Role of Circulating Tumor Cells (CTC), Androgen Receptor Full Length (AR-FL) and Androgen Receptor Splice Variant 7 (AR-V7) in a Prospective Cohort of Castration-Resistant Metastatic Prostate Cancer Patients

Circulating tumor cells (CTC), androgen receptor full-length (AR-FL), and androgen receptor splice variant 7 (AR-V7) are prognostic in patients (pts) with metastatic castration-resistant prostate cancer (mCRPC). AR-V7 seems to predict resistance to androgen-receptor signaling inhibitors (ARSi)

Two calix[4]pyrroles as potential therapeutics for castration-resistant prostate cancer

Macrocyclic compounds meso-(p-acetamidophenyl)-calix[4]pyrrole and meso-(m-acetamidophenyl)-calix[4]pyrrole have previously been reported to exhibit cytotoxic properties towards lung cancer cells. Here, we report pre-clinical in vitro and in vivo studies showing that these calixpyrrole derivatives can inhibit cell growth in both PC3 and DU145 prostatic cancer cell lines. We explored the impact of these compounds on programmed cell death, as well as their ability to inhibit cellular invasion. In this study we have demonstrated the safety of these macrocyclic compounds by cytotoxicity tests on ex-vivo human peripheral blood mononuclear cells (PBMCs), and by in vivo subcutaneous administration. Preliminary in vivo tests demonstrated no hepato-, no nephro- and no genotoxicity in Balb/c mice compared to controls treated with cisplatin. These findings suggest these calixpyrroles might be novel therapeutic tools for the treatment of prostate cancer and of particular interest for the treatment of androgen-independent castration-resistant prostate cancer

Fraisinib: a calixpyrrole derivative reducing A549 cell-derived NSCLC tumor in vivo acts as a ligand of the glycine-tRNA synthase, a new molecular target in oncology

Background and purpose: Lung cancer is the leading cause of death in both men and women, constituting a major public health problem worldwide. Non-small-cell lung cancer accounts for 85%–90% of all lung cancers. We propose a compound that successfully fights tumor growth in vivo by targeting the enzyme GARS1.Experimental approach: We present an in-depth investigation of the mechanism through which Fraisinib [meso-(p-acetamidophenyl)-calix(4)pyrrole] affects the human lung adenocarcinoma A549 cell line. In a xenografted model of non-small-cell lung cancer, Fraisinib was found to reduce tumor mass volume without affecting the vital parameters or body weight of mice. Through a computational approach, we uncovered that glycyl-tRNA synthetase is its molecular target. Differential proteomics analysis further confirmed that pathways regulated by Fraisinib are consistent with glycyl-tRNA synthetase inhibition.Key results: Fraisinib displays a strong anti-tumoral potential coupled with limited toxicity in mice. Glycyl-tRNA synthetase has been identified and validated as a protein target of this compound. By inhibiting GARS1, Fraisinib modulates different key biological processes involved in tumoral growth, aggressiveness, and invasiveness.Conclusion and implications: The overall results indicate that Fraisinib is a powerful inhibitor of non-small-cell lung cancer growth by exerting its action on the enzyme GARS1 while displaying marginal toxicity in animal models. Together with the proven ability of this compound to cross the blood–brain barrier, we can assess that Fraisinib can kill two birds with one stone: targeting the primary tumor and its metastases “in one shot.” Taken together, we suggest that inhibiting GARS1 expression and/or GARS1 enzymatic activity may be innovative molecular targets for cancer treatment

The role of nuclear matrix proteins binding to matrix attachment regions (Mars) in prostate cancer cell differentiation.

In tumor progression definite alterations in nuclear matrix (NM) protein composition as well as in chromatin structure occur. The NM interacts with chromatin via specialized DNA sequences called matrix attachment regions (MARs). In the present study, using a proteomic approach along with a two-dimensional Southwestern assay and confocal laser microscopy, we show that the differentiation of stabilized human prostate carcinoma cells is marked out by modifications both NM protein composition and bond between NM proteins and MARs. Well-differentiated androgen-responsive and slowly growing LNCaP cells are characterized by a less complex pattern and by a major number of proteins binding MAR sequences in comparison to 22Rv1 cells expressing androgen receptor but androgen-independent. Finally, in the poorly differentiated and strongly aggressive androgen-independent PC3 cells the complexity of NM pattern further increases and a minor number of proteins bind the MARs. Furthermore, in this cell line with respect to LNCaP cells, these changes are synchronous with modifications in both the nuclear distribution of the MAR sequences and in the average loop dimensions that significantly increase. Although the expression of many NM proteins changes during dedifferentiation, only a very limited group of MAR-binding proteins seem to play a key role in this process. Variations in the expression of poly (ADP-ribose) polymerase (PARP) and special AT-rich sequence-binding protein-1 (SATB1) along with an increase in the phosphorylation of lamin B represent changes that might trigger passage towards a more aggressive phenotype. These results suggest that elucidating the MAR-binding proteins that are involved in the differentiation of prostate cancer cells could be an important tool to improve our understanding of this carcinogenesis process, and they could also be novel targets for prostate cancer therapy

A proteomic approach to investigate the modification in the proteome of the cytoplasmatic compartment of Balb/3T3 cells after exposure to gold nanoparticles (AuNPs)

Nanoparticles are widely used in consumer products. However, information about the exposure of the consumer to nanoparticles and their potential health effects is very limited. Nanoparticles may move inside the human body in different manners (via inhalation, ingestion or skin contact), cross the cell membranes and accumulate for long periods of time. Emerging approaches in the area of exposure to nanomaterials and assessment of human health effects combine the use of in vitro cell systems and advanced analytical techniques to study the perturbation of the proteome. The appropriate use of these approaches has the potential to provide information on the possible de-regulation of essential physiological cellular processes. In the present study, we investigated the modification in the proteome of the cytoplasmatic compartment of the Balb/3T3 mouse fibroblast cell line after exposure to 5 and 15 nm gold nanoparticles (AuNPs) for 72 h. Protein separation by two-dimensional gel electrophoresis (2DE) followed by protein identification high-resolution mass spectrometry (MS) allowed us to study the differentially expressed proteome in order to explore underlying cellular mechanisms. Differentially expressed proteins were found to cover a range of functions including stress response, cell metabolism, cell growth and cytoskeleton organization. Remarkably, even small differences in particle size (10 nm) seemed to differently affect biological mechanisms. These findings consolidate existing knowledge and permit to get more insight to the cell mechanisms affected by AuNPs exposure. Our activities in the area of exposure to nanomaterials and potential health effects for the consumer are essential to support EU policy implementation.JRC.I.1-Chemical Assessment and Testin

DNA loop organization in LNCaP and PC3 cells.

<p>(A) Representative nucleoids stained either by DAPI (blue) to visualize only total DNA (left panels) or by halo-FISH to highlight the XmnI sequence (red) and counterstained with DAPI to detect total DNA (blue).The bar corresponds to 10 µm. (B) Scatter plot showing the distribution of DNA halo size. Horizontal lines indicate the mean values obtained measuring for each cell line at least 100 nucleoids. The average DNA halo size ± SE was 6.8±0.2 for LNCaP cells and 7.5±0.2 for PC3 cells, respectively (P = 0.009). The bottom panel shows the frequency distribution of the halo radii grouped in intervals of 2 µm. (C) A schematic model of the interrelationship between the loops and the NM in the dedifferentiation of PCa cells. In more-differentiated cells (LNCaP) the NM is well organized with several proteins bound to MAR sequences. In PC3, where some structural regularities of the NM disappear, a smaller number of protein species bound the MARs and so a larger DNA loop is anchored to the NM.</p

NM proteins binding the XmnI sequence in PCa cell lines.

<p>(A) Representative Deep Purple-stained 1D gel and the corresponding SWB. The arrows on the right indicate the three principal bands arising in 1D SWB, each of which corresponds to several spots in 2D as evident in (D), (F) and (H). (B) The comparison between the relative quantity of XmnI binding to NM proteins in the different cell lines. Ordinate represents the mean±SE of the relative amounts of XmnI as determined by quantitative analysis of three different preparations. The decrease in 22Rv1 and PC3 with respect LNCaP cells was significant (*P = 0.004, **P<10⁻⁵). (C, E and G) Representative 2D silver-stained gel maps and (D, F and H) SWB of NM proteins extracted from LNCaP (C, D), 22Rv1 (E, F) and PC3 (G, H) cells. The proteins identified are highlighted in red boxes. The three arrows show the three groups of proteins pointed out in (A). L, lamin; h, hnRNP, fr, fragments.</p

Spatial distribution of PARP and SATB1 in relation to DNA or XmnIsequence.

<p>(A, D) Whole cells stained by dual-color immunofluorescence. (B, E) NM prepared <i>in situ</i> and stained by immuno-FISH. (A, B) Confocal microscope analysis of the localization of PARP (green) and DNA or XmnI sequence (blue). (D, E) Localization of SATB1 (green) and DNA or XmnI sequence (blue). In the bottom of panels B and E the intensity profile line scans, performed between the white crosses of the NM as indicated on confocal merge images in B and E, are shown. The ordinate represents the fluorescence intensity in arbitrary units, the abscissa represents the distance in pixels. The bars correspond to 5 µm. (C, F) Scatter plots showing quantification analyses of the colocalization of PARP/DNA (a), PARP/XmnI (b), SATB1/DNA (a), or SATB1/XmnI (b), respectively. R corresponds to Pearson’s correlation coefficient; M1 to the fraction of protein being studied overlapping the DNA or XmnI and M2 the fraction of DNA or XmnI overlapping the protein. Horizontal lines show the mean values±SE of 20 fields (122–226 total NMs) replicated in two different experiments (*P≤0.03, **P<0.001).</p

Spatial distribution and phosphorylation level of lamin B in the NM.

<p>(A) Representative confocal microscope images of lamin B (red) and XmnI sequence (blue) in the NM extracted <i>in situ</i> and stained by immuno-FISH. The bars correspond to 5 µm. (B) Magnified section of 2D-PAGE stained with SYPRO Ruby (a, c) or Pro-Q Diamond that selectively stains only phosphoproteins (b, d). The arrowheads indicate the various isoforms of lamin B. The same color corresponds to the same isoform in two cell lines. In PC3 cells, the non-phosphorylated peptide present in LNCaP cells disappeared (red arrowheads).</p

Specificity of the antibodies used in this study for WB and SWB analysis.

<p>Specificity of the antibodies used in this study for WB and SWB analysis.</p