The effect of genetic variants of SLC22A18 on proliferation, migration, and invasion of colon cancer cells.

Solute carrier family (SLC) transporters are expressed in the digestive system and play important roles in maintaining physiological functions in the body. In addition, SLC transporters act as oncoproteins or tumor-suppressor proteins during the development, progression, and metastasis of various digestive system cancers. SLC22A18, a member of the SLC22 gene family, is an orphan transporter with an unknown endogenous substrate. Previous study revealed that SLC22A18 is downregulated in colorectal cancer tissues and that it acts as a suppressor in colorectal cancer, although the effects of SLC22A18 variants on colon cancer cell proliferation, migration, and invasion are unknown. Therefore, in this study, we identified SLC22A18 variants found in multiple populations by searching public databases and determined the in vitro effects of these missense variations on transporter expression and cancer progression. Our results indicated that three missense SLC22A18 variants-p.Ala6Thr, p.Arg12Gln, and p.Arg86His-had significantly lower cell expression than the wild type, possibly owing to intracellular degradation. Furthermore, these three variants caused significantly higher proliferation, migration, and invasion of colon cancer cells than the wild type. Our findings suggest that missense variants of SLC22A18 can potentially serve as biomarkers or prognostic tools that enable clinicians to predict colorectal cancer progression.

FDA Public Workshop Summary-Addressing Challenges in Inhaled Antifungal Drug Development.

Allergic bronchopulmonary aspergillosis and invasive fungal diseases represent distinct infectious entities that cause significant morbidity and mortality. Currently, administered inhaled antifungal therapies are unapproved, have suboptimal efficacy, and are associated with considerable adverse reactions. The emergence of resistant pathogens is also a growing concern. Inhaled antifungal development programs are challenged by inadequate nonclinical infection models, highly heterogenous patient populations, low prevalence rates of fungal diseases, difficulties defining clinical trial enrollment criteria, and lack of robust clinical trial endpoints. On September 25, 2020, the US Food and Drug Administration (FDA) convened a workshop with experts in pulmonary medicine and infectious diseases from academia, industry, and other governmental agencies. Key discussion topics included regulatory incentives to facilitate development of inhaled antifungal drugs and combination inhalational devices, limitations of existing nonclinical models and clinical trial designs, patient perspectives, and industry insights.

Functional characterization of ABCA4 genetic variants related to Stargardt disease.

The ATP-binding cassette subfamily 4 (ABCA4), a transporter, is localized within the photoreceptors of the retina, and its genetic variants cause retinal dystrophy. Despite the clinical importance of the ABCA4 transporter, a few studies have investigated the function of each variant. In this study, we functionally characterized ABCA4 variants found in Korean patients with Stargardt disease or variants of the ABCA4 promoter region. We observed that four missense variants-p.Arg290Gln, p.Thr1117Ala, p.Cys1140Trp, and p.Asn1588Tyr-significantly decreased ABCA4 expression on the plasma membrane, which could be due to intracellular degradation. There are four major haplotypes in the ABCA4 proximal promoter. We observed that the H1 haplotype (c.-761C>A) indicated significantly increased luciferase activity compared to that of the wild-type, whereas the H3 haplotype (c.-1086A>C) indicated significantly decreased luciferase activity (P < 0.01 and 0.001, respectively). In addition, c.-900A>T in the H2 haplotype exhibited significantly increased luciferase activity compared with that of the wild-type. Two transcription factors, GATA-2 and HLF, were found to function as enhancers of ABCA4 transcription. Our findings suggest that ABCA4 variants in patients with Stargardt disease affect ABCA4 expression. Furthermore, common variants of the ABCA4 proximal promoter alter the ABCA4 transcriptional activity, which is regulated by GATA-2 and HLF transcription factors.

Generation of a protective murine monoclonal antibody against the stem of influenza hemagglutinins from group 1 viruses and identification of resistance mutations against it.

Vaccines that elicit broadly cross-neutralizing antibodies, including antibodies that target the conserved stem of hemagglutinin (HA), are being developed as a strategy for next-generation influenza vaccines that protect against influenza across multiple years. However, efficient induction of cross-neutralizing antibodies remains a challenge, and potential escape mutations have not been well characterized. Here we elicited cross-neutralizing antibodies by immunizing animals with the hemagglutinins from H5 and H9 subtype influenza A viruses that are sensitive to neutralization by stem antibodies. We further isolated and characterized an HA stem monoclonal antibody 4C2 that broadly neutralizes group 1 influenza viruses and identified HA mutations that reduced sensitivity to stem antibodies. Our results offer insights for next-generation influenza vaccine strategies for inducing cross-neutralizing antibodies.

MiR-657/ATF2 Signaling Pathway Has a Critical Role in Spatholobus suberectus Dunn Extract-Induced Apoptosis in U266 and U937 Cells.

Though Spatholobus suberectus Dunn (SSD) has been reported to have anti-virus, anti-osteoclastogenesis, and anti-inflammation activities, its underlying anti-cancer mechanism has never been elucidated in association with the role of miR-657 in endoplasmic reticulum (ER) stress-related apoptosis to date. SSD treatment exerted cytotoxicity in U266 and U937 cells in a dose-dependent manner. Also, apoptosis-related proteins such as PARP, procaspase-3, and Bax were regulated by SSD treatment. Furthermore, Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay revealed that a number of apoptotic bodies were increased by SSD. Interestingly, the ER stress-related proteins such as p-ATF2 and CHOP were elevated by SSD. Interestingly, reactive oxygen species (ROS) generation and cytotoxicity by SSD treatment were significantly reduced by N-Acetyl-L-cysteine (NAC). Among the microRNAs (miRNAs) regulated by SSD treatment, miR-657 was most significantly reduced by SSD treatment. However, an miR-657 mimic reversed SSD-induced apoptosis by the attenuation of the expression of p-ATF2, CHOP, and PARP cleavage. Overall, these findings provide scientific evidence that miR657 is an onco-miRNA targeting the ER stress signal pathway and SSD induces apoptosis via the inhibition of miR-657, ROS, and the activation of p-ATF2 and CHOP as a potent anti-cancer agent for myeloid-originated hematological cancer.

Neutralizing Antibody Responses to Homologous and Heterologous H1 and H3 Influenza A Strains After Vaccination With Inactivated Trivalent Influenza Vaccine Vary With Age and Prior-year Vaccination.

BACKGROUND: Prior influenza immunity influences the homologous neutralizing antibody responses elicited by inactivated influenza vaccines (IIV), but neutralizing antibody responses to heterologous strains have not been extensively characterized. METHODS: We analyzed neutralizing antibody titers in individuals aged 1-88 who received the 2009-2010 season IIV before infection by or vaccination against the 2009 pandemic H1N1 virus. Neutralization titers to homologous and heterologous past, recent, and advanced H1 and H3 strains, as well as H2, H5, and H7 strains, were measured using influenza hemagglutinin pseudoviruses. We performed exploratory analyses based on age, prior-year IIV, and prevaccination titer, without controlling for Type I errors. RESULTS: IIV elicited neutralizing antibodies to past and advanced H1 and H3 strains, as well as to an H2 strain in individuals who were likely infected early in life. The neutralization of avian subtype viruses was rare, and there was no imprinting of neutralization responses to novel avian subtype viruses based on the influenza group. Compared to adults, children had higher seroresponse rates to homologous and heterologous strains, and their sera generated larger antigenic distances among strains. Seroresponse rates to homologous and heterologous strains were lower in subjects vaccinated with prior-year IIV, though postimmunization titers were generally high. CONCLUSIONS: IIV elicited neutralizing antibodies to heterologous H1 and H3 strains in all ages groups, but titers and seroresponse rates were usually higher in children. Prior-year vaccination with the same strains tended to blunt IIV neutralization responses to all strains in young and old age groups, yet postimmunization titers were high.

Activation of ER Stress-Dependent miR-216b Has a Critical Role in Salviamiltiorrhiza Ethanol-Extract-Induced Apoptosis in U266 and U937 Cells.

Although Salviamiltiorrhiza has been reported to have anti-cancer mechanisms, such as caspase activation, cell cycle arrest, an anti-angiogenesis effect, and Bcl-2 family regulation, its underlying mechanism of endoplasmic reticulum (ER) stress-mediated apoptosis has never been demonstrated. Thus, in this current study, ER stress-related apoptosis via miR-216b of the ethanol extract of Salviamiltiorrhiza (SM) is elucidated for the first time. SM treatment inhibited the viability of U266 and U937 cells in a concentration-dependent manner. However, SM-exposed Raw264.7 cells were intact compared to U266 or U937 cells. Treatment with SM significantly elevated the generation of reactive oxygen species (ROS). The anti-proliferative effect of SM was reversed by pretreatment with the ROS scavenger, N-acetyl-l-cysteine (NAC), compared to cells treated only with SM. Also, SM treatment increased the ER stress by elevation of phosphorylated activating transcription factor 4 (p-ATF4), phosphorylated eukaryotic Initiation Factor 2 (p-eIF2), and phosphorylated protein kinase RNA-like endoplasmic reticulum kinase (p-PERK) expression. Caspase-3 and Poly (ADP-ribose) polymerase (PARP) were cleaved and CCAAT-enhancer-binding protein homologous protein (CHOP) was activated by SM treatment. PARP cleavage and CHOP activation were attenuated by NAC pretreatment. Furthermore, SM increased the tumor suppressor, miR-216b, and suppressed its target, c-Jun. miR-216b inhibitor attenuated the apoptotic effect of SM. Taken together, SM treatment induced apoptosis through regulation of miR-216b and ROS/ER stress pathways. SM could be a potential drug for treatment of multiple myeloma and myeloid leukemia.

miR-211 Plays a Critical Role in Cnidium officinale Makino Extract-Induced, ROS/ER Stress-Mediated Apoptosis in U937 and U266 Cells.

Though Cnidium officinale Makino (COM) was known to have anti-angiogenic, anti-oxidant, neuroprotective, and anti-cancer effects, the underlying anticancer mechanism of COM using endoplasmic reticulum (ER) stress and miRNA remained unclear until now. Thus, in the current study, the inhibitory mechanism of COM in lymphoma and multiple myeloma (MM) cells was elucidated. COM exerted cytotoxicity in U937 and U266 but not Raw264.7 cells. COM treatment increased the expression of ER stress-related proteins such as p-protein kinase RNA-like endoplasmic reticulum kinase (p-PERK), p-eukaryotic initiation factor (p-eIF2α), and activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP). COM also cleaved poly (ADP-ribose) polymerase (PARP) in a dose-dependent manner in both cells. Also, reactive oxygen species (ROS) generation was elevated by COM treatment. Conversely, the apoptotic effect of COM treatment was blocked by N-acetyl-l-cysteine (NAC) pretreatment. Also, the pro-survival miRNA, miR-211 was decreased by COM treatment in U937 and U266 cells. miR-211 mimic attenuated COM-induced apoptosis. Taken together, these results support the scientific evidence that COM induces apoptosis via ROS generation/CHOP activation and miR-211 suppression in U937 and U266 cells.

Conformational Stability of the Hemagglutinin of H5N1 Influenza A Viruses Influences Susceptibility to Broadly Neutralizing Stem Antibodies.

Vaccines that elicit broadly neutralizing antibodies to the conserved stem of hemagglutinin (HA) are being developed as universal influenza vaccines that protect against influenza across multiple years. However, different influenza virus strains, even those in the same subtype with identical stem sequences, can vary in susceptibility to broadly neutralizing stem antibodies, and the reasons are not understood. Here we studied potential mechanisms underlying the differing sensitivities of a panel of H5N1 HA pseudoviruses to broadly neutralizing stem antibodies. We found that greater HA conformational stability, as measured by thermal inactivation and pH triggering of conformational changes, correlates with reduced neutralization sensitivity and antibody binding to HA under neutral- and low-pH conditions. Our data indicate that the conformational stability of HA is an important attribute of susceptibility to broadly neutralizing stem antibodies and is influenced by residues outside the stem antibody epitopes.IMPORTANCE The influenza virus surface glycoprotein hemagglutinin (HA) mediates virus attachment and membrane fusion between virus and host cells, allowing the viral core to enter the host cell cytoplasm for replication. Fusion occurs when HA undergoes low-pH-induced-conformational changes during endocytosis. Broadly neutralizing antibodies targeted to the conserved stem region of HA interfere with conformational changes required for fusion. Vaccines that elicit such antibodies are being developed as novel universal influenza vaccines for multiyear protection. We investigated why H5N1 HAs from different strains differ in their sensitivity to broadly neutralizing stem antibodies despite having conserved epitopes. We report that HA conformational stability due to residues outside the antibody binding site accounted for much of the variation in susceptibility to neutralization by stem antibodies. These findings highlight the importance of nonepitope residues in influencing neutralization sensitivity to stem antibodies and the complexities in developing universal vaccines targeting conserved epitopes in the HA stem.

Lee, J.E. et al. Ethanol Extract of Oldenlandia diffusa Herba Attenuates Scopolamine-Induced Cognitive Impairments in Mice via Activation of BDNF, P-CREB and Inhibition of Acetylcholinesterase.

The authors wish to make the following corrections to this paper [1].[...].

Ethanol Extract of Oldenlandia diffusa Herba Attenuates Scopolamine-Induced Cognitive Impairments in Mice via Activation of BDNF, P-CREB and Inhibition of Acetylcholinesterase.

Though Oldenlandia diffusa Herba (ODH) has been known to exhibit anti-cancer and anti-inflammatory effects, its anti-amnestic effect has never been reported so far. The aim of this present study was to elucidate the anti-amnestic effect of ODH. ODH pretreatment significantly reduced escape latency of scopolamine treated Institute of Cancer Research (ICR) mice compared to untreated control groups in a Morris water maze test. Similarly, the passive avoidance test showed that ODH treatment recovered the scopolamine induced amnesia in the ICR mouse model. Concentration of Ach in brains of ODH treated mice was increased compared to that of scopolamine treated mice. In addition, activity of acetylcholinesterase (AChE) was notably decreased by ODH. The protein expression of brain-derived neurotrophic factor (BDNF) and phospho-cAMP response element-binding protein (p-CREB) (Ser133) was increased in ODH pretreated group compared to control group. Consistently, immunohistochemistry (IHC) revealed the elevated expression of brain-derived neurotrophic factor (BDNF) and p-CREB in brains of ODH treated mice compared to the control group. Overall, these findings suggest that ODH has anti-amnestic potential via activation of BDNF and p-CREB and inhibition of AChE in mice with scopolamine induced amnesia.

Review of Natural Product-Derived Compounds as Potent Antiglioblastoma Drugs.

Common care for glioblastoma multiforme (GBM) is a surgical resection followed by radiotherapy and temozolomide- (TMZ-) based chemotherapy. Unfortunately, these therapies remain inadequate involving severe mortality and recurrence. Recently, new approaches discovering combinations of multiple inhibitors have been proposed along with the identification of key driver mutations that are specific to each patient. To date, this approach is still limited by the lack of effective therapy. Hopefully, novel compounds derived from natural products are suggested as potential solutions. Inhibitory effects of natural products on angiogenesis and metastasis and cancer suppressive effect of altering miRNA expression are provident discoveries. Angelica sinensis accelerates apoptosis by their key substances influencing factors of apoptosis pathways. Brazilin displays antitumor features by making influence on reactive oxygen species (ROS) intensity. Sargassum serratifolium, flavonoids, and so on have antimetastasis effect. Ficus carica controls miRNA that inhibits translation of certain secretory pathway proteins during the UPR. Serratia marcescens and patupilone (EPO 906) are physically assessed materials through clinical trials related to GBM progression. Consequently, our review puts emphasis on the potential of natural products in GBM treatment by regulating multiple malignant cancer-related pathway solving pending problem such as reducing toxicity and side effect.

Gold nanoparticles as a potent radiosensitizer in neutron therapy.

The purpose of this study was to investigate the potential of gold nanoparticles as radiosensitizer for use in neutron therapy against hepatocellular carcinoma. The hepatocellular carcinoma cells lines Huh7 and HepG2 were irradiated with γ and neutron radiation in the presence or absence of gold nanoparticles. Effects were evaluated by transmission electron microscopy, cell survival, cell cycle, DNA damage, migration, and invasiveness. Gold nanoparticles significantly enhanced the radiosensitivity of Huh7 and HepG2 cells to γ-rays by 1.41- and 1.16-fold, respectively, and by 1.80- and 1.35-fold to neutron radiation, which has high linear energy transfer. Accordingly, exposure to neutron radiation in the presence of gold nanoparticles induced cell cycle arrest, DNA damage, and cell death to a significantly higher extent, and suppressed cell migration and invasiveness more robustly. These effects are presumably due to the ability of gold nanoparticles to amplify the effective dose from neutron radiation more efficiently. The data suggest that gold nanoparticles may be clinically useful in combination therapy against hepatocellular carcinoma by enhancing the toxicity of radiation with high linear energy transfer.

Tumor treating fields inhibit glioblastoma cell migration, invasion and angiogenesis.

Treatment with alternating electric fields at an intermediate frequency (100-300 kHz), referred to as tumor treating fields (TTF) therapy, inhibits cancer cell proliferation. In the present study, we demonstrated that TTF application suppressed the metastatic potential of U87 and U373 glioblastoma cell lines via the NF-kB, MAPK and PI3K/AKT signaling pathways. Wound-healing and transwell assays showed that TTF suppressed cell migration and invasion compared with controls. Soft agar and three-dimensional culture assays showed that TTF inhibited both anchorage-dependent (cell proliferation) and anchorage-independent (colony formation) GBM cell growth. TTF dysregulated epithelial-to-mesenchymal transition-related genes, such as vimentin and E-cadherin, which partially accounted for TTF inhibition of cell migration and invasion. We further demonstrated that TTF application suppressed angiogenesis by downregulating VEGF, HIF1α and matrix metalloproteinases 2 and 9. TTF also inhibited NF-kB transcriptional activity. Collectively, our findings show that TTF represents a promising novel anti-invasion and anti-angiogenesis therapeutic strategy for use in GBM patients.

Biological effect of an alternating electric field on cell proliferation and synergistic antimitotic effect in combination with ionizing radiation.

Alternating electric fields at an intermediate frequency (100~300 kHz), referred to as tumour-treating fields (TTF), are believed to interrupt the process of mitosis via apoptosis and to act as an inhibitor of cell proliferation. Although the existence of an antimitotic effect of TTF is widely known, the proposed apoptotic mechanism of TTF on cell function and the efficacy of TTF are controversial issues among medical experts. To resolve these controversial issues, a better understanding of the underlying molecular mechanisms of TTF on cell function and the differences between the effects of TTF alone and in combination with other treatment techniques is essential. Here, we report experimental evidence of TTF-induced apoptosis and the synergistic antimitotic effect of TTF in combination with ionizing radiation (IR). For these experiments, two human Glioblastoma multiforme (GBM) cells (U373 and U87) were treated either with TTF alone or with TTF followed by ionizing radiation (IR). Cell apoptosis, DNA damage, and mitotic abnormalities were quantified after the application of TTF, and their percentages were markedly increased when TTF was combined with IR. Our experimental results also suggested that TTF combined with IR synergistically suppressed both cell migration and invasion, based on the inhibition of MMP-9 and vimentin.

Induced pluripotent stem cells for modeling of pediatric neurological disorders.

The pathophysiological mechanisms underlying childhood neurological disorders have remained obscure due to a lack of suitable disease models reflecting human pathogenesis. Using induced pluripotent stem cell (iPSC) technology, various neurological disorders can now be extensively modeled. Specifically, iPSC technology has aided the study and treatment of early-onset pediatric neurodegenerative diseases such as Rett syndrome, Down syndrome, Angelman syndrome. Prader-Willi syndrome, Friedreich's ataxia, spinal muscular atrophy (SMA), fragile X syndrome, X-linked adrenoleukodystrophy (ALD), and SCN1A gene-related epilepsies. In this paper, we provide an overview of various gene delivery systems for generating iPSCs, the current state of modeling early-onset neurological disorders and the ultimate application of these in vitro models in cell therapy through the correction of disease-specific mutations.

Emodin inhibits proinflammatory responses and inactivates histone deacetylase 1 in hypoxic rheumatoid synoviocytes.

Chronic inflammation of rheumatoid arthritis (RA) is promoted by proinflammatory cytokines and closely linked to angiogenesis. In the present study, we investigated the anti-inflammatory effects of emodin (1,3,8-trihydroxy-6-methyl-anthraquinone) isolated from the root of Rheum palmatum L. in interleukin 1 beta (IL-1ß) and lipopolysaccharide (LPS)-stimulated RA synoviocytes under hypoxia. Emodin significantly inhibited IL-1ß and LPS-stimulated proliferation of RA synoviocytes in a dose-dependent manner under hypoxic condition. Also, enzyme linked immunosorbent assay (ELISA) revealed that emodin significantly reduced the production of pro-inflammatory cytokines [tumor necrosis factor-alpha (TNF-α), IL-6 and IL-8], mediators [prostagladin E(2) (PGE(2)), matrix metalloproteinase (MMP)-1 and MMP-13] and vascular endothelial growth factor (VEGF) as an angiogenesis biomarker in IL-1ß and LPS-treated synoviocytes under hypoxia. Consistently, emodin attenuated the expression of cyclooxygenase 2 (COX-2), VEGF, hypoxia inducible factor 1 alpha (HIF-1α), MMP-1 and MMP-13 at mRNA level in IL-1ß and LPS-treated synoviocytes under hypoxia. Furthermore, emodin reduced histone deacetylase (HDAC) activity as well as suppressed the expression of HDAC1, but not HDAC2 in IL-1ß and LPS-treated synoviocytes under hypoxia. Overall, these findings suggest that emodin inhibits proinflammatory cytokines and VEGF productions, and HDAC1 activity in hypoxic RA synoviocytes.

The genome-wide expression profile of 1,2,3,4,6-penta-O-galloyl-ß-D-glucose-treated MDA-MB-231 breast cancer cells: molecular target on cancer metabolism.

1,2,3,4,6-penta-O-galloyl-beta-D-glucose (PGG), a polyphenolic compound isolated from Rhus chinensis Mill. PGG has been known to have anti-tumor, anti-angiogenic and anti-diabetic activities. The present study revealed another underlying molecular target of PGG in MDA-MB-231 breast cancer cells by using Illumina Human Ref-8 expression BeadChip assay. Through the Beadstudio v3 micro assay program to compare the identified genes expressed in PGG-treated MDA-MB-231 cells with untreated control, we found several unique genes that are closely associated with pyruvate metabolism, glycolysis/gluconeogenesis and tyrosine metabolism, including PC, ACSS2, ACACA, ACYP2, ALDH3B1, FBP1, PRMT2 and COMT. Consistent with microarray data, real-time RT-PCR confirmed the significant down-regulation of these genes at mRNA level in PGG-treated MDA-MB-231 cells. Our findings suggest the potential of PGG as anticancer agent for breast cancer cells by targeting cancer metabolism genes.

Inhibition of JAK1/STAT3 signaling mediates compound K-induced apoptosis in human multiple myeloma U266 cells.

Signal transducer and activator of transcription 3 (STAT3) is an oncogenic transcription factor implicated in carcinogenesis. Here, the role of STAT3 pathway in the antitumor activity of an active ginseng saponin metabolite compound K (CK) was investigated in human multiple myeloma U266 cells. CK increased the cytotoxicity, accumulated the sub-G1 DNA population, cleaved poly (ADP-ribose) polymerase (PARP) and activated caspase-3 in U266 cells. Interestingly, CK inhibited phosphorylation of STAT3 and its upstream activators, the Janus activated kinase 1 (JAK1), but not JAK2. Furthermore, CK enhanced the expression of protein tyrosine phosphatase (PTP) SHP-1, but not PTEN. Additionally, CK down-regulated STAT3 target genes bcl-x(L), bcl-2, survivin, cyclin E and cyclin D1. Conversely, PTP inhibitor pervanadate reversed CK-mediated STAT3 inactivation and cleavages of caspase-3 and PARP. Overall, our findings demonstrate that JAK1/STAT3 signaling mediates CK-induced apoptosis in U266 cells and also suggest the chemopreventive potential of CK for treatment of multiple myeloma.