MO-OH-Nap and SAHA induce distinct patterns of gene expression in the multiple myeloma cell line U266.. Homo sapiens

Tropolones are small organic compounds with metal-directing moieties. Functionally, tropolones inhibit the proliferation of cancer cell lines, possibly through the inhibition of metalloenzymes such as select histone deacetylases (HDACS). Pan-HDAC inhibitors are therapeutically beneficial in the treatment of multiple myeloma albeit with significant side-effects likely owning to their lack of specificity. It was therefore hypothesized that tropolones might have anti-myeloma activities. To this end, a series of novel a-substituted tropolones were evaluated for cytotoxic effects in multiple myeloma cells. While all tested tropolones showed some level of toxicity, MO-OH-Nap had consistently low EC50 values between 1-11 mM in all three cell lines tested and was used for subsequent experiments. MO-OH-Nap was found to induce apoptosis in a concentration-dependent manner. Time course experiments demonstrated that MO-OH-Nap promotes caspase cleavage in a time-frame that was distinct from the pan-HDAC inhibitor SAHA. Furthermore, MO-OH-Nap- and SAHA-treated cells possess unique gene expression patterns, suggesting they promote apoptosis via different mechanisms. In particular, MO-OH-Nap increases the expression of markers associated with endoplasmic reticulum stress and the unfolded protein response. Synergistic cytotoxic effects were observed when cells were treated with the combination of MO-OH-Nap and the proteasome inhibitor bortezomib. However, treatment with MO-OH-Nap did not abrogate the bortezomib-induced increase in aggresomes, consistent with an HDAC6-independent mechanism for the observed synergy. Collectively, these finding support further investigation into the usefulness of a-substituted tropolones as anti-myeloma agents. Overall design: U266 cells were treated with DMSO (24 hours), SAHA (24 hours), MO-OH-Nap (24 hours) or MO-OH-Nap (48 hours). Experiments were performed in triplicate for each of the four treatment groups, yielding a total of 12 samples Following 24 or 48 hr incubations, RNA was extracted and used for microarray analysis with the Illumina HumanHT-12 v4 Expression BeadChip. Data were analyzed with R language and Bioconductor packages. Microarray data normalization, background correction and quality control were carried out with the Bioconductor package “lumi”. The R package “limma” was used to perform differential gene expression analysis of the normalized microarray data. The significant differentially expressed genes (DEG) had a p-value less than 0.05 and fold change with a cut off of 1.5.