Yeast-derived particulate beta-glucan induced angiogenesis via regulating PI3K/Src and ERK1/2 signaling pathway.

The importance of ß-glucan from S. cerevisiae in angiogenesis has not been well studied. We investigated whether ß-glucan induces angiogenesis through PI3K/Src and ERK1/2 signaling pathway in HUVECs. We identified that ß-glucan induced phosphorylation of PI3K, Src, Akt, eNOS, and ERK1/2. Subsequently, we found that this phosphorylation increased the viability of HUVECs. We also observed that stimulation of ß-glucan promoted the activity of MEF2 and MEF2-dependent pro-angiogenic genes, including EGR2, EGR3, KLF2, and KLF4. Additionally, the role of ß-glucan in angiogenesis was confirmed using in vitro and ex vivo experiments including cell migration, capillary-like tube formation and mouse aorta ring assays. To determine the effect of ß-glucan on the PI3K/Akt/eNOS and ERK1/2 signaling pathway, PI3K inhibitor wortmannin and ERK1/2 inhibitor SCH772984 were used. Through the Matrigel plug assay, we confirmed that ß-glucan significantly increased angiogenesis in vivo. Taken together, our study demonstrates that ß-glucan promotes angiogenesis via through PI3K and ERK1/2 signaling pathway.

HDAC5-mediated exosomal Maspin and miR-151a-3p as biomarkers for enhancing radiation treatment sensitivity in hepatocellular carcinoma.

BACKGROUND: Tumor-derived exosomes are critical elements of the cell-cell communication response to various stimuli. This study aims to reveal that the histone deacetylase 5 (HDAC5) and p53 interaction upon radiation in hepatocellular carcinoma intricately regulates the secretion and composition of exosomes. METHODS: We observed that HDAC5 and p53 expression were significantly increased by 2 Gy and 4 Gy radiation exposure in HCC. Normal- and radiation-derived exosomes released by HepG2 were purified to investigate the exosomal components. RESULTS: We found that in the radiation-derived exosome, exosomal Maspin was notably increased. Maspin is known as an anti-angiogenic gene. The expression of Maspin was regulated at the cellular level by HDAC5, and it was elaborately regulated and released in the exosome. Radiation-derived exosome treatment caused significant inhibition of angiogenesis in HUVECs and mouse aortic tissues. Meanwhile, we confirmed that miR-151a-3p was significantly reduced in the radiation-derived exosome through exosomal miRNA sequencing, and three HCC-specific exosomal miRNAs were also decreased. In particular, miR-151a-3p induced an anti-apoptotic response by inhibiting p53, and it was shown to induce EMT and promote tumor growth by regulating p53-related tumor progression genes. In the HCC xenograft model, radiation-induced exosome injection significantly reduced angiogenesis and tumor size. CONCLUSIONS: Our present findings demonstrated HDAC5 is a vital gene of the p53-mediated release of exosomes resulting in tumor suppression through anti-cancer exosomal components in response to radiation. Finally, we highlight the important role of exosomal Maspin and mi-151a-3p as a biomarker in enhancing radiation treatment sensitivity. Therapeutic potential of HDAC5 through p53-mediated exosome modulation in radiation treatment of hepatocellular carcinoma.

Generation of genetically engineered mice for lung cancer with mutant EGFR.

Although epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) have shown dramatic response and improvement in treating lung cancer with mutant EGFR, the emergence of drug resistance remains a major problem. In particular, some mutations including T790 M and C797S have been recognized as mechanisms of acquired resistance because they weaken binding affinity to drugs. To date, many attempts have been made to develop a new drug for overcoming acquired resistance to EGFR-TKIs, including secondary mutations. However, an appropriate animal model to evaluate in vivo efficacy during novel drug development remains lacking. In this study, we generated a novel transgenic mouse model that conditionally expresses human EGFRL858R/T790M/C797S and firefly luciferase using Cas9-mediated homology-independent targeted integration. Using a lung-specific Sftpc-CreERT2 mouse line, we induced expression of both the human EGFRL858R/T790M/C797S transgene and firefly luciferase in the lungs of adult mice. The expression of these genes and lung cancer occurrence was monitored using an in vivo imaging system and magnetic resonance imaging, respectively. Overall, our mouse model can be utilized to develop new drugs for overcoming C797S-mediated resistance to osimertinib; further, such knock-in systems for expressing oncogenes may be applied to study tumorigenesis and the development of other targeted agents.

The reversible fourth-generation EGFR tyrosine kinase inhibitor OBX02-011 overcomes C797S-mediated resistance in lung cancer.

Osimertinib is an irreversible third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) that was initially developed to overcome the EGFR T790M mutation and is used as a standard therapy in patients with advanced non-small cell lung cancer (NSCLC) with EGFR-activating mutations. Despite the remarkable initial efficacy, osimertinib, like other EGFR-TKIs, is limited by the emergence of acquired resistance. As the EGFR mutation C797S has been identified as a key driver of acquired resistance to osimertinib, development of a drug that targets this clinically relevant mutation could help improve patient outcomes. Here, we report the discovery and preclinical efficacy of OBX02-011, a reversible fourth-generation EGFR TKI that overcomes the EGFR C797S mutation. Compared to approved EGFR TKIs, OBX02-011 showed potent anticancer effects and inhibited EGFR-related signaling in various models, including those harboring the EGFR C797S mutation. Additionally, in transgenic mouse models (EGFRL858R/T790M/C797S), OBX02-011 treatment effectively inhibited tumor growth and EGFR activity, leading to enhanced survival. Collectively, these results suggest that OBX02-011 may be a promising new EGFR TKI to overcome C797S-mediated resistance in NSCLC.

Immortalization of primary marmoset skin fibroblasts by CRISPR-Cas9-mediated gene targeting.

Immortalized cell lines can be used for diverse in vitro experiments, providing invaluable data before conducting in vivo studies Callithrix jacchus, the common marmoset, is a non-human primate model utilized for studying various human diseases. However, only a few immortalized marmoset cell lines are currently available. In the present study, we reveal that CRISPR-Cas9-mediated targeting of the p53 gene or CDKN2A locus is an effective means for immortalizing primary marmoset skin fibroblasts. In addition to frameshift mutations that result in premature stop codons, in-frame mutations potentially destroying the DNA-binding motif of p53 are frequently detected in immortalized cells. Like Cdkn2a-deficient mouse cells, CDKN2A-deficient marmoset cells express wild-type p53 proteins normally respond to genotoxic stresses, including adriamycin and etoposide. Taken together, these findings indicate that Cas9- mediated gene targeting of the p53 gene or CDKN2A locus is an effective tool for establishing immortalized marmoset cell lines with defined genetic alterations.

A nonbiodegradable scaffold-free cell sheet of genome-engineered mesenchymal stem cells inhibits development of acute kidney injury.

Cell therapy using genome-engineered stem cells has emerged as a novel strategy for the treatment of kidney diseases. By exploiting genome editing technology, human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) secreting an angiogenic factors or an anti-inflammatory factor were generated for therapeutic application in acute kidney injury. Junction polymerase chain reaction analysis verified zinc finger nucleases-assisted integration of the desired gene into the hUC-MSCs. Flow cytometry and differentiation assays indicated that genome editing did not affect the differentiation potential of these mesenchymal stem cells. Protein measurement in conditioned media with the use of ELISA and immunoblotting revealed the production and secretion of each integrated gene product. For cell therapy in the bilateral ischemia-reperfusion mouse model of acute kidney injury, our innovative scaffold-free cell sheets were established using a non-biodegradable temperature-responsive polymer. One of each type of scaffold-free cell sheets of either the angiogenic factor vascular endothelial grown factor or angiopoietin-1, or the anti-inflammatory factor erythropoietin, or α-melanocyte-stimulating hormone-secreting hUC-MSCs was applied to the decapsulated kidney surface. This resulted in significant amelioration of kidney dysfunction in the mice with acute kidney injury, effects that were superior to intravenous administration of the same genome-engineered hUC-MSCs. Thus, our scaffold-free cell sheets of genome-engineered mesenchymal stem cells provides therapeutic effects by inhibiting acute kidney injury via angiogenesis or anti-inflammation.

miR-34c-5p and CaMKII are involved in aldosterone-induced fibrosis in kidney collecting duct cells.

Mineralocorticoids trigger a profibrotic process in the kidney. In mouse cortical collecting duct cells, the present study addressed two main questions: 1) what are microRNAs (miRNAs) and their target genes that are changed by aldosterone? and 2) what do miRNAs, in response to aldosterone, regulate regarding signaling pathways related to fibrosis? A microarray chip assay was done in cells in the absence or presence of aldosterone treatment (10-6 M; 3 days). The candidate miRNAs were identified by the criteria of >30% of fold change among the significantly changed miRNAs ( P < 0.05). Twenty-nine miRNAs were upregulated (>1.3-fold), and 27 miRNAs were downregulated (<0.7-fold). Putative target genes of identified miRNAs were associated with 74 Kyoto Encyclopedia of Genes and Genomes pathways. Among them, the wingless-related integration site (Wnt) signaling pathway was highly ranked, where 15 mature miRNAs were observed. These miRNAs were further analyzed by real-time quantitative PCR, and among them, miR-130b-3p, miR-34c-5p, and miR-146a-5p were selected. Through the identification of putative target genes of these three miRNAs, mRNA and protein expression of the Ca2+/calmodulin-dependent protein kinase type II ß-chain ( Camk2b) gene (a target gene of miR-34c-5p) were found to be increased significantly in aldosterone-treated cells, where fibronectin (FN) and α-smooth muscle actin were induced. When CaMKIIß small interfering RNA or the miR-34c-5p mimic was transfected, aldosterone-induced FN expression was significantly attenuated, along with reduced CaMKIIß protein expression. A luciferase reporter assay revealed a decrease of CaMKIIß translation in cells transfected with miRNA mimics of miR-34c-5p. In conclusion, aldosterone-induced downregulation of miR-34c-5p in the Wnt signaling pathway and a consequent increase of CaMKIIß expression are likely to be involved in aldosterone-induced fibrosis.