei Med J.  2020 Jul;61(7):587-596. 10.3349/ymj.2020.61.7.587.

Synergistic Antitumor Effects of Combined Treatment with HSP90 Inhibitor and PI3K/mTOR Dual Inhibitor in Cisplatin-Resistant Human Bladder Cancer Cells

Affiliations
  • 1Department of Urology, Myunggok Medical Research Institute, Konyang University College of Medicine, Daejeon, Korea
  • 2Department of Medicine, Graduate School of Yonsei University College of Medicine, Seoul, Korea
  • 3School of Dentistry, Pusan National University, Yangsan, Korea
  • 4Department of Urology, Urological Science Institute, Yonsei University College of Medicine, Seoul, Korea
  • 5Department of Pharmacology, College of Medicine, Konyang University, Daejeon, Korea
  • 6Department of Nuclear Medicine, Yonsei University College of Medicine, Seoul, Korea
  • 7Department of Pathology, Yonsei University College of Medicine, Seoul, Korea
  • 8Division of Medical Oncology, Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Korea

Abstract

Purpose
The current study aimed to investigate the synergistic antitumor effect of combined treatment with 17-DMAG (HSP90 inhibitor) and NVP-BEZ235 (PI3K/mTOR dual inhibitor) on cisplatin-resistant human bladder cancer cells.
Materials and Methods
Human bladder cancer cells exhibiting cisplatin resistance (T24R2) were exposed to escalating doses of 17-DMAG (2.5–20 nM) with or without NVP-BEZ236 (0.5–4 μM) in combination with cisplatin. Antitumor effects were assessed by CCK-8 analysis. Based on the dose-response study, synergistic interactions between the two regimens were evaluated using clonogenic assay and combination index values. Flow cytometry and Western blot were conducted to analyze mechanisms of synergism.
Results
Dose- and time-dependent antitumor effects for 17-DMAG were observed in both cisplatin-sensitive (T24) and cisplatin- resistant cells (T24R2). The antitumor effect of NVP-BEZ235, however, was found to be self-limiting. The combination of 17- DMAG and NVP-BEZ235 in a 1:200 fixed ratio showed a significant antitumor effect in cisplatin-resistant bladder cancer cells over a wide dose range, and clonogenic assay showed compatible results with synergy tests. Three-dimensional analysis revealed strong synergy between the two drugs with a synergy volume of 201.84 μM/mL2%. The combination therapy resulted in G1-phase cell cycle arrest and caspase-dependent apoptosis confirmed by the Western blot.
Conclusion
HSP90 inhibitor monotherapy and in combination with the PI3K/mTOR survival pathway inhibitor NVP-BEZ235 shows a synergistic antitumor effect in cisplatin-resistant bladder cancers, eliciting cell cycle arrest at the G1 phase and induction of caspase-dependent apoptotic pathway.

Keyword

Bladder cancer; Cisplatin; NVP-BEZ235; 17-DMAG
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