Panobinostat synergizes with bortezomib to induce endoplasmic reticulum stress and ubiquitinated protein accumulation in renal cancer cells

Abstract

Background: Inducing endoplasmic reticulum (ER) stress is a novel strategy used to treat malignancies. Inhibition of histone deacetylase (HDAC) 6 by the HDAC inhibitor panobinostat hinders the refolding of unfolded proteins by increasing the acetylation of heat shock protein 90. We investigated whether combining panobinostat with the proteasome inhibitor bortezomib would kill cancer cells effectively by inhibiting the degradation of these unfolded proteins, thereby causing ubiquitinated proteins to accumulate and induce ER stress.

Methods: Caki-1, ACHN, and 769-P cells were treated with panobinostat and/or bortezomib. Cell viability, clonogenicity, and induction of apoptosis were evaluated. The in vivo efficacy of the combination was evaluated using a murine subcutaneous xenograft model. The combination-induced ER stress and ubiquitinated protein accumulation were assessed.

Results: The combination of panobinostat and bortezomib induced apoptosis and inhibited renal cancer growth synergistically (combination indexes <1). It also suppressed colony formation significantly (p <0.05). In a murine subcutaneous tumor model, a 10-day treatment was well tolerated and inhibited tumor growth significantly (p <0.05). Enhanced acetylation of the HDAC6 substrate alpha-tubulin was consistent with the suppression of HDAC6 activity by panobinostat, and the combination was shown to induce ER stress and ubiquitinated protein accumulation synergistically.

Conclusions: Panobinostat inhibits renal cancer growth by synergizing with bortezomib to induce ER stress and ubiquitinated protein accumulation. The current study provides a basis for testing the combination in patients with advanced renal cancer.

Figure 1

The combination of panobinostat and bortezomib inhibited renal cancer growth effectively. A, MTS assay results (mean ± SD, n = 6) after cells were treated for 48 hours either with bortezomib or panobinostat alone or with bortezomib and panobinostat together. B, Colony formation assay results (mean ± SD, n = 3) after 1–2 week incubation in control media (C) or media containing 50 nM panobinostat (P) and/or 10 nM bortezomib (B). *p = 0.0495; **p = 0.0463.

Figure 2

The combination of panobinostat and bortezomib suppressed tumor growth in vivo. A murine subcutaneous tumor model was made using Caki-1 cells, and the control group received intraperitoneal injections of DMSO, while other groups received either panobinostat (2 mg/kg) or bortezomib (60 μg/kg) or both. Injections were given once a day, 5 days a week, for 2 weeks. The 10-day treatment was well tolerated and suppressed tumor growth significantly (mean ± SE; p = 0.0283 at day 12).

Figure 3

The combination of panobinostat and bortezomib induced apoptosis in renal cancer cells. Cells were treated for 48 hours with 50 nM panobinostat with or without 10 nM bortezomib. The combination increased the annexin-V-FITC fluorescence intensity (A) and increased the number of the cells in the sub-G1 fraction (B). Relative annexin-V-FITC fluorescence intensity (control = 1) is shown in the insets. White, control; red, treated. The percentage of cells in the sub-G1 fraction is shown in the graph. Representative results are shown.

Figure 4

The combination therapy induced ER stress and histone acetylation in renal cancer cells. The 48-hour treatment with the combination of panobinostat and bortezomib induced ER stress synergistically as indicated by the increased expression of GRP78, HSP70, ERp44, and Ero1-Lα (A). It also caused ubiquitinated protein accumulation in all the cell lines synergistically and enhanced histone and also α-tubulin acetylation in Caki-1 and ACHN cells. In 769-P cells, the combination enhanced the acetylation of α-tubulin but not that of histone (B). The dashed lines in the Caki-1 results in parts A and B indicate that the order of the bands has been rearranged from the original gel.

Figure 5

Histone acetylation was a consequence of ubiquitinated protein accumulation. A, 48-hour treatment with panobinostat caused dose-dependent histone acetylation in all the cell lines but did not cause ubiquitinated protein accumulation. B, 48-hour treatment with bortezomib, on the other hand, caused both histone acetylation and ubiquitinated protein accumulation in Caki-1 and ACHN cells and caused only ubiquitinated protein accumulation in 769-P cells.