Exploring the Therapeutic Effects of Atractylodes macrocephala Koidz against Human Gastric Cancer.

Atractylodes macrocephala Koidz (AMK) is a traditional herbal medicine used for thousands of years in East Asia to improve a variety of illnesses and conditions, including cancers. This study explored the effect of AMK extract on apoptosis and tumor-grafted mice using AGS human gastric adenocarcinoma cells. We investigated the compounds, target genes, and associated diseases of AMK using the Traditional Chinese Medical Systems Pharmacy (TCMSP) database platform. Cell viability assay, cell cycle and mitochondrial depolarization analysis, caspase activity assay, reactive oxygen species (ROS) assay, and wound healing and spheroid formation assay were used to investigate the anti-cancer effects of AMK extract on AGS cells. Also, in vivo studies were conducted using subcutaneous xenografts. AMK extract reduced the viability of AGS cells and increased the sub-G1 cell fraction and the mitochondrial membrane potential. Also, AMK extract increased the production of ROS. AMK extract induced the increased caspase activities and modulated the mitogen-activated protein kinases (MAPK). In addition, AMK extract effectively inhibited AGS cell migration and led to a notable reduction in the growth of AGS spheroids. Moreover, AMK extract hindered the growth of AGS xenograft tumors in NSG mice. Our results suggest that AMK has anti-cancer effects by promoting cell cycle arrest and inhibiting the proliferation of AGS cancer cells and a xenograft model through apoptosis. This study could provide a novel approach to treat gastric cancer.

The lysosome as a novel therapeutic target of EGFR-mediated tumor inflammation.

EGFR-mediated tumors have been targeted to overcome several different malignant cancers. EGFR overexpression and mutations are directly related to the malignancy, which makes the therapy more complicated. One reason for the malignancy is the induction of AP1 followed by inflammation via IL-6 secretion. Current therapeutic strategies to overcome EGFR-mediated tumors are tyrosine kinase inhibitors (TKIs), anti-EGFR monoclonal antibodies, and the combination of these two agents with classic chemotherapy or immune checkpoint inhibitors (ICIs). Although the strategies are straightforward and have shown promising efficacy in several studies, there are still hurdles to overcoming the adverse effects and limited efficacy. This study reviews the current therapeutic strategies to target EGFR family members, how they work, and their effects and limitations. We also suggest developing novel strategies to target EGFR-mediated tumors in a novel approach. A lysosome is the main custodial staff to discard unwanted amounts of EGFR and other receptor tyrosine kinase molecules. Targeting this organelle may be a new approach to overcoming EGFR-mediated cancers.

High-Grade Endometrial Stromal Sarcoma: Molecular Alterations and Potential Immunotherapeutic Strategies.

Endometrial stromal tumor (EST) is an uncommon and unusual mesenchymal tumor of the uterus characterized by multicolored histopathological, immunohistochemical, and molecular features. The morphology of ESTs is similar to normal endometrial stromal cells during the proliferative phase of the menstrual cycle. ESTs were first classified into benign and malignant based on the number of mitotic cells. However, recently WHO has divided ESTs into four categories: endometrial stromal nodules (ESN), undifferentiated uterine sarcoma (UUS), low-grade endometrial stromal sarcoma (LG-ESS), and high-grade endometrial stromal sarcoma (HG-ESS). HG-ESS is the most malignant of these categories, with poor clinical outcomes compared to other types. With advances in molecular biology, ESTs have been further classified with morphological identification. ESTs, including HG-ESS, is a relatively rare type of cancer, and the therapeutics are not being developed compared to other cancers. However, considering the tumor microenvironment of usual stromal cancers, the advance of immunotherapy shows auspicious outcomes reported in many different stromal tumors and non-identified uterine cancers. These studies show the high possibility of successful immunotherapy in HG-ESS patients in the future. In this review, we are discussing the background of ESTs and the BCOR and the development of HG-ESS by mutations of BCOR or other related genes. Among the gene mutations of HG-ESSs, BCOR shows the most common mutations in different ways. In current tumor therapies, immunotherapy is one of the most effective therapeutic approaches. In order to connect immunotherapy with HG-ESS, the understanding of tumor microenvironment (TME) is required. The TME of HG-ESS shows the mixture of tumor cells, vessels, immune cells and non-malignant stromal cells. Macrophages, neutrophils, dendritic cells and natural killer cells lose their expected functions, but rather show pro-tumoral functions by the matricellular proteins, extracellular matrix and other complicated environment in TME. In order to overcome the current therapeutic limitations of HG-ESS, immunotherapies should be considered in addition to the current surgical strategies. Checkpoint inhibitors, cytokine-based immunotherapies, immune cell therapies are good candidates to be considered as they show promising results in other stromal cancers and uterine cancers, while less studied because of the rarity of ESTs. Based on the advance of knowledge of immune therapies in HG-ESS, the new strategies can also be applied to the current therapies and also in other ESTs.

Targeting lncRNAs of colorectal cancers with natural products.

Non-coding RNA (ncRNA) is one of the functional classes of RNA that has a regulatory role in various cellular processes, such as modulation of disease onset, progression, and prognosis. ncRNAs, such as microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs), have been actively studied in recent years. The change in ncRNA levels is being actively studied in numerous human diseases, especially auto-immune disorders and cancers; however, targeting and regulating ncRNA with natural products to cure cancer has not been fully established. Recently many groups reported the relationship between ncRNA and natural products showing promising effects to serve as additional therapeutic approaches to cure cancers. This mini-review summarizes the aspects of lncRNAs related to cancer biology focusing on colorectal cancers that natural products can target.

Anticancer Effects of Propionic Acid Inducing Cell Death in Cervical Cancer Cells.

Recent studies found that short-chain fatty acids (SCFAs), which are produced through bacterial fermentation in the gastrointestinal tract, have oncoprotective effects against cervical cancer. The most common SCFAs that are well known include acetic acid, butyric acid, and propionic acid, among which propionic acid (PA) has been reported to induce apoptosis in HeLa cells. However, the mechanism in which SCFAs suppress HeLa cell viability remain poorly understood. Our study aims to provide a more detailed look into the mechanism of PA in HeLa cells. Flow cytometry analysis revealed that PA induces reactive oxygen species (ROS), leading to the dysfunction of the mitochondrial membrane. Moreover, PA inhibits NF-κB and AKT/mTOR signaling pathways and induces LC3B protein levels, resulting in autophagy. PA also increased the sub-G1 cell population that is characteristic of cell death. Therefore, the results of this study propose that PA inhibits HeLa cell viability through a mechanism mediated by the induction of autophagy. The study also suggests a new approach for cervical cancer therapeutics.

Structure of SARS-CoV-2 Spike Glycoprotein for Therapeutic and Preventive Target.

The global crisis caused by the coronavirus disease 2019 (COVID-19) led to the most significant economic loss and human deaths after World War II. The pathogen causing this disease is a novel virus called the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). As of December 2020, there have been 80.2 million confirmed patients, and the mortality rate is known as 2.16% globally. A strategy to protect a host from SARS-CoV-2 is by suppressing intracellular viral replication or preventing viral entry. We focused on the spike glycoprotein that is responsible for the entry of SARS-CoV-2 into the host cell. Recently, the US Food and Drug Administration/EU Medicines Agency authorized a vaccine and antibody to treat COVID-19 patients by emergency use approval in the absence of long-term clinical trials. Both commercial and academic efforts to develop preventive and therapeutic agents continue all over the world. In this review, we present a perspective on current reports about the spike glycoprotein of SARS-CoV-2 as a therapeutic target.

Anticancer Activity of Lesbicoumestan in Jurkat Cells via Inhibition of Oxidative Stress-Mediated Apoptosis and MALT1 Protease.

This study explores the potential anticancer effects of lesbicoumestan from Lespedeza bicolor against human leukemia cancer cells. Flow cytometry and fluorescence microscopy were used to investigate antiproliferative effects. The degradation of mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) was evaluated using immunoprecipitation, Western blotting, and confocal microscopy. Apoptosis was investigated using three-dimensional (3D) Jurkat cell resistance models. Lesbicoumestan induced potent mitochondrial depolarization on the Jurkat cells via upregulated expression levels of mitochondrial reactive oxygen species. Furthermore, the underlying apoptotic mechanisms of lesbicoumestan through the MALT1/NF-κB pathway were comprehensively elucidated. The analysis showed that lesbicoumestan significantly induced MALT1 degradation, which led to the inhibition of the NF-κB pathway. In addition, molecular docking results illustrate how lesbicoumestan could effectively bind with MALT1 protease at the latter's active pocket. Similar to traditional 2D cultures, apoptosis was markedly induced upon lesbicoumestan treatment in 3D Jurkat cell resistance models. Our data support the hypothesis that lesbicoumestan is a novel inhibitor of MALT1, as it exhibited potent antiapoptotic effects in Jurkat cells.

The Progression of SARS Coronavirus 2 (SARS-CoV2): Mutation in the Receptor Binding Domain of Spike Gene.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) is a positive-sense single-stranded RNA (+ssRNA) that causes coronavirus disease 2019 (COVID-19). The viral genome encodes twelve genes for viral replication and infection. The third open reading frame is the spike (S) gene that encodes for the spike glycoprotein interacting with specific cell surface receptor - angiotensin converting enzyme 2 (ACE2) - on the host cell membrane. Most recent studies identified a single point mutation in S gene. A single point mutation in S gene leading to an amino acid substitution at codon 614 from an aspartic acid 614 into glycine (D614G) resulted in greater infectivity compared to the wild type SARS-CoV2. We were interested in investigating the mutation region of S gene of SARS-CoV2 from Korean COVID-19 patients. New mutation sites were found in the critical receptor binding domain (RBD) of S gene, which is adjacent to the aforementioned D614G mutation residue. This specific sequence data demonstrated the active progression of SARS-CoV2 by mutations in the RBD of S gene. The sequence information of new mutations is critical to the development of recombinant SARS-CoV2 spike antigens, which may be required to improve and advance the strategy against a wide range of possible SARS-CoV2 mutations.

Vav1 is Essential for HIF-1α Activation via a Lysosomal VEGFR1-Mediated Degradation Mechanism in Endothelial Cells.

The vascular response to hypoxia and ischemia is essential for maintaining homeostasis during stressful conditions and is particularly critical for vital organs such as the heart. Hypoxia-inducible factor-1 (HIF-1) is a central regulator of the response to hypoxia by activating transcription of numerous target genes, including vascular endothelial growth factor (VEGF). Here we identify the guanine nucleotide exchange factor (GEF) Vav1, a regulator of the small Rho-GTPase and cell signaling in endothelial cells, as a key vascular regulator of hypoxia. We show that Vav1 is present in the vascular endothelium and is essential for HIF-1 activation under hypoxia. So, we hypothesized that Vav1 could be a key regulator of HIF-1 signaling. In our findings, Vav1 regulates HIF-1α stabilization through the p38/Siah2/PHD3 pathway. In normoxia, Vav1 binds to vascular endothelial growth factor receptor 1 (VEGFR1), which directs Vav1 to lysosomes for degradation. In contrast, hypoxia upregulates Vav1 protein levels by inhibiting lysosomal degradation, which is analogous to HIF-1α regulation by hypoxia: both proteins are constitutively produced and degraded in normoxia allowing for a rapid response when stress occurs. Consequently, hypoxia rapidly stabilizes Vav1, which is required for HIF-1α accumulation. This shows that Vav1 is the key mediator controlling the stabilization of HIF1α in hypoxic conditions. With this finding, we report a novel pathway to stabilize HIF-1, which shows a possible reason why clinical trials targeting HIF-1 has not been effective. Targeting Vav1 can be the new approach to overcome hypoxic tumors.

The Rho/Rac Guanine Nucleotide Exchange Factor Vav1 Regulates Hif-1α and Glut-1 Expression and Glucose Uptake in the Brain.

Vav1 is a Rho/Rac (Ras-related C3 botulinum toxin substrate) guanine nucleotide exchange factor expressed in hematopoietic and endothelial cells that are involved in a wide range of cellular functions. It is also stabilized under hypoxic conditions when it regulates the accumulation of the transcription factor HIF (Hypoxia Inducible Factor)-1α, which activates the transcription of target genes to orchestrate a cellular response to low oxygen. One of the genes induced by HIF-1α is GLUT (Glucose Transporter)-1, which is the major glucose transporter expressed in vessels that supply energy to the brain. Here, we identify a role for Vav1 in providing glucose to the brain. We found that Vav1 deficiency downregulates HIF-1α and GLUT-1 levels in endothelial cells, including blood-brain barrier cells. This downregulation of GLUT-1, in turn, reduced glucose uptake to endothelial cells both in vitro and in vivo, and reduced glucose levels in the brain. Furthermore, endothelial cell-specific Vav1 knock-out in mice, which caused glucose uptake deficiency, also led to a learning delay in fear conditioning experiments. Our results suggest that Vav1 promotes learning by activating HIF-1α and GLUT-1 and thereby distributing glucose to the brain. We further demonstrate the importance of glucose transport by endothelial cells in brain functioning and reveal a potential new axis for targeting GLUT-1 deficiency syndromes and other related brain diseases.

Oxygen Tension Regulates Lysosomal Activation and Receptor Tyrosine Kinase Degradation.

Oxygen sensing is crucial for adaptation to variable habitats and physiological conditions. Low oxygen tension, or hypoxia, is a common feature of solid tumors, and hypoxic tumors are often more aggressive and resistant to therapy. Here we show that, in cultured mammalian cells, hypoxia suppressed lysosomal acidification/activation and receptor tyrosine kinase (RTK) degradation. Hypoxia down-regulated mTORc1, reducing its ability to activate transcription factor EB (TFEB), a master regulator of V-ATPase, the lysosomal proton pump. Hypoxia prevented epidermal growth factor receptor (EGFR) degradation in tumor tissues, whereas activation of lysosomes enhanced tumor cell response to anti-EGFR treatment. Our results link oxygen tension and lysosomal activity, provide a molecular explanation of the malignant phenotype associated with hypoxic tumors, and suggest activation of lysosomes may provide therapeutic benefit in RTK-targeted cancer therapy.

Neutrophilic Granule Protein Is a Novel Murine LPS Antagonist.

Neutrophilic granule protein (NGP) was previously reported as a granular protein of neutrophils in mouse, but the function has not been known clearly. We found the presence of the possible signal peptide in NGP and validated this protein is circulating in the bloodstream. In our findings, NGP is being modified post-translationally in Golgi apparatus and endoplasmic reticulum, which is a universal character of secretory molecules with a signal peptide. The secreted NGP protein could be detected both in vitro and in vivo. NGP has sequence similarity with an antimicrobial protein cathelicidin, and we observed the aspect of inflammation of NGP. Interestingly, NGP interacts with the complex of LPS and LPS binding protein (LBP). This interaction blocks the binding of the complex of LPS and LBP to TLR4 and the downstream inflammatory signals. Furthermore, the inhibitory function of NGP against the inflammatory effect of LPS could be observed in both in vitro and in vivo. With these findings, we report NGP is a novel secretory protein to mask LPS and inhibit its function.

Interleukin-33 and ST2 Signaling in Tumor Microenvironment.

Interleukin-33 (IL-33) is one of the members of the IL-1 family of cytokines and a ligand of ST2 and IL-1 receptor accessory protein (IL-1RAcP) that is known to affect Th2 inflammatory response with partial effects on Th1 responses. This cytokine is released by epithelial and smooth muscle cells of the airway system during their injury by several environmental stimuli, such as allergens, viruses, helminths, and pollutants. IL-33 is an alarmin that acts as an endogenous danger signal, and it has been known to affect various types of cells, such as mast cells, basophils, eosinophils, T cells, and specific subsets of innate lymphoid cells (ILCs). In recent findings, this cytokine is believed to have a critical role in several types of cancers, such as lung cancer, liver cancer, and head and neck squamous cell cancer. The expression of IL-33/ST2 in cancer tissues shows a close association with tumor growth and tumor progression in several types of cancer, suggesting the IL-33/ST2 pathway as a potential target for therapy.

Proinsulin Shares a Motif with Interleukin-1α (IL-1α) and Induces Inflammatory Cytokine via Interleukin-1 Receptor 1.

Although it has been established that diabetes increases susceptibility to infections, the role of insulin (INS) in the immune response is unknown. Here, we investigated the immunological function of INS. Proinsulin dimer (pINSd) was a potent immune stimulus that induced inflammatory cytokines, but mature INS was unable to induce an immune response. An affinity-purified rabbit polyclonal antibody raised against mature IL-1α recognized IL-1α and pINS but failed to detect mature INS and IL-1ß. Analysis of the pINS sequence revealed the existence of an INS/IL-1α motif in the C-peptide of pINS. Surprisingly, the INS/IL-1α motif was recognized by monoclonal antibody raised against IL-1α. Deleting the INS/IL-1α motif in pINSd and IL-1α changed their activities. To investigate the pINSd receptor, the reconstitution of IL-1 receptor 1 (IL-1R1) in Wish cells restored pINSd activity that was reversed by an IL-1R antagonist. These data suggested that pINSd needs IL-1R1 for inflammatory cytokine induction. Mouse embryo fibroblast cells of IL-1R1-deficient mice further confirmed that pINSd promotes immune responses through IL-1R1.

Inhibition of Nitric Oxide Production in BV2 Microglial Cells by Triterpenes from Tetrapanax papyriferus.

It is well known that activated microglia produce nitric oxide (NO), which has an important role in the pathophysiology of several neurodegenerative diseases such as Alzheimer's disease. In the course of searching for novel therapeutic agents from medicinal plants against neuroinflammatory diseases, the methanolic extract of Tetrapanax papyriferus was found to have significant NO inhibitory activity in lipopolysaccharide (LPS)-stimulated BV2 microglia cells. Nine oleanane-type triterpenes, including two new compounds, epipapyriogenin C-3-O-ß-d-glucopyranoside (6) and 11-O-butylpapyrioside LIIc (9), were isolated from the leaves and stems of Tetrapanax papyriferus. The structures of these compounds were elucidated with 1D- and 2D-NMR and MS data. Among these Δ(11,13) oleanane-type triterpenes, compound 3 showed significant NO inhibitory activity in BV-2 cells, reducing the LPS-induced expression of COX-2 and pro-inflammatory cytokines such as TNF-α and IL-6. Compounds 7 and 9 also showed NO inhibitory activities among the Δ(12) oleanane-type triterpene saponins. These results show that oleanane-type triterpenes isolated from T. papyriferus could be a potential natural resource of NO inhibitors used in the treatment of neurodegenerative disorders.

Reconstitution of ST2 (IL-1R4) specific for IL-33 activity; no suppression by IL-1Ra though a common chain IL-1R3 (IL-1RAcP) shared with IL-1.

Interleukin-33 (IL-33) receptors are composed of ST2 (also known as IL-1R4), a ligand binding chain, and IL-1 receptor accessory protein (IL-1RAcP, also known as IL-1R3), a signal transducing chain. IL-1R3 is a common receptor for IL-1α, and IL-1ß, IL-33, and three IL-36 isoforms. A549 human lung epithelial cells are highly sensitive to IL-1α and IL-1ß but not respond to IL-33. The lack of responsiveness to IL-33 is due to ST2 expression. ST2 was stably transfected into A549 cells to reconstitute its activity. RT-PCR and FACS analysis confirmed ST2 expression on the cell surface of A549/ST2 cells. Upon IL-33 stimulation, A549/ST2 cells induced IL-8 and IL-6 production in a dose dependent manner while A549/mock cells remained unresponsive. There was no difference in IL-1α and IL-1ß activity in A549/ST2 cells compared to A549/mock cells despite the fact that IL-33 shares IL-1R3 with IL-1α/ß. IL-33 activated inflammatory signaling molecules in a time- and dose-dependent manner. Anti-ST2 antibody and soluble recombinant ST2-Fc abolished IL-33-induced IL-6 and IL-8 production in A549/ST2 cells but the IL-1 receptor antagonist failed to block IL-33-induced cytokines. This result demonstrates for the first time the reconstitution of ST2 in A549 human lung epithelial cell line and verified its function in IL-33-mediated cytokine production and signal transduction.

Myocardial pressure overload induces systemic inflammation through endothelial cell IL-33.

Hypertension increases the pressure load on the heart and is associated with a poorly understood chronic systemic inflammatory state. Interleukin 33 (IL-33) binds to membrane-bound ST2 (ST2L) and has antihypertrophic and antifibrotic effects in the myocardium. In contrast, soluble ST2 appears to act as a decoy receptor for IL-33, blocking myocardial and vascular benefits, and is a prognostic biomarker in patients with cardiovascular diseases. Here we report that a highly local intramyocardial IL-33/ST2 conversation regulates the heart's response to pressure overload. Either endothelial-specific deletion of IL33 or cardiomyocyte-specific deletion of ST2 exacerbated cardiac hypertrophy with pressure overload. Furthermore, pressure overload induced systemic circulating IL-33 as well as systemic circulating IL-13 and TGF-beta1; this was abolished by endothelial-specific deletion of IL33 but not by cardiomyocyte-specific deletion of IL33. Our study reveals that endothelial cell secretion of IL-33 is crucial for translating myocardial pressure overload into a selective systemic inflammatory response.

Extracellular forms of IL-37 inhibit innate inflammation in vitro and in vivo but require the IL-1 family decoy receptor IL-1R8.

Similar to IL-1α and IL-33, IL-1 family member IL-37b translocates to the nucleus and is associated with suppression of innate and adaptive immunity. Here we demonstrate an extracellular function of the IL-37 precursor and a processed form. Recombinant IL-37 precursor reduced LPS-induced IL-6 by 50% (P < 0.001) in highly inflammatory human blood-derived M1 differentiated macrophages derived from selective subjects but not M2 macrophages. In contrast, a neutralizing monoclonal anti-IL-37 increased LPS-induced IL-6, TNFα and IL-1ß (P < 0.01). The suppression by IL-37 was consistently observed at low picomolar but not nanomolar concentrations. Whereas LPS induced a 12-fold increase in TNFα mRNA, IL-37 pretreatment decreased the expression to only 3-fold over background (P < 0.01). Mechanistically, LPS-induced p38 and pERK were reduced by IL-37. Recombinant IL-37 bound to the immobilized ligand binding α-chain of the IL-18 receptor as well as to the decoy receptor IL-1R8. In M1 macrophages, LPS increased the surface expression of IL-1R8. Compared with human blood monocytes, resting M1 cells express more surface IL-1R8 as well as total IL-1R8; there was a 16-fold increase in IL-1R8 mRNA levels when pretreated with IL-37. IL-37 reduced LPS-induced TNFα and IL-6 by 50-55% in mouse bone marrow-derived dendritic cells, but not in dendritic cells derived from IL-1R8-deficient mice. In mice subjected to systemic LPS-induced inflammation, pretreatment with IL-37 reduced circulating and organ cytokine levels. Thus, in addition to a nuclear function, IL-37 acts as an extracellular cytokine by binding to the IL-18 receptor but using the IL-1R8 for its anti-inflammatory properties.

IL-32γ overexpression accelerates streptozotocin (STZ)-induced type 1 diabetes.

Interleukin-32 (IL-32) is a cytokine produced by T lymphocytes, natural killer (NK) cells, monocytes and epithelial cells. There are five splicing variants (α, ß, γ, δ, and ε) and IL-32γ is the most active isoform. We generated human IL-32γ transgenic (IL-32γ TG) mice, displaying a high level of IL-32γ expression in the pancreas. We investigated the effect of IL-32γ on streptozotocin (STZ)-induced type 1 diabetes model using IL-32γ TG mice. After a suboptimal diabetogenic dose of STZ administration, IL-32γ TG mice showed significantly increased blood glucose level comparing with that of wild type (WT) mice at day 5. Inflammatory cytokines levels such as, IL-6, TNFα, IFNγ and IL-1ß, in pancreas and liver lysates were accessed by a specific cytokine ELISA. The proinflammatory cytokines were significantly enhanced in the pancreas of IL-32γ TG mice comparing to that of WT mice whereas those cytokines levels in liver of IL-32γ TG and WT mice were not changed by STZ. These data indicate that the overexpression of IL-32γ contributes to initial islet ß-cells injury and inflammation in pancreas and aggravates STZ-induced type 1 diabetes.