Phomoxanthone A Targets ATP Synthase.

Phomoxanthone A is a naturally occurring molecule and a powerful anti-cancer agent, although its mechanism of action is unknown. To facilitate the determination of its biological target(s), we used affinity-based labelling using a phomoxanthone A probe. Labelled proteins were pulled down, subjected to chemoproteomics analysis using LC-MS/MS and ATP synthase was identified as a likely target. Mitochondrial ATP synthase was validated in cultured cells lysates and in live intact cells. Our studies show sixty percent inhibition of ATP synthase by 260 µM phomoxanthone A.

Sunitinib-Loaded Chondroitin Sulfate Hydrogels as a Novel Drug-Delivery Mechanism for the Treatment of Pancreatic Neuroendocrine Tumors.

BACKGROUND: Pancreatic neuroendocrine tumors (PanNETs) are increasingly common. Experts debate whether small tumors should be resected. Tumor destruction via injection of cytotoxic agents could offer a minimal invasive approach to this controversy. We hypothesize that a new drug delivery system comprising chondroitin sulfate (CS) hydrogels loaded with sunitinib (SUN) suppresses tumor growth in PanNET cells. METHODS: Injectable hydrogels composed of CS modified with methacrylate groups (MA) were fabricated and loaded with SUN. Loading target was either 200 µg (SUN200-G) or 500 µg (SUN500-G) as well as sham hydrogel with no drug loading (SUN0-G). SUN release from hydrogels was monitored in vitro over time and cytotoxicity induced by the released SUN was evaluated using QGP-1 and BON1 PanNET cell lines. QGP-1 xenografts were developed in 35 mice and directly injected with 25 µL of either SUN200-G, SUN500-G, SUN0-G, 100 µL of Sunitinib Malate (SUN-inj), or given 40 mg/kg/day oral sunitinib (SUN-oral). RESULTS: SUN-loaded CSMA hydrogel retained complete in vitro cytotoxicity toward the QGP-1 PanNET and BON-1 PanNET cell lines for 21 days. Mouse xenograft models with QGP-1 PanNETs showed a significant delay in tumor growth in the SUN200/500-G, SUN-inj and SUN-oral groups compared with SUN0-G (p = 0.0014). SUN500-G hydrogels induced significantly more tumor necrosis than SUN0-G (p = 0.04). There was no difference in tumor growth delay between SUN200/500G, SUN-inj, and SUN-oral. CONCLUSIONS: This study demonstrates that CSMA hydrogels loaded with SUN suppress PanNETs growth. This drug delivery could approach represents a novel way to treat PanNETs and other neoplasms via intratumoral injection.

A high-resolution protein architecture of the budding yeast genome.

The genome-wide architecture of chromatin-associated proteins that maintains chromosome integrity and gene regulation is not well defined. Here we use chromatin immunoprecipitation, exonuclease digestion and DNA sequencing (ChIP-exo/seq)1,2 to define this architecture in Saccharomyces cerevisiae. We identify 21 meta-assemblages consisting of roughly 400 different proteins that are related to DNA replication, centromeres, subtelomeres, transposons and transcription by RNA polymerase (Pol) I, II and III. Replication proteins engulf a nucleosome, centromeres lack a nucleosome, and repressive proteins encompass three nucleosomes at subtelomeric X-elements. We find that most promoters associated with Pol II evolved to lack a regulatory region, having only a core promoter. These constitutive promoters comprise a short nucleosome-free region (NFR) adjacent to a +1 nucleosome, which together bind the transcription-initiation factor TFIID to form a preinitiation complex. Positioned insulators protect core promoters from upstream events. A small fraction of promoters evolved an architecture for inducibility, whereby sequence-specific transcription factors (ssTFs) create a nucleosome-depleted region (NDR) that is distinct from an NFR. We describe structural interactions among ssTFs, their cognate cofactors and the genome. These interactions include the nucleosomal and transcriptional regulators RPD3-L, SAGA, NuA4, Tup1, Mediator and SWI-SNF. Surprisingly, we do not detect interactions between ssTFs and TFIID, suggesting that such interactions do not stably occur. Our model for gene induction involves ssTFs, cofactors and general factors such as TBP and TFIIB, but not TFIID. By contrast, constitutive transcription involves TFIID but not ssTFs engaged with their cofactors. From this, we define a highly integrated network of gene regulation by ssTFs.

Development of a stacked, porous silk scaffold neuroblastoma model for investigating spatial differences in cell and drug responsiveness.

Development of in vitro, preclinical cancer models that contain cell-driven microenvironments remains a challenge. Engineering of millimeter-scale, in vitro tumor models with spatially distinct regions that can be independently assessed to study tumor microenvironments has been limited. Here, we report the use of porous silk scaffolds to generate a high cell density neuroblastoma (NB) model that can spatially recapitulate changes resulting from cell and diffusion driven changes. Using COMSOL modeling, a scaffold holder design that facilitates stacking of thin, 200 µm silk scaffolds into a thick, bulk millimeter-scale tumor model (2, 4, 6, and 8 stacked scaffolds) and supports cell-driven oxygen gradients was developed. Cell-driven oxygen gradients were confirmed through pimonidazole staining. Post-culture, the stacked scaffolds were separated for analysis on a layer-by-layer basis. The analysis of each scaffold layer demonstrated decreasing DNA and increasing expression of hypoxia related genes (VEGF, CAIX, and GLUT1) from the exterior scaffolds to the interior scaffolds. Furthermore, the expression of hypoxia related genes at the interior of the stacks was comparable to that of a single scaffold cultured under 1% O2 and at the exterior of the stacks was comparable to that of a single scaffold cultured under 21% O2. The four-stack scaffold model underwent further evaluation to determine if a hypoxia activated drug, tirapazamine, induced reduced cell viability within the internal stacks (region of reduced oxygen) as compared with the external stacks. Decreased DNA content was observed in the internal stacks as compared to the external stacks when treated with tirapazamine, which suggests the internal scaffold stacks had higher levels of hypoxia than the external scaffolds. This stacked silk scaffold system presents a method for creating a single culture model capable of generating controllable cell-driven microenvironments through different stacks that can be individually assessed and used for drug screening.

Three-Dimensional, Scaffolded Tumor Model to Study Cell-Driven Microenvironment Effects and Therapeutic Responses.

Development of novel therapeutics is limited by a lack of accurate preclinical models for testing, specifically the inability of traditional 2D culture (monolayer) to accurately mimic in vivo tumors. In this work, lyophilized silk fibroin scaffolds were used to develop 3D neuroblastoma models (scaffolded NB) using multiple neuroblastoma cell lines (SK-N-AS, KELLY, and SH-SY5Y). Cells grown on scaffolds in low (1%) and ambient (21%) oxygen were compared to traditional monolayer cell culture. Monolayer cultures under low oxygen conditions exhibited increased expression of hypoxia-related genes such as VEGF, CAIX, and GLUT1. Scaffolded NB exhibited increased hypoxia-related gene expression under both low and ambient oxygen conditions. Pimonidazole staining confirmed the presence of hypoxic regions in the scaffolded NB. Cytokine secretion in the monolayer and scaffolded NB suggested differential secretion of cytokines due to both oxygen concentration (ex. VEGF, CCL3, and uPAR) and scaffolded culture (ex. IL-8, GM-CSF, and ITAC). Response to etoposide, a standard chemotherapeutic, demonstrated a reduced cytotoxicity in scaffolded culture as compared to monolayer culture regardless of oxygen concentration. However, use of a hypoxia-activated therapeutic, tirapazamine, exhibited cytotoxicity under scaffolded, ambient oxygen conditions and under monolayer and scaffolded, low oxygen conditions. Overall, this culture system provides a platform to study neuroblastoma and to assess the impact of hypoxia on tumor-relevant pathways and environments to aid in development of novel targeted therapeutics.