detection-anti-mda5-autoantibodies-using-hela-cells

Detection of Anti-MDA5 Autoantibodies Using HeLa Cells and Immunocytochemistry with Light Microscopy

Anti-MDA5 autoantibodies are critical biomarkers for dermatomyositis (DM), amyopathic dermatomyositis (CADM), and polymyositis (PM), particularly in ...

China Medical University

China Medical University Hospital

In the past decade, no significant improvement has been made in chemotherapy for osteosarcoma (OS). To develop improved agents against OS, we screened 70 species of medicinal plants and treated two human OS cell lines with different agent concentrations. We then examined cell viability using the MTT assay. Results showed that a candidate plant, particularly the rhizomes of Anemone altaica Fisch. ex C. A. Mey aqueous extract (AAE), suppressed the viability of HOS and U2OS cells in a concentration-dependent manner. Flow cytometry analysis revealed that AAE significantly increased the amount of cell shrinkage (Sub-G1 fragments) in HOS and U2OS cells. Moreover, AAE increased cytosolic cytochrome c and Bax, but decreased Bcl-2. The amount of cleaved caspase-3 and poly-(ADP-ribose) polymerase-1 (PARP-1) were significantly increased. AAE suppressed the growth of HOS and U2OS through the intrinsic apoptotic pathway. Data suggest that AAE is cytotoxic to HOS and U2OS cells and has no significant influence on human osteoblast hFOB cells. The high mRNA levels of apoptosis-related factors (PPP1R15A, SQSTM1, HSPA1B, and DDIT4) and cellular proliferation markers (SKA2 and BUB1B) were significantly altered by the AAE treatment of HOS and U2OS cells. Results show that the anticancer activity of AAE could up-regulate the expression of a cluster of genes, especially those in the apoptosis-related factor family and caspase family. Thus, AAE has great potential as a useful therapeutic drug for human OS.

Anemone altaica Induces Apoptosis in Human Osteosarcoma Cells.

The tyrosine kinase Src is involved in the progression of many cancers. Moreover, inhibiting Src activity has been shown to obstruct several signaling pathways regulated by the EGFR. Thus, Src is a valuable target molecule in drug development. The purpose of this study was to identify compounds that directly or indirectly modulate Src to suppress lung cancer cell growth and motility and to investigate the molecular mechanisms underlying the effects of these compounds.

AC-93253 iodide, a novel Src inhibitor, suppresses NSCLC progression by modulating multiple Src-related signaling pathways.

The tumorous niche may drive the plasticity of heterogeneity and cancer stemness, leading to drug resistance and metastasis, which is the main reason of treatment failure in most cancer patients. The aim of this study was to establish a tumor microenvironment (TME)-based screening to identify drugs that can specifically target cancer stem cells (CSCs) and cancer-associated fibroblasts (CAFs) in the TME.  Lung cancer patient-derived cancer cell and CAFs were utilized to mimic the TME and reproduce the stemness properties of CSCs  and develop a high-throughput drug screening platform with phenotypical parameters. Limiting dilution assay, sphere-forming and ALDH activity assay were utilized to measure the cancer stemness characteristics.  patient-derived xenograft (PDX) models and single-cell RNA sequencing were used to evaluate the mechanisms of the compounds in CSCs and CAFs.  The TME-based drug screening platform could comprehensively evaluate the response of cancer cells, CSCs and CAFs to different treatments. Among the 1,524 compounds tested, several drugs were identified to have anti-CAFs, anticancer and anti-CSCs activities. Aloe-emodin and digoxin both show anticancer and anti-CSCs activity  and , which was further confirmed in the lung cancer PDX model. The combination of digoxin and chemotherapy improved therapeutic efficacy. The single-cell transcriptomics analysis revealed that digoxin could suppress the CSCs subpopulation in CAFs-cocultured cancer cells and cytokine production in CAFs.  The TME-based drug screening platform provides a tool to identify and repurpose compounds targeting cancer cells, CSCs and CAFs, which may accelerate drug development and therapeutic application for lung cancer patients.

Tumor microenvironment-based screening repurposes drugs targeting cancer stem cells and cancer-associated fibroblasts.

Although the heterogeneity of high-density lipoprotein-cholesterol (HDL-c) composition is associated with atherosclerotic cardiovascular risk, the link between electronegative subfractions of HDL-c and atherosclerosis in rheumatoid arthritis (RA) remains unknown. We examined the association of the percentage of the most electronegative subfraction of HDL-c (H5%) and RA-related atherosclerosis. Using anion-exchange purification/fast-protein liquid chromatography, we demonstrated significantly higher H5% in patients (median, 7.2%) than HC (2.8%,  < 0.005). Multivariable regression analysis revealed H5% as a significant predictor for subclinical atherosclerosis. We subsequently explored atherogenic role of H5 using cell-based assay. The results showed significantly higher levels of IL-1β and IL-8 mRNA in H5-treated (mean ± SD, 4.45 ± 1.22 folds, 6.02 ± 1.43-folds, respectively) than H1-treated monocytes (0.89 ± 0.18-folds, 1.03 ± 0.26-folds, respectively, both  < 0.001). In macrophages, H5 upregulated the mRNA and protein expression of IL-1β and IL-8 in a dose-dependent manner, and their expression levels were significantly higher than H1-treated macrophages (all  < 0.001). H5 induced more foam cell formation compared with H1-treated macrophages ( < 0.005). In addition, H5 has significantly lower cholesterol efflux capacity than H1 ( < 0.005). The results of nanoLC-MS/MS approach reveal that the best discriminator between high-H5% and normal-H5% is Apo(a), the main constituent of Lp(a). Moreover, Lp(a) level is a significant predictor for high-H5%. These observations suggest that H5 is involved in RA-related atherosclerosis.

The Potential Role of Electronegative High-Density Lipoprotein H5 Subfraction in RA-Related Atherosclerosis.

Dysregulated epidermal growth factor receptor (EGFR) expression is frequently observed in non-small cell lung cancer (NSCLC) growth and metastasis. Despite recent successes in the development of tyrosine kinase inhibitors (TKIs), inevitable resistance to TKIs has led to urgent calls for novel EGFR inhibitors. Herein, we report a rational workflow used to identify novel EGFR-TKIs by combining hybrid ligand- and structure-based pharmacophore models. Three types of models were developed in this workflow, including 3D QSAR-, common feature-, and structure-based EGFR-TK domain-containing pharmacophores. A National Cancer Institute (NCI) compound dataset was adopted for multiple-stage pharmacophore-based virtual screening (PBVS) of various pharmacophore models. The six top-scoring compounds were identified through the PBVS pipeline coupled with molecular docking. Among these compounds, NSC609077 exerted a significant inhibitory effect on EGFR activity in gefitinib-resistant H1975 cells, as determined by an enzyme-linked immunosorbent assay (ELISA). Further investigations showed that NSC609077 inhibited the anchorage-dependent growth and migration of lung cancer cells. Furthermore, NSC609077 exerted a suppressive effect on the EGFR/PI3K/AKT pathway in H1975 cells. In conclusion, these findings suggest that hybrid virtual screening may accelerate the development of targeted drugs for lung cancer treatment.

Hybrid Pharmacophore- and Structure-Based Virtual Screening Pipeline to Identify Novel EGFR Inhibitors That Suppress Non-Small Cell Lung Cancer Cell Growth.

The etiology underlying cases of palindromic rheumatism (PR) not associated with other rheumatic diseases in patients who are seronegative for rheumatoid factor and anti-cyclic citrullinated peptide (seronegative PR) is unclear. We aimed to investigate the immune cells and genes involved.

Assessing the Immune Cell Subset and Genetic Mutations in Patients With Palindromic Rheumatism Seronegative for Rheumatoid Factor and Anti-Cyclic Citrullinated Peptide.

An efficient host immune response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19) appears to be crucial for controlling and resolving this viral infection. However, many studies have reported autoimmune characteristics in severe COVID-19 patients. Moreover, clinical observations have revealed that COVID-19-associated acute distress respiratory syndrome shares many features in common with inflammatory myopathy including interstitial lung disease (ILD), most particularly rapidly progressive (RP)-ILD. This study explored this phenomenon by seeking to identify and characterize myositis-specific and related autoantibodies in 25 COVID-19 patients with mild or severe symptoms. Line blot analysis with the EUROLINE Myopathies Ag kit identified 9 (36%) patients with COVID-19 with one or more autoantibodies against several myositis-related antigens (Jo-1, Ku, Mi-2β, PL-7, PL-12, PM-Scl 75, PM-Scl 100, Ro-52, and SRP); no anti-MDA5 antibodies were detected. As the presence of antibodies identified by line blots was unrelated to disease severity, we further characterized the autoantibodies by radioimmunoassay, in which [ S]methionine-labeled K562 cellular antigens were precipitated and visualized by gel electrophoresis. This result was confirmed by an immunoprecipitation assay and immunoblotting; 2 patients exhibited anti-Ku70 and anti-Ku80 antibodies. Our data suggest that it is necessary to use more than one method to characterize and evaluate autoantibodies in people recovered from COVID-19, in order to avoid misinterpreting those autoantibodies as diagnostic markers for autoimmune diseases.

Screening and characterization of myositis-related autoantibodies in COVID-19 patients.

First-generation tyrosine kinase inhibitors (TKIs) have been associated with good responses in non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR)-sensitizing mutations. However, this therapeutic strategy inevitably promotes resistance to TKIs. This study aimed to investigate the functional role and mechanism of proscillaridin A in NSCLC with or without EGFR mutations. Cellular function assays showed that proscillaridin A could inhibit cell proliferation, migration and invasion in vitro independent of EGFR mutation status. Real-time PCR of the human chromosome 17 α-satellite region revealed that proscillaridin A significantly suppressed tumour micrometastasis in vivo. In immunofluorescence experiments, we found that proscillaridin A decreased filopodia length in NSCLC cells. Furthermore, proscillaridin A also downregulated EGFR-Src-mediated cytoskeleton-related pathways, including FAK-paxillin signalling, which has been shown to promote cell filopodia formation by regulating small G-proteins. Therefore, we used the GST-PBD pull-down assay to demonstrate that proscillaridin A could decrease Cdc42 activity. Moreover, survival analyses of 591 lung adenocarcinoma patients from the GEO database indicated that the expression levels of  and the risk score of the gene signature based on these two factors were negatively correlated with overall survival and could be used as independent prognostic factors. In conclusion, we speculate that proscillaridin A inhibits lung cancer cell growth and motility by regulating EGFR-Src-associated pathways.

Proscillaridin A inhibits lung cancer cell growth and motility through downregulation of the EGFR-Src-associated pathway.

The role of zinc finger protein 334 (ZNF334) in immunological processes remains unknown. We identified a ZNF334 truncation mutation (p.Thr399fs) in a rare case of late-onset cold-induced autoinflammatory disease with elevated TNF-α, IL-1β, IL-6, and extracellular heat shock protein 90 (eHsp90) plasma levels and progressive sensorineural hearing loss. Using patient-derived monocytes and CRISPR/Cas9-edited THP-1 monocytes with a ZNF334 truncation mutation, we discovered that the mutation reduced the interaction between ZNF334 and Hsp90, diminished the endogenous levels of the cold stress regulators Hsp90 and transient receptor potential melastatin 8 (TRPM8), disrupted ER protein folding response and redox homeostasis, and increased cold-induced NF-κB activation and secretion of the proinflammatory TRPM8+ mitochondria-containing extracellular vesicles in monocytes. Long-term cold avoidance alleviated the patient's cold-induced symptoms. In addition, treatment of ZNF334-truncated THP-1 cells with an Hsp90 inhibitor prevented cold-induced TNF and NLRP3 upregulations. Our findings suggest ZNF334 as an essential regulator of cold-induced inflammation and oxidative stress, and Hsp90 ATPase inhibitors might be effective in the treatment of autoinflammatory diseases induced by repeated mild cold exposure.

Article	Year
Anemone altaica Induces Apoptosis in Human Osteosarcoma Cells. The American journal of Chinese medicine\| PubMed ID: 26224029	2015
AC-93253 iodide, a novel Src inhibitor, suppresses NSCLC progression by modulating multiple Src-related signaling pathways. Journal of hematology & oncology\| PubMed ID: 29132432	2017
Tumor microenvironment-based screening repurposes drugs targeting cancer stem cells and cancer-associated fibroblasts. Theranostics\| PubMed ID: 34646392	2021
The Potential Role of Electronegative High-Density Lipoprotein H5 Subfraction in RA-Related Atherosclerosis. International journal of molecular sciences\| PubMed ID: 34768851	2021
Hybrid Pharmacophore- and Structure-Based Virtual Screening Pipeline to Identify Novel EGFR Inhibitors That Suppress Non-Small Cell Lung Cancer Cell Growth. International journal of molecular sciences\| PubMed ID: 35408854	2022
Assessing the Immune Cell Subset and Genetic Mutations in Patients With Palindromic Rheumatism Seronegative for Rheumatoid Factor and Anti-Cyclic Citrullinated Peptide. Arthritis & rheumatology (Hoboken, N.J.)\| PubMed ID: 35819819	2023
Screening and characterization of myositis-related autoantibodies in COVID-19 patients. Clinical and translational science\| PubMed ID: 36271647	2023
Proscillaridin A inhibits lung cancer cell growth and motility through downregulation of the EGFR-Src-associated pathway. American journal of cancer research\| PubMed ID: 38058797	2023
ZNF334 truncation mutation drives cold-induced autoinflammation. EMBO molecular medicine\| PubMed ID: 41168503	2025

Yi-Hua Lai

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