The MUC1 Ectodomain: A Novel and Efficient Target for Gold Nanoparticle Clustering and Vapor Nanobubble Generation

MUC1 is a large, heavily glycosylated transmembrane glycoprotein that is proposed to create a protective microenvironment in many adenocarcinomas. Here we compare MUC1 and the well studied cell surface receptor target, EGFR, as gold nanoparticle (AuNP) targets and their subsequent vapor nanobubble generation efficacy in the human epithelial cell line, HES. Although EGFR and MUC1 were both highly expressed in these cells, TEM and confocal images revealed MUC1 as a superior target for nanoparticle intracellular accumulation and clustering. The MUC1-targeted AuNP intracellular clusters also generated significantly larger vapor nanobubbles. Our results demonstrate the promising opportunities MUC1 offers to improve the efficacy of targeted nanoparticle based approaches

Ponatinib Activates an Inflammatory Response in Endothelial Cells via ERK5 SUMOylation

Ponatinib is a multi-targeted third generation tyrosine kinase inhibitor (TKI) used in the treatment of chronic myeloid leukemia (CML) patients harboring the Abelson (Abl)-breakpoint cluster region (Bcr) T315I mutation. In spite of having superb clinical efficacy, ponatinib triggers severe vascular adverse events (VAEs) that significantly limit its therapeutic potential. On vascular endothelial cells (ECs), ponatinib promotes EC dysfunction and apoptosis, and inhibits angiogenesis. Furthermore, ponatinib-mediated anti-angiogenic effect has been suggested to play a partial role in systemic and pulmonary hypertension via inhibition of vascular endothelial growth factor receptor 2 (VEGFR2). Even though ponatinib-associated VAEs are well documented, their etiology remains largely unknown, making it difficult to efficiently counteract treatment-related adversities. Therefore, a better understanding of the mechanisms by which ponatinib mediates VAEs is critical. In cultured human aortic ECs (HAECs) treated with ponatinib, we found an increase in nuclear factor NF-kB/p65 phosphorylation and NF-kB activity, inflammatory gene expression, cell permeability, and cell apoptosis. Mechanistically, ponatinib abolished extracellular signal-regulated kinase 5 (ERK5) transcriptional activity even under activation by its upstream kinase mitogen-activated protein kinase kinase 5α (CA-MEK5α). Ponatinib also diminished expression of ERK5 responsive genes such as Krüppel-like Factor 2/4 (klf2/4) and eNOS. Because ERK5 SUMOylation counteracts its transcriptional activity, we examined the effect of ponatinib on ERK5 SUMOylation, and found that ERK5 SUMOylation is increased by ponatinib. We also found that ponatibib-mediated increased inflammatory gene expression and decreased anti-inflammatory gene expression were reversed when ERK5 SUMOylation was inhibited endogenously or exogenously. Overall, we propose a novel mechanism by which ponatinib up-regulates endothelial ERK5 SUMOylation and shifts ECs to an inflammatory phenotype, disrupting vascular homeostasis

Improved Cellular Specificity of Plasmonic Nanobubbles versus Nanoparticles in Heterogeneous Cell Systems

The limited specificity of nanoparticle (NP) uptake by target cells associated with a disease is one of the principal challenges of nanomedicine. Using the threshold mechanism of plasmonic nanobubble (PNB) generation and enhanced accumulation and clustering of gold nanoparticles in target cells, we increased the specificity of PNB generation and detection in target versus non-target cells by more than one order of magnitude compared to the specificity of NP uptake by the same cells. This improved cellular specificity of PNBs was demonstrated in six different cell models representing diverse molecular targets such as epidermal growth factor receptor, CD3 receptor, prostate specific membrane antigen and mucin molecule MUC1. Thus PNBs may be a universal method and nano-agent that overcome the problem of non-specific uptake of NPs by non-target cells and improve the specificity of NP-based diagnostics, therapeutics and theranostics at the cell level

The Transcription Factor ETV5 Mediates BRAFV600E-Induced Proliferation and TWIST1 Expression in Papillary Thyroid Cancer Cells

The ETS family of transcription factors is involved in several normal remodeling events and pathological processes including tumor progression. ETS transcription factors are divided into subfamilies based on the sequence and location of the ETS domain. ETV5 (Ets variant gene 5; also known as ERM) is a member of the PEA3 subfamily. Our meta-analysis of normal, benign, and malignant thyroid samples demonstrated that ETV5 expression is upregulated in papillary thyroid cancer and was predominantly associated with BRAF V600E or RAS mutations. However, the precise role of ETV5 in these lesions is unknown. In this study, we used the KTC1 cell line as a model for human advanced papillary thyroid cancer (PTC) because the cells harbor the heterozygous BRAF (V600E) mutation together with the C250T TERT promoter mutation. The role of ETV5 in PTC proliferation was tested using RNAi followed by high-throughput screening. Signaling pathways driving ETV5 expression were identified using specific pharmacological inhibitors. To determine if ETV5 influences the expression of epithelial-to-mesenchymal (EMT) markers in these cells, an EMT PCR array was used, and data were confirmed by qPCR and ChIP-qPCR. We found that ETV5 is critical for PTC cell growth, is expressed downstream of the MAPK pathway, and directly upregulates the transcription factor TWIST1, a known marker of intravasation and metastasis. Increased ETV5 expression could therefore be considered as a marker for advanced PTCs and a possible future therapeutic target

Cell models and conditions of their treatment with NP and laser pulses.

<p>Cell models and conditions of their treatment with NP and laser pulses.</p

Parameters of PNBs generated around gold NP clusters in water for gold nanoshells.

<p>A: PNB generation threshold fluence of the excitation laser pulse as function of NP cluster size (measured through optical scattering amplitude of NP cluster image for individual clusters); B: PNB lifetime and scattering brightness as function of the NP cluster size (measured through optical scattering amplitude of NP cluster image) at specific fluence of the excitation pulse (778 nm, 22 mJ/cm²).</p

Cell population-averaged levels of optical scattering signals obtained for individual target (solid bar) and non-target (hollow bar) cells in six cell models represented by target/non-target cells/molecular targets:

<p>Squamous cell carcinoma, HN31/NOM9/EGFR (treated with 50 nm NS-Panitumumab conjugates); Lung cancer, A549/Fibroblast/EGFR (treated with 60 nm NSP-C225 conjugates); Epithelial cancer, HES/HS5/MUC1 (treated with 60 nm NSP-214D4 conjugates); Prostate cancer, C2-4B/HS5/PSMA (treated with 60 nm NSP-anti-PSMA conjugates); Leukemia, J32/JRT3-T3.5/CD3 and human T-cells, T-cell/BM/CD3 (treated with 60 nm NSP-OKT3 conjugates) for: Row A (red): gold NP amplitude of scattering image of gold NPs (a metric for the uptake of NPs by cells; Row B (purple): time-resolved scattering image amplitudes of PNBs; Row C (blue): PNB lifetimes. The ratio of the signals for target/non-target cell is shown for each parameter and cell model and indicates the cellular specificity of NPs (row A) and PNBs (rows B,C).</p

Images and signals of gold NPs and PNBs in co-culture of target (HN31, labeled with Green Fluorescent Protein for identification) and non-target (NOM9) cells identically treated with 60 nm gold NSP-C225 conjugates (specific to EGFR that is overexpressed in target cells).

<p>A: overlay of bright field, fluorescent and scattering images shows target cells (green) and gold NPs (red) that can be found in both types of cells (the arrows show NP clusters in non-target cells); B: time-resolved scattering image of the same field shows PNB images (bright white spots) only in target cells; C,D: optical scattering time-responses of individual target (C) and non-target (D) cells show the PNB-specific signal only for target cell and the definition of the PNB lifetime of PNBs; time is measured from the moment of the exposure to the excitation laser pulse.</p

Influence of targeting vectors on NP scattering amplitude (red) and PNB lifetime (blue) in individual target (solid bars) and non-target (hollow bars) cells.

<p>A: Target (HN31) and non-target (NOM9) cells identically treated with bare 60 nm gold NSPs and NSP-Panitumumab conjugates (antibody specific to EGFR that is overexpressed in HN31 cells); B: Effects of EGFR-specific antibodies C225 and Panitumumab as targeting vectors in HN31 cell model show 5 different combinations of the two antibodies; C: Effects of single and dual targeting antibodies against PSMA and EGFR (C225) in C4-2B cell model applied in combination with dual simultaneous optical excitation (so called rainbow PNB method) show synergistic enhancement of PNB lifetime in the rainbow mode.</p