Improved in vivo targeting of BCL-2 phenotypic conversion through hollow gold nanoshell delivery.

Although new cancer therapeutics are discovered at a rapid pace, lack of effective means of delivery and cancer chemoresistance thwart many of the promising therapeutics. We demonstrate a method that confronts both of these issues with the light-activated delivery of a Bcl-2 functional converting peptide, NuBCP-9, using hollow gold nanoshells. This approach has shown not only to increase the efficacy of the peptide 30-fold in vitro but also has shown to reduce paclitaxel resistant H460 lung xenograft tumor growth by 56.4%.

Quantification of glioblastoma progression in zebrafish xenografts: Adhesion to laminin alpha 5 promotes glioblastoma microtumor formation and inhibits cell invasion.

Glioblastoma (GBM) is a deadly disease due to its ability to quickly invade and destroy brain tissue. Slowing or stopping GBM cell progression is crucial to help those inflicted with the disease. Our lab created an embryo-larval zebrafish xenograft model as a tool to study human GBM progression in an observable brain environment. The zebrafish brain is a dynamic and complex environment providing an optimal setting for studying GBM cell progression. Here we demonstrate the ability of our model to quantitate GBM proliferation, dispersal, blood vessel association, microtumor formation, and individual cell invasion by evaluating the importance of an extracellular matrix protein, laminin alpha 5 (lama5), on U251MG cell progression. Lama5 has been implicated in cancer cell survival, proliferation and invasion and is a known adhesion site for GBM cells. While lama5 is highly expressed in endothelial cells in the brain, it is unknown how lama5 affects GBM behavior. Using a lama5 morpholino, we discovered that lama5 decreased U251MG dispersal by 23% and doubles the formation of blood vessel dependent microtumors. Despite lama5 being a known attachment site for GBM, lama5 expression had no effect on U251MG association with blood vessels. Analysis of individual U251MG cells revealed lama5 significantly lowered invasion as mobile U251MG cells traveled 32.5  µm less, invaded 5.0 µm/hr slower and initiated invasion 60% few times per cell.

11-Cl-BBQ, a select modulator of AhR-regulated transcription, suppresses lung cancer cell growth via activation of p53 and p27Kip1.

Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor and functions as a tumour suppressor in different cancer models. In the present study, we report detailed characterization of 11-chloro-7H-benzimidazo[2,1-a]benzo[de]iso-quinolin-7-one (11-Cl-BBQ) as a select modulator of AhR-regulated transcription (SMAhRT) with anti-cancer actions. Treatment of lung cancer cells with 11-Cl-BBQ induced potent and sustained AhR-dependent anti-proliferative effects by promoting G1 phase cell cycle arrest. Investigation of 11-Cl-BBQ-induced transcription in H460 cells with or without the AhR expression by RNA-sequencing revealed activation of p53 signalling. In addition, 11-Cl-BBQ suppressed multiple pathways involved in DNA replication and increased expression of cyclin-dependent kinase inhibitors, including p27Kip1 , in an AhR-dependent manner. CRISPR/Cas9 knockout of individual genes revealed the requirement for both p53 and p27Kip1 for the AhR-mediated anti-proliferative effects. Our results identify 11-Cl-BBQ as a potential lung cancer therapeutic, highlight the feasibility of targeting AhR and provide important mechanistic insights into AhR-mediated-anticancer actions.

The DevTox Germ Layer Reporter Platform: An Assay Adaptation of the Human Pluripotent Stem Cell Test.

Environmental chemical exposures are a contributing factor to birth defects affecting infant morbidity and mortality. The USA EPA is committed to developing new approach methods (NAMs) to detect chemical risks to susceptible populations, including pregnant women. NAM-based coverage for cellular mechanisms associated with early human development could enhance identification of potential developmental toxicants (DevTox) for new and existing data-poor chemicals. The human pluripotent stem cell test (hPST) is an in vitro test method for rapidly identifying potential human developmental toxicants that employs directed differentiation of embryonic stem cells to measure reductions in SOX17 biomarker expression and nuclear localization. The objective of this study was to expand on the hPST principles to develop a model platform (DevTox GLR) that utilizes the transgenic RUES2-GLR cell line expressing fluorescent reporter fusion protein biomarkers for SOX17 (endoderm marker), BRA (mesoderm marker), and SOX2 (ectoderm and pluripotency marker). Initial assay adaption to definitive endoderm (DevTox GLR-Endo) was performed to emulate the hPST SOX17 endpoint and enable comparative evaluation of concordant chemical effects. Assay duration was reduced to two days and screening throughput scaled to 384-well format for enhanced speed and efficiency. Assay performance for 66 chemicals derived from reference and training set data resulted in a balanced accuracy of 72% (79% sensitivity and 65% specificity). The DevTox GLR-Endo assay demonstrates successful adaptation of the hPST concept with increased throughput, shorter assay duration, and minimal endpoint processing. The DevTox GLR model platform expands the in vitro NAM toolbox to rapidly identify potential developmental hazards and mechanistically characterize toxicant effects on pathways and processes associated with early human development.

The Zebrafish Xenograft Models for Investigating Cancer and Cancer Therapeutics.

In order to develop new cancer therapeutics, rapid, reliable, and relevant biological models are required to screen and validate drug candidates for both efficacy and safety. In recent years, the zebrafish (Danio rerio) has emerged as an excellent model organism suited for these goals. Larval fish or immunocompromised adult fish are used to engraft human cancer cells and serve as a platform for screening potential drug candidates. With zebrafish sharing ~80% of disease-related orthologous genes with humans, they provide a low cost, high-throughput alternative to mouse xenografts that is relevant to human biology. In this review, we provide background on the methods and utility of zebrafish xenograft models in cancer research.

Induction of apoptosis and suppression of tumor growth by Nur77-derived Bcl-2 converting peptide in chemoresistant lung cancer cells.

Resistance to chemotherapy is a major cause of treatment failure and poor overall survival in patients with lung cancer. Identification of molecular targets present in resistant cancer cells is essential for addressing therapeutic resistance and prolonging lung cancer patient survival. Members of the B-cell lymphoma 2 (Bcl-2) family of proteins are associated with chemotherapeutic resistance. In this study, we found that pro-survival protein Bcl-2 is upregulated in paclitaxel resistant cells, potentially contributing to chemotherapy resistance. To exploit the increase in Bcl-2 expression for targeting therapy resistance, we investigated the effects of a peptide derived from the nuclear receptor Nur77 that converts Bcl-2 from an anti-apoptotic protein to a pro-apoptotic protein. The Nur77 derived peptide preferentially induced apoptosis in paclitaxel-resistant cancer cells with high expression of Bcl-2. This peptide also induced apoptosis of multidrug resistant H69AR lung cancer cells that express Bcl-2 and inhibited their growth in 3D spheroids. The Nur77 peptide strongly suppressed the growth of paclitaxel-resistant lung cancer cells in a zebrafish xenograft tumor model. Taken together, our data supports a new strategy for treating lung cancers that acquire resistance to chemotherapy through overexpression of Bcl-2.