Human colorectal cancer-on-chip model to study the microenvironmental influence on early metastatic spread.

Colorectal cancer (CRC) progression is a complex process that is not well understood. We describe an in vitro organ-on-chip model that emulates in vivo tissue structure and the tumor microenvironment (TME) to better understand intravasation, an early step in metastasis. The CRC-on-chip incorporates fluid flow and peristalsis-like cyclic stretching and consists of endothelial and epithelial compartments, separated by a porous membrane. On-chip imaging and effluent analyses are used to interrogate CRC progression and the resulting cellular heterogeneity. Mass spectrometry-based metabolite profiles are indicative of a CRC disease state. Tumor cells intravasate from the epithelial channel to the endothelial channel, revealing differences in invasion between aggressive and non-aggressive tumor cells. Tuning the TME by peristalsis-like mechanical forces, the epithelial:endothelial interface, and the addition of fibroblasts influences the invasive capabilities of tumor cells. The CRC-on-chip is a tunable human-relevant model system and a valuable tool to study early invasive events in cancer.

A Proteolytic Site-Directed Affinity Label to Inhibit the Human ATP-Dependent Protease Caseinolytic Complex XP.

Human caseinolytic protease component X and P (hClpXP) is a heterooligomeric ATP-dependent protease. The hClpX subunit catalyzes ATP hydrolysis whereas the hClpP subunit catalyzes peptide bond cleavage. In this study, we generated a peptidyl chloromethyl ketone (dansyl-FAPAL-CMK) that inhibited the hClpP subunit through alkylation of the catalytic His122, which was detected by LC-MS. This inhibitor is composed of a peptide sequence derived from a hydrolyzed peptide product of a substrate cleaved by hClpXP. Binding of FAPAL positions the electrophilic chloromethyl ketone moiety near His122 where alkylation occurs. Dansyl FAPAL-CMK exhibits selectivity for hClpXP over other ATP-dependent proteases such as hLon and the 26S proteasome and abolishes hClpXP activity in HeLa cell lysate. Using the fluorogenic peptide substrate FR-Cleptide as reporter, we detected biphasic inhibition time courses; this supports a slow-binding, time-dependent, covalent inhibition mechanism that is often found in active-site directed affinity labels. Because this inhibitor reacts only with hClpXP but not hLon or the proteasome, it has the potential to serve as a chemical tool to help validate endogenous protein substrates of hClpXP in cell lysate, thereby benefiting investigation of the physiological functions of hClpXP in different cell types or tissue samples.

A Selective Fluorogenic Peptide Substrate for the Human Mitochondrial ATP-Dependent Protease Complex ClpXP.

The goal of this work is to identify differences in the substrate determinants of two human mitochondrial matrix ATP-dependent proteases, human ClpXP (hClpXP) and human Lon (hLon). This information allows the generation of protease-specific peptide substrates that can be used as chemical biology tools to investigate the physiological functions of hClpXP. These enzymes play a role in protein quality control, but currently the physiological functions of human ClpXP are not well defined. In this study, the degradation profile of casein, an alanine positional scanning decapeptide library, and a specific peptide sequence found in an endogenous substrate of bacterial ClpXP by hClpXP as well as hLon were examined. Based on our findings, we generated a specific fluorogenic peptide substrate, FR-Cleptide, for hClpXP with a kcat of 2.44±0.15 s-1 and Km =262±43 µM, respectively. The FR-Cleptide substrate was successfully used to identify a leucine methyl ketone as a potent lead inhibitor, and to detect endogenous hClpXP activity in HeLa cell lysate. We propose that the fluorogenic peptide substrate is a valuable tool for quantitatively monitoring the activity of hClpXP in cell lysate, as well as mechanistic characterization of hClpXP. The peptide-based chemical tools developed in this study will complement the substrates developed for human Lon in aiding the investigation of the physiological functions of the respective protease.

Tracking Decitabine Incorporation into Malignant Myeloid Cell DNA in vitro and in vivo by LC-MS/MS with Enzymatic Digestion.

The DNA hypomethylating agents decitabine and 5-azacytidine are the only two drugs approved for treatment of all subtypes of the myeloid malignancy myelodysplastic syndromes (MDS). The key to drug activity is incorporation into target cell DNA, however, a practical method to measure this incorporation is un-available. Here, we report a sensitive and specific LC-MS/MS method to simultaneously measure decitabine incorporation and DNA hypomethylation. A stable heavy isotope of 2'-deoxycytidine was used as an internal standard and one-step multi-enzyme digestion was used to release the DNA bound drug. Enzyme-released decitabine along with other mononucleosides were separated by a reverse-phase C18 column and quantified by mass spectrometry using multiple-reaction-monitoring (MRM) mode, with a lower limit of quantitation at 1.00 nM. In vitro studies demonstrated dosage and time-dependent incorporation of decitabine into myeloid leukemia cell DNA that correlated with extent of DNA hypomethylation. When applied to clinical samples serially collected from MDS patients treated with decitabine, the method again demonstrated correlation between decitabine DNA-incorporation and DNA hypomethylation. This novel assay to measure the intended molecular pharmacodynamic effect of decitabine therapy can therefore potentially provide insights into mechanisms underlying sensitivity versus resistance to therapy.

Analysis of oxygen-18 labeled phosphate to study positional isotope experiments using LC-QTOF-MS.

A method is proposed in this paper for the determination of oxygen-18 labeled phosphate so that positional isotope experiments using sensitive and rapid liquid chromatography-QTOF-mass spectrometry (LC-QTOF-MS) experiments can be carried out. The positional isotope exchange technique is a useful tool in understanding the mechanisms and kinetics of many enzyme-catalyzed reactions. Detection of the positions and concentration of these exchanged isotopes is the key. Gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance imaging are commonly used analytical techniques for measurement of 18O/16O, 31P and 15N isotope enrichment. Since these techniques either require a time-consuming derivatization step or have a limited sensitivity, an LC and accurate mass-based method for monitoring 18O/16O exchange was developed and compared with a standard GC-MS method. Our results showed that the LC-QTOF-MS method developed was not only as accurate as the standard GC-MS method, but also a sensitive and robust analytical platform for the simultaneous determination of isotope enrichment and the analysis of positional isotopes without chemical derivation. The LC-QTOF-MS method developed was successfully applied to the measurement of 18O/16O in the reversibility study of ATP hydrolysis by Lon proteases.

Microwave-assisted enzymatic hydrolysis of DNA for mass spectrometric analysis: A new strategy for accelerated hydrolysis.

Study of DNA base composition, DNA adducts and modification in its primary structure is a subject of interest in different fields of scientific research. Various methods like immunochemistry, capillary electrophoretic separation, chromatographic separation coupled with mass spectrometric (LC-MS/MS) detection have been developed for DNA analysis. During the past decade LC-MS/MS has emerged as a more sensitive and selective technique and now frequently used for the analysis of DNA. The workflow for the DNA analysis include DNA extraction, hydrolysis of DNA into mononucleosides and analysis. Though high-throughput methods are available for the analysis DNA hydrolysis oftentimes is a rate limiting step. Conventional enzymatic hydrolysis of DNA using a multienzyme mixture will take a minimum of 6-17 h to complete the hydrolysis. In this work, we developed an accelerated enzymatic hydrolysis of DNA using microwave-technology for the first time. 1.00 µg of calf thymus DNA was used to demonstrate the microwave assisted enzymatic hydrolysis of DNA. The resultant mononucleosides were separated on Atlantis T3 (50 × 2.1 mm i.d.; 3 µ particle size) C18 column and analyzed on ABSCIEX QTRAP 5500 LC/MS system. The sample through put and recovery of microwave-assisted digestion were compared with the conventional enzymatic hydrolysis. Efficient digestion of DNA with a performance similar to that obtained by the conventional overnight digestion procedure was attained in just 30 min with a hydrolysis yield of ≥90%. Furthermore, our method was found to be much more accurate and easier to perform. Thus, this new application of microwave technology to DNA enzymatic digestion will facilitate the application of DNA analysis in biological and clinical research.

Novel Protein Disulfide Isomerase Inhibitor with Anticancer Activity in Multiple Myeloma.

Multiple myeloma cells secrete more disulfide bond-rich proteins than any other mammalian cell. Thus, inhibition of protein disulfide isomerases (PDI) required for protein folding in the endoplasmic reticulum (ER) should increase ER stress beyond repair in this incurable cancer. Here, we report the mechanistically unbiased discovery of a novel PDI-inhibiting compound with antimyeloma activity. We screened a 30,355 small-molecule library using a multilayered multiple myeloma cell-based cytotoxicity assay that modeled disease niche, normal liver, kidney, and bone marrow. CCF642, a bone marrow-sparing compound, exhibited a submicromolar IC50 in 10 of 10 multiple myeloma cell lines. An active biotinylated analog of CCF642 defined binding to the PDI isoenzymes A1, A3, and A4 in MM cells. In vitro, CCF642 inhibited PDI reductase activity about 100-fold more potently than the structurally distinct established inhibitors PACMA 31 and LOC14. Computational modeling suggested a novel covalent binding mode in active-site CGHCK motifs. Remarkably, without any further chemistry optimization, CCF642 displayed potent efficacy in an aggressive syngeneic mouse model of multiple myeloma and prolonged the lifespan of C57BL/KaLwRij mice engrafted with 5TGM1-luc myeloma, an effect comparable to the first-line multiple myeloma therapeutic bortezomib. Consistent with PDI inhibition, CCF642 caused acute ER stress in multiple myeloma cells accompanied by apoptosis-inducing calcium release. Overall, our results provide an illustration of the utility of simple in vivo simulations as part of a drug discovery effort, along with a sound preclinical rationale to develop a new small-molecule therapeutic to treat multiple myeloma. Cancer Res; 76(11); 3340-50. ©2016 AACR.