HSP70 is a molecular chaperone localized in the cytoplasm but associated with the regulation of the UPR by forming a stable protein complex selleck chem inhibitor with the cytosolic domain of inositol requiring enzyme 1. Ero1 L regulates oxidative protein folding by selectively oxidizing protein disulfide isomer ase, one of the key players in the control of disulfide bond formation. ERp44 forms mixed disulfides with Ero1 L and may be involved in the control of oxidative protein folding. The increased expression of these ER stress related proteins thus confirmed that ER stress was induced by the combination of panobinostat and bortezomib. Acetylation of tubulin, one of the important sub strates of HDAC6, is consistent with the inhibition of HDAC6 by panobinostat.

Interestingly, panobinostat itself did not cause marked ER stress even though it inhibited HDAC6 function. This may be because the unfolded proteins increased by panobinostat can be degraded immediately by the proteasome if its function is not suppressed. This explanation is consistent with the result that panobinostat induced marked ER stress only when combined with bortezomib. The combination induced ubiquitinated protein ac cumulation synergistically. This is because panobinostat increased unfolded proteins, which were then ubiquiti nated, and bortezomib inhibited their degradation. The ubiquitinated protein accumulation is also in accordance with the above discussed enhanced ER stress induced by the combination because ER stress is induced by the accu mulation of unfolded proteins in the cell, and many of these unfolded proteins are ubiquitinated.

Not only are ubiquitinated proteins themselves toxic to tumor cells, some of them may be important molecules for cancer cell survival that have lost their function because of unfolding and ubiquitination, presumably leading to the inhibition of multiple signal transduction pathways. Fur thermore, the inhibition of NF kB is thought to play an important role in the combination therapy with pano binostat and bortezomib because of the accumulation of undegraded IkB, a suppressor of NF kB. Jiang XJ et al. reported that the combination of panobinostat and bortezomib activated caspases and down regulated antiapoptotic proteins such as XIAP and Bcl 2 through inhibition of the AKT and NF kB pathways.

The com bination is thus thought to inhibit cancer growth by diverse mechanisms other than the induction of ER stress and ubiquitinated protein accumulation. In Caki 1 and ACHN cells the combination of panobi nostat and bortezomib not only caused ubiquitinated pro tein accumulation but also enhanced histone acetylation. In these cell lines, panobinostat alone caused histone acetylation but not ubiquitinated protein accumulation, whereas bortezomib alone induced both ubiquitinated protein accumulation and histone Carfilzomib acetylation.