Trafficking of Full-Length and N-Terminally Truncated Cathepsin B in Human Colorectal Carcinoma Cells

Cathepsin B is an endo-lysosomal cysteine protease. However, its increased expression and altered localization to the extracellular space, to mitochondria, or to the nucleus has been linked to tumor progression. In the present study, we show enhanced levels of cathepsin B in adenocarcinoma tissue in comparison to adjacent normal colon. Additionally, cathepsin B was observed in the nuclear compartment of mucosal cells in adenocarcinoma tissue samples and in the nuclei of the colorectal carcinoma cell line HCT116. Accordingly, a distinct 40-kDa form of cathepsin B was detected in HCT116 cells, which is proposed to represent a specific form lacking the signal peptide and parts of the propeptide. Trafficking studies with an EGFP-tagged N-terminally truncated form, mimicking the 40-kDa form, demonstrated accumulation in aggresome-like inclusion bodies, while EGFP-tagged full-length cathepsin B revealed regular sorting to endo-lysosomes. We conclude that the identity of nuclear cathepsin B in colorectal adenocarcinoma (in situ) and in carcinoma cells (in vitro) cannot be attributed to either full-length or 40-kDa N-terminally truncated cathepsin B forms. Hence, future studies are needed to demonstrate which form/s of cathepsin B may be sorted to the nuclei of colorectal carcinoma cells, and whether redundant regulation of related cathepsin expression occurs.

Effect of KLF17 overexpression on epithelial–mesenchymal transition of gastric cancer cells

Objective To investigate Krüppel-like factor 17 ( KLF17) expression in normal and gastric cancer tissues and cell lines. Methods Levels of KLF17 mRNA and protein in GES-1 normal gastric mucosal cells, and NCI-N87, SGC-7901, BGC-823 and HGC-27 gastric cancer cells were analysed by quantitative polymerase chain reaction (qPCR) and western blot. Differences in KLF17 expression between gastric cancer and adjacent tissues were analysed by qPCR and immunohistochemistry. Invasion/migration effects of KLF17 overexpression in BGC-823 and HGC-27 cells were analysed by wound-healing and Transwell chamber assays. Changes in expression of KLF17 and epithelial–mesenchymal transition (EMT)-related genes (matrix metalloproteinase [MMP]-9, vimentin and E-cadherin) were analysed in BGC-823 and HGC-27 cells before and after transfection using qPCR and western blot. Transforming growth factor (TGF)-β1, Smad family member (Smad)2/3 and phosphorylated-Smad2/3 levels in BGC-823 and HGC-27 cells were assessed by qPCR and western blot. Results KLF17 expression was lower in gastric cancer versus adjacent tissues, and in gastric cancer cell lines versus GES-1 normal gastric mucosal cells, and was positively correlated with degree of cancer-cell differentiation. Wound-healing and Transwell assays showed decreased migration and invasion ability of BGC-823 and HGC-27 cells transfected to overexpress KLF17. KLF17 overexpression was associated with decreased MMP-9 and vimentin in BGC-823 and HGC-27 cancer cells, and increased KLF17 and E-cadherin. KLF17 overexpression also resulted in decreased levels of TGF-β1 and p-Smad2/3 in BGC-823 and HGC-27 cancer cells. Conclusion KLF17 is poorly expressed in gastric cancer tissues and cell lines. KLF17 overexpression might inhibit EMT via the TGF-β/Smad pathway, thereby reducing gastric cancer cell invasion and migration. Therefore, KLF17 may become a novel target for treating gastric cancer.

In Vivo Evaluation of Micronucleus Frequencies in Buccal Mucosal Cells of Orthodontic Patients with and Without Fluoride Use

Introduction: Fluoride agents to prevent white spot lesions are used often during orthodontic treatment. The beneficial effects of fluoride, when consumed within permissible limits on dental structures, are well known. Their implications on underlying biological tissues, however, are unknown. Mouthwashes and dentifrices with fluorides are associated with metal ion release into the mouth with possible cell genotoxicity. Since these cariostatic agents are frequently used during orthodontic therapy, a deeper understanding of the effects of fluoride on oral tissues was considered necessary. Methodology: Three groups of patients (30 each)—group 1 (untreated controls), group 2 (non-fluoridated), and group 3 (Fluoridated) were analyzed. Patients in groups 2 and 3 were bonded with the same bracket prescription and treated with similar archwire sequences. Buccal mucosal cells at 4 specific time periods (before treatment, 1 week, 30 days, and 6 months) were collected, using a wooden tongue depressor, and assessed for any nuclear abnormalities. Comparisons of changes were made with an untreated control group and also between the non-fluoridated and fluoridated groups. Relevant conclusions were drawn after analysis of the results. Results: Greater number of nuclei were observed at the 30-day time interval in the fluoridated group, which was statistically significant at P < .001. Conclusion: Use of fluoridated oral hygiene products in patients undergoing fixed orthodontic treatment with NiTi archwires could increase the risk of micronuclei formation in buccal mucosal cells.

The Association of EBV and Gastric Cancer

The present study aimed to investigate the clinicopathological features of EBV positive and EBV negative gastric cancer patients and the expression levels of proliferative, apoptotic and cell signaling proteins in tissues samples from these patients. The biological role of EBV infection was assessed in gastric cancer. Results: EBV was localized in the nuclei of gastric cancer cells (positive rate 6.86%). The infection rate of EBV in normal gastric mucosal cells, which were adjacent to cancer tissues, was 0. The difference noted was significant (P = 0. 023). The expression levels of caspase-3 (P = 0.0423), FASL (P = 0.00297) and cyclin D1 (P = 0.0345) proteins were significantly different in EBV positive and negative gastric cancer tissues. When the parameters gender, age, Lauren classification, histological grade, early and advanced tumor stage, vascular and nerve invasion, TNM grade and survival status were compared, the maximum tumor diameter, number of lymph node metastasis, caspase-8, Ki67 and P53 protein expression did not reveal significant differences. Bcl-2 protein expression was positive in only one gastric cancer cell sample and negative in the other gastric cancer cell samples as well as in the corresponding normal gastric mucosal epithelial cells. However, significant differences were noted with regard to the positive expression of Bcl-2 in the immune cells of gastric cancer and adjacent tissues (P = 1.17749E-39). The expression levels of Bcl-2 in the immune cells of EBV positive and EBV negative gastric cancer tissues were not significantly different. The expression levels of caspase-8, caspase-3, FASL, Ki67, cyclin D1 and P53 proteins in gastric cancer cells were significantly different compared to those of normal gastric mucosal cells derived from adjacent tissues (P < 0.05). These findings were noted in both EBV positive and/or EBV negative gastric cancer cases (P < 0.05). The survival time of the patients with EBV positive gastric cancer was higher than that of the patients with EBV negative gastric cancer, whereas the differences were not significantly different. The aforementioned results suggested that the EBV virus may directly infect cancerous cells but not normal gastric mucosal cells. With the exception of caspase-3, the expression levels of the proteins FASL and cyclin D1 were closely associated with EBV-positive gastric cancer. EBV did not have a specific effect on the expression of the signaling molecules associated with proliferation and apoptosis of gastric cancer cells. Its effect on gastric cancer cells may be associated with other factors and requires further discussion. No significant differences were noted in the clinicopathological features of EBV positive compared to those of EBV negative gastric cancer patients. However, the prognosis of EBV positive gastric cancer patients was better than that of EBV negative gastric cancer patients. The mechanism of action associated with these processes requires further verification.

Quyu Shengxin Decoction Alleviates DSS-Induced Ulcerative Colitis in Mice by Suppressing RIP1/RIP3/NLRP3 Signalling

Purpose. To study the therapeutic effect of Quyu (QY) Shengxin (SX) decoction (QYSXD) in mice with dextran sulfate sodium- (DSS-) induced ulcerative colitis and to investigate the effects of QYSXD on the regulation of the receptor-interacting protein kinase 1 (RIP1)/receptor-interacting protein kinase 3 (RIP3)/nucleotide-binding oligomerization domain-like receptor family pyrin domain protein 3 (NLRP3) signaling pathway. Method. Thirty-six mice were randomly divided into the following 6 groups: the experimental group (QYSX group), the model group (DSS group), the positive control group (5-aminosalicylic acid (5-ASA) group), the control group, the first component group (QY group), and the second component group (SX group). Each group included 6 mice. Ulcerative colitis (UC) was induced in the mice by providing 3.5% DSS in drinking water. The mice were weighed every day to evaluate the disease activity index (DAI). After 7 days, the mice were sacrificed, and colonic tissues were obtained for colon length measurement. The morphological changes in the colon and the pathological scores of the mice in each group were observed by hematoxylin-eosin (HE) staining. The messenger ribonucleic acid (mRNA) and protein expression levels of RIP1, RIP3, dynamin-related protein 1 (Drp1), NLRP3, cysteinyl aspartate specific proteinase-1 (caspase-1), interleukin (IL)-1β, and IL-18 in the colon tissues of the mice in each group were detected and compared by real-time quantitative reverse transcription PCR (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA). The levels of RIP1, RIP3, NLRP3, IL-1β, and IL-8 in the colonic mucosa were detected by ELISA. Western blotting was used to compare the protein expression of Drp1, caspase-1, mitochondrial fission protein 1 (FIS1), and mitophagy-associated protein light chain 3a/b (LC3a/b) among groups. The levels of reactive oxygen species (ROS) in the colonic mucosal cells were compared by immunofluorescence. Results. Compared with those in the DSS group, the mice with DSS-induced colitis in the QYSX group exhibited clearly higher body weights ( P < 0.05 ) and DAI scores ( P < 0.05 ). The colon lengths of the mice in the QYSX group were longer than those in the DSS group ( P < 0.05 ), and the pathological score of the QYSX group was lower than that of the DSS group ( P < 0.05 ). The RIP1, RIP3, Drp1, IL-1β, IL-18, and caspase-1 mRNA levels in the QYSX, 5-ASA, SX, and QY groups were significantly lower than those in the DSS group ( P < 0.05 ), but there were no differences between the QYSX group and the 5-ASA group. The levels of RIP1, RIP3, NLRP3, IL-1β, and IL-18 in the QYSX group were lower than those in the DSS group ( P < 0.01 ). The levels of Drp1, caspase-1, FIS1, and LC3a/b in the QYSX group and the 5-ASA group were lower than those in the DSS group ( P < 0.05 ). The levels of ROS in the colonic mucosal cells in the QYSX, 5-ASA, and QY groups were lower than those in the DSS group ( P < 0.05 ). Conclusion. QYSXD has certain therapeutic effects on DSS-induced colitis in mice and may be as effective as 5-ASA. QY and SX decoctions also have certain effects on colitis; however, these decoctions are not as beneficial as QYSXD. QYSXD may ameliorate colitis by inhibiting the expression of RIP1/RIP3/NLRP3 pathway-related proteins and reversing mitochondrial dysfunction to control inflammation.

Effects of mobile phone radiation on buccal mucosal cells based on specific absorption rate: a cross sectional study

Background: The usage of mobile phone had been increased drastically which created a major health concern among the people about the radiation emitted from mobile phones. Hence this study was aimed to evaluate the Micronuclei (MN) frequency in exfoliated oral mucosal cells in high and low mobile users based on Specific absorption rate (SAR) value.Methods: The total of 60 subjects were divided into two major groups: low SAR mobile phone users and high SAR mobile phone users. Further, Subjects who use mobile phone for more than 3 hours a week was considered as high talk time users and less than 3 hours a week was low talk time users. The buccal mucosa cells extracted by slightly scraping the oral cavity with a wooden spatula. For staining, Giemsa stain was used. Micronuclei were evaluated in 1000 cells per individual at the microscope.Results: The result show prolonged talk time may interfere with the development of micronuclei in individuals who use mobile phone for more than 3 hours per week rather than high SAR value showing a significant increase in frequency of micronuclei formation.Conclusions: The study showed mobile phone radiation had adverse effects on buccal mucosal cells.

The expression of RIPK3 is associated with cell turnover of gastric mucosa in the mouse and human stomach

Necroptosis is a novel manner of programmed cell death and important for tissue development, homeostasis, damage, and repair. Activation of receptor-interacting protein kinase 3 (RIPK3), a key member of receptor-interacting protein family in contributing significantly to necroptosis, in tissues is a hallmark of cells dying by necroptosis. However, there are few studies that examine the expression of RIPK3 in the glandular cells of stomachs under physiological condition. We have therefore conducted this study to immunohistochemically characterize the key element of necroptosis, RIPK3, in the mouse and human stomach. Results showed that RIPK3 positive cells could be observed in the surface mucosal cells, granular cells, and lamina propria cells in both mouse and human stomach tissues. Ratios of PCNA/RIPK3 positive cells in the glandular cells were ~ 2.1 in mouse and ~ 4.15 in human sections respectively. Morphological and double immunofluorescence analysis confirmed that these RIPK3 positive cells were mucous, parietal and lamina propria cells. Our results indicate that the expression of RIPK3 in different cell types might contribute to cell turnover of gastric mucosa in the mouse and human stomach under physiological condition.

CLN8 Mutations Presenting with a Phenotypic Continuum of Neuronal Ceroid Lipofuscinosis—Literature Review and Case Report

CLN8 is a ubiquitously expressed membrane-spanning protein that localizes primarily in the ER, with partial localization in the ER-Golgi intermediate compartment. Mutations in CLN8 cause late-infantile neuronal ceroid lipofuscinosis (LINCL). We describe a female pediatric patient with LINCL. She exhibited a typical phenotype associated with LINCL, except she did not present spontaneous myoclonus, her symptoms occurrence was slower and developed focal sensory visual seizures. In addition, whole-exome sequencing identified a novel homozygous variant in CLN8, c.531G>T, resulting in p.Trp177Cys. Ultrastructural examination featured abundant lipofuscin deposits within mucosal cells, macrophages, and monocytes. We report a novel CLN8 mutation as a cause for NCL8 in a girl with developmental delay and epilepsy, cerebellar syndrome, visual loss, and progressive cognitive and motor regression. This case, together with an analysis of the available literature, emphasizes the existence of a continuous spectrum of CLN8-associated phenotypes rather than a sharp distinction between them.