Vorinostat Eliminates Multicellular Resistance of Mesothelioma 3D Spheroids via Restoration of Noxa Expression

When grown in 3D cultures as spheroids, mesothelioma cells acquire a multicellular resistance to apoptosis that resembles that of solid tumors. We have previously found that resistance to the proteasome inhibitor bortezomib in 3D can be explained by a lack of upregulation of Noxa, the pro-apoptotic BH3 sensitizer that acts via displacement of the Bak/Bax-activator BH3-only protein, Bim. We hypothesized that the histone deacetylase inhibitor vorinostat might reverse this block to Noxa upregulation in 3D. Indeed, we found that vorinostat effectively restored upregulation of Noxa protein and message and abolished multicellular resistance to bortezomib in the 3D spheroids. The ability of vorinostat to reverse resistance was ablated by knockdown of Noxa or Bim, confirming the essential role of the Noxa/Bim axis in the response to vorinostat. Addition of vorinostat similarly increased the apoptotic response to bortezomib in another 3D model, the tumor fragment spheroid, which is grown from human mesothelioma ex vivo. In addition to its benefit when used with bortezomib, vorinostat also enhanced the response to cisplatin plus pemetrexed, as shown in both 3D models. Our results using clinically relevant 3D models show that the manipulation of the core apoptotic repertoire may improve the chemosensitivity of mesothelioma. Whereas neither vorinostat nor bortezomib alone has been clinically effective in mesothelioma, vorinostat may undermine chemoresistance to bortezomib and to other therapies thereby providing a rationale for combinatorial strategies

Absolute Configuration and Biological Properties of Enantiomers of CFTR Inhibitor BPO-27

We previously reported benzopyrimido-pyrrolo-oxazinedione (BPO) inhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel and showed their efficacy in a model of polycystic kidney disease. Here, we separated the enantiomers of lead compound BPO-27, (1), which contains a single chiral center, and determined their absolute configuration, activity and metabolic stability. Following separation by chiral supercritical fluid chromatography, the R enantiomer, as determined by x-ray crystallography, inhibited CFTR chloride conductance with IC50 ~ 4 nM, while S enantiomer was inactive. In vitro metabolic stability in hepatic microsomes showed both enantiomers as stable, with <5 % metabolism in 4 h. Following bolus interperitoneal administration in mice, serum (R)-1 decayed with t1/2 ~ 1.6 h and gave sustained therapeutic concentrations in kidney

Differential regulation of NFAT5 by NKCC2 isoforms in medullary thick ascending limb (mTAL) cells

The effects of Na+-K+-2Cl− cotransporter type 2 (NKCC2) isoforms on the regulation of nuclear factor of activated T cells isoform 5 (NFAT5) were determined in mouse medullary thick ascending limb (mTAL) cells exposed to high NaCl concentration. Primary cultures of mTAL cells and freshly isolated mTAL tubules, both derived from the outer medulla (outer stripe>inner stripe), express NKCC2 isoforms A and F. The relative expression of NKCC2A mRNA was approximately twofold greater than NKCC2F in these preparations. The abundance of NKCC2A mRNA, but not NKCC2F mRNA, increased approximately twofold when mTAL cells were exposed for 2 h to a change in osmolality from 300 to 500 mosmol/kgH2O, produced with NaCl. Total NKCC2 protein expression also increased. Moreover, a 2.5-fold increase in NFAT5 mRNA accumulation was observed after cells were exposed to 500 mosmol/kgH2O for 4 h. Laser-scanning cytometry detected a twofold increase in endogenous NFAT5 protein expression in response to high NaCl concentration. Pretreatment with the loop diuretic bumetanide dramatically reduced transcriptional activity of the NFAT5-specific reporter construct TonE-Luc in mTAL cells exposed to high NaCl. Transient transfection of mTAL cells with shRNA vectors targeting NKCC2A prevented increases in NFAT5 mRNA abundance and protein expression and inhibited NFAT5 transcriptional activity in response to hypertonic stress. Silencing of NKCC2F mRNA did not affect NFAT5 mRNA accumulation but partially inhibited NFAT5 transcriptional activity. These findings suggest that NKCC2A and NKCC2F exhibit differential effects on NFAT5 expression and transcriptional activity in response to hypertonicity produced by high NaCl concentration

Tumor necrosis factor-α is an endogenous inhibitor of Na+-K+-2Cl− cotransporter (NKCC2) isoform A in the thick ascending limb

The effects of TNF gene deletion on renal Na+-K+-2Cl− cotransporter (NKCC2) expression and activity were determined. Outer medulla from TNF−/− mice exhibited a twofold increase in total NKCC2 protein expression compared with wild-type (WT) mice. This increase was not observed in TNF−/− mice treated with recombinant human TNF (hTNF) for 7 days. Administration of hTNF had no effect on total NKCC2 expression in WT mice. A fourfold increase in NKCC2A mRNA accumulation was observed in outer medulla from TNF−/− compared with WT mice; NKCC2F and NKCC2B mRNA accumulation was similar between genotypes. The increase in NKCC2A mRNA accumulation was attenuated when TNF−/− mice were treated with hTNF. Bumetanide-sensitive O2 consumption, an in vitro correlate of NKCC2 activity, was 2.8 ± 0.2 nmol·min−1·mg−1 in medullary thick ascending limb tubules from WT, representing ∼40% of total O2 consumption, whereas, in medullary thick ascending limb tubules from TNF−/− mice, it was 5.6 ± 0.3 nmol·min−1·mg−1, representing ∼60% of total O2 consumption. Administration of hTNF to TNF−/− mice restored the bumetanide-sensitive component to ∼30% of total O2 consumption. Ambient urine osmolality was higher in TNF−/− compared with WT mice (2,072 ± 104 vs. 1,696 ± 153 mosmol/kgH2O, P < 0.05). The diluting ability of the kidney, assessed by measuring urine osmolality before and after 1 h of water loading also was greater in TNF−/− compared with WT mice (174 ± 38 and 465 ± 81 mosmol/kgH2O, respectively, P < 0.01). Collectively, these findings suggest that TNF plays a role as an endogenous inhibitor of NKCC2 expression and function

Microfluidics platform for measurement of volume changes in immobilized intestinal enteroids

Intestinal enteroids are ex vivo primary cultured single-layer epithelial cell spheroids of average diameter ∼150 μm with luminal surface facing inward. Measurement of enteroid swelling in response to secretagogues has been applied to genetic testing in cystic fibrosis and evaluation of drug candidates for cystic fibrosis and secretory diarrheas. The current measurement method involves manual addition of drugs and solutions to enteroids embedded in a Matrigel matrix and estimation of volume changes from confocal images of fluorescently stained enteroids. We developed a microfluidics platform for efficient trapping and immobilization of enteroids for quantitative measurement of volume changes. Multiple enteroids are trapped in a “pinball machine-like” array of polydimethylsiloxane posts for measurement of volume changes in unlabeled enteroids by imaging of an extracellular, high-molecular weight fluorescent dye. Measurement accuracy was validated using slowly expanding air bubbles. The method was applied to measure swelling of mouse jejunal enteroids in response to an osmotic challenge and cholera toxin-induced chloride secretion. The microfluidics platform allows for parallel measurement of volume changes on multiple enteroids during continuous superfusion, without an immobilizing matrix, and for quantitative volume determination without chemical labeling or assumptions about enteroid shape changes during swelling

Absolute Configuration and Biological Properties of Enantiomers of CFTR Inhibitor BPO-27

We previously reported benzopyrimido-pyrrolo-oxazinedione (BPO) inhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel and showed their efficacy in a model of polycystic kidney disease. Here, we separated the enantiomers of lead compound BPO-27 (<b>1</b>), which contains a single chiral center, and determined their absolute configuration, activity, and metabolic stability. Following separation by chiral supercritical fluid chromatography, the <i>R</i> enantiomer, as determined by X-ray crystallography, inhibited CFTR chloride conductance with IC₅₀ ≈ 4 nM, while <i>S</i> enantiomer was inactive. In vitro metabolic stability in hepatic microsomes showed both enantiomers as stable, with <5% metabolism in 4 h. Following bolus interperitoneal administration in mice, serum (<i>R</i>)-<b>1</b> decayed with <i>t</i>_1/2 ≈ 1.6 h and gave sustained therapeutic concentrations in kidney

Vorinostat restores Noxa mRNA of spheroids to the level of monolayers.

<p>M28 and REN monolayers and spheroids were treated with bortezomib (100 nM), vorinostat (5 µM) or their combination for 4 h and Noxa mRNA was measured by qRT-PCR. After treatment with bortezomib, Noxa mRNA increased significantly in monolayers but was significantly lower in spheroids. The addition of vorinostat to bortezomib led to an increase in Noxa mRNA levels in spheroids to the same level as in monolayers. (* p<0.05, significant increase compared to bortezomib alone; † p<0.05, significantly less than in the monolayer exposed to same treatment; n = 3).</p