RNA-Seq based transcriptome analysis reveals the molecular mechanism of triterpenoid biosynthesis in Glycyrrhiza glabra

Licorice is a frequently-used medicinal plant worldwide. Two triterpenoids, 18α-glycyrrhizic acid (18α-GC) and 18β-glycyrrhizic acid (18β-GC), are the key medicinal components accumulated in licorice. Biosynthesis of triterpenoids is a complex process that involves many secondary metabolic pathways. In this study, we tried to identify the key enzymes and pathways for the triterpenoid biosynthesis in licorice by analyzing the gene expression patterns in samples containing different GC levels. Glycyrrhizia glabra (one of the original species used as licorice in Chinese Pharmacopoeia) seeds were irradiated by X-ray and cultivated for one year, and samples with different GC contents were selected by HPLC analysis. RNA-Seq was performed to determine the gene expression in three X-ray irradiated G. glabra samples (H1, H2, and H3) with the highest GC content and one control G. glabra sample (L1) with the lowest GC content. 28.44 Gb raw data was generated and 47.7 million, 45.4 million, 43.3 million, and 45.9 million clean reads were obtained in samples H1, H2, H3, and L1, respectively. Approximately 48.53% of genes were annotated searching against GO and KEGG databases. A total of 1376 core differentially expressed genes (DEGs) were identified, which mainly enriched in phenylpropanoid metabolism, glycometabolism, plant circadian rhythm, and terpenoid biosynthetic pathway. 15 core DEGs selected from the 1376 DEGs were further verified by qRT-PCR, which confirmed that the RNA-Seq results were accurate and reliable. This study provides a basis for future functional genes mining and molecular regulatory mechanism elucidation of triterpenoid biosynthesis in licorice.     

Growth stimulating activity secreted by human anaplastic thyroid carcinoma cells.

In order to clarify the mechanism of rapid growth of anaplastic thyroid carcinoma, growth stimulating activity produced by the cancer cells in culture was studied. A cell line (HTh7) established from a biopsy specimen of anaplastic thyroid carcinoma was used throughout the study. Growth stimulating activity was determined as an activity to increase 3H-thymidine incorporation in rat thyroid cell line (FRTL5). Conditioned medium of HTh7 cells contained significant growth stimulating activity for FRTL5 cells. The activity was separated into two fractions with heparin agarose gel: heparin-binding and heparin-non-binding. In the medium, the heparin-non-binding activity was much greater than the heparin-binding one. The heparin-non-binding activity was acid stable. It was partially purified with gel filtration in an acidic condition followed by reverse phase HPLC. In gel filtration with a Sephacryl S-200 column, the activity was eluted later than the elution volume of cytochrome c (MW 12400) as several separated peaks. In reverse phase HPLC, however, the activity in these peaks was eluted as a single peak. The retention time of the active peak was almost the same as that of recombinant IGF-I. When measured by specific RIAs, the conditioned media concentrated 20 times contained both 0.35 ng/ml of IGF-I and 5.21 ng/ml of IGF-II. As for the heparin-binding mitogenic activity, when applied to heparin affinity HPLC column and eluted with a linear gradient of NaCl, the activity came out as one major peak with approximately 1.0 M NaCl.(ABSTRACT TRUNCATED AT 250 WORDS)     

Side population cells in anaplastic thyroid cancer and normal thyroid

Comparison of studies of cells derived from normal and pathological tissues of the same organ can be fraught with difficulties, particular with cancer where a number of different diseases are considered cancer within the same tissue. In the thyroid, there are 4 main types of cancer, three of which arise from follicular epithelial cells; papillary and follicular which are classified as differentiated, and anaplastic which is classified as undifferentiated.One assay that can be utilised for isolation of cancer stem cells is the side population (SP) assay. However, SP studies have been limited in part due to lack of optimal isolation strategies and in the case of anaplastic thyroid cancer (ATC) are further compounded by lack of access to ATC tumors. We have used thyroid cell lines to determine the optimal conditions to isolate viable SP cells. We then compared SP cells and NSP cells (bulk tumour cells without the SP) of a normal thyroid cell line N-thy ori-3-1 and an anaplastic thyroid cancer cell line SW1736 and showed that both SP cell populations displayed higher levels of stem cell characteristics than the NSP. When we compared SP cells of the N-thy ori-3-1 and the SW1736, the SW1736 SP had a higher colony forming potential, expressed higher levels of stem cell markers and CXCR4 and where more migratory and invasive, invasiveness increasing in response to CXCL12.This is the first report showing functional differences between ATC SP and normal thyroid SP and could lead to the identification of new therapeutic targets to treat ATC.     

Antithyroid cancer effects of human neural stem cells expressing therapeutic genes on anaplastic thyroid cancer cells

Stem cells that express therapeutic proteins have been identified to have an anticancer effects on various types of cancer. In the present study study, human neural stem cells (hNSCs) that were genetically engineered to express cytosine deaminase (CD) and human interferon-β (IFN-β) were used for anaplastic thyroid cancer (ATC) treatment owing to their tumor-tropic properties and therapeutic effects. CD is an enzyme that converts 5-fluorocytosine (5-FC), a prodrug, to 5-fluorouracil (5-FU) which is a medication to suppress tumor growth through DNA synthesis inhibition. Also, IFN-β suppresses tumor growth by the induction of apoptotic process. In water soluble tetrazolium salt (WST) assay, SNU-80 cells which are human female ATC cells were cocultured with three cell types including engineered hNSCs such as HB1.F3, HB1.F3.CD, and HB1.F3.CD.IFN-β cells on transwells and treated with 5-FC for 72 hours. Finally, the SNU-80 cell viability was reduced by the coculture with HB1.F3.CD and HB1.F3.CD.IFN-β cells. In dichlorofluorescein diacetate (DCF-DA) and TdT-mediated dUTP nick-end labeling (TUNEL) assays, the production of reactive oxygen species (ROS) and the number of apoptotic cells were increased by HB1.F3.CD and HB1.F3.CD.IFN-β cells in the presence of 5-FC. In Western blot assay, ROS, and apoptosis-related genes were increased in SNU-80 cells when they were cocultured with HB1.F3.CD and HB1.F3.CD.IFN-β cells. In transwell migration assay, hNSCs selectively migrated to SNU-80 cells because hNSCs interacted with chemoattractant factors like SDF-1α, uPAR, and CCR2 secreted by SNU-80 cells. Taken together, engineered hNSCs were revealed to selectively migrate to ATC cells and to inhibit growth as well as to induce apoptosis of ATC cells via ROS production through the actions of transgenes such as CD and IFN-β. Therefore, these engineered hNSCs can be promising candidates for the treatment of metastatic ATC.     

Identification of potential key genes in anaplastic thyroid cancer using bioinformatics analysis

Anaplastic thyroid cancer (ATC) has a high degree of malignancy and poor prognosis. The purpose of this study was to determine differentially expressed genes (DEGs) in ATC through biometric analysis technology, clarify potential interactions between them, and screen genes related to the prognosis of ATC. Using obtained DEGs, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Protein-protein interaction (PPI), and survival analysis were performed. After R integration analysis of the four datasets, 764 DEGs were obtained, i.e., 314 upregulated genes and 450 downregulated genes. Among the hub DEGs obtained from the PPI network, the expression levels of TYMS, FN1, CHRDL1, SDC2, ITGA2, COL1A1, COL9A3, and COL23A1 were associated with ATC prognosis. These results showed that the recurrence-free survival (RFS) of ATC was associated with TYMS, FN1, ITGA2, COL23A1, SDC2, and CHRDL1 statistically significantly in the KM plotter (P<0.05). Thus, the study suggests that TYMS, FN1, ITGA2, COL23A1, SDC2, and CHRDL1 may be used as potential biomarkers of ATC. These findings provide new insights for the detection of novel diagnostic and therapeutic biomarkers for ATC.     

Single-cell RNA-Seq analysis identified kidney progenitor cells from human urine

Dear Editor, Urine passes through the entire kidney and urinary tract system starting from the glomerulus and ending to the urethra.Cells in the kidney and urinary tract could be exfoliated from the epithelium into the urine, while leukocyte could infiltrate from the local tissue into the urine, which makes the urine a useful subject for clinical evaluation of relevant diseases.Among them, renal tubular cells and podocytes have been identified and 2D or 3D cultured from human urine specimens (Oliveira Arcolino et al., 2015;Schutgens et al.,2019).Particularly, kidney stem cell/progenitor cells were successfully recovered from pediatric patient urine and then cultured for kidney regenerative purpose by the Romagnani group.However, they also showed that such cells cannot be recovered from healthy individuals (Lazzeri et al., 2015).It remains unknown whether similar types of progenitor cells can be found in different individuals, either healthy or diseased.     

Epidermal growth factor receptor signaling activates met in human anaplastic thyroid carcinoma cells

Overexpression of Met is a common finding in thyroid carcinomas. Recently, we reported on overexpression and ligand-independent constitutive activation of Met in anaplastic thyroid carcinoma cells. In the present study we have investigated a putative mechanism for this phenomenon. Cell lines with constitutively activated Met expressed both TGF-alpha mRNA and protein. Western blot analysis revealed expression of receptors for epidermal growth factor (EGFR) in all carcinoma cell lines; in tumor cells with elevated levels of TGF-alpha mRNA there was a constitutive tyrosine phosphorylation of the EGFRs. Preincubation of carcinoma cells with suramin decreased EGFR activation and downregulated Met expression as well as the ligand-independent phosphorylation of Met. Similar results were obtained with a EGFR tyrosine kinase inhibitor, AG 1478. The MEK inhibitor U0126 had an even more pronounced effect compared to AG 1478, indicating a Ras/MAPK-mediated signal in the regulation of Met expression and activation. Inhibition of EGFR signaling also decreased proliferation of the anaplastic thyroid carcinoma cells. Thus, aberrant activation of EGFRs may lead to an overexpression and activation of Met, which may be of importance for the malignant phenotype of anaplastic thyroid carcinomas.     

Single-nucleus transcriptome analysis reveals cell-type-specific molecular signatures across reward circuitry in the human brain

1. Download : Download high-res image (190KB)
2. Download : Download full-size image

     

Potential antitumor activity of digitoxin and user-designed analog administered to human lung cancer cells

BackgroundCardiac glycosides (CGs), such as digitoxin, are traditionally used for treatment of congestive heart failure; recently they also gained attention for their anticancer properties. Previous studies showed that digitoxin and a synthetic L-sugar monosaccharide analog treatment decreases cancer cell proliferation, increases apoptosis, and pro-adhesion abilities; however, no reports are available on their potential to alter lung cancer cell cytoskeleton structure and reduce migratory ability. Herein, we investigated the anticancer effects of digitoxin and its analog, digitoxigenin-α-L-rhamnoside (D6MA), to establish whether cytoskeleton reorganization and reduced motility are drug-induced cellular outcomes.MethodsWe treated non-small cell lung carcinoma cells (NSCLCs) with sub-therapeutic, therapeutic, and toxic concentrations of digitoxin and D6MA respectively, followed by both single point and real-time assays to evaluate changes in cellular gene and protein expression, adhesion, elasticity, and migration.ResultsDigitoxin and D6MA induced a decrease in matrix metalloproteinases expression via altered focal adhesion signaling and a suppression of the phosphoinositide 3-kinases / protein kinase B pathway which lead to enhanced adhesion, altered elasticity, and reduced motility of NSCLCs. Global gene expression analysis identified dose-dependent changes to nuclear factor kappa-light-chain-enhancer, epithelial tumor, and microtubule dynamics signaling.ConclusionsOur study demonstrates that digitoxin and D6MA can target antitumor signaling pathways to alter NSCLC cytoskeleton and migratory ability to thus potentially reduce their tumorigenicity.SignificanceDiscovering signaling pathways that control cancer's cell phenotype and how such pathways are affected by CG treatment will potentially allow for active usage of synthetic CG analogs as therapeutic agents in advanced lung conditions.     

Destructive thyrotoxicosis in a patient with anaplastic thyroid cancer

A 55-year-old man was admitted to our hospital with an anterior neck tumor, hoarseness, and dysphagia that had continued for a few weeks. He was diagnosed as anaplastic thyroid cancer by fine-needle aspiration cytology. He was treated by external radiation and chemotherapy, but left hemothorax developed and he died of respiratory failure on the 76th day in hospital. On admission, the levels of serum free triiodothyronine (FT3), free thyroxine (FT4), and TSH were 12.8 pg/ml, 4.2 ng/dl, and 0 microU/ml, respectively. The simultaneous thyroidal I-131 uptake rate was 1.2% at 24 hours. The levels of free thyroid hormones fell gradually without antithyroid drugs to result in hypothyroidism (FT3 0.8 pg/ml, FT4 0 ng/dl, and TSH 36 microU/ml). The rapid growth of anaplastic thyroid cancer seemed to be responsible for destructive thyrotoxicosis followed by hypothyroidism in this patient.