Guanabenz Acetate Induces Endoplasmic Reticulum Stress–Related Cell Death in Hepatocellular Carcinoma Cells

Background Development of chemotherapeutics for the treatment of advanced hepatocellular carcinoma (HCC) has been lagging. Screening of candidate therapeutic agents by using patient-derived preclinical models may facilitate drug discovery for HCC patients. Methods Four primary cultured HCC cells from surgically resected tumor tissues and six HCC cell lines were used for high-throughput screening of 252 drugs from the Prestwick Chemical Library. The efficacy and mechanisms of action of the candidate anti-cancer drug were analyzed via cell viability, cell cycle assays, and western blotting. Results Guanabenz acetate, which has been used as an antihypertensive drug, was screened as a candidate anti-cancer agent for HCC through a drug sensitivity assay by using the primary cultured HCC cells and HCC cell lines. Guanabenz acetate reduced HCC cell viability through apoptosis and autophagy. This occurred via inhibition of growth arrest and DNA damage-inducible protein 34, increased phosphorylation of eukaryotic initiation factor 2α, increased activating transcription factor 4, and cell cycle arrest. Conclusions Guanabenz acetate induces endoplasmic reticulum stress–related cell death in HCC and may be repositioned as an anti-cancer therapeutic agent for HCC patients

Donepezil Regulates LPS and Aβ-Stimulated Neuroinflammation through MAPK/NLRP3 Inflammasome/STAT3 Signaling

The acetylcholinesterase inhibitors donepezil and rivastigmine have been used as therapeutic drugs for Alzheimer’s disease (AD), but their effects on LPS-and Aβ-induced neuroinflam-matory responses and the underlying molecular pathways have not been studied in detail in vitro and in vivo. In the present study, we found that 10 or 50 μM donepezil significantly decreased the LPS-induced increases in the mRNA levels of a number of proinflammatory cytokines in BV2 mi-croglial cells, whereas 50 μM rivastigmine significantly diminished only LPS-stimulated IL-6 mRNA levels. In subsequent experiments in primary astrocytes, donepezil suppressed only LPS-stimulated iNOS mRNA levels. To identify the molecular mechanisms by which donepezil regulates LPS-induced neuroinflammation, we examined whether donepezil alters LPS-stimulated proin-flammatory responses by modulating LPS-induced downstream signaling and the NLRP3 inflam-masome. Importantly, we found that donepezil suppressed LPS-induced AKT/MAPK signaling, the NLRP3 inflammasome, and transcription factor NF-kB/STAT3 phosphorylation to reduce neuroin-flammatory responses. In LPS-treated wild-type mice, a model of neuroinflammatory disease, donepezil significantly attenuated LPS-induced microglial activation, microglial density/morphol-ogy, and proinflammatory cytokine COX-2 and IL-6 levels. In a mouse model of AD (5xFAD mice), donepezil significantly reduced Aβ-induced microglial and astrocytic activation, density, and mor-phology. Taken together, our findings indicate that donepezil significantly downregulates LPS-and Aβ-evoked neuroinflammatory responses in vitro and in vivo and may be a therapeutic agent for neuroinflammation-associated diseases such as AD. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.1

Dental Hygienist Job Analysis for Item Development for the Korean Dental Hygienists' Licensing Examination

This study aimed to develop standard items for the Korean Dental Hygienists' Licensing Examination; these items were also earmarked for use in developing the curriculum for dental hygienists, and in writing a job description, based on a job analysis using the Developing A Curriculum Method (DACUM). It also aimed to understand the significance and frequency of task elements that dental hygienists perform. Data were collected by means of a mail survey, in the form of self-entry, from a sample of dental hygienists registered with the Korean Dental Hygienists' Association. In all, 260 responses were analyzed. The tasks of dental hygienists were divided into four categories, 93 tasks, and 494 task elements. There were 281 elements (61%) that scored higher than 3.5 in significance, in the 4-scale items, and 480 elements (98%) that scored higher than 3.0. There were 30 elements (6%) that scored higher than 3.5 in frequency, and 140 elements (29%) that scored higher than 3.0 in frequency. Overall, 130 out of 494 elements (27%) scored higher than 3.0 for both significance and frequency. Therefore, those 130 elements should be included as items in the Korean Dental Hygienists' Licensing Examination. The results can also be used for curriculum development and as the basis of a job description for dental hygienists

Donepezil ameliorates Aβ pathology but not tau pathology in 5xFAD mice

Abstract The cholinesterase inhibitor donepezil is used to improve Aβ pathology and cognitive function in patients with Alzheimer’s disease (AD). However, the impact of donepezil on tau pathology is unclear. Thus, we examined the effects of donepezil on Aβ and tau pathology in 5xFAD mice (a model of AD) in this study. We found that intraperitoneal injection of donepezil (1 mg/kg, i.p.) exhibited significant reductions in Aβ plaque number in the cortex and hippocampal DG region. In addition, donepezil treatment (1 mg/kg, i.p.) reduced Aβ-mediated microglial and, to a lesser extent, astrocytic activation in 5xFAD mice. However, neither intraperitoneal/oral injection of donepezil nor oral injection of rivastigmine altered tau phosphorylation at Thr212/Ser214 (AT100), Thr396, and Thr231 in 5xFAD mice. Surprisingly, we observed that intraperitoneal/oral injection of donepezil treatment significantly increased tau phosphorylation at Thr212 in 5xFAD mice. Taken together, these data suggest that intraperitoneal injection of donepezil suppresses Aβ pathology but not tau pathology in 5xFAD mice

CD49f(high) cells retain sphere-forming and tumor-initiating activities in human gastric tumors.

Identification of gastric tumor-initiating cells (TICs) is essential to explore new therapies for gastric cancer patients. There are reports that gastric TICs can be identified using the cell surface marker CD44 and that they form floating spheres in culture, but we could not obtain consistent results with our patient-derived tumor xenograft (PDTX) cells. We thus searched for another marker for gastric TICs, and found that CD49f(high) cells from newly-dissected gastric cancers formed tumors with histological features of parental ones while CD49f(low) cells did not when subcutaneously injected into immunodeficient mice. These results indicate that CD49f, a subunit of laminin receptors, is a promising marker for human gastric TICs. We established a primary culture system for PDTX cells where only CD49f(high) cells could grow on extracellular matrix (ECM) to form ECM-attaching spheres. When injected into immunodeficient mice, these CD49f(high) sphere cells formed tumors with histological features of parental ones, indicating that only TICs could grow in the culture system. Using this system, we found that some sphere-forming TICs were more resistant than gastric tumor cell lines to chemotherapeutic agents, including doxorubicin, 5-fluorouracil and doxifluridine. There was a patient-dependent difference in the tumorigenicity of sphere-forming TICs and their response to anti-tumor drugs. These results suggest that ECM plays an essential role for the growth of TICs, and that this culture system will be useful to find new drugs targeting gastric TICs

Case description and tumorigenic activity of CD49f^high and CD49f^low tumor cells.

***<p>P<0.001 by Chi-square test.</p

Sphere cells form tumors with histological features of parental ones.

<p>Light micrographs of tumors formed by subcutaneous injection of sphere cells into NOD-SCID mice (right panels), which are obtained by cultures of (A) unsorted HGC-1, (B) unsorted HGC-2 and (C) unsorted HGC-4 tumor cells. Histological features of tumors formed in mice correspond well with those of parental ones (left panels). Tissue specimens are stained with hematoxylin and eosin. Scale bars represent 50 µm.</p

Tumorigenicity of sphere cells obtained by culture of unsorted cells (number of tumors formed in NOD-SCID mice/number of injection of tumor cells).

**<p>P<0.01;</p>*<p>P<0.05 by Chi-square test;</p><p>–not determined.</p