Deep Residual Learning-Based Classification with Identification of Incorrect Predictions and Quantification of Cellularity and Nuclear Morphological Features in Digital Pathological Images of Common Astrocytic Tumors.

Interobserver variations in the pathology of common astrocytic tumors impact diagnosis and subsequent treatment decisions. This study leveraged a residual neural network-50 (ResNet-50) in digital pathological images of diffuse astrocytoma, anaplastic astrocytoma, and glioblastoma to recognize characteristic pathological features and perform classification at the patch and case levels with identification of incorrect predictions. In addition, cellularity and nuclear morphological features, including axis ratio, circularity, entropy, area, irregularity, and perimeter, were quantified via a hybrid task cascade (HTC) framework and compared between different characteristic pathological features with importance weighting. A total of 95 cases, including 15 cases of diffuse astrocytoma, 11 cases of anaplastic astrocytoma, and 69 cases of glioblastoma, were collected in Taiwan Hualien Tzu Chi Hospital from January 2000 to December 2021. The results revealed that an optimized ResNet-50 model could recognize characteristic pathological features at the patch level and assist in diagnosis at the case level with accuracies of 0.916 and 0.846, respectively. Incorrect predictions were mainly due to indistinguishable morphologic overlap between anaplastic astrocytoma and glioblastoma tumor cell area, zones of scant vascular lumen with compact endothelial cells in the glioblastoma microvascular proliferation area mimicking the glioblastoma tumor cell area, and certain regions in diffuse astrocytoma with too low cellularity being misrecognized as the glioblastoma necrosis area. Significant differences were observed in cellularity and each nuclear morphological feature among different characteristic pathological features. Furthermore, using the extreme gradient boosting (XGBoost) algorithm, we found that entropy was the most important feature for classification, followed by cellularity, area, circularity, axis ratio, perimeter, and irregularity. Identifying incorrect predictions provided valuable feedback to machine learning design to further enhance accuracy and reduce errors in classification. Moreover, quantifying cellularity and nuclear morphological features with importance weighting provided the basis for developing an innovative scoring system to achieve objective classification and precision diagnosis among common astrocytic tumors.

Efficiency of PGK1 proteins delivered to the brain via a liposomal system through intranasal route administration for the treatment of spinocerebellar ataxia type 3.

A patient-friendly and efficient treatment method for patients with spinocerebellar ataxia type 3 (SCA3) was provided through a nose-to-brain liposomal system. Initially, PGK1 was overexpressed in HEK 293-84Q-GFP diseased cells (HEK 293-84Q-GFP-PGK1 cells) to confirm its effect on the diseased protein polyQ. A decrease in polyQ expression was demonstrated in HEK 293-84Q-GFP-PGK1 cells compared to HEK 293-84Q-GFP parental cells. Subsequently, PGK1 was encapsulated in a liposomal system to evaluate its therapeutic efficiency in SCA3. The optimized liposomes exhibited a significantly enhanced positive charge, facilitating efficient intracellular protein delivery to the cells. The proteins were encapsulated within the liposomes using an optimized method involving a combination of heat shock and sonication. The liposomal system was further demonstrated to be deliverable to the brain via intranasal administration. PGK1/liposomes were intranasally delivered to SCA3 mice, which subsequently exhibited an amelioration of motor impairment, as assessed via the accelerated rotarod test. Additionally, fewer shrunken morphology Purkinje cells and a reduction in polyQ expression were observed in SCA3 mice that received PGK1/liposomes but not in the untreated, liposome-only, or PGK1-only groups. This study provides a non-invasive route for protein delivery and greater delivery efficiency via the liposomal system for treating neurodegenerative diseases.

Targeting the Axl and mTOR Pathway Synergizes Immunotherapy and Chemotherapy to Butylidenephthalide in a Recurrent GBM.

Background: The role of inherent tumor heterogeneity and an immunosuppressive microenvironment in therapeutic resistance has been determined to be of importance for the better management of glioblastoma multiforme (GBM). Some studies have suggested that combined drugs with divergent mechanisms may be promising in treating recurrent GBM. Methods: Intracranial sustained (Z)-n-butylidenephthalide [(Z)-BP] delivery through Cerebraca Wafers (CWs) to eliminate unresectable brain tumors was combined with the administration of temozolomide (TMZ), pembrolizumab, and cytokine-induced killer (CIK) cells for treating a patient with recurrent glioblastoma. Neurological adverse events and wound healing delay were monitored for estimating tolerance and efficacy. Response Assessment in Neuro-Oncology criteria were applied to evaluate progression-free survival (PFS); further, the molecular characteristics of GBM tissues were analyzed, and the underlying mechanism was investigated using primary culture. Results: Intracerebral (Z)-BP in residual tumors could not only inhibit cancer stem cells but also increase interferon gamma levels in serum, which then led to the regression of GBM and an immune-responsive microenvironment. Targeting receptor tyrosine kinases, including Axl and epidermal growth factor receptor (EGFR), and inhibiting the mechanistic target of rapamycin (mTOR) through (Z)-BP were determined to synergize CIK cells in the presence of pembrolizumab and TMZ in recurrent GBM. Therefore, this well-tolerated regimen could simultaneously block multiple cancer pathways, which allowed extended PFS and improved quality of life for 22 months. Conclusion: Given the several unique functions of (Z)-BP, greater sensitivity of chemotherapy and the synergism of pembrolizumab and CIK cells could have affected the excellent prognosis seen in this patient with recurrent GBM.

Interstitial Control-Released Polymer Carrying a Targeting Small-Molecule Drug Reduces PD-L1 and MGMT Expression in Recurrent High-Grade Gliomas with TMZ Resistance.

In recurrent glioblastoma, Gliadel wafer implantation after surgery has been shown to result in incomplete chemical removal of residual tumor and development of brain edema. Furthermore, temozolomide (TMZ) resistance caused by O6-methylguanine-DNA-methyltransferase (MGMT) activation and programmed cell death-ligand 1 (PD-L1) expression leads to immune-cold lesions that result in poorer prognosis. Cerebraca wafer, a biodegradable polymer containing (Z)-n-butylidenephthalide (BP), is designed to eliminate residual tumor after glioma resection. An open-label, one-arm study with four dose cohorts, involving a traditional 3 + 3 dose escalation clinical trial, of the Cerebraca wafer combined with TMZ on patients with recurrent high-grade glioma, was conducted. Of the 12 patients who receive implantation of Cerebraca wafer, there were no drug-related adverse events (AEs) or serious AEs (SAEs). The median overall survival (OS) of patients receiving low-dose Cerebraca wafer was 12 months in the group with >25% wafer coverage of the resected tumor, which is longer than OS duration in previously published studies (Gliadel wafer, 6.4 months). Patients who received high-dose Cerebraca wafer treatment had not yet died at the data cut-off date; a 100% progression-free survival (PFS) rate at six month was achieved, indicating the median OS of cohort IV was more than 17.4 months. In vitro study of the primary cells collected from the patients revealed that the IC50 of BP against tumor stem cells was four times lower than that of bis-chloroethylnitrosourea (BCNU). A synergistic effect between BP and TMZ was demonstrated by a reduction in MGMT expression. Furthermore, BP inhibited PD-L1 expression, thereby activating T-cell cytotoxicity and increasing interferon-gamma (IFN-γ) secretion. The better therapeutic effect of Cerebraca wafer on recurrent high-grade glioma could occur through re-sensitization of TMZ and reduction of PD-L1.

Telomeres and Cancer.

Telomeres cap the ends of eukaryotic chromosomes and are indispensable chromatin structures for genome protection and replication. Telomere length maintenance has been attributed to several functional modulators, including telomerase, the shelterin complex, and the CST complex, synergizing with DNA replication, repair, and the RNA metabolism pathway components. As dysfunctional telomere maintenance and telomerase activation are associated with several human diseases, including cancer, the molecular mechanisms behind telomere length regulation and protection need particular emphasis. Cancer cells exhibit telomerase activation, enabling replicative immortality. Telomerase reverse transcriptase (TERT) activation is involved in cancer development through diverse activities other than mediating telomere elongation. This review describes the telomere functions, the role of functional modulators, the implications in cancer development, and the future therapeutic opportunities.

Approach to the Connection between Meconium Consistency and Adverse Neonatal Outcomes: A Retrospective Clinical Review and Prospective In Vitro Study.

Whether meconium-stained amniotic fluid (MSAF) serves as an indicator of fetal distress is under debate; however, the presence of MSAF concerns both obstetricians and pediatricians because meconium aspiration is a major contributor to neonatal morbidity and mortality, even with appropriate treatment. The present study suggested that thick meconium in infants might be associated with poor outcomes compared with thin meconium based on chart reviews. In addition, cell survival assays following the incubation of various meconium concentrations with monolayers of human epithelial and embryonic lung fibroblast cell lines were consistent with the results obtained from chart reviews. Exposure to meconium resulted in the significant release of nitrite from A549 and HEL299 cells. Medicinal agents, including dexamethasone, L-Nω-nitro-arginine methylester (L-NAME), and NS-398 significantly reduced the meconium-induced release of nitrite. These results support the hypothesis that thick meconium is a risk factor for neonates who require resuscitation, and inflammation appears to serve as the primary mechanism for meconium-associated lung injury. A better understanding of the relationship between nitrite and inflammation could result in the development of promising treatments for meconium aspiration syndrome (MAS).

Differentiation of Human Pluripotent Stem Cells Into Specific Neural Lineages.

Human pluripotent stem cells (hPSCs) are sources of several somatic cell types for human developmental studies, in vitro disease modeling, and cell transplantation therapy. Improving strategies of derivation of high-purity specific neural and glial lineages from hPSCs is critical for application to the study and therapy of the nervous system. Here, we will focus on the principles behind establishment of neuron and glia differentiation methods according to developmental studies. We will also highlight the limitations and challenges associated with the differentiation of several "difficult" neural lineages and delay in neuronal maturation and functional integration. To overcome these challenges, we will introduce strategies and novel technologies aimed at improving the differentiation of various neural lineages to expand the application potential of hPSCs to the study of the nervous system.

Coactivation of GSK3ß and IGF-1 Attenuates Amyotrophic Lateral Sclerosis Nerve Fiber Cytopathies in SOD1 Mutant Patient-Derived Motor Neurons.

Amyotrophic lateral sclerosis (ALS) is a progressive nervous system disease that causes motor neuron (MN) degeneration and results in patient death within a few years. To recapitulate the cytopathies of ALS patients' MNs, SOD1G85R mutant and corrected SOD1G85G isogenic-induced pluripotent stem cell (iPSC) lines were established. Two SOD1 mutant ALS (SOD1G85R and SOD1D90A), two SOD1 mutant corrected (SOD1G85G and SOD1D90D), and one sporadic ALS iPSC lines were directed toward MNs. After receiving ~90% purity for MNs, we first demonstrated that SOD1G85R mutant ALS MNs recapitulated ALS-specific nerve fiber aggregates, similar to SOD1D90A ALS MNs in a previous study. Moreover, we found that both SOD1 mutant MNs showed ALS-specific neurite degenerations and neurotransmitter-induced calcium hyperresponsiveness. In a small compound test using these MNs, we demonstrated that gastrodin, a major ingredient of Gastrodia elata, showed therapeutic effects that decreased nerve fiber cytopathies and reverse neurotransmitter-induced hyperresponsiveness. The therapeutic effects of gastrodin applied not only to SOD1 ALS MNs but also to sporadic ALS MNs and SOD1G93A ALS mice. Moreover, we found that coactivation of the GSK3ß and IGF-1 pathways was a mechanism involved in the therapeutic effects of gastrodin. Thus, the coordination of compounds that activate these two mechanisms could reduce nerve fiber cytopathies in SOD1 ALS MNs. Interestingly, the therapeutic role of GSK3ß activation on SOD1 ALS MNs in the present study was in contrast to the role previously reported in research using cell line- or transgenic animal-based models. In conclusion, we identified in vitro ALS-specific nerve fiber and neurofunctional markers in MNs, which will be useful for drug screening, and we used an iPSC-based model to reveal novel therapeutic mechanisms (including GSK3ß and IGF-1 activation) that may serve as potential targets for ALS therapy.

Surviving the Rookie Virus, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2): The Immunopathology of a SARS-CoV2 Infection.

Until July 29th, the number of confirmed coronavirus (COVID-19) cases worldwide has risen to over 16 million, within which 655 k deaths. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) emerges as the 11th global pandemic disease, showing the highest infectivity and lowest infection fatality rate. In this review, we compare the immunopathology among SARS-CoV, Middle East respiratory syndrome coronavirus, and SARS-CoV2. SARS-CoV2 is similar to SARS-CoV; it can cause lymphocytopenia and a rising granulocyte count. Here we point out the human body and concentrated society make for an excellent incubator for virus evolution. Most research energies put into developing the SARS-CoV2 vaccine are trying to block virus infection. Sixty-five percent of severe patients die with multiple organ failure, inflammation, and cytokine storm, which indicates that the patient's immune system maintains functionality. Finding a way to trigger the specific T cell subset and plasmablast in our body is the best shot to get away with SARS-CoV2.

Recent Findings on Cell-Based Therapies for COVID19-Related Pulmonary Fibrosis.

COVID-19 has spread worldwide, including the United States, United Kingdom, and Italy, along with its site of origin in China, since 2020. The virus was first found in the Wuhan seafood market at the end of 2019, with a controversial source. The clinical symptoms of COVID-19 include fever, cough, and respiratory tract inflammation, with some severe patients developing an acute and chronic lung injury, such as acute respiratory distress syndrome (ARDS) and pulmonary fibrosis (PF). It has already claimed approximately 300 thousand human lives and the number is still on the rise; the only way to prevent the infection is to be safe till vaccines and reliable treatments develop. In previous studies, the use of mesenchymal stem cells (MSCs) in clinical trials had been proven to be effective in immune modulation and tissue repair promotion; however, their efficacy in treating COVID-19 remains underestimated. Here, we report the findings from past experiences of SARS and MSCs, and how SARS could also induce PF. Such studies may help to understand the rationale for the recent cell-based therapies for COVID-19.

Induced Pluripotent Stem Cell (iPSC)-Based Neurodegenerative Disease Models for Phenotype Recapitulation and Drug Screening.

Neurodegenerative diseases represent a significant unmet medical need in our aging society. There are no effective treatments for most of these diseases, and we know comparatively little regarding pathogenic mechanisms. Among the challenges faced by those involved in developing therapeutic drugs for neurodegenerative diseases, the syndromes are often complex, and small animal models do not fully recapitulate the unique features of the human nervous system. Human induced pluripotent stem cells (iPSCs) are a novel technology that ideally would permit us to generate neuronal cells from individual patients, thereby eliminating the problem of species-specificity inherent when using animal models. Specific phenotypes of iPSC-derived cells may permit researchers to identify sub-types and to distinguish among unique clusters and groups. Recently, iPSCs were used for drug screening and testing for neurologic disorders including Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), spinocerebellar atrophy (SCA), and Zika virus infection. However, there remain many challenges still ahead, including how one might effectively recapitulate sporadic disease phenotypes and the selection of ideal phenotypes and for large-scale drug screening. Fortunately, quite a few novel strategies have been developed that might be combined with an iPSC-based model to solve these challenges, including organoid technology, single-cell RNA sequencing, genome editing, and deep learning artificial intelligence. Here, we will review current applications and potential future directions for iPSC-based neurodegenerative disease models for critical drug screening.

Induced Pluripotent Stem Cells: A Powerful Neurodegenerative Disease Modeling Tool for Mechanism Study and Drug Discovery.

Many neurodegenerative diseases are progressive, complex diseases without clear mechanisms or effective treatments. To study the mechanisms underlying these diseases and to develop treatment strategies, a reliable in vitro modeling system is critical. Induced pluripotent stem cells (iPSCs) have the ability to self-renew and possess the differentiation potential to become any kind of adult cell; thus, they may serve as a powerful material for disease modeling. Indeed, patient cell-derived iPSCs can differentiate into specific cell lineages that display the appropriate disease phenotypes and vulnerabilities. In this review, we highlight neuronal differentiation methods and the current development of iPSC-based neurodegenerative disease modeling tools for mechanism study and drug screening, with a discussion of the challenges and future inspiration for application.

Migration/Invasion of Malignant Gliomas and Implications for Therapeutic Treatment.

Malignant tumors of the central nervous system (CNS) are among cancers with the poorest prognosis, indicated by their association with tumors of high-level morbidity and mortality. Gliomas, the most common primary CNS tumors that arise from neuroglial stem or progenitor cells, have estimated annual incidence of 6.6 per 100,000 individuals in the USA, and 3.5 per 100,000 individuals in Taiwan. Tumor invasion and metastasis are the major contributors to the deaths in cancer patients. Therapeutic goals including cancer stem cells (CSC), phenotypic shifts, EZH2/AXL/TGF-ß axis activation, miRNAs and exosomes are relevant to GBM metastasis to develop novel targeted therapeutics for GBM and other brain cancers. Herein, we highlight tumor metastasis in our understanding of gliomas, and illustrate novel exosome therapeutic approaches in glioma, thereby paving the way towards innovative therapies in neuro-oncology.

Targeting Cellular Stress Mechanisms and Metabolic Homeostasis by Chinese Herbal Drugs for Neuroprotection.

Traditional Chinese medicine has been practiced for centuries in East Asia. Herbs are used to maintain health and cure disease. Certain Chinese herbs are known to protect and improve the brain, memory, and nervous system. To apply ancient knowledge to modern science, some major natural therapeutic compounds in herbs were extracted and evaluated in recent decades. Emerging studies have shown that herbal compounds have neuroprotective effects or can ameliorate neurodegenerative diseases. To understand the mechanisms of herbal compounds that protect against neurodegenerative diseases, we summarize studies that discovered neuroprotection by herbal compounds and compound-related mechanisms in neurodegenerative disease models. Those compounds discussed herein show neuroprotection through different mechanisms, such as cytokine regulation, autophagy, endoplasmic reticulum (ER) stress, glucose metabolism, and synaptic function. The interleukin (IL)-1ß and tumor necrosis factor (TNF)-α signaling pathways are inhibited by some compounds, thus attenuating the inflammatory response and protecting neurons from cell death. As to autophagy regulation, herbal compounds show opposite regulatory effects in different neurodegenerative models. Herbal compounds that inhibit ER stress prevent neuronal death in neurodegenerative diseases. Moreover, there are compounds that protect against neuronal death by affecting glucose metabolism and synaptic function. Since the progression of neurodegenerative diseases is complicated, and compound-related mechanisms for neuroprotection differ, therapeutic strategies may need to involve multiple compounds and consider the type and stage of neurodegenerative diseases.

An integrated microfluidic system for screening of phage-displayed peptides specific to colon cancer cells and colon cancer stem cells.

Affinity reagents recognizing biomarkers specifically are essential components of clinical diagnostics and target therapeutics. However, conventional methods for screening of these reagents often have drawbacks such as large reagent consumption, the labor-intensive or time-consuming procedures, and the involvement of bulky or expensive equipment. Alternatively, microfluidic platforms could potentially automate the screening process within a shorter period of time and reduce reagent and sample consumption dramatically. It has been demonstrated recently that a subpopulation of tumor cells known as cancer stem cells possess high drug resistance and proliferation potential and are regarded as the main cause of metastasis. Therefore, a peptide that recognizes cancer stem cells and differentiates them from other cancer cells will be extremely useful in early diagnosis and target therapy. This study utilized M13 phage display technology to identify peptides that bind, respectively, to colon cancer cells and colon cancer stem cells using an integrated microfluidic system. In addition to positive selection, a negative selection process was integrated on the chip to achieve the selection of peptides of high affinity and specificity. We successfully screened three peptides specific to colon cancer cells and colon cancer stem cells, namely, HOLC-1, HOLC-2, and COLC-1, respectively, and their specificity was measured by the capture rate between target, control, and other cell lines. The capture rates are 43.40 ± 7.23%, 45.16 ± 7.12%, and 49.79 ± 5.34% for colon cancer cells and colon cancer stem cells, respectively, showing a higher specificity on target cells than on control and other cell lines. The developed technique may be promising for early diagnosis of cancer cells and target therapeutics.

Shear-induced endothelial mechanotransduction: the interplay between reactive oxygen species (ROS) and nitric oxide (NO) and the pathophysiological implications.

Hemodynamic shear stress, the blood flow-generated frictional force acting on the vascular endothelial cells, is essential for endothelial homeostasis under normal physiological conditions. Mechanosensors on endothelial cells detect shear stress and transduce it into biochemical signals to trigger vascular adaptive responses. Among the various shear-induced signaling molecules, reactive oxygen species (ROS) and nitric oxide (NO) have been implicated in vascular homeostasis and diseases. In this review, we explore the molecular, cellular, and vascular processes arising from shear-induced signaling (mechanotransduction) with emphasis on the roles of ROS and NO, and also discuss the mechanisms that may lead to excessive vascular remodeling and thus drive pathobiologic processes responsible for atherosclerosis. Current evidence suggests that NADPH oxidase is one of main cellular sources of ROS generation in endothelial cells under flow condition. Flow patterns and magnitude of shear determine the amount of ROS produced by endothelial cells, usually an irregular flow pattern (disturbed or oscillatory) producing higher levels of ROS than a regular flow pattern (steady or pulsatile). ROS production is closely linked to NO generation and elevated levels of ROS lead to low NO bioavailability, as is often observed in endothelial cells exposed to irregular flow. The low NO bioavailability is partly caused by the reaction of ROS with NO to form peroxynitrite, a key molecule which may initiate many pro-atherogenic events. This differential production of ROS and RNS (reactive nitrogen species) under various flow patterns and conditions modulates endothelial gene expression and thus results in differential vascular responses. Moreover, ROS/RNS are able to promote specific post-translational modifications in regulatory proteins (including S-glutathionylation, S-nitrosylation and tyrosine nitration), which constitute chemical signals that are relevant in cardiovascular pathophysiology. Overall, the dynamic interplay between local hemodynamic milieu and the resulting oxidative and S-nitrosative modification of regulatory proteins is important for ensuing vascular homeostasis. Based on available evidence, it is proposed that a regular flow pattern produces lower levels of ROS and higher NO bioavailability, creating an anti-atherogenic environment. On the other hand, an irregular flow pattern results in higher levels of ROS and yet lower NO bioavailability, thus triggering pro-atherogenic effects.

Regulation of shear-induced nuclear translocation of the Nrf2 transcription factor in endothelial cells.

BACKGROUND: Vascular endothelial cells (ECs) constantly experience fluid shear stresses generated by blood flow. Laminar flow is known to produce atheroprotective effects on ECs. Nrf2 is a transcription factor that is essential for the antioxidant response element (ARE)-mediated induction of genes such as heme-oxygenase 1 (HO-1). We previously showed that fluid shear stress increases intracellular reactive oxygen species (ROS) in ECs. Moreover, oxidants are known to stimulate Nrf2. We thus examined the regulation of Nrf2 in cultured human ECs by shear stress. RESULTS: Exposure of human umbilical vein endothelial cells (HUVECs) to laminar shear stress (12 dyne/cm2) induced Nrf2 nuclear translocation, which was inhibited by a phosphatidylinositol 3-kinase (PI3K) inhibitor, a protein kinase C (PKC) inhibitor, and an antioxidant agent N-acetyl cysteine (NAC), but not by other protein kinase inhibitors. Therefore, PI3K, PKC, and ROS are involved in the signaling pathway that leads to the shear-induced nuclear translocation of Nrf2. We also found that shear stress increased the ARE-binding activity of Nrf2 and the downstream expression of HO-1. CONCLUSION: Our data suggest that the atheroprotective effect of laminar flow is partially attributed to Nrf2 activation which results in ARE-mediated gene transcriptions, such as HO-1 expression, that are beneficial to the cardiovascular system.

Rho/Rhotekin-mediated NF-kappaB activation confers resistance to apoptosis.

Rhotekin (RTKN), the gene coding for the Rho effector, RTKN, was shown to be overexpressed in human gastric cancer (GC). In this study, we further showed that RTKN is expressed at a low level in normal cells and is overexpressed in many cancer-derived cell lines. The function of RTKN as an effector protein in Rho GTPase-mediated pathways regulating apoptosis was investigated. By transfection and expression of RTKN in cells that expressed endogenous RTKN at a low basal level, we showed that RTKN overexpression conferred cell resistance to apoptosis induced by serum deprivation or treatment with sodium butyrate, and the increased resistance correlated to the level of RTKN. Conversely, reducing RTKN expression by small interfering RNAs greatly sensitized cells to apoptosis. The RTKN-mediated antiapoptotic effect was blocked by the nuclear factor-kappaB (NF-kappaB) inhibitors, curcumin or parthenolide, but not by the phosphatidylinositol 3'-OH-kinase inhibitor, LY294002, or the MAP kinase inhibitor, PD98059. Reporter gene assays and electrophoretic mobility shift assay confirmed that RTKN overexpression led to constitutive activation of NF-kappaB through the phosphorylation of IkappaB by IKKbeta. By using the RTKN truncation mutants, we showed that RTKN mediated Rho activity eliciting signaling pathway to activate NF-kappaB, with a concomitant induction of expression of the NF-kappaB antiapoptotic genes, cIAP-2, BCl-xL, A1, and A20. Consistent with these data, RTKN-expressing cells showed increased chemoresistance to 5-fluorouracil and paclitaxol, and the resistance was greatly attenuated by NF-kappaB inhibitor. In conclusion, overactivated Rho/RTKN/NF-kappaB signaling pathway through overexpression of RTKN may play a key role in gastric tumorigenesis by conferring cells resistance to apoptosis, and this signaling pathway may serve as an important target for novel therapeutic approaches to the treatment of human GC.

Overexpression of rho effector rhotekin confers increased survival in gastric adenocarcinoma.

Like many epithelial-derived cancers, gastric cancer (GC) results from a multistep tumorigenic process. However, the detailed mechanisms involved in GC formation are poorly characterized. Using an ordered differential display method, we have identified rhotekin (RTKN), the gene coding for the Rho effector, RTKN, as one of the genes differentially expressed in human GC. Northern analysis using human multiple tissue blots showed that RTKN is predominantly expressed in the kidney and spinal cord, and, to a lesser degree, in the thyroid, tongue, liver, brain, prostate, trachea, and stomach. RT-PCR analysis confirmed that RTKN was overexpressed in most (5/7; 71%) GC examined. By analyzing the Stanford Microarray Database for the expression profiles of gastric tissues, we also found a progressional increase in RTKN expression in nonneoplastic mucosa, GC, and then lymph node metastases (p < 0.005 by Jonckheere-Terpstra test), suggesting that RTKN expression correlates with GC progression. The role of RTKN in the pathogenic development of GC was investigated by transfection and expression of RTKN in AGS gastric cells, which express endogenous RTKN at a low basal level. Flow-cytometric analysis showed that RTKN-transfected AGS cells were significantly more resistant than vector-transfected cells to apoptosis upon treatment with sodium butyrate. To explore the mechanisms underlying RTKN-mediated cell survival, a reporter assay was performed. Since the NF-kappaB activation is known to promote cell survival and Rho GTPase may lead to NF-kappaB activation, we transfected AGS cells with the RTKN expression vector along with a pNF-kappaB-Luc reporter plasmid. Our results showed that overexpression of RTKN induced robust activation of NF-kappaB, and RTKN-mediated NF-kappaB activation was suppressed significantly by C3 transferase, an inhibitor of the small GTPase Rho. We conclude that Rho/RTKN-mediated NF-kappaB activation leading to cell survival may play a key role in gastric tumorigenesis. This study provides original documentation for the overrepresentation of the Rho GTPase effector rhotekin in human cancer and its links to cancer formation.