Plasma-activated medium ameliorates the chemoresistance of human lung adenocarcinoma cells mediated via downregulation of claudin-2 expression.

Plasma-activated medium (PAM) has various biological activities including anticancer and antimicrobial. However, the effect on chemoresistance in cancer cells has not been clarified in detail. Solid cancer cells form a microenvironment in the body and acquire resistance against anticancer drugs. So far, we reported that claudin-2 (CLDN2), a component of tight junctions, suppresses the anticancer drug-induced cytotoxicity of spheroids that mimic in vivo tumors. Here, we found that the protein level of CLDN2 is downregulated by the sublethal concentration of PAM in human lung adenocarcinoma-derived A549 and PC-3 cells. A cycloheximide pulse-chase assay showed that PAM accelerates the degradation of CLDN2 protein. The PAM-induced reduction of CLDN2 protein was inhibited by a lysosome inhibitor, indicating PAM may enhance the lysosomal degradation of CLDN2. The paracellular permeability to doxorubicin (DXR), an anthracycline antitumor drug, was enhanced by PAM. In the spheroids, the accumulation and toxicity of DXR were enhanced by PAM. In addition, oxidative stress and the expression of nuclear factor erythroid 2-related factor 2, one of the key factors for the acquisition of chemoresistance, were attenuated by PAM. The improvement effect of PAM on chemoresistance was suppressed by the exogenous CLDN2 overexpression. These results indicate that PAM has the ability to downregulate CLDN2 expression and may become an adjuvant drug against lung adenocarcinoma.

Acrolein suppresses anticancer drug-induced toxicity mediated by activating claudin-1 and Nrf2 axis in a spheroid model of human lung squamous cell carcinoma cells.

Tobacco smoke contains various carcinogenic ingredients such as nicotine, acrolein, and benzopyrene; however, their effects on cancer treatment are not fully understood. Claudin-1 (CLDN1), a component of tight junctions, is involved in the increased resistance to anticancer drugs. In this study, we found that acrolein increases the mRNA and protein levels of CLDN1 in RERF-LC-AI cells derived from human lung squamous cell carcinoma (SCC). Acrolein increased the p-extracellular signal-regulated kinase (ERK) 1/2 levels without affecting the p-Akt level. The acrolein-induced elevation of CLDN1 expression was attenuated by U0126, a mitogen-activated protein kinase kinas (MEK) inhibitor. These results indicate that the activation of MEK/ERK pathway is involved in the acrolein-induced elevation of CLDN1 expression. In a spheroid model, acrolein suppressed the accumulation and toxicity of doxorubicin (DXR), which were rescued by CLDN1 silencing. The acrolein-induced effects were also observed in lung SCC-derived EBC-1 and LK-2 cells. Acrolein also increased the expression level of nuclear factor erythroid 2-related factor 2 (Nrf2), a transcription factor that regulates antioxidant and detoxifying genes, which were inhibited by CLDN1 silencing. In spheroid cells, the levels of reactive oxygen species were enhanced by acrolein, which was inhibited by CLDN1 silencing. Taken together, acrolein may reduce the anticancer drug-induced toxicity in human lung SCC cells mediated by high CLDN1 expression followed by the upregulation of Nrf2 signaling pathway.

Knockout of Rnf213 Ameliorates Cerebral Ischemic-reperfusion Injury by Inhibiting Neuronal Apoptosis Through the Akt/GSK-3ß/ß-catenin/Bcl-2 Pathway.

Previous studies by us and others have shown that RING finger protein 213 (RNF213) is associated with cerebrovascular disease and systemic vasculopathy. Indeed, Rnf213 mRNA expression is increased in cerebral ischemia reperfusion injury (CIRI). The purpose of the present study was to investigate the role of Rnf213 in CIRI. Using the middle cerebral artery occlusion (MCAO) model, we confirmed that the expression of RNF213 protein was significantly upregulated in neurons in the ischemic penumbra. Rnf213 knockout mice were successfully generated using CRISPR/Cas9 technology. According to TTC staining and Bederson neurological scale, removal of Rnf213 decreased brain infarct volume and improved neurological deficit score, although the restoration of cerebral blood flow after MCAO was similar in WT and Rnf213-/- mice. In addition, the levels of p-Akt, p-GSK-3ß, ß-catenin and Bcl-2 were significantly increased 24 h after MCAO in the ischemic penumbra of the Rnf213-/- mice compared to WT mice, indicating that Rnf213 removal may ameliorate neuronal apoptosis by regulating the Akt/GSK-3ß/ß-catenin/Bcl-2 signaling pathway. Taken together, our study reveals that Rnf213 regulates neuronal apoptosis in CIRI, therefore impacting on brain infarct volume in brain ischemia.

Elevation of Anticancer Drug Toxicity by Caffeine in Spheroid Model of Human Lung Adenocarcinoma A549 Cells Mediated by Reduction in Claudin-2 and Nrf2 Expression.

Claudin-2 (CLDN2), a component of tight junctions, is abnormally expressed in human lung adenocarcinoma tissue. CLDN2 contributes to chemoresistance in human lung adenocarcinoma-derived A549 cells, and it may be a target for cancer therapy. Here, we found that coffee ingredients, namely caffeine and theobromine, decreased the protein level of CLDN2 in human lung adenocarcinoma-derived A549 cells. In contrast, other components, such as theophylline and chlorogenic acid, had no effect. These results indicate that the 7-methyl group in methylxanthines may play a key role in the reduction in CLDN2 expression. The caffeine-induced reduction in the CLDN2 protein was inhibited by chloroquine, a lysosome inhibitor. In a protein-stability assay using cycloheximide, CLDN2 protein levels decreased faster in caffeine-treated cells than in vehicle-treated cells. These results suggest that caffeine accelerates the lysosomal degradation of CLDN2. The accumulation and cytotoxicity of doxorubicin were dose-dependently increased, which was exaggerated by caffeine but not by theophylline in spheroids. Caffeine decreased nuclear factor-erythroid 2-related factor 2 (Nrf2) levels without affecting hypoxia-inducible factor-1α levels. Furthermore, caffeine decreased the expression of Nrf2-targeted genes. The effects of caffeine on CLDN2 expression and anticancer-drug-induced toxicity were also observed in lung adenocarcinoma RERF-LC-MS cells. We suggest that caffeine enhances doxorubicin-induced toxicity in A549 spheroids mediated by the reduction in CLDN2 and Nrf2 expression.

Urine cytological study in patients with clinicopathologically confirmed neuronal intranuclear inclusion disease.

Objective: The diagnosis of neuronal intranuclear inclusion disease (NIID) is currently based on CGG repeat expansion in the 5'UTR of the NOTCH2NLC gene, or p62-positive intranuclear inclusions in skin biopsy. The purpose of this study is to explore the value of non-invasive pathological findings in urine sediment cells from NIID patients. Materials and methods: Ten patients with clinically suspected NIID were enrolled for skin biopsy and gene screening. Morning urine (500 ml) was collected from each patient, and cell sediment was obtained by centrifugation. Urine cytology, including Giemsa staining, p62 immunostaining, and electron microscopic examination, were conducted on cell sediment. Results: The main clinical symptoms of 10 patients included episodic disturbance of consciousness, cognitive impairment, tremor, limb weakness, and so on. Cerebral MRI showed that 9 patients had linear DWI high signal in the corticomedullary junction. Genetic testing found that the number of CGG repeat ranged from 96 to 158 in the NOTCH2NLC gene. Skin biopsy revealed that all patients showed p62-positive intranuclear inclusions in 18.5 ± 6.3% of the duct epithelial cells of sweat gland. In contrast, urine sediment smears revealed that only 3 patients had p62 positive intranuclear inclusions in 3.5 ± 1.2% of the sedimentary cells. Ultrastructural examinations showed that intranuclear inclusions were also identified in the cell sediment of the 3 patients. Conclusion: Urine cytology may be a new and non-invasive pathological diagnosis technique for some NIID patients, although the positive rate is not as high as that of skin biopsy, which is a sensitive and reliable pathological method for NIID.

Genetic spectrum in a cohort of patients with distal hereditary motor neuropathy.

BACKGROUND: Distal hereditary motor neuropathy (dHMN) is a heterogeneous group of diseases characterized by exclusive degeneration of peripheral motor nerves, while only 20.0-47.8% of dHMN patients are genetically identified. Recently, GGC expansion in the 5'UTR of NOTCH2NLC has been associated with dHMN. Accordingly, short tandem repeat (STR) should be further explored in genetically unsolved patients with dHMN. METHODS: A total of 128 patients from 90 unrelated families were clinically diagnosed as dHMN, and underwent a comprehensively genetic screening. Skin biopsies were conducted with routine protocols. RESULTS: Most patients showed chronic distal weakness of lower limbs (121/128), while 20 patients initially had asymmetrical involvements, 14 had subclinical sensory abnormalities, 11 had pyramidal impairments, five had cerebellar disturbance, and four had hyperCKmia. The rate of genetic detection was achieved in 36.7% (33/90), and the rate increased to 46.7% (42/90) if patients with variants uncertain significance were included. The most common causative genes included chaperone-related genes (8/33, 24.2%), tRNA synthetase genes (4/33, 12.1%), and cytoskeleton-related genes (4/33, 12.1%). Additionally, two dominant inherited families were attributed to abnormal expansion of GGC repeats in the 5'UTR of NOTCH2NLC; and a patient with dHMN and cerebellar symptoms had CAG repeat expansion in the ATXN2 gene. Skin biopsy from patients with GGC expansion in NOTCH2NLC revealed typical intranuclear inclusions on histological and ultrastructural examinations. INTERPRETATIONS: This study further extends the genetic heterogeneity of dHMN. Given some dHMN patients may be associated with nucleotides repeat expansion, STR screening is necessary to perform in genetically unsolved patients.

The 16MΔvjbR as an ideal live attenuated vaccine candidate for differentiation between Brucella vaccination and infection.

Brucellosis brings great economic burdens for developing countries. Live attenuated vaccines are the most efficient means for prevention and control of animal Brucellosis. However, the difficulties of differentiating of infection from vaccine immunization, which is essential for eradication programs, limit their applications. Therefore, the development of a vaccine that could differentiate infection from immunization will overcome the limitations and get extensive application. VjbR is a quorum sensing regulator involving in Brucella's intracellular survival. The vjbR∷Tn5 mutants have been proven effective against wild type strain challenge, implying its possibility of use in vaccine candidate development. To further evaluate this candidate gene, in the present study, the antigenicity of purified recombinant VjbR protein was analyzed. Antibodies to Brucella melitensis VjbR could be detected in sera from patients and animals with brucellosis but not in control ones, implying the potential use of this protein as a diagnostic antigen. Then a vjbR mutant of B. melitensis 16M was constructed by replacing the vjbR with kanamycin gene. The mutant showed reduced survival in macrophage and mice. Vaccination of BALB/c mice with 16MΔvjbR conferred significant protective immunity against B. melitensis strain 16M challenges, being equivalent to which induced by the license vaccine Rev.1. The vjbR deletion mutant elicited an anti-Brucella-specific immunoglobulin G response and induced the secretion of gamma interferon and interleukin-10. The most importance is that, the use of vjbR mutants as vaccines in association with diagnostic tests based on the VjbR antigen would allow the serological differentiation between infected and vaccinated animals. These results suggest that 16MΔvjbR is an ideal live attenuated vaccine candidate against B. melitensis and deserves further evaluation for vaccine development.