Selective cell killing of BCPAP TSHR-positive vs NSC-34 TSHR-negative cells with TSHR-targeted bionanofluid.

<p><b>A.</b> TSHR expression of BCPAP and NSC-34 cells was determined by Western blot analysis, using TSHR specific antibody. BCPAP were positive for TSHR expression, whereas NSC-34 cells were null. B-ACTIN was used as a loading control. <b>B.</b> BCPAP and NSC-34 cell were incubated α-THSR-, Thyrogen-, and purified Thyrotropin-Thiol-PEG-CNT conjugates. Control conditions included IgG-thiol-PEG-CNTs, PBS and CNT alone. All conditions were performed in 2:1 cell:bionanofluid ratio and 30 second laser exposure. The BCPAP cells showed ~60% to ~73% cell killing with all TSHR targeted bionanofluid conjugates, whereas minimal cell death was observed with the control other conditions. The NSC-34 cell line showed negligible cell death in all conditions.</p

Structure and coupling chemistry of Au-modified MWCNTs.

<p><b>A.</b> Scanning electron microscopy (SEM) images of COOH-functionalized Au-labeled Thiol-Carbon derived bionanofluids, at two different magnifications. Au particles have defined spherical structures, highlighted by the arrowhead. <b>B.</b> EDC-NHS coupling chemistry to attach bio-affinity molecules, whether antibody or protein/mitogen to the Thiol-PEG-CNT. PEGylation of the Thiol-CNT is described in the Materials and Methods.</p

Bionanofluid conjugate stability assessment.

<p><b>A</b>. α-TSHR- and Thyrogen-Thiol-PEG-CNT conjugates were prepared on day 1 and kept at 4°C for up to 21 days. Conjugates activity was assessed by cell killing assay of BCPAP cells (as described above). <b>B.</b> Similarly, α-TSHR- and Thyrogen-Thiol-PEG-CNT conjugates were prepared on day 1 and were kept at -20°C or -80°C for up to 6 weeks. Conjugates activity was assessed by cell killing assay at day 5, day 7, and every week for up to 6 weeks.</p

Concentration and time optimization of TSHR targeting bionanofluid to BCPAP cells.

<p>Included in these experimental conditions are, α-THSR-, Thyrogen-, and purified Thyrotropin-Thiol-PEG-CNT conjugates. Control conditions included PBS and CNT alone. <b>A.</b> Determination of optimal cell to conjugate BioNanofluid ratio to achieve specific maximum targeted BCPAP cell killing. Laser exposure time was 30 seconds for all conditions. Ratios are represented as volume:volume ratios, thus for a 1:1 ratio, 100 μL of cells (of 250,000–350,000 cells per ml) were mixed with 100 μL of Conjugated-BioNanofluid of 2 μg/mL concentration. <b>B.</b> Optimal exposure time determination experiment. BCPAP cells were exposed to laser treatment for 20, 30, and 40 seconds, at a 2:1 cell:conjugated- or unconjugated-Thiol-PEG CNT ratio.</p

Proteomic-Coupled-Network Analysis of T877A-Androgen Receptor Interactomes Can Predict Clinical Prostate Cancer Outcomes between White (Non-Hispanic) and African-American Groups

<div><p>The androgen receptor (AR) remains an important contributor to the neoplastic evolution of prostate cancer (CaP). CaP progression is linked to several somatic AR mutational changes that endow upon the AR dramatic gain-of-function properties. One of the most common somatic mutations identified is Thr877-to-Ala (T877A), located in the ligand-binding domain, that results in a receptor capable of promiscuous binding and activation by a variety of steroid hormones and ligands including estrogens, progestins, glucocorticoids, and several anti-androgens. In an attempt to further define somatic mutated AR gain-of-function properties, as a consequence of its promiscuous ligand binding, we undertook a proteomic/network analysis approach to characterize the protein interactome of the mutant T877A-AR in LNCaP cells under eight different ligand-specific treatments (dihydrotestosterone, mibolerone, R1881, testosterone, estradiol, progesterone, dexamethasone, and cyproterone acetate). In extending the analysis of our multi-ligand complexes of the mutant T877A-AR we observed significant enrichment of specific complexes between normal and primary prostatic tumors, which were furthermore correlated with known clinical outcomes. Further analysis of certain mutant T877A-AR complexes showed specific population preferences distinguishing primary prostatic disease between white (non-Hispanic) vs. African-American males. Moreover, these cancer-related AR-protein complexes demonstrated predictive survival outcomes specific to CaP, and not for breast, lung, lymphoma or medulloblastoma cancers. Our study, by coupling data generated by our proteomics to network analysis of clinical samples, has helped to define real and novel biological pathways in complicated gain-of-function AR complex systems.</p></div

Survival outcomes reflected by Copy Number Variation.

<p>Survival outcomes based on copy number variation (GSE21035) of gene-set 2 and same patients described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113190#pone-0113190-g004" target="_blank">Figure 4</a>, was applied. Patient stratification is based on serum PSA (≥4 ng/mL) and Gleason score (≥7) and combination of serum PSA and Gleason score values.</p

Two dimensional RMSD matrices of the residues of Helix α11 and Helix α12 and loop between them without fitting.

<p>Two dimensional RMSD matrices of the residues of Helix α11 and Helix α12 and loop between them without fitting.</p

MD average structure of T877A mutant of AR ligand-binding domain with eight different ligands.

<p>Docking program WILMA was used to examine structural changes of the ligand-binding domain of the T877A mutation, upon binding to ligand binding. Illustrated is the average structure of T877A-AR mutation ligand binding domain with eight different ligands. <b>A.</b> testosterone, <b>B.</b> DHT, <b>C.</b> R1881, <b>D.</b> MB, <b>E.</b> estrogen (EST), <b>F.</b> progesterone (PGR), <b>G.</b> CPA, <b>H.</b> dexamethasone (DEX). A red box highlights the changes in the helix α 11 loop upon binding to each hormone ligand.</p

Loop structure fluctuations.

<p><b>A.</b> Superimposed average structures of all eight ligand bound receptor complexes. <b>B.</b> Regions of T877A AR-LBD loop between Helixα9 and Helixα10 showed maximum flexibility. <b>C.</b> Expanded view of Helix α11 and Helix α12 regions of T877A AR-LBD bound to eight different ligands studied in this study. Residues of α11 and α12 and loop between them showed higher flexibility compared to other regions of the receptor where T877A is located. Color corresponds to the following ligands: Green - testosterone, Cyan- DHT, Yellow-R1881, Pink- MB, Rose- EST, Grey- PGR, Orange-CPA, Purple-DEX.</p

Characterization of T877A-AR protein interaction and gene expression profiles.

<p><b>A.</b> Quantified protein interaction network of DHT stimulated LNCaP cells. The value of each protein, defined by our label-free quantitative MS, is distinguished by both color and size, and sub-sequent protein-protein interactions. The AR is designated by the black circle. To determine the relationship between protein interaction and gene expression patterns, hierarchical clustering of multi-panel hormone-stimulated LNCaP cells was carried out. <b>B.</b> Interacting proteins identified by mass spectrometry. <b>C.</b> Most variably expressed genes upon ligand stimulation. Although it has been suggested that all hormones used are able to activate androgen-dependent gene transcription with the T877A-AR mutant receptor, there are differences between AR protein complexes and AR gene expression patterns. This cluster analysis illustrates that even the synthetic androgens like Mibolerone (MB) and R1881, have similar gene expression profiles, their protein-interaction complexes are more similar to dexamethasone (DEX) and progesterone (PGR), respectively, than to either natural androgens testosterone and DHT. Moreover, even natural androgens (DHT and testosterone) can be segregated by their protein complexes. This would suggest that there are functions for the AR beyond gene transactivation. Protein and gene expression values are given as a ratio of quantified protein of each stimulation vs. vehicle control stimulation.</p