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Next‐generation tobacco products and nicotine delivery systems such as heat‐not‐burn tobacco products and electronic cigarettes, the usage of which is expected to have a beneficial impact on public health, have gained popularity over the past decade. However, the risks associated with the long‐term use of such products are still incompletely understood. Although the risks of these products should be clarified through epidemiological studies, such studies are normally performed based on each product category, not product‐by‐product. Therefore, investigation of the risk on a product‐by‐product basis is important to provide specific scientific evidence. In the current study, we performed the 40‐day repeated exposure of in vitro human bronchial epithelial tissues to cigarette smoke (CS) or vapor from our proprietary novel tobacco vapor product (NTV). In addition, tissue samples exposed to CS were switched to NTV or CS exposure was stopped at 20 days to reflect a situation where smokers switched to NTV or ceased to smoke. All tissue samples were assessed in terms of toxicity, inflammation and transcriptomic alterations. Tissue samples switched to NTV and the cessation of exposure samples showed recovery from CS‐induced damage although there was a time‐course difference. Moreover, repeated exposure to NTV produced negligible effects on the tissue samples while CS produced cumulative effects. Our results suggest that the use of NTV, including switching to NTV from cigarette smoking, has fewer effects on bronchial epithelial tissues than continuing smoking. We carried out the 40‐day repeated exposure of in vitro bronchial epithelial tissues to cigarette smoke (CS) or vapor from novel tobacco vapor product (NTV) and intermediate switching from CS exposure to NTV exposure. Long‐term exposure to NTV resulted in negligible effect on the tissues. Moreover, the tissues that intermediately switched to NTV exposure showed recovery from CS‐induced damage similar to exposure cessation. These results implied that exposure to NTV showed few effects on bronchial epithelial tissues. [ABSTRACT FROM AUTHOR]

Cigarette smoke (CS) is a complex mixture of chemicals and interacts with various physiological processes. We previously reported that nuclear factor erythroid 2‐related factor 2 (NRF2) was the most sensitive transcription factor to aqueous CS extract (AqCSE) exposure in monolayer cultured human bronchial epithelial cell lines. Recently, in vitro three‐dimensional (3D) culture models have been used to supplement pharmacological and toxicological assessments. Bronchial epithelium models in particular are useful for the evaluation of substances that directly contact the respiratory tract, such as CS. In the present study, we used 3D‐cultured human bronchial epithelial cells (HBECs) to assess activation of transcription factors and relevant gene expression in response to AqCSE, primarily focusing on NRF2 and nuclear factor‐kappa B (NF‐κB) pathways. The 3D‐cultured HBECs exposed to AqCSE showed expression of NRF2 and its nuclear translocation in addition to upregulation of genes related to oxidative stress. Our results suggest that the NRF2 pathway was the dominant pathway when 3D‐cultured HBECs were exposed to AqCSE at a low dose, supporting our previous findings that NRF2 was the most sensitive transcription factor in response to AqCSE. Expression and nuclear translocation of NF‐κB were not increased, although proinflammatory genes were upregulated. However, another inflammation‐related transcription factor, activation protein 1, was induced by AqCSE. Gene classification analysis suggested that induction of the inflammatory response by AqCSE was dependent on NRF2 and activation protein 1 rather than NF‐κB. We assessed activation of transcription factors and relevant signaling pathways when human bronchial epithelial cells in three‐dimensional culture were exposed to aqueous cigarette smoke extract (AqCSE). Our results suggest that the nuclear factor erythroid 2‐related factor 2 (NRF2) pathway is the dominant pathway at low‐dose exposure, supporting our previous findings that NRF2 is the most sensitive transcription factor to AqCSE. Moreover, the AqCSE‐induced inflammatory response is dependent on NRF2 and activation protein 1 rather than nuclear factor‐kappa B. [ABSTRACT FROM AUTHOR]

Radiolabeled antibodies, polyethylene glycol-conjugated (PEGylated) peptides, liposomes, and other materials were investigated as positron-emission tomography (PET) probes. These substances accumulate in tumors but often remain too long in circulation. We investigated the combination of intravenous urokinase injection and its substrate linker as a triggered radioisotope clearance enhancement system to improve imaging contrast. To this end, we synthesized a four-arm PEGylated 64 Cu-bombesin analog tetramer with a urokinase substrate linker. In mouse blood, it was almost perfectly cleaved and degraded into smaller radioactive fragments in vitro with urokinase (≥20,000 IU/mL). In mouse blood circulation, ∼50–65% of the probe was rapidly degraded after the urokinase injection and the radioactive fragments were eliminated mainly from the kidney. In contrast, tumor radioactivity levels did not change, and therefore, the tumors were clearly visualized. The tumor/blood ratio, an indicator of imaging contrast, increased 2.5 times, while elimination of the radioisotope from the blood was enhanced. This approach has the potential to improve imaging contrast using various PET probes. It could also shorten the time required to obtain sufficient contrast and decrease patient radiation exposure. [ABSTRACT FROM AUTHOR]

CCHH-type zinc fingers are among the most common DNA binding motifs found in eukaryotes. In previous report, we substituted the second ligand cysteine residue with aspartic acid, producing Zn(II)-responsive transcription factor; this indicates that a ligand substitution is a possible design target of an engineered zinc finger peptide. Despite the importance of Zn(II) binding with respect to the folding and DNA binding properties of a zinc finger peptide, no study about the effects of ligand substitution on both Zn(II) binding and DNA binding properties has been reported. Here, we substituted a conserved cysteine (C) with other zinc-coordinated amino acid residues, histidine (H), aspartic acid (D), and glutamic acid (E), to create CXHH-type zinc finger peptides (X = C, H, D, and E). The Zn(II)-dependent conformational change was observed in oil peptides; however, the Zn(II) binding affinity and metal coordination geometry of the peptides were different. Gel mobility shift assays showed that the Zn(II)-bound forms of the ligand-substituted derivatives retain DNA binding ability, while the DNA binding affinity decreased in the following manner: CCHH CDHH > CEHH >> CHHH. The DNA binding sequence preferences of the ligand-substituted derivatives were similar to that of the wild type in the contest of the full three-finger DNA-binding domain of transcription factor Zif268. These results indicate that artificial zinc finger proteins with various DNA binding affinities that respond to a diverse range of Zn(II) concentrations can be designed by substituting the Zn(II) ligand. [ABSTRACT FROM AUTHOR]

Proteasome inhibitors are a novel class of molecular-targeted anti-cancer drugs that suppress the degradation of malfolded proteins, trigger endoplasmic reticulum (ER) stress, and activate apoptosis signals. Glucose-regulated protein 78 (GRP78), a major ER chaperone, is one of the most important molecules for transduction of unfolded protein response (UPR) signals. In accordance with past findings that expression of GRP78 is elevated in cancer cells and helps to resist stress-induced apoptosis, GRP78 knockdown could be effective in anticancer therapy. We tested this hypothesis and found that transfection of small interfering RNA (siRNA) targeting GRP78 inhibited the growth of RENCA renal carcinoma cells, in association with elevated gene expression of UPR downstream signaling molecules (CHOP, EDEM1, and ERdj4 mRNA). In addition, the combinatorial effect of GRP78 siRNA with ER stress inducers (tunicamycin, MG132, and 2-deoxyglucose) on survival was measured. Combination of GRP78 siRNA and the ER stress inducers more extensively reduced cell viability than combination with scrambled siRNA. Besides RENCA, B16BL6 melanoma cells were also shown to be sensitive to GRP78 siRNA. These results suggest that GRP78 knockdown might be an effective strategy for cancer therapy targeting UPR-induced apoptosis.