Comparison of Tepotinib, Paclitaxel, or Ramucirumab Efficacy According to the Copy Number or Phosphorylation Status of the MET Gene: Doublet Treatment versus Single Agent Treatment.

Although conventional combination chemotherapies for advanced gastric cancer (GC) increase survival, such therapies are associated with major adverse effects; more effective and less toxic treatments are required. Combinations of different anti-cancer drugs, for example, paclitaxel plus ramucirumab, have recently been used as second-line treatments for advanced GC. This study evaluated how copy number variations of the MET gene, MET mutations, and MET gene and protein expression levels in human GC cells modulate the susceptibility of such cells to single-agent (tepotinib, ramucirumab, or paclitaxel) and doublet (tepotinib-plus-paclitaxel or ramucirumab-plus-paclitaxel treatment regimens. Compared with ramucirumab-plus-paclitaxel, tepotinib-plus-paclitaxel better inhibited the growth of GC cells with MET exon 14 skipping mutations and those lacking MET amplification but containing phosphorylated MET; such inhibition was dose-dependent and associated with cell death. Tepotinib-plus-paclitaxel and ramucirumab-plus-paclitaxel similarly inhibited the growth of GC cells lacking MET amplification or MET phosphorylation, again in a dose-dependent manner, but without induction of cell death. However, tepotinib alone or tepotinib-plus-ramucirumab was more effective against c-MET-positive GC cells (>30 copy number variations) than was ramucirumab or paclitaxel alone or ramucirumab-plus-paclitaxel. These in vitro findings suggest that compared with ramucirumab-plus-paclitaxel, tepotinib-plus-paclitaxel better inhibits the growth of c-MET-positive GC cells, cells lacking MET amplification but containing phosphorylated MET, and cells containing MET mutations. Clinical studies are required to confirm the therapeutic effects of these regimens.

Protective Effect of Ulinastatin on Cognitive Function After Hypoxia.

Ulinastatin (UTI) has neuroprotective properties. Neurologic insults, including hypoxia and use of anesthetic agents, cause postoperative cognitive dysfunction and alter gamma-aminobutyric acid (GABA) function. This study aimed to assess whether UTI could preserve learning and memory using a zebrafish hypoxic behavior model and biomarkers. Zebrafish (6-8 months of age and 2.5-3.5 cm long) were divided into eight groups as follows: phosphate-buffered saline (PBS) control, hypoxia + PBS, UTI (10,000, 50,000, and 100,000 units/kg), and hypoxia with UTI (10,000, 50,000, and 100,000 units/kg) groups. The endpoints of the T-maze experiment included total time, distance moved, and frequency in target or opposite compartment. We also measured the degree of brain infarction using 2,3,5­triphenyltetrazolium chloride staining, assessed SA-ß-galactosidase activity, and examined GABAA receptor expression using real-time polymerase chain reaction. In a dose-dependent manner, UTI affected learning and memory in zebrafish. Despite hypoxia, 100,000 units/kg of UTI preserved preference (time and distance) for the target compartment. More than 50,000 units/kg of UTI also showed reduced hypoxia-induced brain infarction, decreased SA-ß-galactosidase levels, and upregulated GABAA receptors. This study demonstrated that the location of the GABAA receptor is affected by hypoxia or UTI.

Responses to the Tepotinib in Gastric Cancers with MET Amplification or MET Exon 14 Skipping Mutations and High Expression of Both PD-L1 and CD44.

Both MET exon 14 skipping mutation (METex14SM) and high copy-number variation (CNV) lead to enhanced carcinogenesis; additionally, programmed-death ligand 1 (PD-L1) is often upregulated in cancers. In this study, we characterized the expression of MET (including METex14SM), PD-L1, and CD44 in human gastric cancer (GC) cells as well as the differential susceptibility of these cells to tepotinib. Tepotinib treatments inhibited the growth of five GC cells in a dose-dependent manner with a concomitant induction of cell death. Tepotinib treatments also significantly reduced the expression of phospho-MET, total MET, c-Myc, VEGFR2, and Snail protein in SNU620, MKN45, and Hs746T cells. Notably, tepotinib significantly reduced the expression of CD44 and PD-L1 in METex14SM Hs746T cells. By contrast, tepotinib was only slightly active against SNU638 and KATO III cells. Migration was reduced to a greater extent in the tepotinib-treated group than in the control group. Tepotinib may have therapeutic effects on c-MET-amplified GC, a high expression of both PD-L1 and CD44, and METex14SM. Clinical studies are needed to confirm these therapeutic effects.

TRK inhibitors block NFKB and induce NRF2 in TRK fusion-positive colon cancer.

Tropomyosin receptor kinase (TRK) fusion is one of the oncogenic driver causes of colon cancer, and tropomyosin 3-neurotrophic receptor tyrosine kinase 1 (TPM3-NTRK1) fusion has been detected in the KM12SM cell line. In the present study, we investigated anticancer mechanisms in the KM12SM cell line using three different form of dovitinib (dovitinib (free base), dovitinib lactate (mono acid), and dovitinib dilactic acid (diacid)) and four TRK inhibitors (LOXO-101, entrectinib, regorafenib, and crizotinib). Exposure of TRK inhibitors at concentrations of 10 nM resulted in the apoptosis of KM12SM cells, whereas regorafenib had no effect. Treatment with all inhibitors except regorafenib also significantly increased the expression levels of the genes nuclear factor-erythroid 2-related factor 2 (NRF2) and glutamyl cysteine ligase catalytic subunit (GCLC) in KM12SM. These drugs significantly reduced expression of the phosphorylated proteins NFκB and COX-2 in the KM12SM cell line, and significantly attenuated KM12SM cell migration, according to a Transwell migration assay. Together, these results suggest that TRK inhibitors block products of carcinogenesis by negatively regulating the NFκB signaling pathway and positively regulating the antioxidant NRF2 signaling pathway.

RNF43 and PWWP2B inhibit cancer cell proliferation and are predictive or prognostic biomarker for FDA-approved drugs in patients with advanced gastric cancer.

Background: Abnormal regulation of genes has been closely related to gastric cancer. The characterization of gastric cancer has necessitated the development of new therapeutics as well as the identification of prognostic markers to predict the response to novel drugs. In our study, we used RNA sequencing analyses to show that on gastric cancer tissues to identification of gastric cancer prognostic markers. We specifically chose to study RNF43 because it inhibits gastric cancer-related Wnt/ß-catenin signaling by interacting with Wnt receptors. PWWP2B was chosen because it is a gene which is downregulated in gastric cancer. Methods: Utilizing RNA sequencing analysis, we evaluated the mRNA expression profile in gastric cancer patients. Also, we used HAP1 cells which is a human near-haploid cell line derived from the male chronic myelogenous leukemia cell line KBM-7. These cell line has one copy of each gene, ensuring the edited allele will not be masked by additional alleles. We investigated the screening of 1,449 FDA-approved drugs in HAP1, HAP1 RNF43 KO and HAP1 PWWP2B KO cells. RNA sequencing data reveals that RNF43 and PWWP2B expression were down-regulated in recurrence gastric cancer patients. Next, we investigated the anti-cancer effects of selected drugs in RNF43 and PWWP2B down-regulated MKN45 gastric cancer cells and xenograft model. Results: Among these FDA-approved drugs, three drugs (docetaxel trihydrate, pelitinib and uprosertib) showed strong inhibitory effects in RNF43 KO cells and PWWP2B KO cells. In MKN45 xenograft model, tumor volumes were significantly reduced in the docetaxel trihydrate, uprosertib or pelitinib-treated group. Our data demonstrated that RNF43 and PWWP2B are a biomarker that predict recurrence of gastric cancer. Conclusions: Our findings suggest that docetaxel trihydrate, uprosertib and pelitinib could be used as novel therapeutic agents for the prevention and treatment of gastric cancer with a decrease in RNF43 and PWWP2B expression.

Evaluation of 3D Templated Synthetic Vascular Graft Compared with Standard Graft in a Rat Model: Potential Use as an Artificial Vascular Graft in Cardiovascular Disease.

Although the number of vascular surgeries using vascular grafts is increasing, they are limited by vascular graft-related complications and size discrepancy. Current efforts to develop the ideal synthetic vascular graft for clinical application using tissue engineering or 3D printing are far from satisfactory. Therefore, we aimed to re-design the vascular graft with modified materials and 3D printing techniques and also demonstrated the improved applications of our new vascular graft clinically. We designed the 3D printed polyvinyl alcohol (PVA) templates according to the vessel size and shape, and these were dip-coated with salt-suspended thermoplastic polyurethane (TPU). Next, the core template was removed to obtain a customized porous TPU graft. The mechanical testing and cytotoxicity studies of the new synthetic 3D templated vascular grafts (3DT) were more appropriate compared with commercially available polytetrafluoroethylene (PTFE) grafts (ePTFE; standard graft, SG) for clinical use. Finally, we performed implantation of the 3DTs and SGs into the rat abdominal aorta as a patch technique. Four groups of the animal model (SG_7 days, SG_30 days, 3DT_7 days, and 3DT_30 days) were enrolled in this study. The abdominal aorta was surgically opened and sutured with SG or 3DT with 8/0 Prolene. The degree of endothelial cell activation, neovascularization, thrombus formation, calcification, inflammatory infiltrates, and fibrosis were analyzed histopathologically. There was significantly decreased thrombogenesis in the group treated with the 3DT for 30 days compared with the group treated with the SG for 7 and 30 days, and the 3DT for 7 days. In addition, the group treated with the 3DT for 30 days may also have shown increased postoperative endothelialization in the early stages. In conclusion, this study suggests the possibility of using the 3DT as an SG substitute in vascular surgery.

The Effect of Ulinastatin to the Learning and Memory in Zebrafish.

Previous study indicated that Ulinastatin (UTI) increased glutamine uptake by upregulation of glutamate transporters in astrocytes. These glutamate transporters have important role to improve cognitive function in hippocampus. In this study, we wanted to demonstrate whether UTI could improve learning and memory by using zebrafish behavior model and bio-markers. Zebrafish were 6-8 months of age and were 2.5-3.5 cm long. They were divided into four groups by control, 1X PBS-injected control, UTI 10,000, and 50,000 injected group. All PBS and UTI injected zebrafish were anesthetized by Tricainemethanesulphonate. We measured total time, distance moved, and frequency in each compartment of T-maze. We also measured the expression levels of glutamate transporter levels and cognitive bio-markers such as c-fos, c-jun, BDNF. UTI affected the learning and memory in zebrafish in a dose-dependent manner. In 50,000 unit/kg UTI-treated zebrafish, there were increases of time, distance, and frequency in target compartment. In 50,000 unit/kg UTI-treated zebrafish, there was an increase of time in target compartment. There was no difference among control, PBS-injected, and UTI 10,000 unit/kg-treated groups. EAAT4 glutamate transporter, c-fos and BDNF were significantly increased in 50,000 unit/kg UTI-treated group. UTI-enhanced learning and memory in zebrafish. The expressions of EAAT4 glutamate transporter, c- fos and BDNF in zebrafish were highly correlated may play a role.

Anti-thrombotic and anti-inflammatory activity of sulodexide compared to aspirin in the rat model.

BACKGROUND: Although the number of vascular surgeries performed is increasing, the incidence of complications associated with this surgery has not improved and re-operations are frequently required. Thrombosis in a vessel is the most hazardous postoperative complication. OBJECTIVE: The aim of this study was to evaluate the anti-thrombotic and anti-inflammatory effects of sulodexide compared to aspirin in a rat model. METHODS: We divided the animals into three groups (sham (saline), aspirin, and sulodexide). The abdominal aorta was surgically opened and closed, primarily with 8/0 Prolene sutures. Postoperatively, saline, aspirin, or sulodexide was administered by oral gavage for 14 days to the rats. The degree of neovascularization, thrombus, calcification, inflammatory infiltrates, and fibrosis were analyzed histopathologically by hematoxylin and eosin staining. RESULTS: There was no significant difference in the incidence of postoperative thrombogenesis, but less calcification and inflammatory infiltrates were observed in the sulodexide group compared to the aspirin group. Histopathologic score revealed less infiltration of inflammatory cells and mild calcification for the sulodexide group (0.17±0.41 and 1.33±0.52, respectively) compared to the aspirin group (0.67±0.52 and 1.67±0.52, respectively) at days 14. CONCLUSIONS: This study offers the possibility that sulodexide could be used as an aspirin substitute for the postoperative management of vascular patients, with low gastrointestinal discomfort. In addition, it may also offer reduced postoperative calcification and inflammation.

Entrectinib Induces Apoptosis and Inhibits the Epithelial-Mesenchymal Transition in Gastric Cancer with NTRK Overexpression.

Tropomyosin receptor kinase (TRK) and receptor tyrosine kinase (RTK class VII) expression are important in many human diseases, especially cancers, including colorectal, lung, and gastric cancer. Using RNA sequencing analysis, we evaluated the mRNA expression and mutation profiles of gastric cancer patients with neurotropic tropomyosin receptor kinase (NTRK) 1-3 overexpression (defined as a ≥2.0-fold change). Furthermore, we screened eight TRK inhibitors in NCI-N87, SNU16, MKN28, MKN7, and AGS cells. Among these inhibitors, entrectinib showed the highest inhibitory activity; therefore, this drug was selected for analysis of its therapeutic mechanisms in gastric cancer. Entrectinib treatment induced apoptosis in NTRK1-3-expressing and VEGFR2-expressing NCI-N87 and AGS cells, but it had no effect on NTRK1-3-, VEGFR2-, TGFBR1-, and CD274-expressing MKN7 cells. SNU16 and MKN28 cells with low NTRK1-3 expression were not affected by entrectinib. Therefore, a mechanistic study was conducted in NCI-N87 (high expression of NTRK1-3 but mutation of NTRK3), AGS (high expression of NTRK1-3) and MKN28 (low expression of NTRK1-3) gastric cancer cell lines. Entrectinib treatment significantly reduced expression levels of phosphorylated NFκB, AKT, ERK, and ß-catenin in NCI-N87 and AGS cells, whereas it upregulated the expression levels of ECAD in NCI-N87 cells. Together, these results suggest that entrectinib has anti-cancer activity not only in GC cells overexpressing pan NTRK but also in VEGFR2 GC cells via the inhibition of the pan NTRK and VEGFR signaling pathways.

Tepotinib Inhibits the Epithelial-Mesenchymal Transition and Tumor Growth of Gastric Cancers by Increasing GSK3ß, E-Cadherin, and Mucin 5AC and 6 Levels.

Aberrant expression of mucins (MUCs) can promote the epithelial-mesenchymal transition (EMT), which leads to enhanced tumorigenesis. Carcinogenesis-related pathways involving c-MET and ß-catenin are associated with MUCs. In this study, we characterized the expression of EMT-relevant proteins including MET, ß-catenin, and E-cadherin in human gastric cancer (GC) cell lines, and further characterized the differential susceptibility of these cell lines compared with the c-MET inhibitor tepotinib. We assessed the antitumor activity of tepotinib in GC cell lines. The effects of tepotinib on cell viability, apoptotic cell death, EMT, and c-MET and ß-catenin signaling were evaluated by 3-(4,5 dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl-2-(4-sulfophenyl)-2H-tetrazolium (MTS), flow cytometry, Western blotting, and qRT-PCR. The antitumor efficacy was assessed in MKN45 xenograft mice. Tepotinib treatment induced apoptosis in c-MET-amplified SNU620, MKN45, and KATO III cells, but had no effect on c-MET-reduced MKN28 or AGS cells. Tepotinib treatment also significantly reduced the protein levels of phosphorylated and total c-MET, phosphorylated and total ERK, ß-catenin, and c-MYC in SNU620 and MKN45 cells. In contrast, this drug was only slightly active against KATO III cells. Notably, tepotinib significantly reduced the expression of EMT-promoting genes such as MMP7, COX-2, WNT1, MUC5B, and c-MYC in c-MET-amplified GC cells and increased the expression of EMT-suppressing genes such as MUC5AC, MUC6, GSK3ß, and E-cadherin. In a mouse model, tepotinib exhibited good antitumor growth activity along with increased E-cadherin and decreased phosphorylated c-MET (phospho-c-MET) protein levels. Collectively, these results suggest that tepotinib suppresses tumor growth and migration by negatively regulating c-MET-induced EMT. These findings provide new insights into the mechanism by which MUC5AC and MUC6 contribute to GC progression.

Tivantinib inhibits the VEGF signaling pathway and induces apoptosis in gastric cancer cells with c-MET or VEGFA amplification.

Tivantinib has been described as a selective inhibitor of c-Met and is being studied in various types of cancer. In this study, we evaluated the effects of tivantinib on the suppression of gastric cancer (GC) cell migration and apoptosis. We also examined the mechanism of action of tivantinib by oncogenic pathway analysis. We applied an RNA-sequencing approach in 34 GC patients to identify oncogenes that are differentially expressed in GC tissues. To examine the inhibitory effect of tivantinib on GC cells, we conducted apoptosis analysis using an annexin V-APC/PI apoptosis detection kit and trans-well migration assay with human GC cell lines. For oncogenic pathway analysis, Western blot and quantitative real-time PCR analysis were used to detect the expression of proteins and genes before and after tivantinib exposure. In the RNA-sequencing analysis of 34 GC patients, c-Met and VEGFA genes were expressed and positively correlated with each other. Cell migration and apoptosis analysis demonstrated that tivantinib induced the best inhibition effect in SNU620, MKN45 (carries VEGFB mutation), AGS, and MKN28 cells, but not in KATO III (carries VEGFB and VEGFC mutations) cells. Oncogenic pathway analysis showed that tivantinib, in addition to c-Met signaling pathway inhibition, also inhibits VEGF signaling and MYC expression in VEGFA-expressing GC cells. We found that tivantinib has anti-cancer activity not only in GC cells overexpressing c-Met but also in non-c-Met GC cells by inhibition of the VEGF signaling pathway.

Foretinib Inhibits Cancer Stemness and Gastric Cancer Cell Proliferation by Decreasing CD44 and c-MET Signaling.

PURPOSE: CD44 isoforms are highly expressed in cancer stem cells, initiating tumor growth and sustaining tumor self-renewal. Among these isoforms, CD44 variant 9 (CD44v9) is overexpressed in chronic inflammation-induced cancer. CD44 and the mesenchymal-to-epithelial transition (MET) receptor tyrosine kinase are coactivated in some gastric cancers (GCs). In this study, we characterized MET and CD44 expression and signaling in human GC cell lines and analyzed differences in the susceptibility of these lines to foretinib. PATIENTS AND METHODS: We analyzed cell viability and the rate of apoptotic cells using MTS assays and flow cytometry, respectively. Gene and protein expression were assessed by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and immunoblotting, respectively. RESULTS: Foretinib treatment resulted in dose-dependent inhibition of growth in c-MET-amplified MKN45 and SNU620 cells with concomitant induction of apoptosis, but not in c-MET-reduced MKN28 and AGS cells. Foretinib treatment also significantly reduced phosphor-c-MET, phosphor-AKT, beta-catenin, and COX-2 protein expression in MKN45 and SNU620 cells. Interestingly, foretinib significantly reduced CD44, CD44v9, COX-2, OCT3/4, CCND1, c-MYC, VEGFA, and HIF-1a gene expression in CD44 and MET coactivated MKN45 cells and increased CD44s gene expression; in contrast, these drugs were only slightly active against SNU620 cells. CONCLUSION: The results of this study indicate that foretinib could be a therapeutic agent for the prevention or treatment of GCs positive for CD44v9 and c-MET.

INC280 inhibits Wnt/ß-catenin and EMT signaling pathways and its induce apoptosis in diffuse gastric cancer positive for c-MET amplification.

OBJECTIVE: Gastric cancer is more open related to genetic predisposition. In our RNA sequencing study on gastric cancer patients, Runt-related transcription factor-3 (RUNX3) expression was significantly down-regulated in gastric cancer. We showed that decreased levels of RUNX3 are significantly associated with c-MET (r = - 0.4216, P = 0.0130). In addition, c-MET expression is a candidate for targeted therapy in gastric cancer. Therefore, in the present study, the anti-cancer effects of the c-MET inhibitor on gastric cancer cells from positive or negative for c-MET amplification were evaluated. RESULTS: INC280 treatment inhibits growth of a c-MET-amplified MKN45 (RUNX3-positive) and SNU620 (RUNX3-negative) diffuse type cells. Then, INC280 showed the highest inhibition and apoptotic rates with the lowest IC50s in MKN45 cells but not in c-MET-reduced MKN28 (intestinal type) cells. We also showed that INC280 inhibits the WNT signaling pathway and SNAIL expression in MKN45 cells. The data indicate that INC280 could be used as therapeutic agents for the prevention or treatment of diffuse gastric cancer positive for c-MET amplification.

Effect of Estradiol in an Azoxymethane/Dextran Sulfate Sodium-Treated Mouse Model of Colorectal Cancer: Implication for Sex Difference in Colorectal Cancer Development.

PURPOSE: This study demonstrates that estradiol downregulates inflammation and inhibits colorectal cancer (CRC) development in azoxymethane/dextran sulfate sodium (AOM/DSS) mouse model. MATERIALS AND METHODS: AOM/DSS-treated male and female mice were sacrificed at weeks 2, 10, and 16, to assess estrogen effects on colitis and carcinogenesis. Macroscopic and histologic severity of colitis and Western blot and quantitative real-time polymerase chain reaction were evaluated, to measure inflammatory mediators and cytokines. RESULTS: Compared with AOM/DSS-treated male mice (M-AOM/DSS group), AOM/DSS-treated male mice with estradiol administration (M-AOM/DSS+estr group) displayed at week 2 significantly decreased severity of colitis. At weeks 10 and 16, AOM/DSS-treated female mice (F-AOM/DSS group) and the M-AOM/DSS+estr group showed significantly lower tumor multiplicity compared with the M-AOM/DSS group. At week 2, F-AOM/DSS group had a lower level of nuclear factor-κB (NF-κB) expression and higher level of nuclear factor erythroid 2-related factor 2 (Nrf2) expression, compared to the M-AOM/DSS group. At week 2, expression levels of NF-κB and its related mediators decreased in the M-AOM/DSS+estr group, while levels of Nrf2 and Nrf2-related anti-oxidant enzymes increased. In addition, estradiol significantly increased Nod-like receptor protein 3 (NLRP3) inflammasome expressions in AOM/DSS-treated male mice. In contrast, at weeks 10 and 16, Nrf2 and its-related anti-oxidant enzymes and NLRP3 inflammasome were highly expressed in M-AOM/DSS group and in F-AOM/DSS group, who developed cancer. CONCLUSION: The data suggest that estradiol inhibits the initiation of CRC by regulating Nrf2-related pathways. Moreover, these imply the dual role of Nrf2 and NLRP3 inflammasome, including promotion of tumor progression upon tumor initiation.

Effects of 17ß-Estradiol on Colonic Permeability and Inflammation in an Azoxymethane/Dextran Sulfate Sodium-Induced Colitis Mouse Model.

Background/Aims: Intestinal barrier dysfunction is a hallmark of inflammatory bowel diseases (IBDs) such as ulcerative colitis. This dysfunction is caused by increased permeability and the loss of tight junctions in intestinal epithelial cells. The aim of this study was to investigate whether estradiol treatment reduces colonic permeability, tight junction disruption, and inflammation in an azoxymethane (AOM)/dextran sodium sulfate (DSS) colon cancer mouse model. Methods: The effects of 17ß-estradiol (E2) were evaluated in ICR male mice 4 weeks after AOM/DSS treatment. Histological damage was scored by hematoxylin and eosin staining and the levels of the colonic mucosal cytokine myeloperoxidase (MPO) were assessed by enzyme-linked immunosorbent assay (ELISA). To evaluate the effects of E2 on intestinal permeability, tight junctions, and inflammation, we performed quantitative real-time polymerase chain reaction and Western blot analysis. Furthermore, the expression levels of mucin 2 (MUC2) and mucin 4 (MUC4) were measured as target genes for intestinal permeability, whereas zonula occludens 1 (ZO-1), occludin (OCLN), and claudin 4 (CLDN4) served as target genes for the tight junctions. Results: The colitis-mediated induced damage score and MPO activity were reduced by E2 treatment (p＜0.05). In addition, the mRNA expression levels of intestinal barrier-related molecules (i.e., MUC2, ZO-1, OCLN, and CLDN4) were decreased by AOM/DSS-treatment; furthermore, this inhibition was rescued by E2 supplementation. The mRNA and protein expression of inflammation-related genes (i.e., KLF4, NF-κB, iNOS, and COX-2) was increased by AOM/DSS-treatment and ameliorated by E2. Conclusions: E2 acts through the estrogen receptor ß signaling pathway to elicit anti-inflammatory effects on intestinal barrier by inducing the expression of MUC2 and tight junction molecules and inhibiting pro-inflammatory cytokines.

Probiotics reduce repeated water avoidance stress-induced colonic microinflammation in Wistar rats in a sex-specific manner.

The colonic response to stress is greater in female rats than in male rats. The aim of this study was to evaluate the effect of probiotics in the repeated water avoidance stress (rWAS)-induced colonic microinflammation model of Wistar rats in a sex-specific manner. The three groups (no-stress, WAS, and WAS with probiotics) were exposed to r-WAS for 1 h daily for 10 days, and Lactobacillus farciminis was administered by oral gavage for 10 days to animals in the probiotics group. The visceromotor response (VMR) to colorectal distension (CRD) was assessed using a barostat and noninvasive manometry before and after WAS exposure. Immunohistochemistry for mast cells and real-time polymerase chain reaction (RT-PCR) for detection of mucosal cytokines were performed using distal colon tissue after the animals were sacrificed. Significant reduction of VMR to CRD (visceral analgesia) was observed at 60 mmHg in the female WAS group (P = 0.045), but not in males. In addition, the female WAS with probiotics group showed a significantly lower colonic mucosal mast cell count in comparison to the female WAS group (P = 0.013), but this phenomenon was not observed in the male group. The colonic mucosal mRNA levels of interferon-γ (IFNR), tumor necrosis factor-α (TNFA), interleukin (IL) 6, and IL17 were higher in the female WAS group than in the male WAS group. The mRNA levels of IFNR, TNFA, and IL6 were significantly decreased in WAS females who received probiotics (all P < 0.050). In conclusion, rWAS is induced in a sex-specific manner. A 10-day-long treatment with L. farciminis is an effective therapy for rWAS-induced colonic microinflammation in female rates, but not in male rats.

Analysis of Gastric Body Microbiota by Pyrosequencing: Possible Role of Bacteria Other Than Helicobacter pylori in the Gastric Carcinogenesis.

BACKGROUND: Gastric microbiota along with Helicobacter pylori (HP) plays a key role in gastric disease. The aim of our study is to investigate the difference of human gastric microbiota between antrum and body according to disease (control vs. gastric cancer) and HP status. METHODS: Each antrum and body biopsy was collected from 12 subjects at Seoul National University Bundang Hospital. Gastric microbiota was analyzed by bar-coded 454 pyrosequencing of the 16S rRNA gene. Twelve subjects consisted of HP-negative control (n = 2), HP-negative cancer (n = 2), HP-positive control (n = 3), and HP-positive cancer (n = 5). The analysis was focused on non-HP urease-producing bacteria (UB) and non-HP nitrosating or nitroreducing bacteria (NB) between antrum and body. RESULTS: Gastric body samples showed higher diversity compared to gastric antrum mucosa samples but there was no significant difference. The mean of operational taxonomic units was higher in HP(-) cancer than HP(+) cancer (antrum, 273.5 vs. 228.2, P = 0.439; body, 585.5 vs. 183.2, P = 0.053). The number of non-HP UB and non-HP NB was higher in HP(-) cancer groups than the others. These differences were more pronounced in the body (P = 0.051 and P = 0.081, respectively). Analysis of overlap of non-HP UB and non-HP NB revealed the higher composition of Streptococcus pseudopneumoniae, S. parasanguinis, and S. oralis in HP(-) cancer groups than the others, only in the body (P = 0.030) but not in the antrum (P = 0.123). CONCLUSIONS: Higher diversity and higher composition of S. pseudopneumoniae, S. parasanguinis, and S. oralis in HP(-) cancer group than the other groups in the body suggest that analysis of microbiota from body mucosa could be beneficial to identify a role of non-HP bacteria in the gastric carcinogenesis.

Erratum: Analysis of Gastric Body Microbiota by Pyrosequencing: Possible Role of Bacteria Other Than Helicobacter pylori in the Gastric Carcinogenesis.

[This corrects the article on p. 115 in vol. 22, PMID: 28698866.].

Immunotherapy with methyl gallate, an inhibitor of Treg cell migration, enhances the anti-cancer effect of cisplatin therapy.

Foxp3(+) CD25(+)CD4(+) regulatory T (Treg) cells are crucial for the maintenance of immunological self-tolerance and are abundant in tumors. Most of these cells are chemo-attracted to tumor tissues and suppress anti-tumor responses inside the tumor. Currently, several cancer immunotherapies targeting Treg cells are being clinically tested. Cisplatin is one of the most potent chemotherapy drugs widely used for cancer treatment. While cisplatin is a powerful drug for the treatment of multiple cancers, there are obstacles that limit its use, such as renal dysfunction and the development of cisplatin-resistant cancer cells after its use. To minimize these barriers, combinatorial therapies of cisplatin with other drugs have been developed and have proven to be more effective to treat cancer. In the present study, we evaluated the eff ect of the combination therapy using methyl gallate with cisplatin in EL4 murine lymphoma bearing C57BL/6 mice. The combinatorial therapy of methyl gallate and cisplatin showed stronger anti-cancer eff ects than methyl gallate or cisplatin as single treatments. In Treg cell-depleted mice, however, the eff ect of methyl gallate vanished. It was found that methyl gallate treatment inhibited Treg cell migration into the tumor regardless of cisplatin treatment. Additionally, in both the normal and cisplatin-treated tumor-bearing mice, there was no renal toxicity attributed to methyl gallate treatment. These findings suggest that methyl gallate treatment could be useful as an adjuvant method accompanied with cisplatin therapy.

Regulatory T Cells Contribute to the Inhibition of Radiation-Induced Acute Lung Inflammation via Bee Venom Phospholipase A2 in Mice.

Bee venom has long been used to treat various inflammatory diseases, such as rheumatoid arthritis and multiple sclerosis. Previously, we reported that bee venom phospholipase A2 (bvPLA2) has an anti-inflammatory effect through the induction of regulatory T cells. Radiotherapy is a common anti-cancer method, but often causes adverse effects, such as inflammation. This study was conducted to evaluate the protective effects of bvPLA2 in radiation-induced acute lung inflammation. Mice were focally irradiated with 75 Gy of X-rays in the lung and administered bvPLA2 six times after radiation. To evaluate the level of inflammation, the number of immune cells, mRNA level of inflammatory cytokine, and histological changes in the lung were measured. BvPLA2 treatment reduced the accumulation of immune cells, such as macrophages, neutrophils, lymphocytes, and eosinophils. In addition, bvPLA2 treatment decreased inflammasome-, chemokine-, cytokine- and fibrosis-related genes' mRNA expression. The histological results also demonstrated the attenuating effect of bvPLA2 on radiation-induced lung inflammation. Furthermore, regulatory T cell depletion abolished the therapeutic effects of bvPLA2 in radiation-induced pneumonitis, implicating the anti-inflammatory effects of bvPLA2 are dependent upon regulatory T cells. These results support the therapeutic potential of bvPLA2 in radiation pneumonitis and fibrosis treatments.