C-H Functionalization of Poly(ethylene oxide) - Embracing Functionality, Degradability, and Molecular Delivery.

This study presents an organocatalytic C-H functionalization approach for postpolymerization modification (PPM) of poly(ethylene oxide) (PEO). Most of PEO PPM is previously processed at the end hydroxy group, but recent advances in C-H functionalization open a way to modify the backbone position. Structurally diverse carboxylic acids are attached to PEO through a cascade process of radical generation by peroxide and oxidation to oxocarbenium by tertiary butylammonium iodide. Attaching carboxylic acids yields a series of functionalize PEO with acetal units (2-5 mol%) in a backbone, which is not accessible via conventional copolymerization of epoxides. The optimized conditions minimizes the uncontrolled degradation or crosslinking from the highly reactive radical and oxocarbenium intermediate. The newly introduced acetal units bring degradability of PEO as well as delivery of carboxylic acid molecules. Hydrolysis studies with high molecular weight functionalization PEO (Mn = 13.0 kg mol-1) confirm the steady release of fragmented PEO (Mn ∼ 2.0 kg mol-1) and carboxylic acid over days and the process rate is not sensitive to pH variation between pH 5 and 9. The presented method offers a versatile and efficient way to modify PEO with potential energy and medical applications.

Examining the role of moral foundations in promoting intergroup contact willingness.

Improving people's motivation to seek meaningful intergroup contact is considered key to facilitating intergroup harmony. Based on moral foundations theory, this study examines how moral foundations as individual traits predict contact willingness with three minority groups (foreign domestic helpers, LGBT, and Chinese expats) and how moral emotions mediate such associations. We tested our hypotheses based on survey data across Hong Kong and Singapore. We found that care/harm foundation positively predicted contact willingness with foreign domestic helpers and LGBT people, mediated by compassion. Sanctity/degradation foundation negatively predicted contact willingness with LGBT people only in Singapore. Loyalty/betrayal foundation served as a positive predictor of willingness to contact Chinese expats. We also found care/harm foundation to be exclusively associated with compassion and promoted willingness to contact with helpers and LGBT people. Our findings highlight the influence of moral foundations, and possibly norms and intergroup dynamics at the societal level in predicting willingness to contact outgroups.

Oxidative Stress Amplifying Polyprodrug Micelles as Drug Carriers for Combination Anticancer Therapy.

Cancer cells are more vulnerable to reactive oxygen species (ROS)-mediated oxidative stress than normal cells due to disturbed redox balance. It can be postulated that ROS-generating drug carriers exert anticancer actions, leading to combination anticancer therapy with drug payloads. Here, we report a ROS-generating polyprodrug of cinnamaldehyde (CA) that not only serves as a drug carrier but also synergizes with drug payloads. The polyprodrug of CA (pCA) incorporates ROS-generating CA in the backbone of an amphiphilic polymer through an acid-cleavable acetal linkage. pCA could self-assemble with tumor-targeting lipopeptide (DSPE-PEG-RGD) and encapsulate doxorubicin (DOX) to form T-pCAD micelles. At acidic pH, T-pCAD micelles release both CA and DOX to exert synergistic anticancer actions. Animal studies using mouse xenograft models revealed that T-pCAD micelles accumulate in tumors preferentially and suppress the tumor growth significantly. Based on the oxidative stress amplification and acid-responsiveness, ROS-generating pCAD micelles hold tremendous potential as drug carriers for combination anticancer therapy.

Fake news, real risks: How online discussion and sources of fact-check influence public risk perceptions toward nuclear energy.

This study seeks to understand how online discussion, fact-checking, and sources of fact-checks will influence individuals' risk perceptions toward nuclear energy when they are exposed to fake news. Using a 2 × 3 experimental design, 320 participants were randomly assigned to one of the six experimental conditions. Results showed an interaction effect between online discussion and exposure to fact-checking, in which online discussion lowered individuals' risk perception toward nuclear energy when a fact-check was unavailable. Of those who participated in the online discussion, those who viewed a fact-check posted by traditional media have higher risk perception as compared to those who viewed a fact-check posted by a fact-check organization. Our findings indicate that different fact-checking sources can have differential effects on public risk perceptions, depending on whether online discussion is involved. To curb the spread of fake news, different fact-checking strategies will need to be deployed depending on the situation.

Prospero homeobox 1 mediates the progression of gastric cancer by inducing tumor cell proliferation and lymphangiogenesis.

BACKGROUND: Prospero homeobox 1 (PROX1) functions as a tumor suppressor gene or an oncogene in various cancer types. However, the distinct function of PROX1 in gastric cancer is unclear. We determined whether PROX1 affected the oncogenic behavior of gastric cancer cells and investigated its prognostic value in patients with gastric cancer. METHODS: A small interfering RNA against PROX1 was used to silence PROX1 expression in gastric cancer cell lines AGS and SNU638. Expression of PROX1 in gastric cancer tissues was investigated by performing immunohistochemistry. Apoptosis, proliferation, angiogenesis, and lymphangiogenesis were determined by performing the TUNEL assay and immunohistochemical staining for Ki-67, CD34, and D2-40. RESULTS: PROX1 knockdown induced apoptosis by activating cleaved caspase-3, caspase-7, caspase-9, and poly(ADP-ribose) polymerase, and by decreasing the expression of anti-apoptotic proteins Bcl-2 and Bcl-xL. PROX1 knockdown also suppressed tumor cell proliferation. In addition, PROX1 knockdown decreased lymphatic endothelial cell invasion and tube formation and the expression of vascular endothelial growth factor (VEGF)-C and -D and cyclooxygenase (COX)-2. However, PROX1 knockdown only decreased umbilical vein endothelial cell invasion, not tube formation. The mean Ki-67 labeling index and lymphatic vessel density value of PROX1-positive tumors were significantly higher than those of PROX1-negative tumors. However, no significant difference was observed between PROX1 expression and apoptotic index or microvessel density. PROX1 expression was significantly associated with age, cell differentiation, lymph node metastasis, cancer stage, and poor survival. CONCLUSIONS: These results indicate that PROX1 mediates the progression of gastric cancer by inducing tumor cell proliferation and lymphangiogenesis.

70-kDa Heat Shock Protein Downregulates Dynamin in Experimental Stroke: A New Therapeutic Target?

BACKGROUND AND PURPOSE: The 70-kDa heat shock protein (Hsp70) protects brain cells in models of cerebral ischemia. Proteomic screening of mice subjected to middle cerebral artery occlusion identified dynamin as a major downregulated protein in Hsp70-overexpressing mice (Hsp70 transgenic mice). Dynamin-1 is expressed in neurons and participates in neurotransmission, but also transports the death receptor Fas to the cell surface, where it can be bound by its ligand and lead to apoptosis. METHODS: Mice were subjected to distal middle cerebral artery occlusion. Neuro-2a cells were subjected to oxygen glucose deprivation. Hsp70 transgenic and Hsp70-deficient (Hsp70 knockout) mice were compared with wild-type mice for histological and behavioral outcomes. Some mice and neuro-2a cell cultures were given dynasore, a dynamin inhibitor. RESULTS: Hsp70 transgenic mice had better outcomes, whereas Hsp70 knockout mice had worse outcomes compared with wild-type mice. This correlated with decreased and increased dynamin expression, respectively. Dynamin colocalized to neurons and Fas, with higher Fas levels and increased caspase-8 expression. Hsp70 induction in neuro-2a cells was protected from oxygen glucose deprivation, while downregulating dynamin and Fas expression. Further, dynamin inhibition was found to be neuroprotective. CONCLUSIONS: Dynamin may facilitate Fas-mediated apoptotic death in the brain, and Hsp70 may protect by preventing this trafficking. Dynamin should be explored as a new therapeutic target for neuroprotection.

Livin is associated with the invasive and oncogenic phenotypes of human hepatocellular carcinoma cells.

AIM: Livin, a member of the inhibitors of apoptosis proteins, is expressed in variable cancers, and its expression is considered a poor prognostic marker. The aims of this study were to observe the effect of Livin on the behaviors of hepatocellular carcinoma (HCC) cells and to evaluate its expression in HCC tissues and its relation to prognosis. METHODS: The biological effects of Livin on tumor cell behavior were investigated using siRNA in HepG2 and Chang cells. Migration, invasion and proliferation assays were performed. Flow cytometric analyses and western blotting were used to evaluate the impact of Livin on apoptosis and the cell cycle. In addition, western blotting and immunohistochemistry were used to investigate Livin expression in HCC tissues. RESULTS: Livin knockdown suppressed tumor cell migration, invasion and proliferation in HCC cells, and increased the proportion of apoptotic cells as compared with scrambled siRNA-transfected HCC cells. Furthermore, Livin knockdown resulted in the activation of caspases and increased apoptosis. In addition, Livin knockdown modulated cell cycle regulatory protein levels such as decrease of cyclins and cyclin-dependent kinase (CDK) level, and increase of CDK inhibitor (CDKI) level in HCC cells. The Livin protein level was significantly elevated in HCC tissues as compared with normal hepatic tissues. However, Livin expression was not found to be associated with clinicopathological parameters, which included patient survival. CONCLUSION: These results suggest that Livin is associated with invasive and oncogenic phenotypes of human HCC cells.

Tumor-targeted and stimulus-responsive polymeric prodrug nanoparticles to enhance the anticancer therapeutic efficacy of doxorubicin.

Polymeric prodrug nanoparticles have gained increasing attention in the field of anticancer drug delivery because of their dual functions as a drug carrier and a therapeutic agent. Doxorubicin (DOX) is a highly effective chemotherapeutic agent for various cancers but causes cardiotoxicity. In this work, we developed polymeric prodrug (pHU) nanoparticles that serve as both a drug carrier of DOX and a therapeutic agent. The composition of pHU includes antiangiogenic hydroxybenzyl alcohol (HBA) and ursodeoxycholic acid (UDCA), covalently incorporated through hydrogen peroxide (H2O2)-responsive peroxalate. To enhance cancer cell specificity, pHU nanoparticles were surface decorated with taurodeoxycholic acid (TUDCA) to facilitate p-selectin-mediated cancer targeting. TUDCA-coated and DOX-loaded pHU nanoparticles (t-pHUDs) exhibited controlled release of DOX triggered by H2O2, characteristic of the tumor microenvironment. t-pHUDs also effectively suppressed cancer cell migration and vascular endothelial growth factor (VEGF) expression in response to H2O2. In animal studies, t-pHUDs exhibited highly potent anticancer activity. Notably, t-pHUDs, with their ability to accumulate preferentially in tumors due to the p-selectin targeting, surpassed the therapeutic efficacy of equivalent DOX and pHU nanoparticles alone. What is more, t-pHUDs significantly suppressed VEGF expression in tumors and mitigated hepato- and cardiotoxicity of DOX. Given their cancer targeting ability, enhanced therapeutic efficacy and minimized off-target toxicity, t-pHUDs present an innovative and targeted approach with great translational potential as an anticancer therapeutic agent.

The 70 kDa heat shock protein protects against experimental traumatic brain injury.

Traumatic brain injury (TBI) causes disruption of the blood brain barrier (BBB) leading to hemorrhage which can complicate an already catastrophic illness. Matrix metalloproteinases (MMPs) involved in the breakdown of the extracellular matrix may lead to brain hemorrhage. We explore the contribution of the 70 kDa heat shock protein (Hsp70) to outcome and brain hemorrhage in a model of TBI. Male, wildtype (Wt), Hsp70 knockout (Ko) and transgenic (Tg) mice were subjected to TBI using controlled cortical impact (CCI). Motor function, brain hemorrhage and lesion size were assessed at 3, 7 and 14 days. Brains were evaluated for the effects of Hsp70 on MMPs. In Hsp70 Tg mice, CCI led to smaller brain lesions, decreased hemorrhage and reduced expression and activation of MMPs compared to Wt. CCI also significantly decreased right-biased swings and corner turns in the Hsp70 Tg mice. Conversely, Hsp70 Ko mice had significantly increased lesion size, worsened brain hemorrhage and increased expression and activation of MMPs with worsened behavioral outcomes compared to Wt. Hsp70 is protective in experimental TBI. To our knowledge, this is the direct demonstration of brain protection by Hsp70 in a TBI model. Our data demonstrate a new mechanism linking TBI-induced hemorrhage and neuronal injury to the suppression of MMPs by Hsp70, and support the development of Hsp70 enhancing strategies for the treatment of TBI.

Electroencephalography Network Indices as Biomarkers of Upper Limb Impairment in Chronic Stroke.

Alteration of electroencephalography (EEG) signals during task-specific movement of the impaired limb has been reported as a potential biomarker for the severity of motor impairment and for the prediction of motor recovery in individuals with stroke. When implementing EEG experiments, detailed paradigms and well-organized experiment protocols are required to obtain robust and interpretable results. In this protocol, we illustrate a task-specific paradigm with upper limb movement and methods and techniques needed for the acquisition and analysis of EEG data. The paradigm consists of 1 min of rest followed by 10 trials comprising alternating 5 s and 3 s of resting and task (hand extension)-states, respectively, over 4 sessions. EEG signals were acquired using 32 Ag/AgCl scalp electrodes at a sampling rate of 1,000 Hz. Event-related spectral perturbation analysis associated with limb movement and functional network analyses at the global level in the low-beta (12-20 Hz) frequency band were performed. Representative results showed an alteration of the functional network of low-beta EEG frequency bands during movement of the impaired upper limb, and the altered functional network was associated with the degree of motor impairment in chronic stroke patients. The results demonstrate the feasibility of the experimental paradigm in EEG measurements during upper limb movement in individuals with stroke. Further research using this paradigm is needed to determine the potential value of EEG signals as biomarkers of motor impairment and recovery.

Tumor-targeted redox-regulating and antiangiogenic phototherapeutics nanoassemblies for self-boosting phototherapy.

Cancer cells are equipped with abundant antioxidants such as glutathione (GSH) that eliminate reactive oxygen species (ROS) to deteriorate the therapeutic efficacy of photodynamic therapy (PDT). Another challenge in PDT is circumventing PDT-induced hypoxic condition that provokes upregulation of pro-angiogenic factor such as vascular endothelial growth factor (VEGF). It is therefore reasonable to expect that therapeutic outcomes of PDT could be maximized by concurrent delivery of photosensitizers with GSH depleting agents and VEGF suppressors. To achieve cooperative therapeutic actions of PDT with in situ GSH depletion and VEGF suppression, we developed tumor targeted redox-regulating and antiangiogenic phototherapeutic nanoassemblies (tRAPs) composed of self-assembling disulfide-bridged borylbenzyl carbonate (ssBR), photosensitizer (IR780) and tumor targeting gelatin. As a framework of tRAPs, ssBR was rationally designed to form nanoconstructs that serve as photosensitizer carriers with intrinsic GSH depleting- and VEGF suppressing ability. tRAPs effectively depleted intracellular GSH to render cancer cells more vulnerable to ROS and also provoked immunogenic cell death (ICD) of cancer cells upon near infrared (NIR) laser irradiation. In mouse xenograft models, tRAPs preferentially accumulated in tumors and dramatically eradicated tumors with laser irradiation. The design rationale of tRAPs provides a simple and versatile strategy to develop self-boosting phototherapeutic agents with great potential in targeted cancer therapy.

Acid-sensitive stable polymeric micelle-based oxidative stress nanoamplifier as immunostimulating anticancer nanomedicine.

Oxidative stress amplifying compounds could elicit selective killing of cancer cells with minimal toxicity to normal cells and also induce immunogenic cell death (ICD). However, compared to conventional anticancer drugs, oxidative stress amplifying compounds have inferior therapeutic efficacy. It can be postulated that the anticancer therapeutic efficacy and immunostimulating activity of oxidative stress amplifying hybrid prodrug (OSamp) could be fully maximized by employing ultrastable polymeric micelles as drug carriers. In this work, we developed tumour-targeted oxidative stress nanoamplifiers, composed of OSamp, amphiphilic poly(ethylene glycol) methyl ether-block-poly(cyclohexyloxy ethyl glycidyl ether)s (mPEG-PCHGE) and a lipopeptide containing Arg-Gly-Asp (RGD). Tumour targeted OSamp-loaded mPEG-PCHGE (T-POS) micelles exhibited excellent colloidal stability and significant cytotoxicity to cancer cells with the expression of DAMPs (damage-associated molecular patterns). In the syngeneic mouse tumour model, T-POS micelles induced significant apoptotic cell death to inhibit tumour growth without noticeable body weight changes. T-POS micelles also induced ICD and activated adaptive immune responses by increasing the populations of cytotoxic CD4+ and CD8+ T cells. Therefore, these results suggest that T-POS micelles hold great translational potential as immunostimulating anticancer nanomedicine.

Anti-inflammatory properties and pharmacological induction of Hsp70 after brain injury.

The 70-kDa heat shock protein (Hsp70) is thought to protect the brain from a variety of insults. Although the mechanism has been largely limited to its chaperone functions, recent work indicates that Hsp70 also modulates inflammatory pathways. Brain injury and ischemia are associated with an immune response that is largely innate. Hsp70 appears to suppress this response and lead to improved neurological outcome. However, most of this work has relied on the use of genetic mutant models or Hsp70 overexpression using gene transfer or heat stress, thus limiting its translational utility. A few compounds have been studied by various disciplines which, through their ability to inhibit Hsp90, can cause induction of Hsp70. The investigation of Hsp70-inducing pharmacological compounds has obvious clinical implications in terms of potential therapies to mitigate neuroinflammation and lead to neuroprotection from stroke or traumatic brain injury. This review will focus on the inflammation modulating properties of Hsp70, and the current literature surrounding the pharmacological induction in acute neurological injury models with comments on potential applications at the clinical level.

Tumor-targeting oxidative stress nanoamplifiers as anticancer nanomedicine with immunostimulating activity.

Compared to normal cells, cancer cells are more susceptible to insults of prooxidants that generate ROS (reactive oxygen species) or scavenge antioxidants such as glutathione (GSH). Cancer cells undergo immunogenic cell death (ICD) by elevated oxidative stress. Herein, we report rationally designed F-ssPBCA nanoparticles as a tumor-targeting prooxidant, which generates ROS and scavenges GSH simultaneously to cooperatively amplify oxidative stress, leading to ICD. Prooxidant F-ssPBCA nanoparticles are composed of a disulfide-bridged GSH scavenging dimeric prodrug (ssPB) that self-assembles to form nanoconstructs and encapsulates ROS-generating BCA (benzoyloxy cinnamaldehyde). F-ssPBCA nanoparticles significantly elevate oxidative stress to kill cancer cells and also evoke ICD featured by the release of CRT (calreticulin), HMGB-1 (high mobility group box-1), and adenosine triphosphate (ATP). Animal studies revealed that F-ssPBCA nanoparticles accumulate in tumors preferentially and suppress tumor growth effectively. The results of this study demonstrate that prooxidant-mediated oxidative stress elevation is a highly effective strategy to kill cancer cells selectively and even evoke abundant ICD. We anticipate that oxidative stress amplifying F-ssPBCA nanoparticles hold tremendous translational potential as a tumor targeted ICD-inducing anticancer nanomedicine.

A Caenorhabditis elegans Model for Integrating the Functions of Neuropsychiatric Risk Genes Identifies Components Required for Normal Dendritic Morphology.

Analysis of patient-derived DNA samples has identified hundreds of variants that are likely involved in neuropsychiatric diseases such as autism spectrum disorder (ASD) and schizophrenia (SCZ). While these studies couple behavioral phenotypes to individual genotypes, the number and diversity of candidate genes implicated in these disorders highlights the fact that the mechanistic underpinnings of these disorders are largely unknown. Here, we describe a RNAi-based screening platform that uses C. elegans to screen candidate neuropsychiatric risk genes (NRGs) for roles in controlling dendritic arborization. To benchmark this approach, we queried published lists of NRGs whose variants in ASD and SCZ are predicted to result in complete or partial loss of gene function. We found that a significant fraction (>16%) of these candidate NRGs are essential for dendritic development. Furthermore, these gene sets are enriched for dendritic arbor phenotypes (>14 fold) when compared to control RNAi datasets of over 500 human orthologs. The diversity of PVD structural abnormalities observed in these assays suggests that the functions of diverse NRGs (encoding transcription factors, chromatin remodelers, molecular chaperones and cytoskeleton-related proteins) converge to regulate neuronal morphology and that individual NRGs may play distinct roles in dendritic branching. We also demonstrate that the experimental value of this platform by providing additional insights into the molecular frameworks of candidate NRGs. Specifically, we show that ANK2/UNC-44 function is directly integrated with known regulators of dendritic arborization and suggest that altering the dosage of ARID1B/LET-526 expression during development affects neuronal morphology without diminishing aspects of cell fate specification.

Visualizing the metazoan proliferation-quiescence decision in vivo.

Cell proliferation and quiescence are intimately coordinated during metazoan development. Here, we adapt a cyclin-dependent kinase (CDK) sensor to uncouple these key events of the cell cycle in Caenorhabditis elegans and zebrafish through live-cell imaging. The CDK sensor consists of a fluorescently tagged CDK substrate that steadily translocates from the nucleus to the cytoplasm in response to increasing CDK activity and consequent sensor phosphorylation. We show that the CDK sensor can distinguish cycling cells in G1 from quiescent cells in G0, revealing a possible commitment point and a cryptic stochasticity in an otherwise invariant C. elegans cell lineage. Finally, we derive a predictive model of future proliferation behavior in C. elegans based on a snapshot of CDK activity in newly born cells. Thus, we introduce a live-cell imaging tool to facilitate in vivo studies of cell-cycle control in a wide-range of developmental contexts.

All living things are made up of cells that form the different tissues, organs and structures of an organism. The human body, for example, is thought to consist of some 37 trillion cells and harbor over 200 cell types. To maintain a working organism, cells divide to create new cells and replace the ones that have died. Cell division is a tightly controlled process consisting of several steps, and cells continuously face a Shakespearean dilemma of deciding whether to continue dividing (also known as cell proliferation) or to halt the process (known as quiescence). This difficult balancing act is critical during all stages of life, from embryonic development to tissue growth in an adult. Problems in the underlying pathways can result in diseases such as cancer. Cell division is driven by proteins called CDKs, which help cells to complete their cell cycle in the correct sequence. To gain more insight into this complex process, scientists have developed tools for monitoring CDKs. One such tool is a fluorescent biosensor, a molecule that can be inserted into cells that glows and moves in response to CDK activity. The biosensor can be studied and measured in each cell using a microscope. Adikes, Kohrman, Martinez et al. adapted and optimized an existing CDK biosensor to help study cell division and the switch between proliferation and quiescence in two common research organisms, the nematode Caenorhabditis elegans and the zebrafish. Analysis of this biosensor showed that CDK activity at the end of cell division is higher if the cells will divide again but is low if the cells are going to become quiescent. This could suggest that the decision of a cell between proliferation and quiescence may happen earlier than expected. The optimized biosensor is sensitive enough to detect these differences and can even measure variations that influence proliferation in a region on C. elegans that was once thought to be unchanging. The development of this biosensor provides a useful research tool that could be used in other living organisms. Many research questions relate to cell division and so the applications of this tool are wide ranging.

Hypothermia Identifies Dynamin as a Potential Therapeutic Target in Experimental Stroke.

Apoptosis is a cell death pathway that is activated in ischemic stroke. The interaction between Fas and its ligand (FasL) initiates a complex pattern of intracellular events involving the recruitment of specific adaptor proteins and the development of apoptosis. We recently reported that dynamin is increased after experimental stroke, and its inhibition improves neurological outcome. Dynamin has been shown to transport Fas from the endoplasmic reticulum to the cell surface where it can be bound by its ligand, FasL. Hypothermia has been shown to improve outcome in numerous stroke models, and this protection is associated with reduced apoptosis and Fas expression. To explore the contribution of dynamin to hypothermic neuroprotection, we subjected mice to distal middle cerebral artery occlusion (dMCAO) and applied one of two cooling paradigms: one where cooling began at the onset of dMCAO (early hypothermia) and another where cooling began 1 hour later (delayed hypothermia), compared with normothermia (Norm). Both cooling paradigms reduced numbers of apoptotic cells, as well as Fas and dynamin compared with Norm. Fas and dynamin were co-expressed in neurons. Neuronal cultures were exposed to oxygen glucose deprivation. Hypothermia decreased dynamin as well as surface expression of Fas, and this correlated to reduced cell death. The results of this study suggest that dynamin may participate in the Fas-mediated apoptotic pathway, and its reduction may be linked to hypothermic neuroprotection.

Effect of Recepteur d'Origine Nantais expression on chemosensitivity and tumor cell behavior in colorectal cancer.

Recepteur d'Origine Nantais (RON) expression is known to induce oncogenic properties including tumor cell growth, survival, motility, angiogenesis and chemoresistance. In the present study, we evaluated whether RON affects chemosensitivity and oncogenic behavior of colorectal cancer cells and investigated its prognostic value in colorectal cancer. To evaluate the impact of RON on chemosensitivity and tumor cell behavior, we treated colorectal cancer cells with small interfering RNAs specific to RON. This was followed by flow cytometric analyses and migration, Matrigel invasion and endothelial tube formation assays. The expression of RON was investigated by immunohistochemistry in colorectal cancer tissues. TUNEL assay and immunohistochemical staining for CD34 and D2-40 were deployed to determine apoptosis, angiogenesis and lymphangiogenesis. RON knockdown enhanced 5-fluorouracil (FU)-induced apoptosis by upregulating the activities of caspases and expression of proapoptotic genes. Moreover, it enhanced 5-FU-induced cell cycle arrest by decreasing the expression of cyclins and cyclin­dependent kinases and inducing that of p21. Furthermore, RON knockdown augmented the 5-FU-induced inhibition of invasion and migration of colorectal cancer cells. The ß-catenin signaling cascade was blocked by RON knockdown upon 5-FU treatment. RON knockdown also decreased endothelial tube formation and expression of VEGF-A and HIF-1α and increased angiostatin expression. Furthermore, it inhibited lymphatic endothelial cell tube formation and the expression of VEGF-C and COX-2. RON expression was observed to be associated with age, tumor size, lymphovascular and perineural invasion, tumor stage, lymph node and distant metastasis, and poor survival rate. The mean microvessel density value of RON-positive tumors was significantly higher than that of RON-negative ones. These results indicate that RON is associated with tumor progression by inhibiting chemosensitivity and enhancing angiogenesis in colorectal cancer.

Impact of KITENIN on tumor angiogenesis and lymphangiogenesis in colorectal cancer.

Angiogenesis and lymphangiogenesis are involved in the dissemination of tumor cells from solid tumors to regional lymph nodes and various distant sites. KAI1 COOH-terminal interacting tetraspanin (KITENIN) contributes to tumor progression and poor clinical outcomes in various cancers including colorectal cancer. The aim of the present study was to evaluate whether KITENIN affects tumor angiogenesis and lymphangiogenesis in colorectal cancer. A KITENIN small interfering RNA vector was used to silence KITENIN expression in colorectal cancer cell lines including DLD1 and SW480 cells. To evaluate the ability of KITENIN to induce angiogenesis and lymphangiogenesis in human umbilical vein endothelial cells (HUVECs) and lymphatic endothelial cells (HLECs), we performed Matrigel invasion and tube formation assays. Immunohistochemistry was used to determine the expression of KITENIN in colorectal cancer tissues. Angiogenesis and lymphangiogenesis were evaluated by immunostaining with CD34 and D2-40 antibodies. KITENIN silencing inhibited both HUVEC invasion and tube formation in the DLD1 and SW480 cells. KITENIN silencing led to decreased expression of the angiogenic inducers vascular endothelial growth factor (VEGF)-A and hypoxia-inducible factor-1α and increased expression of the angiogenic inhibitor angiostatin. KITENIN silencing did not inhibit either HLEC invasion or tube formation in all tested cells, but it resulted in decreased expression of the lymphangiogenic inducer VEGF-C. KITENIN expression was significantly associated with tumor stage, depth of invasion, lymph node and distant metastases and poor survival. The mean microvessel density was significantly higher in the KITENIN-positive tumors than that in the KITENIN-negative tumors. However, the mean lymphatic vessel density of KITENIN-positive tumors was not significantly higher than that of the KITENIN-negative tumors. These results suggest that KITENIN promotes tumor progression by enhancing angiogenesis in colorectal cancer.