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1.
Hum Exp Toxicol ; 41: 9603271221089257, 2022.
Article in English | MEDLINE | ID: covidwho-1789083

ABSTRACT

BACKGROUND: Remdesivir is an anti-viral drug that inhibits RNA polymerase. In 2020, remdesivir was recognized as the most promising therapeutic agents against coronavirus disease 2019 (COVID-19). However, the effects of remdesivir on cancers have hardly been studied. PURPOSE: Here, we reported that the anti-carcinogenic effect of remdesivir on SKOV3 cells, one of human ovarian cancer cell lines. RESEARCH DESIGN: We anlalyzed the anti-carcarcinogenic effect of remdesivir in SKOV3 cells by performing in vitro cell assay and western blotting. RESULTS: WST-1 showed that remdesivir decreased cell viability in SKOV3 cells. Experiments conducted by Muse Cell Analyzer showed that remdesivir-induced apoptosis in SKOV3 cells. We found that the expression level of FOXO3, Bax, and Bim increased, whereas Bcl-2, caspase-3, and caspase-7 decreased by remdesivir in SKOV3 cells. Furthermore, we observed that intracellular reactive oxygen species (ROS) level increased after treatment of remdesivir in SKOV3 cells. Interestingly, cytotoxicity of remdesivir decreased after treatment of N-Acetylcysteine. CONCLUSION: Taken together, our results demonstrated that remdesivir has an anti-carcinogenic effect on SKOV3 cells vis up-regulation of reactive oxygen species, which suggests that remdesivir could be a promising reagent for treatment of ovarian cancer.


Subject(s)
Anticarcinogenic Agents , COVID-19 , Ovarian Neoplasms , Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Anticarcinogenic Agents/pharmacology , Apoptosis , COVID-19/drug therapy , Cell Line, Tumor , Cell Proliferation , Female , Humans , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/genetics , Ovarian Neoplasms/metabolism , Reactive Oxygen Species/metabolism
2.
Int J Mol Sci ; 23(7)2022 Apr 02.
Article in English | MEDLINE | ID: covidwho-1785742

ABSTRACT

The angiotensin II (Ang II) type 1 receptor (AT1R) is involved in the regulation of blood pressure (through vasoconstriction) and water and ion homeostasis (mediated by interaction with the endogenous agonist). AT1R can also be activated by auto-antibodies (AT1R-Abs), which are associated with manifold diseases, such as obliterative vasculopathy, preeclampsia and systemic sclerosis. Knowledge of the molecular mechanisms related to AT1R-Abs binding and associated signaling cascade (dys-)regulation remains fragmentary. The goal of this study was, therefore, to investigate details of the effects of AT1R-Abs on G-protein signaling and subsequent cell proliferation, as well as the putative contribution of the three extracellular receptor loops (ELs) to Abs-AT1R signaling. AT1R-Abs induced nuclear factor of activated T-cells (NFAT) signaling, which reflects Gq/11 and Gi activation. The impact on cell proliferation was tested in different cell systems, as well as activation-triggered receptor internalization. Blockwise alanine substitutions were designed to potentially investigate the role of ELs in AT1R-Abs-mediated effects. First, we demonstrate that Ang II-mediated internalization of AT1R is impeded by binding of AT1R-Abs. Secondly, exclusive AT1R-Abs-induced Gq/11 activation is most significant for NFAT stimulation and mediates cell proliferation. Interestingly, our studies also reveal that ligand-independent, baseline AT1R activation of Gi signaling has, in turn, a negative effect on cell proliferation. Indeed, inhibition of Gi basal activity potentiates proliferation triggered by AT1R-Abs. Finally, although AT1R containing EL1 and EL3 blockwise alanine mutations were not expressed on the human embryonic kidney293T (HEK293T) cell surface, we at least confirmed that parts of EL2 are involved in interactions between AT1R and Abs. This current study thus provides extended insights into the molecular action of AT1R-Abs and associated mechanisms of interrelated pathogenesis.


Subject(s)
Antibodies , Receptor, Angiotensin, Type 1 , Alanine , Angiotensin II , Antibodies/pharmacology , Cell Proliferation , HEK293 Cells , Humans , Receptor, Angiotensin, Type 1/genetics , Receptor, Angiotensin, Type 1/metabolism
3.
Int J Mol Sci ; 23(7)2022 Apr 01.
Article in English | MEDLINE | ID: covidwho-1785739

ABSTRACT

Di-(2-ethylhexyl) phthalate (DEHP) is a frequently used plasticizer that may be linked to the development of endometriosis, a common gynecological disorder with a profound impact on quality of life. Despite its prevalence, vital access to treatment has often been hampered by a lack of understanding of its pathogenesis as well as reliable disease models. Recently, epithelial-mesenchymal transition (EMT) has been suggested to have a significant role in endometriosis pathophysiology. In this study, we found that DEHP treatment enhanced proliferation, migration, and inflammatory responses, along with EMT and stemness induction in human endometrial and endometriotic cells. The selective transforming growth factor-ß (TGF-ß) receptor type 1/2 inhibitor LY2109761 reversed the DEHP-induced cell proliferation and migration enhancement as well as the increased expression of crucial molecules involved in inflammation, EMT, and stemness, indicating that DEHP-triggered phenomena occur via the TGF-ß/Smad signaling pathway. Our study clearly defines the role of DEHP in the etiology and pathophysiological mechanisms of endometriosis and establishes an efficient disease model for endometriosis using a biomimetic 3D cell culture technique. Altogether, our data provide novel etiological and mechanistic insights into the role of DEHP in endometriosis pathogenesis, opening avenues for developing novel preventive and therapeutic strategies for endometriosis.


Subject(s)
Diethylhexyl Phthalate , Endometriosis , Cell Proliferation , Diethylhexyl Phthalate/metabolism , Diethylhexyl Phthalate/toxicity , Endometriosis/pathology , Epithelial Cells/metabolism , Epithelial-Mesenchymal Transition , Female , Humans , Phthalic Acids , Quality of Life , Signal Transduction , Transforming Growth Factor beta/metabolism , Transforming Growth Factor beta1/metabolism , Transforming Growth Factors/metabolism
4.
Molecules ; 27(3)2022 Jan 28.
Article in English | MEDLINE | ID: covidwho-1686898

ABSTRACT

Cancer is the second most fatal disease worldwide, with colon cancer being the third most prevalent and fatal form of cancer in several Western countries. The risk of acquisition of resistance to chemotherapy remains a significant hurdle in the management of various types of cancer, especially colon cancer. Therefore, it is essential to develop alternative treatment modalities. Naturally occurring alkaloids have been shown to regulate various mechanistic pathways linked to cell proliferation, cell cycle, and metastasis. This review aims to shed light on the potential of alkaloids as anti-colon-cancer chemotherapy agents that can modulate or arrest the cell cycle. Preclinical investigated alkaloids have shown anti-colon cancer activities and inhibition of cancer cell proliferation via cell cycle arrest at different stages, suggesting that alkaloids may have the potential to act as anticancer molecules.


Subject(s)
Alkaloids/chemistry , Alkaloids/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Cell Cycle Checkpoints/drug effects , Colonic Neoplasms/drug therapy , Alkaloids/therapeutic use , Animals , Antineoplastic Agents/therapeutic use , Apoptosis/drug effects , Cell Proliferation/drug effects , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Drug Discovery , Humans
5.
J Cell Mol Med ; 26(4): 1144-1155, 2022 02.
Article in English | MEDLINE | ID: covidwho-1685345

ABSTRACT

High glucose (HG) is one of the basic factors of diabetic nephropathy (DN), which leads to high morbidity and disability. During DN, the expression of glomerular glucose transporter 1 (GLUT1) increases, but the relationship between HG and GLUT1 is unclear. Glomerular mesangial cells (GMCs) have multiple roles in HG-induced DN. Here, we report prominent glomerular dysfunction, especially GMC abnormalities, in DN mice, which is closely related to GLUT1 alteration. In vivo studies have shown that BBR can alleviate pathological changes and abnormal renal function indicators of DN mice. In vitro, BBR (30, 60 and 90 µmol/L) not only increased the proportion of G1 phase cells but also reduced the proportion of S phase cells under HG conditions at different times. BBR (60 µmol/L) significantly reduced the expression of PI3K-p85, p-Akt, p-AS160, membrane-bound GLUT1 and cyclin D1, but had almost no effect on total protein. Furthermore, BBR significantly declined the glucose uptake and retarded cyclin D1-mediated GMC cell cycle arrest in the G1 phase. This study demonstrated that BBR can inhibit the development of DN, which may be due to BBR inhibiting the PI3K/Akt/AS160/GLUT1 signalling pathway to regulate HG-induced abnormal GMC proliferation and the cell cycle, supporting BBR as a potential therapeutic drug for DN.


Subject(s)
Berberine , Diabetes Mellitus , Diabetic Nephropathies , Animals , Berberine/pharmacology , Cell Cycle , Cell Division , Cell Proliferation , Diabetes Mellitus/pathology , Diabetic Nephropathies/pathology , Glucose/metabolism , Glucose Transporter Type 1/genetics , Glucose Transporter Type 1/metabolism , Mesangial Cells/metabolism , Mice , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism
6.
Life Sci ; 295: 120411, 2022 Apr 15.
Article in English | MEDLINE | ID: covidwho-1683412

ABSTRACT

AIMS: Virus-infected host cells switch their metabolism to a more glycolytic phenotype, required for new virion synthesis and packaging. Therefore, we investigated the effect and mechanistic action of glycolytic inhibitor 2-Deoxy-d-glucose (2-DG) on virus multiplication in host cells following SARS-CoV-2 infection. MAIN METHODS: SARS-CoV-2 induced change in glycolysis was examined in Vero E6 cells. Effect of 2-DG on virus multiplication was evaluated by RT-PCR (N and RdRp genes) analysis, protein expression analysis of Nucleocapsid (N) and Spike (S) proteins and visual indication of cytopathy effect (CPE), The mass spectrometry analysis was performed to examine the 2-DG induced change in glycosylation status of receptor binding domain (RBD) in SARS-CoV-2 spike protein. KEY FINDINGS: We observed SARS-COV-2 infection induced increased glucose influx and glycolysis, resulting in selectively high accumulation of the fluorescent glucose analog, 2-NBDG in Vero E6 cells. 2-DG inhibited glycolysis, reduced virus multiplication and alleviated cells from virus-induced cytopathic effect (CPE) in SARS-CoV-2 infected cells. The progeny virions produced from 2-DG treated cells were found unglycosylated at crucial N-glycosites (N331 and N343) of the receptor-binding domain (RBD) in the spike protein, resulting in production of defective progeny virions with compromised infective potential. SIGNIFICANCE: The mechanistic study revealed that the inhibition of SARS-COV-2 multiplication is attributed to 2-DG induced glycolysis inhibition and possibly un-glycosylation of the spike protein, also. Therefore, based on its previous human trials in different types of Cancer and Herpes patients, it could be a potential molecule to study in COVID-19 patients.


Subject(s)
COVID-19/drug therapy , Deoxyglucose/pharmacology , SARS-CoV-2/drug effects , SARS-CoV-2/pathogenicity , Adenosine Triphosphate/metabolism , Animals , Antiviral Agents/pharmacology , COVID-19/metabolism , COVID-19/virology , Cell Proliferation/drug effects , Cell Survival/drug effects , Chlorocebus aethiops , Glucose/metabolism , Glycolysis/drug effects , Glycosylation , Host-Pathogen Interactions/drug effects , Mannose/pharmacology , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells , Virion/drug effects , Virion/pathogenicity , Virus Replication/drug effects
7.
Front Immunol ; 12: 780900, 2021.
Article in English | MEDLINE | ID: covidwho-1662580

ABSTRACT

Mesenchymal stem cells (MSCs) are multipotent adult stem cells present in virtually all tissues; they have potent self-renewal capacity and differentiate into multiple cell types. For many reasons, these cells are a promising therapeutic alternative to treat patients with severe COVID-19 and pulmonary post-COVID sequelae. These cells are not only essential for tissue regeneration; they can also alter the pulmonary environment through the paracrine secretion of several mediators. They can control or promote inflammation, induce other stem cells differentiation, restrain the virus load, and much more. In this work, we performed single-cell RNA-seq data analysis of MSCs in bronchoalveolar lavage samples from control individuals and COVID-19 patients with mild and severe clinical conditions. When we compared samples from mild cases with control individuals, most genes transcriptionally upregulated in COVID-19 were involved in cell proliferation. However, a new set of genes with distinct biological functions was upregulated when we compared severely affected with mild COVID-19 patients. In this analysis, the cells upregulated genes related to cell dispersion/migration and induced the γ-activated sequence (GAS) genes, probably triggered by IFNGR1 and IFNGR2. Then, IRF-1 was upregulated, one of the GAS target genes, leading to the interferon-stimulated response (ISR) and the overexpression of many signature target genes. The MSCs also upregulated genes involved in the mesenchymal-epithelial transition, virus control, cell chemotaxis, and used the cytoplasmic RNA danger sensors RIG-1, MDA5, and PKR. In a non-comparative analysis, we observed that MSCs from severe cases do not express many NF-κB upstream receptors, such as Toll-like (TLRs) TLR-3, -7, and -8; tumor necrosis factor (TNFR1 or TNFR2), RANK, CD40, and IL-1R1. Indeed, many NF-κB inhibitors were upregulated, including PPP2CB, OPTN, NFKBIA, and FHL2, suggesting that MSCs do not play a role in the "cytokine storm" observed. Therefore, lung MSCs in COVID-19 sense immune danger and act protectively in concert with the pulmonary environment, confirming their therapeutic potential in cell-based therapy for COVID-19. The transcription of MSCs senescence markers is discussed.


Subject(s)
COVID-19/immunology , Cell Proliferation/physiology , Inflammation/immunology , Lung/immunology , Mesenchymal Stem Cells/immunology , Regeneration/immunology , Adult , COVID-19/metabolism , Cell Differentiation/immunology , Cell Movement/immunology , Cytoplasm/immunology , Epithelial-Mesenchymal Transition/immunology , Humans , Inflammation/metabolism , Mesenchymal Stem Cells/metabolism , SARS-CoV-2/immunology , Up-Regulation/immunology , Young Adult
8.
Med Oncol ; 39(3): 32, 2022 Jan 20.
Article in English | MEDLINE | ID: covidwho-1633699

ABSTRACT

To investigate the effects of isolated SARS-CoV-2 spike protein on prostate cancer cell survival. The effects of SARS-CoV-2 spike protein on LNCaP prostate cancer cell survival were assessed using clonogenic cell survival assay, quick cell proliferation assay, and caspase-3 activity kits. RT-PCR and immunohistochemistry were performed to investigate underlying molecular mechanisms. SARS-CoV-2 spike protein was found to inhibit prostate cancer cell proliferation as well as promote apoptosis. Further investigation revealed that anti-proliferative effects were associated with downregulation of the pro-proliferative molecule cyclin-dependent kinase 4 (CDK4). The increased rate of apoptosis was associated with the upregulation of pro-apoptotic molecule Fas ligand (FasL). SARS-CoV-2 spike protein inhibits the growth of LNCaP prostate cancer cells in vitro by a two-pronged approach of downregulating the expression of CDK4 and upregulating FasL. The introduction of SARS-CoV-2 spike protein into the body via COVID-19 vaccination may have the potential to inhibit prostate cancer in patients. This potential beneficial association between COVID-19 vaccines and prostate cancer inhibition will require more extensive studies before any conclusions can be drawn about any in vivo effects in a human model.


Subject(s)
COVID-19 Vaccines/immunology , Cell Proliferation/physiology , Prostatic Neoplasms/immunology , Prostatic Neoplasms/pathology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Antibodies, Viral/immunology , Apoptosis/immunology , COVID-19/immunology , Cell Line, Tumor , Cell Survival/immunology , Down-Regulation/immunology , Humans , Male , Up-Regulation/immunology , Vaccination/methods
9.
Int J Mol Sci ; 23(2)2022 Jan 13.
Article in English | MEDLINE | ID: covidwho-1637017

ABSTRACT

Malignant melanoma is still a serious medical problem. Relatively high mortality, a still-growing number of newly diagnosed cases, and insufficiently effective methods of therapy necessitate melanoma research. Tetracyclines are compounds with pleiotropic pharmacological properties. Previously published studies on melanotic melanoma cells ascertained that minocycline and doxycycline exerted an anti-melanoma effect. The purpose of the study was to assess the anti-melanoma potential and mechanisms of action of minocycline and doxycycline using A375 and C32 human amelanotic melanoma cell lines. The obtained results indicate that the tested drugs inhibited proliferation, decreased cell viability, and induced apoptosis in amelanotic melanoma cells. The treatment caused changes in the cell cycle profile and decreased the intracellular level of reduced thiols and mitochondrial membrane potential. The exposure of A375 and C32 cells to minocycline and doxycycline triggered the release of cytochrome c and activated initiator and effector caspases. The anti-melanoma effect of analyzed drugs appeared to be related to the up-regulation of ERK1/2 and MITF. Moreover, it was noticed that minocycline and doxycycline increased the level of LC3A/B, an autophagy marker, in A375 cells. In summary, the study showed the pleiotropic anti-cancer action of minocycline and doxycycline against amelanotic melanoma cells. Considering all results, it could be concluded that doxycycline was a more potent drug than minocycline.


Subject(s)
Antineoplastic Agents/pharmacology , Doxycycline/pharmacology , Minocycline/pharmacology , Apoptosis/drug effects , Autophagy/drug effects , Biomarkers, Tumor , Caspases/metabolism , Cell Cycle/drug effects , Cell Line, Tumor , Cell Proliferation , Cell Survival/drug effects , Dose-Response Relationship, Drug , Humans , Melanoma, Amelanotic , Membrane Potential, Mitochondrial/drug effects
10.
Biosci Rep ; 41(12)2021 12 22.
Article in English | MEDLINE | ID: covidwho-1592575

ABSTRACT

Parasporin-2Aa1 (PS2Aa1) is a toxic protein of 37 KDa (30 kDa, activated form produced by proteolysis) that was shown to be cytotoxic against specific human cancer cells, although its mechanism of action has not been elucidated yet. In order to study the role of some native peptide fragments of proteins on anticancer activity, here we investigated the cytotoxic effect of peptide fragments from domain-1 of PS2Aa1 and one of the loops present in the binding region of the virus spike protein from Alphacoronavirus (HCoV-229E), the latter according to scientific reports, who showed interaction with the human APN (h-APN) receptor, evidence corroborated through computational simulations, and thus being possible active against colon cancer cells. Peptides namely P264-G274, Loop1-PS2Aa, and Loop2-PS2Aa were synthesized using the Fmoc solid-phase synthesis and characterized by mass spectrometry (MS). Additionally, one region from loop 1 of HCoV-229E, Loop1-HCoV-229E, was also synthesized and characterized. The A4W-GGN5 anticancer peptide and 5-fluorouracil (5-FU) were taken as a control in all experiments. Circular dichroism revealed an α-helix structure for the peptides derived from PS2Aa1 (P264-G274, Loop1-PS2Aa, and Loop2-PS2Aa) and ß-laminar structure for the peptide derived from Alphacoronavirus spike protein Loop1-HCoV-229E. Peptides showed a hemolysis percentage of less than 20% at 100 µM concentration. Besides, peptides exhibited stronger anticancer activity against SW480 and SW620 cells after exposure for 48 h. Likewise, these compounds showed significantly lower toxicity against normal cells CHO-K1. The results suggest that native peptide fragments from Ps2Aa1 may be optimized as a novel potential cancer-therapeutic agents.


Subject(s)
Antineoplastic Agents/pharmacology , Colorectal Neoplasms/drug therapy , Endotoxins/pharmacology , Peptide Fragments/pharmacology , Spike Glycoprotein, Coronavirus/pharmacology , Alphacoronavirus , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/toxicity , CD13 Antigens/metabolism , CHO Cells , Cell Line, Tumor , Cell Proliferation/drug effects , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , Cricetulus , Endotoxins/toxicity , Hemolysis/drug effects , Humans , Molecular Docking Simulation , Peptide Fragments/chemical synthesis , Peptide Fragments/toxicity , Protein Conformation, alpha-Helical , Sheep, Domestic , Spike Glycoprotein, Coronavirus/toxicity , Structure-Activity Relationship
11.
J Exp Med ; 219(2)2022 02 07.
Article in English | MEDLINE | ID: covidwho-1594167

ABSTRACT

In rare instances, pediatric SARS-CoV-2 infection results in a novel immunodysregulation syndrome termed multisystem inflammatory syndrome in children (MIS-C). We compared MIS-C immunopathology with severe COVID-19 in adults. MIS-C does not result in pneumocyte damage but is associated with vascular endotheliitis and gastrointestinal epithelial injury. In MIS-C, the cytokine release syndrome is characterized by IFNγ and not type I interferon. Persistence of patrolling monocytes differentiates MIS-C from severe COVID-19, which is dominated by HLA-DRlo classical monocytes. IFNγ levels correlate with granzyme B production in CD16+ NK cells and TIM3 expression on CD38+/HLA-DR+ T cells. Single-cell TCR profiling reveals a skewed TCRß repertoire enriched for TRBV11-2 and a superantigenic signature in TIM3+/CD38+/HLA-DR+ T cells. Using NicheNet, we confirm IFNγ as a central cytokine in the communication between TIM3+/CD38+/HLA-DR+ T cells, CD16+ NK cells, and patrolling monocytes. Normalization of IFNγ, loss of TIM3, quiescence of CD16+ NK cells, and contraction of patrolling monocytes upon clinical resolution highlight their potential role in MIS-C immunopathogenesis.


Subject(s)
COVID-19/complications , Hepatitis A Virus Cellular Receptor 2/metabolism , Interferon-gamma/metabolism , Killer Cells, Natural/immunology , Monocytes/metabolism , Receptors, IgG/metabolism , Systemic Inflammatory Response Syndrome/immunology , T-Lymphocytes/immunology , Adolescent , Alveolar Epithelial Cells/pathology , B-Lymphocytes/immunology , Blood Vessels/pathology , COVID-19/immunology , COVID-19/pathology , Cell Proliferation , Child , Cohort Studies , Complement Activation , Cytokines/metabolism , Enterocytes/pathology , Female , Humans , Immunity, Humoral , Inflammation/pathology , Interferon Type I/metabolism , Interleukin-15/metabolism , Lymphocyte Activation/immunology , Male , Receptors, Antigen, T-Cell/metabolism , SARS-CoV-2/immunology , Superantigens/metabolism , Systemic Inflammatory Response Syndrome/pathology
13.
Chem Biol Interact ; 352: 109776, 2022 Jan 25.
Article in English | MEDLINE | ID: covidwho-1568541

ABSTRACT

Boosting or suppressing our immune system represents an attractive adjunct in the treatment of infections including SARS-CoV-2, cancer, AIDS, malnutrition, age related problems and some inflammatory disorders. Thus, there has been a growing interest in exploring and developing novel drugs, natural or synthetic, that can manipulate our defence mechanism. Many of such studies, reported till date, have been designed to explore effect of the therapeutic on function of macrophages, being a key component in innate immune system. Indeed, RAW264.7, J774A.1, THP-1 and U937 cell lines act as ideal model systems for preliminary investigation and selection of dose for in vivo studies. Several bioassays have been standardized so far where many techniques require high throughput instruments, cost effective reagents and technical assistance that may hinder many scholars to perform a method demanding compilation of available protocols. In this review, we have taken an attempt for the first time to congregate commonly used in vitro immune-modulating techniques explaining their principles. The study detected that among about 40 different assays and more than 150 sets of primers, the methods of cell proliferation by MTT, phagocytosis by neutral red, NO detection by Griess reaction and estimation of expression of TLRs, COX-2, iNOS, TNF-α, IL-6 and IL-1ß by PCR have been the most widely used to screen the therapeutics under investigation.


Subject(s)
Immunity, Innate/immunology , Immunomodulation/immunology , Animals , Cell Line , Cell Line, Tumor , Cell Proliferation/physiology , Humans , Inflammation/immunology , Phagocytosis/immunology
14.
Nature ; 602(7895): 148-155, 2022 02.
Article in English | MEDLINE | ID: covidwho-1556858

ABSTRACT

Immunological memory is a hallmark of adaptive immunity and facilitates an accelerated and enhanced immune response upon re-infection with the same pathogen1,2. Since the outbreak of the ongoing COVID-19 pandemic, a key question has focused on which SARS-CoV-2-specific T cells stimulated during acute infection give rise to long-lived memory T cells3. Here, using spectral flow cytometry combined with cellular indexing of transcriptomes and T cell receptor sequencing, we longitudinally characterized individual SARS-CoV-2-specific CD8+ T cells of patients with COVID-19 from acute infection to 1 year into recovery and found a distinct signature identifying long-lived memory CD8+ T cells. SARS-CoV-2-specific memory CD8+ T cells persisting 1 year after acute infection express CD45RA, IL-7 receptor-α and T cell factor 1, but they maintain low expression of CCR7, thus resembling CD45RA+ effector memory T cells. Tracking individual clones of SARS-CoV-2-specific CD8+ T cells, we reveal that an interferon signature marks clones that give rise to long-lived cells, whereas prolonged proliferation and mechanistic target of rapamycin signalling are associated with clonal disappearance from the blood. Collectively, we describe a transcriptional signature that marks long-lived, circulating human memory CD8+ T cells following an acute viral infection.


Subject(s)
Antigens, Viral/immunology , Biomarkers/metabolism , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , COVID-19/immunology , /metabolism , SARS-CoV-2/immunology , Acute Disease , COVID-19/virology , Cell Proliferation , Clone Cells/cytology , Clone Cells/immunology , Humans , Interferons/immunology , Interleukin-7 Receptor alpha Subunit/metabolism , Leukocyte Common Antigens/metabolism , Longitudinal Studies , Mechanistic Target of Rapamycin Complex 1/metabolism , Receptors, Antigen, T-Cell/metabolism , Receptors, CCR7/metabolism , T Cell Transcription Factor 1/metabolism , Time Factors , Transcriptome
15.
J Cell Mol Med ; 26(1): 235-238, 2022 01.
Article in English | MEDLINE | ID: covidwho-1555067

ABSTRACT

Due to the restrictions in accessing research laboratories and the challenges in providing proper storage and transportation of cells during the COVID-19 pandemic, having an effective and feasible mean to solve these challenges would be of immense help. Therefore, we developed a 3D culture setting of cancer cells using alginate beads and tested its effectiveness in different storage and transportation conditions. The viability and proliferation of cancer cells were assessed using trypan blue staining and quantitative CCK-8 kit, respectively. The developed beads allowed cancer cells survival up to 4 weeks with less frequent maintenance measures such as change of the culture media or subculture of cells. In addition, the recovery of cancer cells and proliferation pattern were significantly faster with better outcomes in the developed 3D alginate beads compared to the standard cryopreservation of cells or the 2D culture conditions. The 3D alginate beads also supported the viability of cells while the shipment at room temperature for a duration of up to 5 days with no humidity or CO2  support. Therefore, 3D culture in alginate beads can be used to store or ship biological cells with ease at room temperature with minimal preparations.


Subject(s)
Alginates/pharmacology , COVID-19/epidemiology , Cell Culture Techniques , Hydrogels/pharmacology , Osteoblasts/drug effects , A549 Cells , Alginates/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Hep G2 Cells , Humans , Hydrogels/chemistry , Osteoblasts/cytology , SARS-CoV-2/pathogenicity , Time Factors
16.
Viruses ; 13(12)2021 12 04.
Article in English | MEDLINE | ID: covidwho-1554971

ABSTRACT

Epidemic RNA viruses seem to arise year after year leading to countless infections and devastating disease. SARS-CoV-2 is the most recent of these viruses, but there will undoubtedly be more to come. While effective SARS-CoV-2 vaccines are being deployed, one approach that is still missing is effective antivirals that can be used at the onset of infections and therefore prevent pandemics. Here, we screened FDA-approved compounds against SARS-CoV-2. We found that atovaquone, a pyrimidine biosynthesis inhibitor, is able to reduce SARS-CoV-2 infection in human lung cells. In addition, we found that berberine chloride, a plant-based compound used in holistic medicine, was able to inhibit SARS-CoV-2 infection in cells through direct interaction with the virion. Taken together, these studies highlight potential avenues of antiviral development to block emerging viruses. Such proactive approaches, conducted well before the next pandemic, will be essential to have drugs ready for when the next emerging virus hits.


Subject(s)
Antiviral Agents/pharmacology , Atovaquone/pharmacology , Berberine/pharmacology , SARS-CoV-2/drug effects , Virus Replication/drug effects , Alveolar Epithelial Cells , Animals , Berberine/chemistry , Cell Proliferation/drug effects , Chlorides/chemistry , Chlorides/pharmacology , Chlorocebus aethiops , Drug Synergism , Humans , Proguanil/pharmacology , Vero Cells , Virion/drug effects
17.
Nucleic Acids Res ; 49(21): 12502-12516, 2021 12 02.
Article in English | MEDLINE | ID: covidwho-1546005

ABSTRACT

Circular RNAs (circRNAs) are noncoding RNAs that exist in all eukaryotes investigated and are derived from back-splicing of certain pre-mRNA exons. Here, we report the application of artificial circRNAs designed to act as antisense-RNAs. We systematically tested a series of antisense-circRNAs targeted to the SARS-CoV-2 genome RNA, in particular its structurally conserved 5'-untranslated region. Functional assays with both reporter transfections as well as with SARS-CoV-2 infections revealed that specific segments of the SARS-CoV-2 5'-untranslated region can be efficiently accessed by specific antisense-circRNAs, resulting in up to 90% reduction of virus proliferation in cell culture, and with a durability of at least 48 h. Presenting the antisense sequence within a circRNA clearly proved more efficient than in the corresponding linear configuration and is superior to modified antisense oligonucleotides. The activity of the antisense-circRNA is surprisingly robust towards point mutations in the target sequence. This strategy opens up novel applications for designer circRNAs and promising therapeutic strategies in molecular medicine.


Subject(s)
Genome, Viral/genetics , RNA, Antisense/genetics , RNA, Circular/genetics , RNA, Viral/genetics , SARS-CoV-2/genetics , Virus Replication/genetics , 5' Untranslated Regions/genetics , Animals , Antiviral Agents/metabolism , Base Sequence , COVID-19/prevention & control , COVID-19/virology , Cell Proliferation/genetics , Chlorocebus aethiops , Drug Design , HeLa Cells , Host-Pathogen Interactions/genetics , Humans , Nucleic Acid Conformation , RNA, Viral/chemistry , RNA-Seq/methods , SARS-CoV-2/physiology , Vero Cells
18.
J Cancer Res Clin Oncol ; 148(3): 599-608, 2022 Mar.
Article in English | MEDLINE | ID: covidwho-1527467

ABSTRACT

INTRODUCTION: Cathepsin L (CTSL) is a kind of the SARS-entry-associated CoV-2's proteases, which plays a key role in the virus's entry into the cell and subsequent infection. We investigated the association between the expression level of CTSL and overall survival in Glioblastoma multiforme (GBM) patients, to better understand the possible route and risks of new coronavirus infection for patients with GBM. METHODS: The expression level of CTSL in GBM was analyzed using TCGA and CGGA databases. The relationship between CTSL and immune infiltration levels was analyzed by means of the TIMER database. The impact of CTSL inhibitors on GBM biological activity was tested. RESULTS: The findings revealed that GBM tissues had higher CTSL expression levels than that of normal brain tissues, which was associated with a significantly lower survival rate in GBM patients. Meanwhile, the expression level of CTSL negatively correlated with purity, B cell and CD8+ T cell in GBM. CTSL inhibitor significantly reduced growth and induced mitochondrial apoptosis. CONCLUSION: According to the findings, CTSL acts as an independent prognostic factor and can be considered as promising therapeutic target for GBM.


Subject(s)
Biomarkers, Tumor/metabolism , COVID-19/pathology , Cathepsin L/metabolism , Dipeptides/pharmacology , Gene Expression Regulation, Enzymologic/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Glioblastoma/pathology , Apoptosis , COVID-19/drug therapy , COVID-19/enzymology , COVID-19/virology , Case-Control Studies , Cell Proliferation , Female , Glioblastoma/drug therapy , Glioblastoma/enzymology , Humans , Male , Middle Aged , Prognosis , SARS-CoV-2/physiology , Survival Rate , Tumor Cells, Cultured
19.
Biosci Rep ; 41(12)2021 12 22.
Article in English | MEDLINE | ID: covidwho-1526113

ABSTRACT

Parasporin-2Aa1 (PS2Aa1) is a toxic protein of 37 KDa (30 kDa, activated form produced by proteolysis) that was shown to be cytotoxic against specific human cancer cells, although its mechanism of action has not been elucidated yet. In order to study the role of some native peptide fragments of proteins on anticancer activity, here we investigated the cytotoxic effect of peptide fragments from domain-1 of PS2Aa1 and one of the loops present in the binding region of the virus spike protein from Alphacoronavirus (HCoV-229E), the latter according to scientific reports, who showed interaction with the human APN (h-APN) receptor, evidence corroborated through computational simulations, and thus being possible active against colon cancer cells. Peptides namely P264-G274, Loop1-PS2Aa, and Loop2-PS2Aa were synthesized using the Fmoc solid-phase synthesis and characterized by mass spectrometry (MS). Additionally, one region from loop 1 of HCoV-229E, Loop1-HCoV-229E, was also synthesized and characterized. The A4W-GGN5 anticancer peptide and 5-fluorouracil (5-FU) were taken as a control in all experiments. Circular dichroism revealed an α-helix structure for the peptides derived from PS2Aa1 (P264-G274, Loop1-PS2Aa, and Loop2-PS2Aa) and ß-laminar structure for the peptide derived from Alphacoronavirus spike protein Loop1-HCoV-229E. Peptides showed a hemolysis percentage of less than 20% at 100 µM concentration. Besides, peptides exhibited stronger anticancer activity against SW480 and SW620 cells after exposure for 48 h. Likewise, these compounds showed significantly lower toxicity against normal cells CHO-K1. The results suggest that native peptide fragments from Ps2Aa1 may be optimized as a novel potential cancer-therapeutic agents.


Subject(s)
Antineoplastic Agents/pharmacology , Colorectal Neoplasms/drug therapy , Endotoxins/pharmacology , Peptide Fragments/pharmacology , Spike Glycoprotein, Coronavirus/pharmacology , Alphacoronavirus , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/toxicity , CD13 Antigens/metabolism , CHO Cells , Cell Line, Tumor , Cell Proliferation/drug effects , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , Cricetulus , Endotoxins/toxicity , Hemolysis/drug effects , Humans , Molecular Docking Simulation , Peptide Fragments/chemical synthesis , Peptide Fragments/toxicity , Protein Conformation, alpha-Helical , Sheep, Domestic , Spike Glycoprotein, Coronavirus/toxicity , Structure-Activity Relationship
20.
Theranostics ; 11(14): 7005-7017, 2021.
Article in English | MEDLINE | ID: covidwho-1524524

ABSTRACT

The tumor suppressor protein p53 remains in a wild type but inactive form in ~50% of all human cancers. Thus, activating it becomes an attractive approach for targeted cancer therapies. In this regard, our lab has previously discovered a small molecule, Inauhzin (INZ), as a potent p53 activator with no genotoxicity. Method: To improve its efficacy and bioavailability, here we employed nanoparticle encapsulation, making INZ-C, an analog of INZ, to nanoparticle-encapsulated INZ-C (n-INZ-C). Results: This approach significantly improved p53 activation and inhibition of lung and colorectal cancer cell growth by n-INZ-C in vitro and in vivo while it displayed a minimal effect on normal human Wi38 and mouse MEF cells. The improved activity was further corroborated with the enhanced cellular uptake observed in cancer cells and minimal cellular uptake observed in normal cells. In vivo pharmacokinetic evaluation of these nanoparticles showed that the nanoparticle encapsulation prolongates the half-life of INZ-C from 2.5 h to 5 h in mice. Conclusions: These results demonstrate that we have established a nanoparticle system that could enhance the bioavailability and efficacy of INZ-C as a potential anti-cancer therapeutic.


Subject(s)
Antineoplastic Agents/pharmacology , Colorectal Neoplasms/drug therapy , Indoles/pharmacology , Lung Neoplasms/drug therapy , Nanoparticles/chemistry , Phenothiazines/pharmacology , Tumor Suppressor Protein p53/metabolism , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/therapeutic use , Biological Availability , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Humans , Indoles/chemistry , Indoles/pharmacokinetics , Indoles/therapeutic use , Mice , Mice, Inbred C57BL , Microscopy, Electron, Transmission , Nanoparticles/toxicity , Nanoparticles/ultrastructure , Phenothiazines/chemistry , Phenothiazines/pharmacokinetics , Phenothiazines/therapeutic use , Spectroscopy, Fourier Transform Infrared , Tumor Suppressor Protein p53/genetics , Xenograft Model Antitumor Assays
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