Erratum: Cedrol, a Sesquiterpene Isolated from Juniperus chinensis, Inhibits Human Colorectal Tumor Growth associated through Downregulation of Minichromosome Maintenance Proteins.

[This corrects the article on p. 221 in vol. 27, PMID: 36713942.].

Induction of Cell Cycle Arrest, Apoptosis, and Reducing the Expression of MCM Proteins in Human Lung Carcinoma A549 Cells by Cedrol, Isolated from Juniperus chinensis.

Proteins related to DNA replication have been proposed as cancer biomarkers and targets for anticancer agents. Among them, minichromosome maintenance (MCM) proteins, often overexpressed in various cancer cells, are recognized both as notable biomarkers for cancer diagnosis and as targets for cancer treatment. Here, we investigated the activity of cedrol, a single compound isolated from Juniperus chinensis, in reducing the expression of MCM proteins in human lung carcinoma A549 cells. Remarkably, cedrol also strongly inhibited the expression of all other MCM protein family members in A549 cells. Moreover, cedrol treatment reduced cell viability in A549 cells, accompanied by cell cycle arrest at the G1 phase, and enhanced apoptosis. Taken together, this study broadens our understanding of how cedrol executes its anticancer activity while demonstrating that cedrol has potential application in the treatment of human lung cancer as an inhibitor of MCM proteins.

The preventive effect of loganin on oxidative stress-induced cellular damage in human keratinocyte HaCaT cells.

Loganin is a type of iridoid glycosides isolated from Corni fructus and is known to have various pharmacological properties, but studies on its antioxidant activity are still lacking. Therefore, in this study, the preventive effect of loganin on oxidative stress-mediated cellular damage in human keratinocyte HaCaT cells was investigated. Our results show that loganin pretreatment in a non-toxic concentration range significantly improved cell survival in hydrogen peroxide (H2O2)-treated HaCaT cells, which was associated with inhibition of cell cycle arrest at the G2/M phase and induction of apoptosis. H2O2-induced DNA damage and reactive oxygen species (ROS) generation were also greatly reduced in the presence of loganin. Moreover, H2O2 treatment enhanced the cytoplasmic release of cytochrome c, upregulation of the Bax/Bcl-2 ratio and degradation of cleavage of poly (ADP-ribose) polymerase, whereas loganin remarkably suppressed these changes. In addition, loganin obviously attenuated H2O2-induced autophagy while inhibiting the increased accumulation of autophagosome proteins, including as microtubule-associated protein 1 light chain 3-II and Beclin-1, and p62, an autophagy substrate protein, in H2O2-treated cells. In conclusion, our current results suggests that loganin could protect HaCaT keratinocytes from H2O2-induced cellular injury by inhibiting mitochondrial dysfunction, autophagy and apoptosis. This finding indicates the applicability of loganin in the prevention and treatment of skin diseases caused by oxidative damage.

Three-Dimensional Skin Tissue Printing with Human Skin Cell Lines and Mouse Skin-Derived Epidermal and Dermal Cells.

Since the skin covers most surfaces of the body, it is susceptible to damage, which can be fatal depending on the degree of injury to the skin because it defends against external attack and protects internal structures. Various types of artificial skin are being studied for transplantation to repair damaged skin, and recently, the production of replaceable skin using three-dimensional (3D) bioprinting technology has also been investigated. In this study, skin tissue was produced using a 3D bioprinter with human skin cell lines and cells extracted from mouse skin, and the printing conditions were optimized. Gelatin was used as a bioink, and fibrinogen and alginate were used for tissue hardening after printing. Printed skin tissue maintained a survival rate of 90% or more when cultured for 14 days. Culture conditions were established using 8 mM calcium chloride treatment and the skin tissue was exposed to air to optimize epidermal cell differentiation. The skin tissue was cultured for 14 days after differentiation induction by this optimized culture method, and immunofluorescent staining was performed using epidermal cell differentiation markers to investigate whether the epidermal cells had differentiated. After differentiation, loricrin, which is normally found in terminally differentiated epidermal cells, was observed in the cells at the tip of the epidermal layer, and cytokeratin 14 was expressed in the lower cells of the epidermis layer. Collectively, this study may provide optimized conditions for bioprinting and keratinization for three-dimensional skin production.

Cedrol, a Sesquiterpene Isolated from Juniperus chinensis, Inhibits Human Colorectal Tumor Growth associated through Downregulation of Minichromosome Maintenance Proteins.

Cedrol, a sesquiterpene alcohol, isolated from Juniperus chinensis has been reported to inhibit minichromosome maintenance (MCM) proteins as cancer biomarkers in human lung cancer in vitro. In the present study, we investigated the anti-cancer activity of cedrol in vitro and in vivo using human colorectal cancer HT29 cells and a human colorectal tumor xenograft model. Cedrol inhibited MCM protein expression and cell growth in HT29 cells, which are associated with G1 arrest and the induction of apoptosis. We demonstrated that cedrol effectively reduced HT29 tumor growth without apparent weight loss in a human tumor xenograft model. Compared with vehicle- and adriamycin-treated tumor tissues, cedrol induced changes in the tumor tissue structure, resulting in a reduced cell density within the tumor parenchyma and reduced vascularization. Moreover, the expression of MCM7, an important subunit of MCM helicase, was significantly suppressed by cedrol in tumor tissue. Collectively, these results suggest that cedrol may act as a potential anti-cancer agent for colorectal cancer by inhibiting MCM protein expression and tumor growth.

The anti-cancer effect of betulinic acid in u937 human leukemia cells is mediated through ROS-dependent cell cycle arrest and apoptosis.

Although previous studies have shown anti-cancer activity of betulinic acid (BA), a pentacyclic triterpenoid, against various cancer lines, the underlying molecular mechanisms are not well elucidated. In this study, we evaluated the mechanisms involved in the anti-cancer efficacy of BA in U937 human myeloid leukemia cells. BA exerted a significant cytotoxic effect on U937 cells through blocking cell cycle arrest at the G2/M phase and inducing apoptosis, and that the intracellular reactive oxygen species (ROS) levels increased after treatment with BA. The down-regulation of cyclin A and cyclin B1, and up-regulation of cyclin-dependent kinase inhibitor p21WAF1/CIP1 revealed the G2/M phase arrest mechanism of BA. In addition, BA induced the cytosolic release of cytochrome c by reducing the mitochondrial membrane potential with an increasing Bax/Bcl-2 expression ratio. BA also increased the activity of caspase-9 and -3, and subsequent degradation of the poly (ADP-ribose) polymerase. However, quenching of ROS by N-acetyl-cysteine, an ROS scavenger, markedly abolished BA-induced G2/M arrest and apoptosis, indicating that the generation of ROS plays a key role in inhibiting the proliferation of U937 cells by BA treatment. Taken together, our results provide a mechanistic rationale that BA exhibits anti-cancer properties in U937 leukemia cells through ROS-dependent induction of cell cycle arrest at G2/M phase and apoptosis.

Alcohol stimulates the proliferation of mouse small intestinal epithelial cells via Wnt signaling.

The intestinal epithelium is one of the fastest renewing tissues in mammals and is a barrier against toxic substances such as alcohol. Excessive alcohol can induce intestinal damage leading to intestinal bowel diseases. Thus, the control of small intestinal epithelial cell (IEC) regeneration is thought to be important for homeostasis in response to epithelium damage. However, reports on how epithelial cells respond to small intestinal damage are scarce. We investigated the effects of alcohol consumption on small intestinal epithelial cells of mice. To verify that alcohol altered the small intestinal epithelium, we used 8-10 weeks old male C57BL/6J mice for models of chronic and binge alcohol consumption (the NIAAA model) in addition to an organoid model. Alcohol promoted the proliferative activity of IECs and intestinal stem cells (ISCs) in intestinal crypts. Alcohol consumption increased expression of the proliferation marker cyclin D1 and activated the p44/42 MAPK (Erk1/2) signaling pathway in small intestinal epithelial cells. The Wnt target genes were markedly increased in IECs from alcohol-treated mice. In the small intestinal organoid model exposed to alcohol, the organoid area and numbers of buds increased with alcohol concentrations up to 0.5% similar to in vivo observations. These results suggest that alcohol consumption stimulates the proliferation of small intestinal epithelial cells via Wnt signaling.

Curcumin Blocks Naproxen-Induced Gastric Antral Ulcerations through Inhibition of Lipid Peroxidation and Activation of Enzymatic Scavengers in Rats.

Curcumin is a polyphenol derived from the plant Curcuma longa, which is used for the treatment of diseases associated with oxidative stress and inflammation. The present study was undertaken to determine the protective effect of curcumin against naproxen-induced gastric antral ulcerations in rats. Different doses (10, 50, and 100 mg/kg) of curcumin or vehicle (curcumin, 0 mg/kg) were pretreated for 3 days by oral gavage, and then gastric mucosal lesions were caused by 80 mg/kg naproxen applied for 3 days. Curcumin significantly inhibited the naproxen-induced gastric antral ulcer area and lipid peroxidation in a dose-dependent manner. In addition, curcumin markedly increased activities of radical scavenging enzymes, such as superoxide dismutase (SOD), catalase, and glutathione peroxidase in a dose-dependent manner. Specifically, 100 mg/kg curcumin completely protected the gastric mucosa against the loss in the enzyme, resulting in a drastic increase of activities of radical scavenging enzymes up to more than the level of untreated normal rats. Histological examination obviously showed that curcumin prevents naproxen-induced gastric antral ulceration as a result of direct protection of the gastric mucosa. These results suggest that curcumin blocks naproxen-induced gastric antral ulcerations through prevention of lipid peroxidation and activation of radical scavenging enzymes, and it may offer a potential remedy of gastric antral ulcerations.

Sorbus rufopilosa Extract Exhibits Antioxidant and Anticancer Activities by Inducing Cell Cycle Arrest and Apoptosis in Human Colon Adenocarcinoma HT29 Cells.

BACKGROUND: Sorbus rufopilosa, a tsema rowan, is a species of the small ornamental trees in the genus Sorbus and the family Rosaceae found in East Asia. The bioactivities of S. rufopilosa have not yet been fully determined. The objective of this study is to evaluate the antioxidant and anticancer effects of ethanol extract of S. rufopilosa (EESR) and to determine the molecular mechanism of its anticancer activity in human colon carcinoma HT29 cells. METHODS: To examine the antioxidant activity of EESR, 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity assay was performed. Inhibitory effect of EESR on cancer cell growth and proliferation was determined by water-soluble tetrazolium salt assay. To investigate the mechanism of EESR-mediated cytotoxicity, HT29 cells were treated with various concentrations of EESR and the induction of cell cycle arrest and apoptosis was analyzed by flow cytometry, 4,6-diamidino-2-phenylindole staining, and Western blot analysis. RESULTS: EESR showed significant antioxidant activity and inhibitory effect on HT29 cell growth in a dose-dependent manner. EESR induced cell cycle arrest at G2/M phase in a dose-dependent manner by modulating cyclin B, cyclin-dependent kinase 1 (CDK1), and CDK inhibitor p21 expression. EESR-induced apoptosis was associated with the upregulation of p53, a death receptor Fas, and a pro-apoptotic protein Bax and the activation of caspase 3, 8, and 9, resulting in the degradation of PARP. CONCLUSIONS: EESR possessing antioxidant activity efficiently inhibits proliferation of HT29 cells by inducing both cell cycle arrest and apoptosis. EESR may be a possible candidate for the anticancer drug development.

Widdrol, a sesquiterpene isolated from Juniperus chinensis, inhibits angiogenesis by targeting vascular endothelial growth factor receptor 2 signaling.

Widdrol is an odorous compound derived from Juniperus chinensis that is widely used in traditional medicine to treat fever, inflammation and cancer. It was previously reported that widdrol has antitumor activity by apoptosis induction in cancer cells in vitro. However, its anti-angiogenic activity remains elusive. In the present study, we investigated the anti­angiogenic activity of widdrol and the molecular mechanisms involved. Widdrol inhibited cell proliferation via G1 phase arrest induction in human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner. Additionally, it was associated with a decreased expression of cyclin-dependent kinase 2 (CDK2) and an increased expression of p21, a CDK inhibitor. Widdrol significantly inhibited the cell migration and tube formation of HUVECs using an in vitro angiogenesis assay. The results showed that widdrol suppressed phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2) and its downstream proteins, such as AKT, focal adhesion kinase (FAK) and endothelial nitric oxide synthase (eNOS). Moreover, widdrol effectively reduced tumor growth and blood vessel formation in colon tumor xenograft mice. Collectively, these results suggested that widdrol may act as a potential anti-angiogenic agent by inhibiting vessel sprouting and growth, which may have implications for angioprevention.

Anti-cancer Activity of Osmanthus matsumuranus Extract by Inducing G2/M Arrest and Apoptosis in Human Hepatocellular Carcinoma Hep G2 Cells.

BACKGROUND: Osmanthus matsumuranus, a species of Oleaceae, is found in East Asia and Southeast Asia. The bioactivities of O. matsumuranus have not yet been fully understood. Here, we studied on the molecular mechanisms underlying anti-cancer effect of ethanol extract of O. matsumuranus (EEOM). METHODS: Inhibitory effect of EEOM on cell growth and proliferation was determined by WST assay in various cancer cells. To investigate the mechanisms of EEOM-mediated cytotoxicity, HepG2 cells were treated with various concentration of EEOM and analyzed the cell cycle arrest and apoptosis induction by flow cytometry, Western blot analysis, 4,6-diamidino-2-phenylindole (DAPI) staining and DNA fragmentation. RESULTS: EEOM showed the cytotoxic activities in a dose-dependent manner in various cancer cell lines but not in normal cells, and HepG2 cells were most susceptible to EEOM-induced cytotoxicity. EEOM induced G2/M arrest in HepG2 cells associated with decreased expression of cyclin-dependent kinase 1 (CDK1), cyclin A and cylcin B, and increased expression of phospho-checkpoint kinase 2, p53 and CDK inhibitor p21. Immunofluorescence staining showed that EEOM-treated HepG2 increased doublet nuclei and condensed actin, resulting in cell rounding. Furthermore, EEOM-mediated apoptosis was determined by Annexin V staining, chromatin condensation and DNA fragmentation. EEOM caused upregulation of FAS and Bax, activation of caspase-3, -8, -9, and fragmentation of poly ADP ribose polymerase. CONCLUSIONS: These results suggest that EEOM efficiently inhibits proliferation of HepG2 cells by inducing both G2/M arrest and apoptosis via intrinsic and extrinsic pathways, and EEOM may be used as a cancer chemopreventive agent in the food or nutraceutical industry.

Dynamic changes in endothelial cell adhesion molecule nepmucin/CD300LG expression under physiological and pathological conditions.

Vascular endothelial cells often change their phenotype to adapt to their local microenvironment. Here we report that the vascular endothelial adhesion molecule nepmucin/CD300LG, which is implicated in lymphocyte binding and transmigration, shows unique expression patterns in the microvascular endothelial cells of different tissues. Under physiological conditions, nepmucin/CD300LG was constitutively and selectively expressed at the luminal surface of the small arterioles, venules, and capillaries of most tissues, but it was only weakly expressed in the microvessels of the splenic red pulp and thymic medulla. Furthermore, it was barely detectable in immunologically privileged sites such as the brain, testis, and uterus. The nepmucin/CD300LG expression rapidly decreased in lymph nodes receiving acute inflammatory signals, and this loss was mediated at least in part by TNF-α. It was also down-regulated in tumors and tumor-draining lymph nodes, indicating that nepmucin/CD300LG expression is negatively regulated by locally produced signals under these circumstances. In contrast, nepmucin/CD300LG was induced in the high endothelial venule-like blood vessels of chronically inflamed pancreatic islets in an animal model of non-obese diabetes. Interestingly, the activated CD4(+) T cells infiltrating the inflamed pancreas expressed high levels of the nepmucin/CD300LG ligand(s), supporting the idea that nepmucin/CD300LG and its ligand interactions are locally involved in pathological T cell trafficking. Taken together, these observations indicate that the nepmucin/CD300LG expression in microvascular endothelial cells is influenced by factor(s) that are locally produced in tissues, and that its expression is closely correlated with the level of leukocyte infiltration in certain tissues.

Involvement of the lysophosphatidic acid-generating enzyme autotaxin in lymphocyte-endothelial cell interactions.

Autotaxin (ATX) is a secreted protein with lysophospholipase D activity that generates lysophosphatidic acid (LPA) from lysophosphatidylcholine. Here we report that functional ATX is selectively expressed in high endothelial venules (HEVs) of both lymph nodes and Peyer's patches. ATX expression was developmentally regulated and coincided with lymphocyte recruitment to the lymph nodes. In adults, ATX expression was independent of HEV-expressed chemokines such as CCL21 and CXCL13, innate immunity signals including those via TLR4 or MyD88, and of the extent of lymphocyte trafficking across the HEVs. ATX expression was induced in venules at sites of chronic inflammation. Receptors for the ATX enzyme product LPA were constitutively expressed in HEV endothelial cells (ECs). In vitro, LPA induced strong morphological changes in HEV ECs. Forced ATX expression caused cultured ECs to respond to lysophosphatidylcholine, up-regulating lymphocyte binding to the ECs in a LPA receptor-dependent manner under both static and flow conditions. Although in vivo depletion of circulating ATX did not affect lymphocyte trafficking into the lymph nodes, we surmise, based on the above data, that ATX expressed by HEVs acts on HEVs in situ to facilitate lymphocyte binding to ECs and that ATX in the general circulation does not play a major role in this process. Tissue-specific inactivation of ATX will verify this hypothesis in future studies of its mechanism of action.

Nepmucin/CLM-9, an Ig domain-containing sialomucin in vascular endothelial cells, promotes lymphocyte transendothelial migration in vitro.

Nepmucin/CLM-9 is an Ig domain-containing sialomucin expressed in vascular endothelial cells. Here we show that, like CD31, nepmucin was localized to interendothelial contacts and to vesicle-like structures along the cell border and underwent intracellular recycling. Functional analyses showed that nepmucin mediated homotypic and heterotypic cell adhesion via its Ig domain. Nepmucin-expressing endothelial cells showed enhanced lymphocyte transendothelial migration (TEM), which was abrogated by anti-nepmucin mAbs that block either homophilic or heterophilic binding. Notably, the mAbs that inhibited homophilic binding blocked TEM without affecting lymphocyte adhesion. These results suggest that endothelial nepmucin promotes lymphocyte TEM using multiple adhesion pathways.

Nepmucin, a novel HEV sialomucin, mediates L-selectin-dependent lymphocyte rolling and promotes lymphocyte adhesion under flow.

Lymphocyte trafficking to lymph nodes (LNs) is initiated by the interaction between lymphocyte L-selectin and certain sialomucins, collectively termed peripheral node addressin (PNAd), carrying specific carbohydrates expressed by LN high endothelial venules (HEVs). Here, we identified a novel HEV-associated sialomucin, nepmucin (mucin not expressed in Peyer's patches [PPs]), that is expressed in LN HEVs but not detectable in PP HEVs at the protein level. Unlike conventional sialomucins, nepmucin contains a single V-type immunoglobulin (Ig) domain and a mucin-like domain. Using materials affinity-purified from LN lysates with soluble L-selectin, we found that two higher molecular weight species of nepmucin (75 and 95 kD) were decorated with oligosaccharides that bind L-selectin as well as an HEV-specific MECA-79 monoclonal antibody. Electron microscopic analysis showed that nepmucin accumulates in the extended luminal microvillus processes of LN HEVs. Upon appropriate glycosylation, nepmucin supported lymphocyte rolling via its mucin-like domain under physiological flow conditions. Furthermore, unlike most other sialomucins, nepmucin bound lymphocytes via its Ig domain, apparently independently of lymphocyte function-associated antigen 1 and very late antigen 4, and promoted shear-resistant lymphocyte binding in combination with intercellular adhesion molecule 1. Collectively, these results suggest that nepmucin may serve as a dual-functioning PNAd in LN HEVs, mediating both lymphocyte rolling and binding via different functional domains.

In vitro differentiation of mouse embryonic stem cells: enrichment of endodermal cells in the embryoid body.

Embryonic stem (ES) cells have the potential to differentiate into all three germ layers, providing new perspectives not only for embryonic development but also for the application in cell replacement therapies. Even though the formation of an embryoid body (EB) in a suspension culture has been the most popular method to differentiate ES cells into a wide range of cells, not much is known about the characteristics of EB cells. To this end, we investigated the process of EB formation in the suspension culture of ES cells at weekly intervals for up to 6 weeks. We observed that the central apoptotic area is most active in the first week of EB formation and that the cell adhesion molecules, except beta-catenin, are highly expressed throughout the examination period. The sequential expression of endodermal genes in EBs during the 6-week culture correlated closely with that of normal embryo development. The outer surface of EBs stained positive for alpha-fetoprotein and GATA-4. When isolated from the 2-week-old EB by trypsin treatment, these endodermal lineage cells matured in vitro into hepatocytes upon stimulation with various hepatotrophic factors. In conclusion, our results demonstrate that endodermal cells can be retrieved from EBs and matured into specific cell types, opening new therapeutic usage of these in vitro differentiated cells in the cell replacement therapy of various diseases.