Hyaluronan synthases (HAS1-3) and hyaluronidases (HYAL1-2) in the accumulation of hyaluronan in endometrioid endometrial carcinoma

<p>Abstract</p> <p>Background</p> <p>Hyaluronan accumulation correlates with the degree of malignancy in many solid tumor types, including malignant endometrial carcinomas. To elucidate the mechanism of hyaluronan accumulation, we examined the expression levels of the hyaluronan synthases (<it>HAS1</it>, <it>HAS2 </it>and <it>HAS3</it>) and hyaluronidases (<it>HYAL1 </it>and <it>HYAL2</it>), and correlated them with hyaluronan content and HAS1-3 immunoreactivity.</p> <p>Methods</p> <p>A total of 35 endometrial tissue biopsies from 35 patients, including proliferative and secretory endometrium (n = 10), post-menopausal proliferative endometrium (n = 5), complex atypical hyperplasia (n = 4), grade 1 (n = 8) and grade 2 + 3 (n = 8) endometrioid adenocarcinomas were divided for gene expression by real-time RT-PCR, and paraffin embedded blocks for hyaluronan and HAS1-3 cytochemistry.</p> <p>Results</p> <p>The mRNA levels of <it>HAS1-3 </it>were not consistently changed, while the immunoreactivity of all HAS proteins was increased in the cancer epithelium. Interestingly, <it>HAS3 </it>mRNA, but not HAS3 immunoreactivity, was increased in post-menopausal endometrium compared to normal endometrium (p = 0.003). The median of <it>HYAL1 </it>mRNA was 10-fold and 15-fold lower in both grade 1 and grade 2+3 endometrioid endometrial cancers, as compared to normal endometrium (p = 0.004-0.006), and post-menopausal endometrium (p = 0.002), respectively. <it>HYAL2 </it>mRNA was also reduced in cancer (p = 0.02) and correlated with <it>HYAL1</it> (r = 0.8, p = 0.0001). There was an inverse correlation between <it>HYAL1 </it>mRNA and the epithelial hyaluronan staining intensity (r = -0.6; P = 0.001).</p> <p>Conclusion</p> <p>The results indicated that <it>HYAL1 </it>and <it>HYAL2 </it>were coexpressed and significantly downregulated in endometrioid endometrial cancer and correlated with the accumulation of hyaluronan. While immunoreactivity for HASs increased in the cancer cells, tumor mRNA levels for <it>HAS</it>s were not changed, suggesting that reduced turnover of HAS protein may also have contributed to the accumulation of hyaluronan.</p

Expression of Hyaluronan Synthases (HAS1–3) and Hyaluronidases (HYAL1–2) in Serous Ovarian Carcinomas: Inverse Correlation between HYAL1 and Hyaluronan Content

<p>Abstract</p> <p>Background</p> <p>Hyaluronan, a tumor promoting extracellular matrix polysaccharide, is elevated in malignant epithelial ovarian tumors, and associates with an unfavorable prognosis. To explore possible contributors to the accumulation of hyaluronan, we examined the expression of hyaluronan synthases (<it>HAS1</it>, <it>HAS2 </it>and <it>HAS3</it>) and hyaluronidases (<it>HYAL1 </it>and <it>HYAL2</it>), correlated with hyaluronidase enzyme activity hyaluronan content and HAS1–3 immunoreactivity.</p> <p>Methods</p> <p>Normal ovaries (n = 5) and 34 serous epithelial ovarian tumors, divided into 4 groups: malignant grades 1+2 (n = 10); malignant grade 3 (n = 10); borderline (n = 4) and benign epithelial tumors (n = 10), were analyzed for mRNA by real-time RT-PCR and compared to hyaluronidase activity, hyaluronan staining, and HAS1–3 immunoreactivity in tissue sections of the same specimens.</p> <p>Results</p> <p>The levels of <it>HAS2 </it>and <it>HAS3 </it>mRNA (<it>HAS1 </it>was low or absent), were not consistently increased in the carcinomas, and were not significantly correlated with HAS protein or hyaluronan accumulation in individual samples. Instead, the median of <it>HYAL1 </it>mRNA level was 69% lower in grade 3 serous ovarian cancers compared to normal ovaries (P = 0.01). The expression of <it>HYAL1</it>, but not <it>HYAL2</it>, significantly correlated with the enzymatic activity of tissue hyaluronidases (r = 0.5; P = 0.006). An inverse correlation was noted between <it>HYAL1 </it>mRNA and the intensity of hyaluronan staining of the corresponding tissue sections (r = -0.4; P = 0.025).</p> <p>Conclusion</p> <p>The results indicate that in serous epithelial ovarian malignancies <it>HAS </it>expression is not consistently elevated but <it>HYAL1 </it>expression is significantly reduced and correlates with the accumulation of hyaluronan. (233 words)</p

High-Resolution Confocal Fluorescence Imaging of Serine Hydrolase Activity in Cryosections - Application to Glioma Brain Unveils Activity Hotspots Originating from Tumor-Associated Neutrophils

Background Serine hydrolases (SHs) are a functionally diverse family of enzymes playing pivotal roles in health and disease and have emerged as important therapeutic targets in many clinical conditions. Activity-based protein profiling (ABPP) using fluorophosphonate (FP) probes has been a powerful chemoproteomic approach in studies unveiling roles of SHs in various biological systems. ABPP utilizes cell/tissue proteomes and features the FP-warhead, linked to a fluorescent reporter for in-gel fluorescence imaging or a biotin tag for streptavidin enrichment and LC-MS/MS-based target identification. Existing ABPP approaches characterize global SH activity based on mobility in gel or MS-based target identification and cannot reveal the identity of the cell-type responsible for an individual SH activity originating from complex proteomes. Results Here, by using an activity probe with broad reactivity towards the SH family, we advance the ABPP methodology to glioma brain cryosections, enabling for the first time high-resolution confocal fluorescence imaging of global SH activity in the tumor microenvironment. Tumor-associated cell types were identified by extensive immunohistochemistry on activity probe-labeled sections. Tissue-ABPP indicated heightened SH activity in glioma vs. normal brain and unveiled activity hotspots originating from tumor-associated neutrophils (TANs), rather than tumor-associated macrophages (TAMs). Thorough optimization and validation was provided by parallel gel-based ABPP combined with LC-MS/MS-based target verification. Conclusions Our study advances the ABPP methodology to tissue sections, enabling high-resolution confocal fluorescence imaging of global SH activity in anatomically preserved complex native cellular environment. To achieve global portrait of SH activity throughout the section, a probe with broad reactivity towards the SH family members was employed. As ABPP requires no a priori knowledge of the identity of the target, we envisage no imaginable reason why the presently described approach would not work for sections regardless of species and tissue source.Peer reviewe

High-Resolution Confocal Fluorescence Imaging of Serine Hydrolase Activity in Cryosections - Application to Glioma Brain Unveils Activity Hotspots Originating from Tumor-Associated Neutrophils

Background Serine hydrolases (SHs) are a functionally diverse family of enzymes playing pivotal roles in health and disease and have emerged as important therapeutic targets in many clinical conditions. Activity-based protein profiling (ABPP) using fluorophosphonate (FP) probes has been a powerful chemoproteomic approach in studies unveiling roles of SHs in various biological systems. ABPP utilizes cell/tissue proteomes and features the FP-warhead, linked to a fluorescent reporter for in-gel fluorescence imaging or a biotin tag for streptavidin enrichment and LC-MS/MS-based target identification. Existing ABPP approaches characterize global SH activity based on mobility in gel or MS-based target identification and cannot reveal the identity of the cell-type responsible for an individual SH activity originating from complex proteomes. Results Here, by using an activity probe with broad reactivity towards the SH family, we advance the ABPP methodology to glioma brain cryosections, enabling for the first time high-resolution confocal fluorescence imaging of global SH activity in the tumor microenvironment. Tumor-associated cell types were identified by extensive immunohistochemistry on activity probe-labeled sections. Tissue-ABPP indicated heightened SH activity in glioma vs. normal brain and unveiled activity hotspots originating from tumor-associated neutrophils (TANs), rather than tumor-associated macrophages (TAMs). Thorough optimization and validation was provided by parallel gel-based ABPP combined with LC-MS/MS-based target verification. Conclusions Our study advances the ABPP methodology to tissue sections, enabling high-resolution confocal fluorescence imaging of global SH activity in anatomically preserved complex native cellular environment. To achieve global portrait of SH activity throughout the section, a probe with broad reactivity towards the SH family members was employed. As ABPP requires no a priori knowledge of the identity of the target, we envisage no imaginable reason why the presently described approach would not work for sections regardless of species and tissue source.Peer reviewe

Hyaluronan Export through Plasma Membranes Depends on Concurrent K+ Efflux by Kir Channels

Hyaluronan is synthesized within the cytoplasm and exported into the extracellular matrix through the cell membrane of fibroblasts by the MRP5 transporter. In order to meet the law of electroneutrality, a cation is required to neutralize the emerging negative hyaluronan charges. As we previously observed an inhibiting of hyaluronan export by inhibitors of K+ channels, hyaluronan export was now analysed by simultaneously measuring membrane potential in the presence of drugs. This was done by both hyaluronan import into inside-out vesicles and by inhibition with antisense siRNA. Hyaluronan export from fibroblast was particularly inhibited by glibenclamide, ropivacain and BaCl2 which all belong to ATP-sensitive inwardly-rectifying Kir channel inhibitors. Import of hyaluronan into vesicles was activated by 150 mM KCl and this activation was abolished by ATP. siRNA for the K+ channels Kir3.4 and Kir6.2 inhibited hyaluronan export. Collectively, these results indicated that hyaluronan export depends on concurrent K+ efflux

Interstitial cell migration: integrin-dependent and alternative adhesion mechanisms

Adhesion and migration are integrated cell functions that build, maintain and remodel the multicellular organism. In migrating cells, integrins are the main transmembrane receptors that provide dynamic interactions between extracellular ligands and actin cytoskeleton and signalling machineries. In parallel to integrins, other adhesion systems mediate adhesion and cytoskeletal coupling to the extracellular matrix (ECM). These include multifunctional cell surface receptors (syndecans and CD44) and discoidin domain receptors, which together coordinate ligand binding with direct or indirect cytoskeletal coupling and intracellular signalling. We review the way that the different adhesion systems for ECM components impact cell migration in two- and three-dimensional migration models. We further discuss the hierarchy of these concurrent adhesion systems, their specific tasks in cell migration and their contribution to migration in three-dimensional multi-ligand tissue environments

The Implementation of Guide Conversation Method in Teaching Self Introduction to Improve Students’ Speaking Skills. Rilla Syahida Yusup; 187010037

This research is entitled “The Implementation of Guide Conversation Method in Teaching Self-Introduction in Improving Students’ Speaking Skills”. The guide conversation is expected to increase students’ confidence and enable students to speak in public with others with fun learning process. The researcher conducts this research to know how the procedure of implementation of guide conversation method in self-introduction learning, to know what the teaching equipment used to implement the guide conversation method in self-introduction learning, to know how the result of the implementation of guide conversation method in self�introduction learning, and to know what the students’ responses towards the implementation of guide conversation method in improving students’ speaking skills. The population in this research is the 10th grade students of Pasundan 2 Senior High School Bandung, and the sample of this research is 10th grade IPS 2. This research through several steps, carried outby referring to the lesson plan. The data are obtained through qualitative method with the interview, observation and tests in the classroom during learning process. This research uses the oral test concerning to four aspects speaking skills such as pronunciation, fluency, accuracy, and vocabulary. The scoring aspect based on Brown’s scale assessment. After the researcher gets the mean from the pre-test and post-test, then researcher calculates the mean. Guide conversation method helps students’ speaking skill enhancement with 57,5%. The responses of the students are good. They can be easy to understand and more active during learning process. They can speak actively and enjoy, so that students’ confidence increase. Keywords: Guide Conversation Method, Self-Introduction, Speaking Skill

Melanoma cell-derived factors stimulate hyaluronan synthesis in dermal fibroblasts by upregulating HAS2 through PDGFR-PI3K-AKT and p38 signaling

In many cancers hyaluronan content is increased, either by tumor cells or the surrounding stromal cells and this increased hyaluronan content correlates with unfavorable clinical prognosis. In the present work, we studied the effects of melanoma cell (aggressive melanoma cell line C8161)-derived factors on fibroblast hyaluronan synthesis, intracellular signaling, MMP expression and invasion. Treatment of the fibroblast cultures with melanoma cell conditioned medium (CM) caused accumulation of hyaluronan in the culture medium and formation of thick pericellular hyaluronan coat and hyaluronan cables. The expression of Has2 was increased approximately 20-fold by the C8161 melanoma cell CM, while Has1 and Has3 were increased twofold. Knock-down of Has2 expression with siRNA showed that Has2 was responsible for the increased hyaluronan synthesis induced by the melanoma cell CM. To find out the signaling routes, which led to Has2 upregulation, the phosphorylation profiles of 46 kinases were screened with phosphokinase array kit. Melanoma cell CM treatment strongly induced a rapid phosphorylation of p38, JNK, AKT, CREB, HSP27, STAT3 and cJUN. Treatment of the fibroblasts with specific inhibitors of PI3K, AKT and p38 reduced the melanoma cell CM-induced hyaluronan secretion, while the inhibitor of PDGFR totally blocked it. In addition, siRNA for PDGFRα/β inhibited Has2 upregulation in melanoma cell CM-treated fibroblasts. In parallel with the increased hyaluronan synthesis the melanoma cell CM-treated fibroblasts showed spindle shape, numerous long cell protrusions, enhanced MMP expression and increased invasion into collagen-Cultrex matrix. siRNA blocking of Has2 or PDGFRα/β expression reversed the stimulatory effect of melanoma cell CM on fibroblast invasion. PDGF secreted by melanoma cells thus mediated fibroblasts activation, with HAS2 upregulation as a major factor in the fibroblast response. This effect on stromal matrix is suggested to favor tumor growth

Advanced nanoengineered—customized point-of-care tools for prostate-specific antigen

Change in the level of human prostate-specific antigen (PSA) is a major element in the development and progression of prostate cancer (PCa). Most of the methodologies are currently restricted to their application in routine clinical screening due to the scarcity of adequate screening tools, false reading, long assay time, and cost. Innovative techniques and the integration of knowledge from a variety of domains, such as materials science and engineering, are needed to provide sustainable solutions. The convergence of precision point-of-care (POC) diagnostic techniques, which allow patients to respond in real time to changes in PSA levels, provides promising possibilities for quantitative and quantitative detection of PSA. This solution could be interesting and relevant for use in PCa diagnosis at the POC. The approaches enable low-cost real-time detection and are simple to integrate into user-friendly sensor devices. This review focuses on the investigations, prospects, and challenges associated with integrating engineering sciences with cancer biology to develop nanotechnology-based tools for PCa diagnosis. This article intends to encourage the development of new nanomaterials to construct high-performance POC devices for PCa detection. Finally, the review concludes with closing remarks and a perspective forecast. Graphical abstract: [Figure not available: see fulltext.