Presentation Authors: Akinori Sato, Takako Asano, Makoto Isono, Kazuki Okubo*, Tokorozawa, Japan
Introduction: Inducing endoplasmic reticulum (ER) stress is a novel approach to cancer treatment. The next-generation proteasome inhibitor oprozomib acts against cancer by increasing the amount of ubiquitinated protein and thereby causing ER stress, but its efficacy as a single agent is limited especially in solid tumors. We thought that inhibition of the intracellular drug-degrading enzyme CYP3A4 by cobicistat would enhance oprozomib&[prime]s activity and thereby kill bladder cancer cells effectively.
Methods: The viability and clonogenicity of bladder cancer cells (J82, T24, UMUC3) treated with oprozomib (25-50 nM) and/or cobicistat (20-40 ÂµM) were assessed by MTS assay and colony formation assay. Apoptosis was assayed using flow cytometry. Western blotting was used to evaluate the induction of ER stress (expression of ER stress markers), protein ubiquitination, histone acetylation, and the expression of the autophagy marker light chain 3-II, AMP-activated protein kinase (AMPK), eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1), and histone deacetylases (HDACs).
Results: The combination of oprozomib and cobicistat inhibited the growth of bladder cancer cells synergistically (combination indexes < 1). It also suppressed colony formation significantly (P < 0.05) and induced robust apoptosis synergistically: either 50 nM oprozomib or 40 ÂµM cobicistat caused slight to moderate increases in the amount of annexin V-positive cells, but the combination increased it drastically (up to 89.1% annexin V-positive cells). The combination was shown to cause ubiquitinated protein accumulation and thereby induce ER stress synergistically. Furthermore, we found that the excessively accumulated ubiquitinated proteins aggregated and thereby caused autophagy. The combination-induced ER stress caused dephosphorylation of 4EBP1 by increasing the expression of mammalian target of rapamycin (mTOR) inhibitor AMPK, showing that the combination also inhibited the mTOR pathway. Interestingly, we also found that the combination increased histone acetylation synergistically by decreasing the expression of HDACs 1, 3, and 6.
Conclusions: The combination of oprozomib and cobicistat inhibits bladder cancer growth by inducing ER stress synergistically. Histone acetylation is also an important mechanism of the combination&[prime]s action.