Insulin/IGF and Sex Hormone Axes in Human Endometrium and Associations with Endometrial Cancer Risk Factors

Given an ordered set of points and an ordered set of geometric objects in the plane, we are interested in finding a non-crossing matching between point-object pairs. In this paper, we address the algorithmic problem of determining whether a non-crossing matching exists between a given point-object pair. We show that when the objects we match the points to are finite point sets, the problem is NP-complete in general, and polynomial when the objects are on a line or when their size is at most 2. When the objects are line segments, we show that the problem is NP-complete in general, and polynomial when the segments form a convex polygon or are all on a line. Finally, for objects that are straight lines, we show that the problem of finding a min-max non-crossing matching is NP-complete. © 2012 Elsevier B.V.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Molecular, pathological, radiological, and immune profiling of non-brainstem pediatric high-grade glioma from the HERBY phase II randomized trial

The HERBY trial was a phase II open-label, randomized, multicenter trial evaluating bevacizumab (BEV) in addition to temozolomide/radiotherapy in patients with newly diagnosed non-brainstem high-grade glioma (HGG) between the ages of 3 and 18 years. We carried out comprehensive molecular analysis integrated with pathology, radiology, and immune profiling. In post-hoc subgroup analysis, hypermutator tumors (mismatch repair deficiency and somatic POLE/POLD1 mutations) and those biologically resembling pleomorphic xanthoastrocytoma ([PXA]-like, driven by BRAF_V600E or NF1 mutation) had significantly more CD8+ tumor-infiltrating lymphocytes, and longer survival with the addition of BEV. Histone H3 subgroups (hemispheric G34R/V and midline K27M) had a worse outcome and were immune cold. Future clinical trials will need to take into account the diversity represented by the term ‘‘HGG’’ in the pediatric population

Microtubule configuration and its relationship to sperm morphology in mammalian spermiogenesis

The acrosome is a secretory vesicle located in the mammalian sperm head. Its main function is to transport hydrolytic enzymes. These enzymes will dissolve the zona pellucid, assisting the sperm in fertilizing the egg. The acrosome is assembled in the haploid spermatid during spermiogenesis, but many of its enzymes are already synthesized in pachytene spermatocytes during the early phase of spermatogenesis. Haploid spermatids have developed unique mechanism(s) to assure the proper localization/orientation of the acrosome, attachment near the nucleus, and the targeting of acrosomal proteins towards this vesicle. During spermiogenesis, these germ cells undergo dramatic transformations in shape and intracellular distribution of organelles, and the configuration of microtubules seems to be involved with each specific step. In this context, it seems microtubules are essential, for the assembly and formation of the acrosome during spermiogenesis. Microtabules are also involved in guiding pro

TACE/ADAM17 is involved in germ cell apoptosis during rat spermatogenesis

The pathways leading to male germ cell apoptosis in vivo are poorly understood, but are highly relevant for the comprehension of sperm production regulation by the testis. In this work, we show the evidence of a mechanism where germ cell apoptosis is induced through the inactivation and shedding of the extracellular domain of KIT (c-kit) by the protease TACE/a disintegrin and metalloprotease 17 (ADAM17) during the first wave of spermatogenesis in the rat. We show that germ cells undergoing apoptosis lacked the extracellular domain of the KIT receptor. TACE/ADAM17, a membrane-bound metalloprotease, was highly expressed in germ cells undergoing apoptosis as well. On the contrary, cell surface presence of ADAM10, a closely related metalloprotease isoform, was not associated with apoptotic germ cells. Pharmacological inhibition of TACE/ADAM17, but not ADAM10, significantly prevented germ cell apoptosis in the male pubertal rat. Induction of TACE/ADAM17 by the phorbol-ester phorbol 12-myri

Insulin-Like Growth Factor 2 Silencing Restores Taxol Sensitivity in Drug Resistant Ovarian Cancer

<div><p>Drug resistance is an obstacle to the effective treatment of ovarian cancer. We and others have shown that the insulin-like growth factor (IGF) signaling pathway is a novel potential target to overcome drug resistance. The purpose of this study was to validate IGF2 as a potential therapeutic target in drug resistant ovarian cancer and to determine the efficacy of targeting IGF2 <i>in vivo</i>. An analysis of The Cancer Genome Atlas (TCGA) data in the serous ovarian cancer cohort showed that high IGF2 mRNA expression is significantly associated with shortened interval to disease progression and death, clinical indicators of drug resistance. In a genetically diverse panel of ovarian cancer cell lines, the IGF2 mRNA levels measured in cell lines resistant to various microtubule-stabilizing agents including Taxol were found to be significantly elevated compared to the drug sensitive cell lines. The effect of IGF2 knockdown on Taxol resistance was investigated <i>in vitro</i> and <i>in vivo</i>. Transient IGF2 knockdown significantly sensitized drug resistant cells to Taxol treatment. A Taxol-resistant ovarian cancer xenograft model, developed from HEY-T30 cells, exhibited extreme drug resistance, wherein the maximal tolerated dose of Taxol did not delay tumor growth in mice. Blocking the IGF1R (a transmembrane receptor that transmits signals from IGF1 and IGF2) using a monoclonal antibody did not alter the response to Taxol. However, stable IGF2 knockdown using short-hairpin RNA in HEY-T30 effectively restored Taxol sensitivity. These findings validate IGF2 as a potential therapeutic target in drug resistant ovarian cancer and show that directly targeting IGF2 may be a preferable strategy compared with targeting IGF1R alone.</p></div

IGF2 knockdown restores Taxol sensitivity.

<p>(A) Taxol sensitivity after IGF2 knockdown by siRNA. HEY-T30 and A2780-T15 cells were transfected with control nontargeting siRNA (siCTRL) or siRNAs targeting IGF2 (siIGF2-1, siIGF2-2, siIGF2-3), and treated with DMSO or Taxol at the approximate IC₅₀ (100 nM for HEY-T30 and 15 nM for A2780-T15). The surviving fraction was reduced after IGF2 siRNA transfection compared to control siRNA transfection. The effect of Taxol was significantly enhanced in both HEY-T30 and A2780-T15 by IGF2 siRNA transfection compared with control siRNA transfection. Bars represent the mean of four independent experiments (each done in duplicate)±SEM; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; Two-way ANOVA with Bonferroni posttest. (B) IGF2 mRNA after stable knockdown with shRNA. HEY-T30 cells were transfected with shRNA targeting IGF2 by plasmid transfection (shIGF2-p) or lentiviral infection (shIGF2-v), or with a control vector containing the scrambled version of the IGF2 targeting sequence (shScrambled). Clonal stably-transfected cell lines were used for all experiments. IGF2 mRNA levels are depicted in bars±SEM. The shIGF2-p and shIGF2-v cell lines have low IGF2 mRNA levels similar to HEY, whereas HEY-T30 and shScrambled cell lines had several-fold higher mRNA expression levels. (n>6 independent experiments, each done in triplicate) *p<0.05; One-way ANOVA with Bonferroni posttest. (C) Cytotoxicity assays in cell lines with IGF2 shRNA. The indicated cell lines were treated with serial dilutions of Taxol, and sensitivity to Taxol determined by the sulforhodamine B assay. The dose-response curves are shown on the left, with the mean±SEM go the surviving fraction of cells relative to untreated cells shown at the indicated Taxol concentrations. The mean IC₅₀ values±SEM for the indicated cell lines and drugs are shown in the table below. HEY T30 shIGF2-p and HEY-T30 shIGF2-v were significantly more sensitive to Taxol, ixabepilone, and vinblastine compared to control transfected HEY-T30 (shScrambled) or untransfected HEY-T30. The efficacy of IGF2 knockdown at restoring sensitivity to Taxol was similar to NVP-AEW541 treatment. Only 1.6 fold sensitization to doxorubicin and no effect on cisplatin sensitivity were observed in shIGF2 lines compared to shScrambled or untransfected HEY-T30. Asterisks denote the statistical significance when comparing the IC₅₀ for the indicated shIGF2 cell line versus shScrambled, where *p<0.05, **p<0.01, ****p<0.0001; Two-way ANOVA with Bonferroni posttest. (n = 5 independent experiments each done in six replicates). (D) Xenograft growth with Taxol treatment of HEY-T30 shIGF2-p and HEY-T30 shScrambled. Female athymic nude mice were subcutaneously injected with 1 million HEY-T30 shScrambled or HEY-T30 shIGF2-p cells and tumors were allowed to grow to an average volume of 120 mm³. Mice were then treated with either D5W (vehicle) or Taxol at the MTD (described in 2D). Tumors were measured every three days, and data points show the mean tumor volume±SEM for each group at each time point. Two independent experiments were done for a total of 8–10 animals per group. shIGF2-p xenografts responded to Taxol treatment (triangle, dashed black line) and showed significantly tumor growth suppression compared to treatment with D5W (circle, solid black line); at day 12, **p<0.01 for shIGF2-p Taxol vs shIGF2-p D5W; One-way ANOVA with Bonferroni posttest. In contrast, HEY-T30 shScrambled xenografts did not respond to Taxol (+, dashed purple line) and continued growing at a similar rate as the vehicle-treated HEY-T30 shScrambled group (x, solid purple line). No significant difference in tumor size was observed between D5W-treated shIGF2-p and shScrambled xenografts at day 12. Representative tumor xenografts were excised on day 13 after treatment for further analysis. (E, F) IGF2 immunohistochemical staining of xenografts. Excised tumor xenografts (n = 2 animals per group) on day 13 after treatment initiation were formalin-fixed, paraffin embedded and sections stained with hematoxylin and eosin or with an anti-human IGF2 antibody. Staining was evaluated by the pathologist blinded to the groups and the mean of H-scores±SEM calculated and graphed (E). (F) Shows representative HEY-T30 shScrambled and HEY-T30 shIGF2 xenograft sections stained with hematoxylin and eosin (H&E) or with anti-IGF2 antibody (right panels). HEY-T30 shIGF2-p xenografts showed lower IGF2 expression when compared to HEY-T30 shScrambled.</p

High IGF2 expression is associated with early recurrence, poor survival, and drug resistance.

<p>IGF2 expression and ovarian cancer survival. Using the cBioPortal for Cancer Genomics to analyze the data from the Cancer Genome Atlas study of ovarian serous cystadenocarcinoma, we compared the progression-free survival and overall survival in patients with high tumor levels of IGF2 mRNA (greater than 1.6 standard deviations above the mean; gray line) and those with normal tumor levels of IGF2 mRNA (all other patients; black line). Patients with high IGF2 mRNA levels had significantly shortened progression-free survival (A) and overall survival (B). (C) IGF2 expression in sensitive and resistant cell lines. By RT-qPCR, we measured the IGF2 mRNA level in six drug resistant ovarian carcinoma cell lines and their three cell lines of origin. All drug resistant cell lines have significantly higher IGF2 mRNA expression compared to their sensitive cell line of origin. Bars show the mean±SEM of IGF2 mRNA expression score for at least two independent experiments for each cell line, each done in triplicate, and the symbol above the bar indicates the statistical significance comparing that resistant cell line with its parental counterpart; *p<0.05, ***p<0.001, ****p<0.0001 by One-way ANOVA with Bonferroni posttest. (D) ABCB1 expression. ABCB1 mRNA expression levels were measured by RT-qPCR in HEY, HEY-T30, A2780 and A2780-T15. HEY-T30 has higher ABCB1 mRNA expression compared to the other cell lines. Bars show the mean±SEM of ABCB1 mRNA expression score for at least four independent experiments for each cell line, each done in triplicate, (E) ABCB1 DNA copy number. By qPCR performed on genomic DNA, HEY-T30 has a six-fold increase in ABCB1 DNA copy number indicating gene amplification. Bars show the mean±SEM of two independent experiments, each done in triplicate.</p

IGF1R-specific inhibition does not alter Taxol resistance.

<p>(A) Combination treatment using Taxol and IMC-A12 in mice with HEY-T30 xenografts. HEY-T30 xenografts were grown as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100165#pone-0100165-g002" target="_blank">Figure 2D</a>, then treated with Taxol (black circles), according to the same dose/schedule described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100165#pone-0100165-g002" target="_blank">Figure 2D</a>, or with IMC-A12 (intraperitoneal injection of 40 mg/kg three times per week, grey *), or with combination treatment of Taxol and IMC-A12 (grey x). Data points show the mean tumor volume±SEM for each treatment group (n = 8 animals per group). No significant differences in tumor volume were observed with IMC-A12 or combined Taxol/IMC-A12 treatment compared to D5W or Taxol alone. This experiment was repeated a second independent time with similar results. (B) Effect of IMC-A12 on Taxol sensitivity, evaluated by SRB cytotoxicity assay. In 96-well plates, cells were treated with serial dilutions of Taxol alone (solid lines) or together with a fixed dose of 10 µg/ml IMC-A12 (dashed lines). Dose response curves show the mean of the surviving fraction±SEM of cells relative to untreated cells at the indicated Taxol concentrations (n = 7 independent experiments, each done in six replicates). No significant Taxol sensitization was observed in the presence of IMC-A12. (C) Effect of NVP-AEW541 on Taxol sensitivity, evaluated by SRB cytotoxicity assay. In 96-well plates, cells were treated with serial dilutions of Taxol alone (solid lines) or together with a fixed dose of 1 µM NVP-AEW541 (dashed lines). Dose response curves show the mean±SEM of the surviving fraction of cells relative to untreated cells at the indicated Taxol concentrations. The bar graphs depict the IC₅₀ of Taxol for the indicated cell lines and drug treatments, and show the mean±SEM calculated from at least four independent experiments (each done in six replicates). NVP-AEW541 sensitizes A2780-T15 to Taxol. ***p<0.001, ****p<0.0001; One-way ANOVA with Bonferroni posttest. (D) ABCB1 (p-glycoprotein) function in HEY and HEY-T30 in the presence and absence of NVP-AEW541. Cells were incubated with labeled Taxol (Oregon Green 488 Taxol, bis-acetate) in the presence or absence of NVP-AEW541 or verapamil. The ABCB1-overexpressing HEY-T30 cells (light gray shaded graph) retain less labeled Taxol than HEY cells (unfilled black outline). Verapamil, a known p-glycoprotein inhibitor, markedly increases the retention of labeled Taxol in HEY-T30 cells (and to a lesser degree in HEY cells), as demonstrated by the rightward shift of the peaks. Shown in the right panel with the dashed black line, treatment with 1 µM NVP-AEW541 does not appear to affect retention of labeled Taxol in HEY-T30 cells. Two independent experiments were done with similar results.</p

Characterization of the drug resistance phenotype.

<p>Proliferation kinetics of cell lines. Cells were grown in complete media with the indicated concentration of Taxol in 6-well dishes, and cells from duplicate wells counted every 24 hours. (A) HEY-T30 cells in the presence or absence of 30 nM Taxol proliferate more slowly than HEY cells (p<0.05, Repeated Measures Two-way ANOVA, Bonferroni posttest). (B) A2780-T15 in the presence of 2 nM and 15 nM Taxol grow similarly to A2780. However, in the absence of Taxol, A2780-T15 proliferates more slowly, suggesting Taxol dependence (p<0.01, Two-way ANOVA, Bonferroni posttest). Each growth curve represents the mean of at least two independent experiments each done in duplicates, each point is represented as the mean±SEM. (C) IC₅₀’s of chemotherapeutic drugs in sensitive and resistant cell lines. In 96-well plates, cells were treated with serial dilutions of the indicated drugs and the concentration of 50% proliferation inhibition (IC₅₀) determined using the SRB cytotoxicity assay. HEY-T30 are cross-resistant to ixabepilone and to vinblastine. There is modest cross-resistance to doxorubicin but not to CDDP. A2780-T15 are cross-resistant to ixabepilone, and modestly cross-resistant to doxorubicin and CDDP, but more sensitive to vinblastine. Data are presented as the mean±SEM of at least three independent experiments performed with six replicates. P-values calculated by unpaired t-test. (D, E) Comparison of HEY-T30 and HEY xenograft response to Taxol. Following subcutaneous injection of HEY-T30 or HEY cells, mice were divided into treatment groups of 6 animals each. When the average xenograft volume reached 120 mm³, Taxol treatment was administered at the maximal tolerated dose (MTD) of 20 mg/kg intraperitoneally every three days for five treatments (treatment days: 0, 3, 6, 9, 12), resulting in a cumulative dose of 100 mg/kg. Control animals received intraperitoneal injections of the diluent (5% dextrose in water; D5W) according to the same schedule. Tumors were measured every three days, and mean tumor volumes±SEM for each group shown at each time point. HEY xenografts responded to Taxol treatment, which potently suppressed tumor growth (Fig. 2D; dashed blue line). HEY-T30 xenografts did not respond to Taxol treatment (Fig. 2E; dashed orange line) and grew at a similar rate as vehicle-treated HEY-T30 xenografts. HEY Taxol vs HEY D5W on day 19, p<0.001; unpaired t-test.</p