Flavonoids Isolated from Rumex aquaticus Exhibit Neuroprotective and Neurorestorative Properties by Enhancing Neurite Outgrowth and Synaptophysin

Mice lacking ALK activity have previously been reported to exhibit subtle behavioral phenotypes. In this study of ALK of loss of function mice we present data supporting a role for ALK in hypogonadotropic hypogonadism in male mice. We observed lower level of serum testosterone at P40 in ALK knock-out males, accompanied by mild disorganization of seminiferous tubules exhibiting decreased numbers of GATA4 expressing cells. These observations highlight a role for ALK in testis function and are further supported by experiments in which chemical inhibition of ALK activity with the ALK TKI crizotinib was employed. Oral administration of crizotinib resulted in a decrease of serum testosterone levels in adult wild type male mice, which reverted to normal levels after cessation of treatment. Analysis of GnRH expression in neurons of the hypothalamus revealed a significant decrease in the number of GnRH positive neurons in ALK knock-out mice at P40 when compared with control littermates. Thus, ALK appears to be involved in hypogonadotropic hypogonadism by regulating the timing of pubertal onset and testis function at the upper levels of the hypothalamic-pituitary gonadal axis

Targeted Disruption of ALK Reveals a Potential Role in Hypogonadotropic Hypogonadism

Mice lacking ALK activity have previously been reported to exhibit subtle behavioral phenotypes. In this study of ALK of loss of function mice we present data supporting a role for ALK in hypogonadotropic hypogonadism in male mice. We observed lower level of serum testosterone at P40 in ALK knock-out males, accompanied by mild disorganization of seminiferous tubules exhibiting decreased numbers of GATA4 expressing cells. These observations highlight a role for ALK in testis function and are further supported by experiments in which chemical inhibition of ALK activity with the ALK TKI crizotinib was employed. Oral administration of crizotinib resulted in a decrease of serum testosterone levels in adult wild type male mice, which reverted to normal levels after cessation of treatment. Analysis of GnRH expression in neurons of the hypothalamus revealed a significant decrease in the number of GnRH positive neurons in ALK knock-out mice at P40 when compared with control littermates. Thus, ALK appears to be involved in hypogonadotropic hypogonadism by regulating the timing of pubertal onset and testis function at the upper levels of the hypothalamic-pituitary gonadal axis

Cytokine profiling of tumor interstitial fluid of the breast and its relationship with lymphocyte infiltration and clinicopathological characteristics

The tumor microenvironment is composed of many immune cell subpopulations and is an important factor in the malignant progression of neoplasms, particularly breast cancer (BC). However, the cytokine networks that coordinate various regulatory events within the BC interstitium remain largely uncharacterized. Moreover, the data obtained regarding the origin of cytokine secretions, the levels of secretion associated with tumor development, and the possible clinical relevance of cytokines remain controversial. Therefore, we profiled 27 cytokines in 78 breast tumor interstitial fluid (TIF) samples, 43 normal interstitial fluid (NIF) samples, and 25 matched serum samples obtained from BC patients with Luminex xMAP multiplex technology. Eleven cytokines exhibited significantly higher levels in the TIF samples compared with the NIF samples: interleukin (IL)-7, IL-10, fibroblast growth factor-2, IL-13, interferon (IFN)γ-inducible protein (IP-10), IL-1 receptor antagonist (IL-1RA), platelet-derived growth factor (PDGF)-β, IL-1β, chemokine ligand 5 (RANTES), vascular endothelial growth factor, and IL-12. An immunohistochemical analysis further demonstrated that IL-1RA, IP-10, IL-10, PDGF-β, RANTES, and VEGF are widely expressed by both cancer cells and tumor-infiltrating lymphocytes (TILs), whereas IP-10 and RANTES were preferentially abundant in triple-negative breast cancers (TNBCs) compared to Luminal A subtype cancers. The latter observation corresponds with the high level of TILs in the TNBC samples. IL-1β, IL-7, IL-10, and PDGFβ also exhibited a correlation between the TIF samples and matched sera. In a survival analysis, high levels of IL-5, a hallmark TH2 cytokine, in the TIF samples were associated with a worse prognosis. These findings have important implications for BC immunotherapy research

Sites in the genome of frequent cancer focal deletions.

<p>(A) Average DNA segment size as a function of position on chromosome 1 is displayed. (B) the 9,401 focal deletions that were not telomeric were binned into 2 Mb intervals and used to generate a frequency distribution across the genome. The most frequently affected genomic interval corresponded to the <i>CDKN2A/B</i> locus and the next six most frequently affected genomic intervals are indicated with red arrows and the relevant gene.</p

Deletion properties that distinguish common fragile site genes from tumor suppressor genes.

<p>Scatter dot plots of values for statistics that distinguish focal deletions affecting common fragile site (CFS) genes (orange) from tumor suppressor genes (blue). (A) “RNA/DNA Correlation,” which corresponds to the Pearson’s correlation coefficient of log2-transformed DNA copy number values and relative RNA expression values for the 115 tumor samples with both array CGH and RNA expression profiling data, (B) DNA Copy Number is the median value of segmented DNA copy number values. (C) Cell Line Proportion” which provides a relative measure of how frequently deletions for a given gene are found in cell lines rather than primary tumors. Panels (D) and (E) show the number of deletions found in each gene within a 10 Mb region centered on the <i>MACROD2</i> locus (D) and TP53 and <i>MAP2K4</i> loci (E). (F) Box and whisker plots of “Deletion Isolation,” which measures how frequently neighboring genes are deleted relative to the featured gene.</p

Deletion size and deletion separation can distinguish common fragile sites from tumor suppressor genes.

<p>(A) Box and whisker plots for “Deletion Size,” which measures the median size of deletions within a 2-Mb window centered on the gene, common fragile site genes (orange) and tumor suppressor genes (blue) (B) Box and whisker plots for “Deletion Separation,” which measures the separation (non-overlapping) of deletions within a 2-Mb window centered on the gene, (C) The co-deletion tendency of common fragile site genes (orange) and tumor suppressor genes (blue) relative to the co-deletion of genes of different classes (gray).</p

Validation of <i>MACROD2</i> as a common fragile site gene.

<p>(A) A schematic of the experiment, which used a custom tiling array to determine if aphidicolin induction of replicative stress could generate focal deletions at three loci (<i>A2BP1</i>, <i>MACROD2</i>, and <i>FHIT</i>) in colon epithelial cancer cells. No deletions affecting <i>A2BP1</i> were observed in the 8 examined clones; 4 out of 8 clones had deletions affecting <i>MACROD2</i>; and 2 clones had deletions affecting <i>FHIT</i>. (B) An example of an induced focal deletion affecting the <i>MACROD2</i> locus. The <i>x</i>-axis represents an approximately 1-Mb region spanning <i>MACROD2</i>. The blue lines represent the normalized DNA copy number values, and the orange lines represent the segmented DNA copy number values. (C) An example of an induced focal deletion affecting <i>FHIT</i>. (D) An example of an unaffected locus (the HLA locus on chromosome 6p).</p

The boundaries of focal deletions that affect the <i>A2BP1</i>, <i>MACROD2</i>, <i>CDKN2A/B</i>, and <i>PTEN</i> genes.

<p>Each of the four panels shows (top) an ideogram of the chromosome on which the featured gene is located and (below) an expanded view of a 4-Mb region centered on the featured gene, showing other genes in the area (location based on the UCSC Genome Browser). If the gene is large enough, the exon-intron structure and/or transcriptional direction of the gene are indicated. For most genes, particularly in panels C and D, the genes are shown as rectangles due to their smaller size. Shown below each expanded region are horizontal bars indicating the extent and boundaries of individual deletions in colon tumors (Panels A and B; <i>A2BP1</i> and <i>MACROD2</i>, respectively), lung tumors (Panel C; <i>CDKN2A/B</i>), or multiple tumor types (Panel D; <i>PTEN</i>). The featured genes in Panels C and D are highlighted in green.</p

Expression and tumor suppressive properties of <i>A2BP1</i> and <i>MACROD2</i>.

<p>(A) Threshold PCR cycles for RT-PCR detection of <i>ACTB</i> (control) and <i>A2BP1</i> in two different samples of normal brain tissue, four different samples of normal colon tissue, and 19 colon cancer cell lines. Values below 40 indicate no signal detection. (B) The effect of forced ectopic expression of <i>A2BP1</i> on tumor formation of colon cancer cell line HCT-15, which harbors a 250-kb deletion within <i>A2BP1</i>. Detection of expression by immunoblotting using a polyclonal antibody that recognizes the A2BP1 protein <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066264#pone.0066264-Zhang1" target="_blank">[38]</a> is shown in the insert. Lack of tumor suppressive effects were also observed for the colon cancer cell lines HCT-116 and SW480, both harboring deletions in <i>A2BP1</i>. (C) The expression of <i>MACROD2</i> in colon cancer cell lines as determined by TaqMan RT-PCR using four different probes (three to coding sequences and one to a 3′ UTR, all unaffected by deletions), comparing cell lines that harbor deletions within the <i>MACROD2</i> gene to ones that do not. Relative expression was calculated by the ΔC_T method using <i>GADPH</i> expression levels as the reference. (D) The expression of <i>MACROD2</i> in colon cancer cell lines as determined by immunoblotting using an antibody to MacroD2.</p