iTRAQ-Based Proteomic Analysis of Polyploid Giant Cancer Cells and Budding Progeny Cells Reveals Several Distinct Pathways for Ovarian Cancer Development

<div><p>Polyploid giant cancer cells (PGCCs) are a morphologically distinct subgroup of human tumor cells with increased nuclear size or multiple nuclei, but they are generally considered unimportant because they are presumed to be nondividing and thus nonviable. We have recently shown that these large cancer cells are not only viable but also can divide asymmetrically and yield progeny cancer cells with cancer stem-like properties via budding division. To further understand the molecular events involved in the regulation of PGCCs and the generation of their progeny cancer cells, we comparatively analyzed the proteomic profiles of PGCCs, PGCCs with budding daughter cells, and regular control cancer cells from the HEY and SKOv3 human ovarian cancer cell lines with and without CoCl₂. We used a high-throughput iTRAQ-based proteomic methodology coupled with liquid chromatography-electrospray ionization tandem mass spectroscopy to determine the differentiated regulated proteins. We performed Western blotting and immunohistochemical analyses to validate the differences in the expression patterns of a variety of proteins between PGCCs or budding PGCCs and regular cancer cells identified by iTRAQ approach and also a selected group of proteins from the literature. The differentially regulated proteins included proteins involved in response to hypoxia, stem cell generation, chromatin remodeling, cell-cycle regulation, and invasion and metastasis. In particular, we found that HIF-1alpha and its known target STC1 are upregulated in PGCCs. In addition, we found that a panel of stem cell-regulating factors and epithelial-to-mesenchymal transition regulatory transcription factors were upregulated in budding PGCCs, whereas expression of the histone 1 family of nucleosomal linker proteins was consistently lower in PGCCs than in control cells. Thus, proteomic expression patterns provide valuable insight into the underlying mechanisms of PGCC formation and the relationship between PGCCs and cancer stem cells in patients with ovarian cancers.</p> </div

Unique Conformation in a Natural Interruption Sequence of Type XIX Collagen Revealed by Its High-Resolution Crystal Structure

Naturally occurring interruptions in nonfibrillar collagen play key roles in molecular flexibility, collagen degradation, and ligand binding. The structural feature of the interruption sequences and the molecular basis for their functions have not been well studied. Here, we focused on a G5G type natural interruption sequence G-POALO-G from human type XIX collagen, a homotrimer collagen, as this sequence possesses distinct properties compared with those of a pathological similar Gly mutation sequence in collagen mimic peptides. We determined the crystal structures of the host–guest peptide (GPO)₃-GPOALO-(GPO)₄ to 1.03 Å resolution in two crystal forms. In these structures, the interruption zone brings localized disruptions to the triple helix and introduces a light 6–8° bend with the same directional preference to the whole molecule, which may correspond structurally to the first physiological kink site in type XIX collagen. Furthermore, at the G5G interruption site, the presence of Ala and Leu residues, both with free N–H groups, allows the formation of more direct and water-mediated interchain hydrogen bonds than in the related Gly → Ala structure. These could partly explain the difference in thermal stability between the different interruptions. In addition, our structures provide a detailed view of the dynamic property of such an interrupted zone with respect to hydrogen bonding topology, torsion angles, and helical parameters. Our results, for the first time, also identified the binding of zinc to the end of the triple helix. These findings will shed light on how the interruption sequence influences the conformation of the collagen molecule and provide a structural basis for further functional studies

Tumor metastasis-related protein expression in HEY PGCCs alone, HEY PGCCs with budding daughter cells, control HEY cells, SKOv3 PGCCs alone, and control SKOv3 cells.

<p>(A) Western blot analysis confirming tumor metastasis-related protein expression in these cells. (B) Immunohistochemical stain of cathepsin B expression in tumor cells derived from control HEY cells and HEY PGCCs (20×).</p

Western blot analysis of stem cell-related proteins expressed in HEY PGCCs alone, HEY PGCCs with budding daughter cells, control HEY cells, SKOv3 PGCCs alone, and control SKOv3 cells.

<p>(A) Stem cell-related protein expression. (<b>B</b>). EMT-related protein expression in HEY PGCCs alone, HEY PGCCs with budding daughter cells, control HEY cells, SKOv3 PGCCs alone, and control SKOv3 cells. (C) Immunohistochemical stain showing the EMT-related protein expression in tumor cells derived from control HEY cells and HEY PGCCs (20×).</p

Control ovarian cancer cells, PGCCs alone, and PGCCs generating daughter cells via budding (10×).

<p>(A) Control HEY cells. (B) HEY PGCCs after treatment with CoCl₂. (C) HEY PGCCs generating daughter cells via budding (black arrows). (D) Control SKOv3 cells. (E) SKOv3 PGCCs after treatment with CoCl₂. (F) SKOv3 PGCCs generating daughter cells via budding (black arrows).</p

Western blot analysis of (A) kinase-related protein expression and (B) cell cycle-related protein expression in HEY PGCCs alone, HEY PGCCs with budding daughter cells, control HEY cells, SKOv3 PGCCs alone, and control SKOv3 cells.

<p>Western blot analysis of (A) kinase-related protein expression and (B) cell cycle-related protein expression in HEY PGCCs alone, HEY PGCCs with budding daughter cells, control HEY cells, SKOv3 PGCCs alone, and control SKOv3 cells.</p

Western blot analysis of epigenetic modifying proteins in HEY PGCCs alone, HEY PGCCs with budding daughter cells, control HEY cells, SKOv3 PGCCs alone, and control SKOv3 cells.

<p>Western blot analysis of epigenetic modifying proteins in HEY PGCCs alone, HEY PGCCs with budding daughter cells, control HEY cells, SKOv3 PGCCs alone, and control SKOv3 cells.</p

Pd-Catalyzed One-Pot Synthesis of Polysubstituted Acrylamidines from Isocyanides, Diazo Compounds, and Imines

A novel and efficient Pd-catalyzed one-pot reaction of ethyl diazoacetate, isocyanides, and imines for the synthesis of acrylamidines was developed. The multicomponent reaction may have occurred through an unpredicted ring-opening process of the ketenimine–imine [2 + 2] intermediate to form the acrylamidine products

Pd-Catalyzed One-Pot Synthesis of Polysubstituted Acrylamidines from Isocyanides, Diazo Compounds, and Imines

A novel and efficient Pd-catalyzed one-pot reaction of ethyl diazoacetate, isocyanides, and imines for the synthesis of acrylamidines was developed. The multicomponent reaction may have occurred through an unpredicted ring-opening process of the ketenimine–imine [2 + 2] intermediate to form the acrylamidine products

Validation of gene signature.

<p>(A) The predictive model constructed from the TCGA training set was applied to an independent TCGA validation set (n = 261) and split the patients into two groups based on the score cutoff of −0.16 as determined by the ROC curve. Thirty five patients are identified to have an explicit response to chemotherapy <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036383#pone.0036383-TCGA1" target="_blank">[22]</a>. (B) The two groups are well separated, with 212 patients in the low-scoring group and 49 in the high-scoring group. (C) Exclusion of patients with no survival data resulted in 109 patients in the low-scoring group and 29 in the high-scoring group. Kaplan-Meier analysis shows patients in the high-scoring group had poorer progression-free survival (<i>P</i> = 0.04). (D) The predictive model as applied to the external data set distinguishes the patients in the low-scoring group from in the high-scoring group; where the low-scoring group consists of the 70.1% patients (171 out of 244) with the highest predictive scores, and the high-scoring group consists of the 29.9% patients (73 out of 244) with the lowest predictive scores (see text for details). (E) Kaplan-Meier analysis shows patients in the high-scoring group had poorer progression-free survival than those in the low-scoring group (<i>P</i><0.0001).</p