Tubulointerstitial nephritis antigen-like 1 from cancer-associated fibroblasts contribute to the progression of diffuse-type gastric cancers through the interaction with integrin ß1.

BACKGROUND: Tumor cells of diffuse-type gastric cancer (DGC) are discohesive and infiltrate into the stroma as single cells or small subgroups, so the stroma significantly impacts DGC progression. Cancer-associated fibroblasts (CAFs) are major components of the tumor stroma. Here, we identified CAF-specific secreted molecules and investigated the mechanism underlying CAF-induced DGC progression. METHODS: We conducted transcriptome analysis for paired normal fibroblast (NF)-CAF isolated from DGC patient tissues and proteomics for conditioned media (CM) of fibroblasts. The effects of fibroblasts on cancer cells were examined by transwell migration and soft agar assays, western blotting, and in vivo. We confirmed the effect of blocking tubulointerstitial nephritis antigen-like 1 (TINAGL1) in CAFs using siRNA or shRNA. We evaluated the expression of TINAGL1 protein in frozen tissues of DGC and paired normal stomach and mRNA in formalin-fixed, paraffin-embedded (FFPE) tissue using RNA in-situ hybridization (RNA-ISH). RESULTS: CAFs more highly expressed TINAGL1 than NFs. The co-culture of CAFs increased migration and tumorigenesis of DGC. Moreover, CAFs enhanced the phosphorylation of focal adhesion kinase (FAK) and mesenchymal marker expression in DGC cells. In an animal study, DGC tumors co-injected with CAFs showed aggressive phenotypes, including lymph node metastasis. However, increased phosphorylation of FAK and migration were reduced by blocking TINAGL1 in CAFs. In the tissues of DGC patients, TINAGL1 was higher in cancer than paired normal tissues and detected with collagen type I alpha 1 chain (COL1A1) in the same spot. Furthermore, high TINAGL1 expression was significantly correlated with poor prognosis in several public databases and our patient cohort diagnosed with DGC. CONCLUSIONS: These results indicate that TINAGL1 secreted by CAFs induces phosphorylation of FAK in DGC cells and promotes tumor progression. Thus, targeting TINAGL1 in CAFs can be a novel therapeutic strategy for DGC.

Characterization of gastric cancer-stimulated signaling pathways and function of CTGF in cancer-associated fibroblasts.

BACKGROUND: Cancer-associated fibroblasts (CAFs) are key components of the tumor microenvironment (TME) that play an important role in cancer progression. Although the mechanism by which CAFs promote tumorigenesis has been well investigated, the underlying mechanism of CAFs activation by neighboring cancer cells remains elusive. In this study, we aim to investigate the signaling pathways involved in CAFs activation by gastric cancer cells (GC) and to provide insights into the therapeutic targeting of CAFs for overcoming GC. METHODS: Alteration of receptor tyrosine kinase (RTK) activity in CAFs was analyzed using phospho-RTK array. The expression of CAFs effector genes was determined by RT-qPCR or ELISA. The migration and invasion of GC cells co-cultured with CAFs were examined by transwell migration/invasion assay. RESULTS: We found that conditioned media (CM) from GC cells could activate multiple receptor tyrosine kinase signaling pathways, including ERK, AKT, and STAT3. Phospho-RTK array analysis showed that CM from GC cells activated PDGFR tyrosine phosphorylation, but only AKT activation was PDGFR-dependent. Furthermore, we found that connective tissue growth factor (CTGF), a member of the CCN family, was the most pronouncedly induced CAFs effector gene by GC cells. Knockdown of CTGF impaired the ability of CAFs to promote GC cell migration and invasion. Although the PDGFR-AKT pathway was pronouncedly activated in CAFs stimulated by GC cells, its pharmacological inhibition affected neither CTGF induction nor CAFs-induced GC cell migration. Unexpectedly, the knockdown of SRC and SRC-family kinase inhibitors, dasatinib and saracatinib, significantly impaired CTGF induction in activated CAFs and the migration of GC cells co-cultured with CAFs. SRC inhibitors restored the reduced expression of epithelial markers, E-cadherin and Zonula Occludens-1 (ZO-1), in GC cells co-cultured with CAFs, as well as CAFs-induced aggregate formation in a 3D tumor spheroid model. CONCLUSIONS: This study provides a characterization of the signaling pathways and effector genes involved in CAFs activation, and strategies that could effectively inhibit it in the context of GC. Video Abstract.

Inhibiting the GAS6/AXL axis suppresses tumor progression by blocking the interaction between cancer-associated fibroblasts and cancer cells in gastric carcinoma.

BACKGROUND: The effects of cancer-associated fibroblasts (CAF) on the progression of gastric carcinoma (GC) has recently been demonstrated. However, agents targeting the interaction between CAF and GC cells have not been applied in a clinical setting. Here, we examined if inhibition for Axl receptor tyrosine kinase (AXL) can suppress CAF-induced aggressive phenotype in GC. METHODS: We investigated the function of CAF-derived growth arrest-specific 6 (GAS6), a major ligand of AXL, on the migration and proliferation of GC cells. The effect of the AXL inhibitor, BGB324, on the CAF-induced aggressive phenotype of GC cells was also investigated. In addition, we performed immunohistochemistry to examine the expression of phosphorylated AXL protein in 175 GC tissues and evaluated its correlation with the prognosis. RESULTS: The qPCR and western blot analysis showed that GAS6 expression was higher in CAF relative to other cells. We found that co-culture with CAF increased the phosphorylation of AXL (P-AXL), differentiation into a mesenchymal-like phenotype, and cell survival in GC cell lines. When the expression of AXL was genetically inhibited in GC cells, the effect of CAF was reduced. BGB324, a small molecule inhibitor of AXL, suppressed the effects of CAF on GC cell lines. In GC tissues, high levels of P-AXL were significantly associated with poor overall survival (P = 0.022). CONCLUSIONS: We concluded that CAF are a major source of GAS6 and that GAS6 promotes an aggressiveness through AXL activation in GC. We suggested that an AXL inhibitor may be a novel agent for GC treatment.

Targeting interleukin-6 as a strategy to overcome stroma-induced resistance to chemotherapy in gastric cancer.

BACKGROUND: Although the tumor stroma in solid tumors like gastric cancer (GC) plays a crucial role in chemo-resistance, specific targets to inhibit the interaction between the stromal and cancer cells have not yet been utilized in clinical practice. The present study aims to determine whether cancer-associated fibroblasts (CAFs), a major component of the tumor stroma, confer chemotherapeutic resistance to GC cells, and to discover potential targets to improve chemo-response in GC. METHODS: To identify CAF-specific proteins and signal transduction pathways affecting chemo-resistance in GC cells, secretome and transcriptome analyses were performed. We evaluated the inhibiting effect of CAF-specific protein in in vivo and in vitro models and investigated the expression of CAF-specific protein in human GC tissues. RESULTS: Secretome and transcriptome data revealed that interleukin-6 (IL-6) is a CAF-specific secretory protein that protects GC cells via paracrine signaling. Furthermore, CAF-induced activation of the Janus kinase 1-signal transducer and activator of transcription 3 signal transduction pathway confers chemo-resistance in GC cells. CAF-mediated inhibition of chemotherapy-induced apoptosis was abrogated by the anti-IL-6 receptor monoclonal antibody tocilizumab in various experimental models. Clinical data revealed that IL-6 was prominently expressed in the stromal portion of GC tissues, and IL-6 upregulation in GC tissues was correlated with poor responsiveness to chemotherapy. CONCLUSIONS: Our data provide plausible evidence for crosstalk between GC cells and CAFs, wherein IL-6 is a key contributor to chemoresistance. These findings suggest the potential therapeutic application of IL-6 inhibitors to enhance the responsiveness to chemotherapy in GC.

Vasorelaxant Effect of Prunus mume (Siebold) Siebold & Zucc. Branch through the Endothelium-Dependent Pathway.

Korean plum (Prunus mume (Siebold) Siebold & Zucc.) has long been used as a health food or herbal medicine in Asia. Previous studies have shown that several plants of the genus Prunus have vasodilatory and antihypertensive effects; we hypothesized that P. mume branches may have a vasorelaxant effect. In this study, we evaluated the effects and action mechanism of 70% ethanol extract of P. mume branch (PMB) on isolated rat aortic rings. Inhibitors such as NG-nitro-l-arginine methyl ester, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, methylene blue, indomethacin, atropine, tetraethylammonium chloride, glibenclamide, 4-aminopyridine and BaCl2 were used to investigate the mechanism of vasodilation responsible for the vascular relaxation. PMB (2-30 µg/mL) induced vasorelaxation in the presence of vascular endothelium, and all inhibitors used in this study affected the degree of relaxation. These results suggest that the vasorelaxant effect of PMB is endothelium-dependent and affects the nitric oxide-cyclic guanosine monophosphate pathway, prostacyclin pathway, muscarinic receptor pathway, and potassium channels. Our study explains that PMB may be another approach to hypertension treatment to reduce the burden of cardiovascular disease.

Gastrokine 1 protein is a potential theragnostic target for gastric cancer.

BACKGROUND: Gastrokine 1 (GKN1) plays important roles in maintaining mucosal homeostasis, and in regulating cell proliferation and differentiation. Here, we determined whether GKN1 is a potential theragnostic marker for gastric cancer. METHODS: We identified GKN1 binding proteins using the protein microarray assay and investigated whether GKN1 is one of the exosomal cargo proteins by western blot, immunoprecipitation, and immunofluorescent assays. Cell proliferation and apoptosis were analyzed by MTT, BrdU incorporation, flow cytometry, and western blot assays. We further validated the functional relevance of exosomal GKN1 in MKN1-injected xenograft mice. The possibility of serum GKN1 as a diagnostic marker for gastric cancer was determined by ELISA assay. RESULTS: In protein microarray assay, GKN1 binding to 27 exosomal proteins was clearly observed. GKN1 was expressed in exosomes derived from HFE-145 gastric epithelial cells by western blot and immunofluorescent assays, but not in exosomes from AGS and MKN1 gastric cancer cells. Exosomes carrying GKN1 inhibited cell proliferation and induced apoptosis in both AGS and MKN1 cells, and exosomes carrying GKN1-treated nude mice-bearing MKN1 xenograft tumors exhibited significantly reduced tumor volume and tumor weight. Silencing of clathrin markedly down-regulated the internalization of exosomal GKN1. Interestingly, serum GKN1 concentrations in patients with gastric cancer were significantly lower than those in healthy individuals and patients with colorectal and hepatocellular carcinomas. CONCLUSIONS: The GKN1 is secreted and internalized in the gastric epithelium by exosome-driven transfer, which inhibits gastric tumorigenesis and supports the clinical application of GKN1 protein in gastric cancer diagnosis and treatment.

Discoidin domain receptor 1 activity drives an aggressive phenotype in gastric carcinoma.

BACKGROUND: Discoidin domain receptor 1 (DDR1), a receptor tyrosine kinase that utilizes collagen as a ligand, is a key molecule in the progression of solid tumors as it regulates the interaction of cancer cells with the tumor stroma. However, the clinical relevance of DDR1 expression in gastric carcinoma is yet to be investigated. Here, we assessed the role of DDR1 in mediating the aggressive phenotype of gastric carcinoma and its potential as a therapeutic target. METHODS: We conducted DDR1 immunohistochemistry using a tissue microarray of 202 gastric carcinoma specimens. We examined the effect of collagen-induced activation of DDR1 on cell signaling, tumorigenesis, and cell migration in gastric cancer cell lines, and tumor growth in a xenograft animal model of gastric cancer. RESULTS: Our results showed that 50.5% of gastric cancer tissues are positive for DDR1 expression, and positive DDR1 expression was significantly correlated with a poor prognosis (P = 0.015). In a subgroup analysis, DDR1 expression was prognostically meaningful only in patients receiving adjuvant treatment (P = 0.013). We also demonstrated that collagen was able to activate DDR1 and increase the clonogenicity and migration of gastric cancer cells. We observed that a DDR1 inhibitor, 7rh benzamide, suppressed tumor growth in gastric cancer xenografts. CONCLUSIONS: Our findings suggest a key role for DDR1 signaling in mediating the aggressive phenotype of gastric carcinoma. Importantly, inhibition of DDR1 is an attractive strategy for gastric carcinoma therapy.

Intratumor stromal proportion predicts aggressive phenotype of gastric signet ring cell carcinomas.

OBJECTIVE: The aim of this study was to evaluate the prognostic significance of the intratumor stromal proportion in gastric signet ring cell (SRC) carcinomas. BACKGROUND: Cancer stroma, as exemplified by cancer-associated fibroblasts (CAFs), plays critical roles in cancer proliferation, invasion, and metastasis. METHODS: One hundred seventy-five SRC carcinoma cases were classified according to the intratumor desmoplastic stromal proportion to then analyze the clinicopathologic characteristics of stroma-rich cases. We also investigated the impact of CAFs on the migration as well as on the phenotypic changes of gastric SRC carcinomas in vitro. Furthermore, we performed RNA sequencing of a pair of CAFs and normal-tissue-associated fibroblasts. RESULTS: Stroma-rich SRC carcinomas (64 of 175 cases, 36.5%) were associated with female patients (P = 0.045), large tumor size (P = 0.007), higher T category (P < 0.001), and the presence of perineural invasion (P = 0.018). Patients with stroma-rich SRC carcinomas had a significantly shorter disease-free survival (P < 0.001) and overall survival (P < 0.001). However, in a subgroup analysis, the prognostic significance of the stromal proportion correlated only with patients with T3/4 disease. From multivariate analysis, the high stromal proportion is an independent prognostic factor to predict worse disease-free survival (hazard ratio 2.288; P = 0.001) and overall survival (hazard ratio 2.503; P = 0.001). We found that CAFs enhanced the migratory abilities of cancer cells through the epithelial-mesenchymal transition, and RNA sequencing results confirmed these findings. CONCLUSIONS: The intratumor stromal proportion could be a useful prognostic biomarker and a potential therapeutic target in gastric SRC carcinomas.

Vasorelaxant effects of Angelica decursiva root on isolated rat aortic rings.

BACKGROUND: Hypertension is one of the most important risk factors for cardiovascular disease (CVD) and a worldwide problem. Despite increases in the development of synthetic drugs for hypertension treatment, the rate of untreated and uncontrolled hypertension remains high. These drugs are effective, but can also cause side effects. Approximately 80% of the world population uses herbal medicines because of their low toxicity and better acceptability by the human body. Therefore, we attempted to identify natural medications for treating hypertension. The 70% ethanol extract of Angelica decursiva root (ADE) shows strong vasorelaxant potential, but no studies have investigated the mechanisms underlying the vasorelaxation effect of A. decursiva. METHODS: Dried root of A. decursiva was identified by DNA sequencing and was extracted once with 1 L 70% ethanol (EtOH) for 3 h in a reflux apparatus at 70 °C. ADE was evaluated for its vasorelaxant effects in rat thoracic aortas. Various inhibitors of ADE-induced vasorelaxation were used. RESULTS: ADE showed vasorelaxant effects on the intact and denuded endothelium of aortic rings pre-contracted with phenylephrine and KCl in Krebs-Henseleit solution. Tetraethylammonium and 4-aminopyridine did not alter ADE-induced vasorelaxation. However, the vasorelaxant effect of ADE was partially inhibited by pre-treatment with glibenclamide an ATP-sensitive K+ channel blocker. Furthermore, ADE concentration-dependently inhibited Ca2+ supplementation-induced vasoconstriction of aortic rings that had been pretreated with phenylephrine or KCl in Ca2+-free Krebs-Henseleit solution. CONCLUSIONS: These results suggest that ADE-induced vasorelaxation occurred in an endothelium-independent manner. The vasorelaxant effects of ADE were correlated with blockade of the KATP channel and inhibition of Ca2+ influx via receptor-operative Ca2+ channels or voltage-dependent Ca2+ channels.

HVC1 ameliorates hyperlipidemia and inflammation in LDLR-/- mice.

BACKGROUND: HVC1 consists of Coptidis Rhizoma (dried rhizome of Coptischinensis), Scutellariae Radix (root of Scutellariabaicalensis), Rhei Rhizoma (rhizome of Rheum officinale), and Pruni Cortex (cortex of Prunusyedoensis Matsum). Although the components are known to be effective in various conditions such as inflammation, hypertension, and hypercholesterolemia, there are no reports of the molecular mechanism of its hypolipidemic effects. METHODS: We investigated the hypolipidemic effect of HVC1 in low-density lipoprotein receptor-deficient (LDLR-/-) mice fed a high-cholesterol diet for 13 weeks. Mice were randomized in to 6 groups: ND (normal diet) group, HCD (high-cholesterol diet) group, and treatment groups fed HCD and treated with simvastatin (10 mg/kg, p.o.) or HVC1 (10, 50, or 250 mg/kg, p.o.). RESULTS: HVC1 regulated the levels of total cholesterol, triglyceride (TG), low-density lipoprotein (LDL) cholesterol, and high-density lipoprotein (HDL) cholesterol in mouse serum. In addition, it regulated the transcription level of the peroxisome proliferator-activated receptors (PPARs), sterol regulatory element-binding proteins (SREBP)-2, 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase, lipoprotein lipase (LPL), apolipoprotein B (apo B), liver X receptor (LXR), and inflammatory cytokines (IL-1ß, IL-6, and TNF-α). Furthermore, HVC1 activated 5' adenosine monophosphate-activated protein kinase (AMPK). CONCLUSION: Our results suggest that HVC1 might be effective in preventing high-cholesterol diet-induced hyperlipidemia by regulating the genes involved in cholesterol and lipid metabolism, and inflammatory responses.

Loss of ACSS2 expression predicts poor prognosis in patients with gastric cancer.

BACKGROUND: Recent studies have demonstrated that acetyl-CoA synthetase 2 (ACSS2) plays a critical role in cancer cell survival; however, the role of ACSS2 in gastric carcinogenesis has not been determined. METHODS: We investigated the expression of ACSS2 in human gastric cancer (GC) tissues using immunohistochemistry, and analyzed its clinicopathological correlation and prognostic relevance. RESULTS: Among 350 GCs, 219 cases (62.6%) were classified as ACSS2-low, whereas 131 cases (37.4%) were ACSS2-high. Loss of ACSS2 expression (ACSS2-low) was more frequently observed in undifferentiated histology (P = 0.002), in cases with MLH1-loss (P = 0.003), and in cases with SIRT3-low (P < 0.001). The ACSS2-low cases showed significantly lower mean disease-free survival (DFS, 68.5 vs. 81.8 months; P = 0.025) and overall survival (OS, 73.5 vs. 86.6 months; P = 0.029). In multivariate analysis, loss of ACSS2 expression was identified as one of the independent prognostic factors predicting worse DFS (HR: 1.547, P = 0.018) and OS (HR: 1.476, P = 0.036). CONCLUSIONS: We revealed that the loss of ACSS2 expression is a reliable independent poor prognostic factor in GC. Our results may expand our understanding of the involvement of glucose metabolism, including the role of ACSS2, in the pathogenesis of GC.

Dichloroacetate attenuates hypoxia-induced resistance to 5-fluorouracil in gastric cancer through the regulation of glucose metabolism.

In this study, we investigated whether gastric cancer with hypoxia-induced resistance to 5-fluorouracil (5-FU) could be re-sensitized following treatment with low-dose dichloroacetate (DCA), an inhibitor of the glycolytic pathway. The expression profiles of hypoxia-inducible factor-1α (HIF-1α) and pyruvate dehydrogenase kinase-1 (PDK-1) were analyzed in tissues from 10 patients with gastric cancer who had different responses to adjuvant 5-FU treatment. For the in vitro assays, cell viability and apoptosis were evaluated with and without treatment with 20mM DCA in the AGS and MKN45 cell lines, as well as in PDK1 knockdown cell lines. The expression levels of HIF-1α and PDK-1 were both elevated in the tumor tissues relative to the normal gastric tissues of most patients who showed recurrence after adjuvant 5-FU treatment. Cellular viability tests showed that these cell lines had a lower sensitivity to 5-FU under hypoxic conditions compared to normoxic conditions. Moreover, the addition of 20mM DCA only increased the sensitivity of these cells to 5-FU under hypoxic conditions, and the resistance to 5-FU under hypoxia was also attenuated in PDK1 knockdown cell lines. In conclusion, DCA treatment was able to re-sensitize gastric cancer cells with hypoxia-induced resistance to 5-FU through the alteration of glucose metabolism.

Investigation of the mechanisms of Angelica dahurica root extract-induced vasorelaxation in isolated rat aortic rings.

BACKGROUND: The root of Angelica dahurica Bentham et Hooker (Umbelliferae) has been used as a traditional medicine for colds, headache, dizziness, toothache, supraorbital pain, nasal congestion, acne, ulcer, carbuncle, and rheumatism in China, Japan, and Korea. Interestingly, it has been used in the treatment of vascular diseases including hypertension. The aim of this study was to provide pharmacological evidence for the anti-hypertensive effect of A. dahurica by investigating the mechanism underlying its vasorelaxant effect. METHODS: The vasorelaxant effects of a 70% methanol extract of the A. dahurica root (ADE) on rat thoracic aorta and its underlying mechanisms were assessed. Isolated rat aortic rings were suspended in organ chambers containing 10 ml Krebs-Henseleit (K-H) solution and placed between 2 tungsten stirrups and connected to an isometric force transducer. Changes in tension were recorded via isometric transducers connected to a data acquisition system. RESULTS: ADE causes concentration-dependent relaxation in both endothelium-intact and endothelium-denuded aortic rings precontracted with phenylephrine (PE; 1 µM) or potassium (KCl; 60 mM) in K-H solution. And pre-treatment with ADE (1 mg/ml) inhibited calcium-induced vasocontraction of aortic rings induced by PE or KCl. However, ADE pre-treatment did not affect the contraction induced by PE or caffeine in Ca(2+)-free K-H solution. CONCLUSIONS: These results suggested that the ADE has vasorelaxant effect and the vasorelaxant activity is mediated by endothelium-independent pathway that includes the blockade of extracellular calcium influx through the receptor-operated Ca(2+) channel and voltage-dependent calcium channel pathways.

Effect of Ampelopsis Radix on wound healing in scalded rats.

BACKGROUND: Ampelopsis Radix has been used as a traditional Korean medicine for the treatment of burns and scalds. However, there has been no scientific research to date on the wound healing properties of Ampelopsis Radix for scald burns. This study aimed to evaluate the healing effect of Ampelopsis japonica root tuber ethanol extract (AJE) on induced cutaneous scald injury in Sprague Dawley (SD) rats. METHODS: Hot water scalds were induced in SD rats, who were then divided into the following 5 groups; 1) control group without treatment, 2) positive control group with 1% Silver sulfadiazine (SSD), 3) Vaseline group, and groups 4) and 5) that used Vaseline containing 5% and 20% AJE, respectively. The ointment was applied topically to the experimental rats, once daily for 21 days, starting at 24 h post induction of the scald injury. Gross examination, measurement of wound size, and histopathological examination were performed. And quantitative measurement of cytokine levels of tumor necrosis factor alpha (TNF-α), interleukin-10 (IL-10), transforming growth factor beta 1 (TGF-ß1), and vascular endothelial growth factor (VEGF) were performed by enzyme-linked immunosorbent assay. RESULTS: Clinical evaluation showed that the AJE and Vaseline groups, rapidly desquamated scab on day 12 post-scalding; in particular, the 20% AJE group achieved the greatest extent of skin recovery. Sizes of scald wound were significantly lower on days 12, 15, 18, and 21 in the AJE treated groups compared to the control groups. Histopathological evaluation showed a well-organized epithelial layer, angiogenesis, tissue granulation and collagen formation with the exception of inflammatory cells in the AJE-treated groups compared to the control groups on day 14, indicating that tissue regeneration had occurred. AJE treatment decreased TNF-α and increased IL-10 levels on days 2 and 14, indicating the anti-inflammatory action of AJE. The AJE groups also showed a decrease in TGF-ß1 levels on day 7 and VEGF on day 14 in the serum of scald inflicted SD rat model. CONCLUSIONS: These results suggest that AJE possesses scald wound healing activity via accelerating the scald wound repair during the inflammation and proliferative phases of the healing process.

Endothelium-Independent Vasorelaxant Effect of Ligusticum jeholense Root and Rhizoma on Rat Thoracic Aorta.

Ligusticum jeholense has been used as the traditional medicine 'Go-Bon' (Chinese name, Gao-ben) in China and Korea. Considering the increased use of medicinal herbs to treat hypertension, in this study, we aimed to investigate the mechanisms of the vasorelaxation effect caused by L. jeholense. We tested the methanol (MeOH) extract of L. jeholense root and rhizoma for vasorelaxant effects; while using an isolated organ-chamber technique, L. jeholense extract (LJE) induced relaxation in the rat aortic rings by stimulating vascular endothelial and smooth muscle cells. LJE showed concentration-dependent relaxant effects on endothelium-intact and endothelium-denuded aortic rings pre-contracted with both phenylephrine (PE) and potassium chloride (KCl) in Krebs-Henseleit (KH) buffer. The vasorelaxant effect of LJE was partly attenuated by pre-treatment with glibenclamide or 4-aminopyridine (4-AP) as K+ channel blockers. Moreover, LJE showed concentration-dependent inhibition of vasoconstriction by Ca2+ supplementation in the aortic rings that were pre-contracted with PE or KCl in Ca2+-free KH buffer. In addition, a combination of LJE and nifedipine, pre-incubated further, decreased PE-induced contractions. The results suggested that LJE-induced vasorelaxation were related to blocking K+ channels and inhibiting entry of extracellular Ca2+ via receptor-operative Ca2+ channels (ROCCs) or voltage-dependent Ca2+ channels (VDCCs).

Vasorelaxant effect of Prunus yedoensis bark.

BACKGROUND: Prunus yedoensis Matsum. is used as traditional medicine-'Yaeng-Pi' or 'Hua-Pi'-in Japan and Korea. However, no studies have examined the pharmacological activities of the P. yedoensis bark. Only the antioxidant and antiviral activities of P. yedoensis fruit and the anti-hyperglycaemic effect of P. yedoensis leaf have been investigated. While studying the antihypertensive effects of several medicinal plants, we found that a methanol extract of P. yedoensis bark (MEPY) had distinct vasorelaxant effects on rat aortic rings. METHODS: The aortic rings were removed from Sprague-Dawley rats and suspended in organ chambers containing 10 ml Krebs-Henseleit solution. The aortic rings were placed between 2 tungsten stirrups and connected to an isometric force transducer. Changes in tension were recorded via isometric transducers connected to a data acquisition system. RESULTS: MEPY relaxed the contraction induced by phenylephrine (PE) both in endothelium-intact and endothelium-denuded aortic rings concentration dependently. However, the vasorelaxant effects of MEPY on endothelium-denuded aortic rings were lower than endothelium-intact aortic rings. The vasorelaxant effects of MEPY on endothelium-intact aortic rings were reduced by pre-treatment with L-NAME, methylene blue, or ODQ. However, pre-treatment with indomethacin, atropine, glibenclamide, tetraethylammonium, or 4-aminopyridine had no affection. In addition, MEPY inhibited the contraction induced by extracellular Ca(2+) in endothelium-denuded rat thoracic aorta rings pre-contracted by PE (1 µM) or KCl (60 mM) in Ca(2+)-free solution. CONCLUSIONS: Our results suggest that MEPY exerts its vasorelaxant effects via the activation of NO formation by means of L-Arg and NO-cGMP pathways and via the blockage of extracellular Ca(2+) channels.

Anti-inflammatory effect of Prunus yedoensis through inhibition of nuclear factor-κB in macrophages.

BACKGROUND: Prunus yedoensis (PY) is a traditional anti-allergy and anti-inflammatory herb medicine used in South Korea. However, until date, little is known regarding its mechanism of action. METHODS: In order to elucidate the mechanism of anti-inflammatory effect of PY, the constituents of PY were analysed by high performance liquid chromatography (HPLC), and nitric oxide (NO) and prostaglandin E2 (PGE2) production were measured enzyme-linked immuno sorbent assay (ELISA). The expression levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and nuclear factor-κB (NF-κB) were also measured by western blotting in lipopolysaccharide (LPS)-induced RAW 264.7 macrophage cells treated with PY. RESULTS: The results indicate that (50, 100 µg/mL) methanol and ethyl acetate fractionation extracts of PY not only inhibited LPS-mediated NO production and iNOS expression, but also decreased LPS-induced PGE2 production and COX-2 expression. The anti-inflammatory effects of PY were also due to the attenuation of nuclear translocation of NF-κB, as evaluated by the use of anti-p50 on nuclear fractions. LPS-induced nuclear translocation of NF-κB decreased significantly by the methanol extract and ethyl acetate fraction of PY. High performance liquid chromatography (HPLC) analyses revealed that methanol extract and ethyl acetate fraction have similar patterns of retention time and peaks. CONCLUSION: Our results demonstrate that methanol extracts and the ethyl acetate fraction of PY have anti-inflammatory properties, thus emphasizing the potential of PY as a natural health product.

Endothelium-independent vasorelaxation effects of Sigesbeckia glabrescens (Makino) Makino on isolated rat thoracic aorta.

The present study aimed to investigate the vasorelaxant effect of the methanol extract of Sigesbeckia glabrescens (Makino) Makino (MESG) on rat aortic rings and mechanism of action. MESG inhibited both noradrenaline bitartrate (NA)- and potassium chloride (KCl)-induced contraction of endothelium-intact aortic rings in a concentration-dependent manner. Removal of the endothelium did not influence the effect of MESG on NA-precontracted aortic rings. Pretreatment with MESG (250 µg/mL) inhibited calcium chloride-induced vasocontraction of NA- or KCl-precontracted endothelium-denuded aortic rings. It also relaxed phorbol-12-myristate-13-acetate-induced contraction of aortic rings in a concentration-dependent manner. In addition, Bay K8644 (an L-type calcium channel opener) vasocontracted in MESG pretreated aortic rings. On the other hand, the inositol 1,4,5-triphosphate receptor, the ryanodine receptor, the Rho-kinase inhibitor (Y-27632), a soluble guanylyl cyclase blocker (1-H-[1,2,4]-oxadiazolo-[4,3a]-quinoxalin-1-one), and K⁺ channel blockers (glybenclamide, tetraethylammonium, and 4-aminopyridine) did not affect the effect of MESG. These results suggested that the mechanism underlying the vasorelaxant effect of MESG is mediated by endothelium-independent pathways. This specifically refers to blockade of the influx of extracellular Ca²âº via receptor-operative Ca²âº channels and voltage-dependent Ca²âº channels and inhibition of a protein kinase C-mediated cellular pathway.

Expression and activity of the na-k ATPase in ischemic injury of primary cultured astrocytes.

Astrocytes are reported to have critical functions in ischemic brain injury including protective effects against ischemia-induced neuronal dysfunction. Na-K ATPase maintains ionic gradients in astrocytes and is suggested as an indicator of ischemic injury in glial cells. Here, we examined the role of the Na-K ATPase in the pathologic process of ischemic injury of primary cultured astrocytes. Chemical ischemia was induced by sodium azide and glucose deprivation. Lactate dehydrogenase assays showed that the cytotoxic effect of chemical ischemia on astrocytes began to appear at 2 h of ischemia. The expression of Na-K ATPase α1 subunit protein was increased at 2 h of chemical ischemia and was decreased at 6 h of ischemia, whereas the expression of α1 subunit mRNA was not changed by chemical ischemia. Na-K ATPase activity was time-dependently decreased at 1, 3, and 6 h of chemical ischemia, whereas the enzyme activity was temporarily recovered to the control value at 2 h of chemical ischemia. Cytotoxicity at 2 h of chemical ischemia was significantly blocked by reoxygenation for 24 h following ischemia. Reoxygenation following chemical ischemia for 1 h significantly increased the activity of the Na-K ATPase, while reoxygenation following ischemia for 2 h slightly decreased the enzyme activity. These results suggest that the critical time for ischemia-induced cytotoxicity of astrocytes might be 2 h after the initiation of ischemic insult and that the increase in the expression and activity of the Na-K ATPase might play a protective role during ischemic injury of astrocytes.

Molecular and Immune Profiling of Syngeneic Mouse Models Predict Response to Immune Checkpoint Inhibitors in Gastric Cancer.

PURPOSE: Appropriate preclinical mouse models are needed to evaluate the response to immunotherapeutic agents. Immunocompetent mouse models have rarely been reported for gastric cancer. Thus, we investigated immunophenotypes and responses to immune checkpoint inhibitor (ICI) in immunocompetent mouse models using various murine gastric cancer cell lines. MATERIALS AND METHODS: We constructed subcutaneous syngeneic tumors with murine gastric cancer cell lines, YTN3 and YTN16, in C57BL/6J mice. Mice were intraperitoneally treated with IgG isotype control or an anti-programmed death-ligand 1 (PD-L1) neutralizing antibody. We used immunohistochemistry to evaluate the tumor-infiltrating immune cells of formalin-fixed paraffin-embedded mouse tumor tissues. We compared the protein and RNA expression between YTN3 and YTN16 cell lines using a mouse cytokine array and RNA sequencing. RESULTS: The mouse tumors revealed distinct histological and molecular characteristics. YTN16 cells showed upregulation of genes and proteins related to immunosuppression, such as Ccl2 (CCL2) and Csf1 (M-CSF). Macrophages and exhausted T cells were more enriched in YTN16 tumors than in YTN3 tumors. Several YTN3 tumors were completely regressed by the PD-L1 inhibitor, whereas YTN16 tumors were unaffected. Although treatment with a PD-L1 inhibitor increased infiltration of T cells in both the tumors, the proportion of exhausted immune cells did not decrease in the non-responder group. CONCLUSION: We confirmed the histological and molecular features of cancer cells with various responses to ICI. Our models can be used in preclinical research on ICI resistance mechanisms to enhance clinical efficacy.