Presentation Authors: Takako Asano*, Akinori Sato, Makoto Isono, Kazuki Okubo, Tokorozawa, Japan
Introduction: Induction of endoplasmic reticulum (ER) stress is a novel strategy used to treat malignancies. The novel proteasome inhibitor ixazomib kills cancer cells by causing unfolded protein accumulation and thereby inducing ER stress, but its efficacy as a single agent is limited especially in solid tumors. Cobicistat is a potent inhibitor of the intracellular drug-degrading enzyme CYP3A and we postulated that combining cobicistat with ixazomib would kill renal cancer cells by inducing ER stress effectively.
Methods: Renal cancer cells (769-P, 786-O, Caki-1) were treated with cobicistat (10-40 ÂµM) and ixazomib (25-200 nM). Cell viability and clonogenicity were assessed by MTS assay and colony formation assay. Flow cytometry was used for cell cycle analysis and annexin V assay. Western blotting was used to evaluate the induction of ER stress and the expression of apoptosis-associated proteins, cell cycle-associated proteins, NOXA, acetylated histone, and the autophagy marker light chain (LC) 3-II. Combination indexes were calculated by the Chou-Talalay method.
Results: The combination of cobicistat and ixazomib inhibited cancer cell growth synergistically (combination indexes < 1) and suppressed colony formation significantly (p < 0.05). The combination decreased the expression of cyclin D1 and cyclin-dependent kinase 4, leading to the accumulation of the cells in the sub-G1 fraction. Drastic increases in the number of annexin-V positive cells (up to 94.6%) and increased expression of cleaved poly(ADP-ribose) polymerase confirmed that the combination induced apoptosis. As expected, ixazomib induced ER stress in a dose-dependent fashion, and cobicistat enhanced this stress synergistically. Increased expression of NOXA confirmed that the combination-induced apoptosis was a result of ER stress. Furthermore, this ER stress induction was essential to the combination&[prime]s cytotoxic action because inhibition of unfolded protein accumulation by the protein synthesis inhibitor cycloheximide markedly attenuated the combination-induced apoptosis. Interestingly, we also found that the cobicistat-ixazomib combination increased the expression of LC3-II, confirming that the unfolded protein accumulation due to the combination induced autophagy.
Conclusions: Cobicistat enhanced ixazomib&[prime]s activity, and the combination inhibited renal cancer growth synergistically.