Korea Research Institute of Bioscience and Biotechnology
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Peroxiredoxin 5 deficiency exacerbates iron overload-induced neuronal death via ER-mediated mitochondrial fission in mouse hippocampus
No full textIron is an essential element for cellular functions, including those of neuronal cells. However, an imbalance of iron homeostasis, such as iron overload, has been observed in several neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. Iron overload causes neuronal toxicity through mitochondrial fission, dysregulation of Ca2+, ER-stress, and ROS production. Nevertheless, the precise mechanisms between iron-induced oxidative stress and iron toxicity related to mitochondria and endoplasmic reticulum (ER) in vivo are not fully understood. Here, we demonstrate the role of peroxiredoxin 5 (Prx5) in iron overload-induced neurotoxicity using Prx5-deficient mice. Iron concentrations and ROS levels in mice fed a high iron diet were significantly higher in Prx5-/- mice than wildtype (WT) mice. Prx5 deficiency also exacerbated ER-stress and ER-mediated mitochondrial fission via Ca2+/calcineurin-mediated dephosphorylation of Drp1 at Serine 637. Moreover, immunoreactive levels of cleaved caspase3 in the CA3 region of the hippocampus were higher in iron-loaded Prx5-/- mice than WT mice. Furthermore, treatment with N-acetyl-cysteine, a reactive oxygen species (ROS) scavenger, attenuated iron overload-induced hippocampal damage by inhibiting ROS production, ER-stress, and mitochondrial fission in iron-loaded Prx5-/- mice. Therefore, we suggest that iron overload-induced oxidative stress and ER-mediated mitochondrial fission may be essential for understanding iron-mediated neuronal cell death in the hippocampus and that Prx5 may be useful as a novel therapeutic target in the treatment of iron overload-mediated diseases and neurodegenerative diseases.
A regulatory noncoding RNA, nc886, suppresses esophageal cancer by inhibiting the AKT pathway and cell cycle progression
No full textnc886 is a regulatory non-coding RNA (ncRNA) whose expression is frequently silenced in malignancies. In the case of esophageal squamous cell carcinoma (ESCC), nc886 silencing is associated with shorter survival of patients, suggesting nc886's tumor suppressor role in ESCC. However, this observation has not been complemented by an in-detail study about nc886's impact on gene expression and cellular phenotypes. Here we have shown that nc886 inhibits AKT, a key protein in a renowned pro-survival pathway in cancer. nc886-silenced cells (nc886- cells) have activated AKT and altered expression of cell cycle genes. nc886- cells tend to have lower expression of CDKN2A and CDKN2C, both of which are inhibitors for cyclin-dependent kinase (CDK), and higher expression of CDK4 than nc886-expressing cells. As a result, nc886- cells are hyperactive in the progression of the G1 to S cell cycle phase, proliferate faster, and are more sensitive to palbociclib, which is a cancer therapeutic drug that targets CDK4/6. Experimentally by nc886 expression and knockdown, we have determined the AKT target genes and cell cycle genes that are controlled by nc886 (nc886-associated gene sets). These gene sets, in combination with pathologic staging and nc886 expression levels, are a vastly superior predictor for the survival of 108 ESCC patients. In summary, our study has elucidated in ESCC how nc886 inhibits cell proliferation to explain its tumor suppressor role and identified gene sets that are of future clinical utility, by predicting patient survival and responsiveness to a therapeutic drug.
Phylogenetic analysis of Aegista quelpartensis using metallothionein gene
No full textMetallothionein (MT) is a metal-binding protein with a small molecular weight that can be induced by factors such as metal ions and chemicals, and is widely present in various organisms. The high cysteine content of MT contributes to the regulation of intracellular metal concentration, detoxification of heavy metals, and it may be induced due to the environment exposed to heavy metals or oxidation stress. MT has been consistently studied as a taxonomic biomarker for various species, including mollusks. Aegista quelpartensis, a native species of Korea, has habitat in Jeju and Jindo island. DNAseq and transcriptome information is registered with NCBI's SRA, but no molecular phylogenetic studies have been conducted. RNA sequencing was performed using Illumina Hiseq 2500 to find the genetic information of A. quelpartensis. The unigene obtained through the de novo assembly was annotated according to the PANM database using the BLAST program, and the MT sequence consisting of 288 bp coding region and 96 amino acid residues was extracted. The bioinformatic analysis verified that A. quelpartensis was grouped in the Stylommatophora clad using the ClustalX and MEGA7 programs. This study confirmed the phylogenetic location of A. quelpartensis and the MT gene is sufficient gene that can be used in phylogenetic studies on Mollusca.
ONECUT2 upregulation is associated with CpG hypomethylation at promoter-proximal DNA in gastric cancer and triggers ACSL5
No full textMany studies have focused on global hypomethylation or hypermethylation of tumor suppressor genes, but less is known about the impact of promoter hypomethylation of oncogenes. We previously showed that promoter methylation may gradually increase or decrease during the transition from gastric mucosa (GM) to intestinal metaplasia (IM) to gastric cancer (GC). In our study, we focused on regional CpG hypomethylation of the promoter-proximal DNA of the transcription factor ONECUT2 (OC2) in IM and GC cells. We validated the hypomethylation of promoter-proximal DNA of OC2 in 160 primary GCs, in which methylation level correlated negatively with OC2 mRNA level. IM and GC cells stained positively for OC2, whereas GM cells did not. Stable transfection of OC2 in GC cells promoted colony formation, cell migration, invasion and proliferation. Moreover, OC2 knockdown with a short hairpin RNA suppressed tumorigenesis in nude mice. In addition, chromatin immunoprecipitation coupled with DNA sequencing and RNA-seq analyses revealed that OC2 triggered ACSL5, which is strongly expressed in IM of the stomach but not in GM, indicating that OC2 and ACSL5 are early-stage biomarkers for GC. We also observed a high correlation between the levels of OC2 and ACSL5 mRNAs in the GENT database These results suggest that epigenetic alteration of OC2 upregulates its expression, which then activates ACSL5; thus, OC2 is induced in IM by epigenetic alteration and triggers ACSL5 expression, and thus OC2 and ACSL5 may cooperatively promote intestinal differentiation and GC progression.
Elucidation of the biosynthetic pathway of vitamin B groups and potential secondary metabolite gene clusters via genome analysis of a marine bacterium Pseudoruegeria sp. M32A2M
No full textThe symbiotic nature of the relationship between algae and marine bacteria is well-studied among the complex microbial interactions. The mutual profit between algae and bacteria occurs via nutrient and vitamin exchange. It is necessary to analyze the genome sequence of a bacterium to predict its symbiotic relationships. In this study, the genome of a marine bacterium, Pseudoruegeria sp. M32A2M, isolated from the south-eastern isles (GeoJe-Do) of South Korea, was sequenced and analyzed. A draft genome (91 scaffolds) of 5.5 Mb with a DNA G+C content of 62.4% was obtained. In total, 5,101 features were identified from gene annotation, and 4,927 genes were assigned to functional proteins. We also identified transcription core proteins, RNA polymerase subunits, and sigma factors. In addition, full flagella-related gene clusters involving the flagellar body, motor, regulator, and other accessory compartments were detected even though the genus Pseudoruegeria is known to comprise non-motile bacteria. Examination of annotated KEGG pathways revealed that Pseudoruegeria sp. M32A2M has the metabolic pathways for all seven vitamin Bs, including thiamin (vitamin B1), biotin (vitamin B7), and cobalamin (vitamin B12), which are necessary for symbiosis with vitamin B auxotroph algae. We also identified gene clusters for seven secondary metabolites including ectoine, homoserine lactone, beta-lactone, terpene, lasso peptide, bacteriocin, and nonribosomal proteins.
Licochalcone C induces cell cycle G1 arrest and apoptosis in human esophageal squamous carcinoma cells by activation of the ROS/MAPK signaling pathway
No full textAlong with changes in dietary habits and lifestyle, the incidence of esophageal cancer is increasing around the world. Since chemotherapy for esophageal cancer has significant side effects, phytochemicals have attracted attention as an alternative medicine. Licochalcone C (LCC) is a flavonoid compound extracted from Licorice, with a variety of clinical uses including anti-cancer, anti-inflammatory and anti-oxidant effects. Treatment with LCC for 48 h significantly decreased cell viability of esophageal squamous cell carcinoma (ESCC) cells in a dose- and time-dependent manner with IC50 values of 28 μM (KYSE 30), 36 μM (KYSE 70), 19 μM (KYSE 410), 28 μM (KYSE 450) and 26 μM (KYSE 510). LCC induced G1 arrest accompanied by decreased cyclin D1 expression and an increase in the levels of p21 and p27. LCC increased the levels of intracellular ROS, cytochrome C release, and multi-caspase activity, and decreased mitochondrial membrane potential. LCC induced the protein expression of ER stress markers (GRP78 and CHOP) and phosphorylation JNK, c-Jun and p38. We investigated the expression of pro-apoptotic and anti-apoptotic proteins to elucidate the mechanism of apoptosis. Our findings contribute to the understanding of apoptosis mechanism underlying LCC in ESCC cells and provide new insights into the potential clinical opportunities of LCC for ESCC treatment.
Protein kinase A catalytic subunit is a molecular switch that promotes the pro-tumoral function of macrophages
No full textAs current therapies benefit only a minority of cancer patients, additional therapeutic targets are needed. Tumor-associated macrophages (TAMs) have attracted attention for improving therapeutic responses, yet regulatory strategies remain elusive. Here, we show that the protein kinase A catalytic subunit (PKA-C) acts as a molecular switch, inducing a pro-tumoral immunosuppressive macrophage phenotype within tumors. In human and murine breast cancer, overactivated PKA in TAMs creates a detrimental microenvironment for cancer progression by inducing vascular endothelial growth factor A (VEGFA), interleukin-10 (IL-10), and macrophage-derived arginase 1 (ARG1) expression. Macrophages with genetic deletion of PKA-C are prone to be pro-inflammatory, suggesting a possible immunotherapeutic target. Delivery of liposomal PKA inhibitor facilitates tumor regression and abrogates pro-tumoral TAM functions in mice. The therapeutic effect of targeting PKA is pronounced when combined with αCTLA-4 antibody, increasing cluster of differentiation 8 (CD8)+GranzymeB+ T cells by about 60-fold. Our findings demonstrate critical roles of TAM PKA-C in tumor progression and suggest that targeting PKA-C efficiently augments cancer treatment responses. ⓒ 2020 The Author(s)Na et al. show that overactivated protein kinase A catalytic subunit beta (PKA-Cβ) of macrophages creates a detrimental microenvironment for the breast cancer progression. Delivery of a liposomal PKA inhibitor reduces pro-tumoral function of macrophages and induces the activation of cytotoxic T lymphocytes, leading to a tumor regression.
A D-glucose- and D-xylose-tolerant GH1 β-glucosidase from Cellulosimicrobium funkei HY-13, a fibrolytic gut bacterium of Eisenia fetida
No full textThe GluM gene (1491-bp) coding for a β-glucosidase comprising a single catalytic glycoside hydrolase family 1 domain from an earthworm (Eisenia fetida)-symbiotic bacterium, Cellulosimicrobium funkei HY-13, was cloned and over-expressed in Escherichia coli BL21. The recombinant histidine-tagged enzyme (rGluM: 56 kDa) displayed the highest cleavage activity toward p-nitrophenyl (pNP)-β-D-glucopyranoside at pH 5.0 and 40 °C. The β-glucosidase activity of rGluM was enhanced over 1.8-fold of its original activity in the presence of 1 mM Ca2+, Ni2+, Mn2+, and Co2+ ions, respectively, while it was highly sensitive to 5 mM N-bromosuccinimide and 1 mM Hg2+. The susceptibility of some pNP-sugar derivatives and D-cellobiose to rGluM was evaluated to be in the order of pNP-β-D-glucopyranoside > pNP-β-D-galactopyranoside > D-cellobiose > pNP-β-D-cellobioside > pNP-β-D-mannopyranoside. The kcat/Km values of rGluM toward pNP-β-D-glucopyranoside, pNP-β-D-galactopyranoside, and D-cellobiose were 302.28, 179.73, and 6.40 mM-1 s-1, respectively. At a concentration below 1.0 M, D-galactose was a potent activator of rGluM with β-glucosidase activity enhanced by approximately 160% in a dose-dependent manner. Moreover, the D-glucose (< 400 mM) and D-xylose (≤ 700 mM) stimulation of rGluM suggests that it can be exploited as a potential biocatalyst to generate D-glucose molecules in D-cellobiose degradation.
The study of differentiation in cancer stem cell by oxidative stress and cytokine
No full text산화스트레스와 사이토카인 조절에 의한 암줄기세포 발생 기전 연구OGM520202
Establishment and characterization of immortalized miniature pig pancreatic cell lines expressing oncogenic K-rasG12D
No full textIn recent decades, many studies on the treatment and prevention of pancreatic cancer have been conducted. However, pancreatic cancer remains incurable, with a high mortality rate. Although mouse models have been widely used for preclinical pancreatic cancer research, these models have many differences from humans. Therefore, large animals may be more useful for the investigation of pancreatic cancer. Pigs have recently emerged as a new model of pancreatic cancer due to their similarities to humans, but no pig pancreatic cancer cell lines have been established for use in drug screening or analysis of tumor biology. Here, we established and characterized an immortalized miniature pig pancreatic cell line derived from primary pancreatic cells and pancreatic cancer-like cells expressing K-rasG12D regulated by the human PTF1A promoter. Using this immortalized cell line, we analyzed the gene expression and phenotypes associated with cancer cell characteristics. Notably, we found that acinar-to-ductal transition was caused by K-rasG12D in the cell line constructed from acinar cells. This may constitute a good research model for the analysis of acinar-to-ductal metaplasia in human pancreatic cancer.