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Cannabidiol (CBD), one of the compounds present in the marijuana plant, has anti-tumor properties, but its mechanism is not well known. This study aimed to evaluate the apoptotic action of CBD in colorectal cancer (CRC) cells, and focused on its effects on the novel pro-apoptotic Noxa-reactive oxygen species (ROS) signaling pathway. CBD experiments were performed using the CRC cell lines HCT116 and DLD-1. CBD induced apoptosis by regulating many pro- and anti-apoptotic proteins, of which Noxa showed significantly higher expression. To understand the relationship between Noxa and CBD-induced apoptosis, Noxa levels were downregulated using siRNA, and the expression of apoptosis markers decreased. After ROS production was blocked, the level of Noxa also decreased, suggesting that ROS is involved in the regulation of Noxa, which along with ROS is a well-known pro-apoptotic signaling agents. As a result, CBD induced apoptosis in a Noxa-and-ROS-dependent manner. Taken together, the results obtained in this study re-demonstrated the effects of CBD treatment in vivo, thus confirming its role as a novel, reliable anticancer drug.

Amplification of fibroblast growth factor receptor2 (FGFR2) has been regarded as a druggable target in gastric cancer (GC). Despite known potential of AZD4547, a selective inhibitor of FGFR 1-3, to suppress tumorigenic effects of activated FGFR2, resistance to the targeted agent has been an unresolved issue. This study was performed to elucidate the mechanism of AZD4547 resistance in GC cells. SNU-16 cells were used to establish an AZD4547-resistant GC cell line, SNU-16R. Elevated phosphorylation of EphB3 was confirmed using the Human Phospho-Receptor Tyrosine Kinase Array kit. A tyrosine kinase inhibitor (TKI) of EphB3 was used to investigate the effects of suppressed EphB3 activity in the SNU-16R cell line. SNU-16R cells exhibited upregulated phosphorylation of EphB3. Treatment of SNU-16R cells with the EphB3 TKI resulted in induction of apoptosis, decreased cellular viability, and cell cycle arrest at sub-G1 phase. SNU-16R cells expressed upregulated levels of N-cadherin, vimentin, Snail, matrix metalloproteinase 2 (MMP-2), and MMP-9, and reduced levels of E-cadherin, characteristic of epithelial to mesenchymal transition (EMT). Matrigel invasion assay also demonstrated the increased invasiveness of SNU-16R cells. EphB3 TKI treatment inhibited EMT of SNU-16R cells. Activation of mammalian target of rapamycin (mTOR) through the Ras-ERK1/2 pathway was suggested as the signal transduction mechanism downstream EphB3 by showing enhanced phosphorylation of Raf-1, MEK1/2, ERK1/2, mTOR and its downstream substrates in SNU-16R cells. As expected, EphB3 TKI decreased phosphorylation of these proteins. Our data suggest phosphorylation of mTOR through signaling by EphB3 is a potential mechanism of AZD4547 resistance in GC cells.

The molecular epidemiology and antimicrobial resistance of Clostridioides difficile were studied in South Korea in 2017 as part of a National Surveillance System. From February to May 2017, all non-duplicate isolates of C. difficile were recovered from patients who were suspected to have C. difficile infection and collected from 6 referral hospitals representing the 6 regions in South Korea. We performed PCRs for the toxin gene, PCR ribotyping, multilocus sequence typing (MLST), antimicrobial susceptibility testing by agar dilution according to the recommendations of the CLSI and detection of antimicrobial resistance genes such as ermB, catD, tetM, vanZ and nimR by PCR. Of 331 C. difficile isolates, 257 (77.6%) were toxigenic and the prevalence of strains producing binary toxin (CDT) was 5.1% (13/257). A total of 52 different ribotype (RT) patterns were found. RT018 was the most common (25.1% of all isolates), and RT014/020, RT002 and RT012 were also common. RT010 was most common non-toxigenic strain. MLST analysis of randomly selected 72 C. difficile isolates identified 46 sequence types (STs), of which three were new and not in the PubMLST library. There was a good correlation between MLST and RT as following: ST1 (RT027), ST8 (RT002), ST11 (RT078), ST17 (RT018), ST35 (RT046), ST37 (RT017), ST42 (RT106), ST53 (RT103), ST81 (RT369), and ST99 (RT070). All toxigenic isolates were susceptible to metronidazole and vancomycin (MIC ≤ 2 mg/L). For rifaximin, 24% of toxigenic isolates were resistant. Of randomly selected 106 toxigenic isolates, resistance rates for ampicillin, cefotetan, clindamycin, imipenem, chloramphenicol, tetracycline, and moxifloxacin were 48%, 46%, 64%, 54%, 0%, 6% and 52% respectively and frequencies of various resistance genes were 62.3% for ermB, 0.9% catD and 10.4% tetM. RTs018, 002, 017 and 369 showed high MICs to various antimicrobial agents and multi-drug resistance was common also.

Background Multifunctional, antiproliferative small molecules are deemed to offer pharmacodynamic and pharmacokinetic benefits over combination therapy, in addition to reducing toxicity, cancer resistance, and therapy costs. In this study, we conducted an in vitro cellular screen of our recently reported series of EGFR/HER2 inhibitors. Methods Cytotoxicity induced by AF8c was confirmed using WST-1 assay. An Annexin V-Fluorescein Isothiocyanate (FITC) Apoptosis Detection Kit, TUNEL assay, and Western blotting were carried out to invastigate apoptosis. A human phosphorylated kinase array was used to identify proteins differentially expressed between control cells and AF8c-treated cell lines. ROS generation was monitored by flow cytometry, confocal microscopy using dihydroethidium (DHE) and MitoSOX. For in vivo tumor xenograft study, four-week-old female BALB/c nude mice were injected subcutaneously with either HT29 Luc+ or HCT116 Luc+ cells (1 × 107 cells in 100 μl PBS). When the tumor had reached approximately 100 mm3 in size, the mice were randomly divided into three groups (n = 8): DMSO-treated, treated with 10 mg/kg AF8c, and treated with 20 mg/kg AF8c. Results Analysis of the AF8c mode of action in CRC cells revealed that it mediates apoptosis via the generation of endoplasmic reticulum (ER) stress and reactive oxygen species (ROS), as well as selective activation of nuclear respiratory factor 2 alpha subunit (Nrf2) and death receptor 5 (DR5), but not DR4. Silencing of DR5 attenuated the expression levels of Nrf2 and partially inhibited AF8c-induced apoptosis. Additionally, upregulation of Nrf2 by AF8c evoked apoptosis through a decrease in antioxidant levels. Treatment of mice with AF8c also resulted in the upregulation of DR5, Nrf2, and CHOP proteins, subsequently leading to a significant decrease in tumor burden. Conclusions AF8c-induced apoptosis may be associated with DR5/Nrf2 activation through ER stress and ROS generation in CRC. These findings indicate that AF8c represents a promising polypharmacological molecule for the treatment of human CRC. Moreover, it could also be a potential starting point for the understanding of the structural features of quinazoline-based ErbB family inhibitors targeting the TRAIL cascade. Legal entity responsible for the study The authors. Funding Has not received any funding. Disclosure All authors have declared no conflicts of interest.

Bone morphogenetic proteins (BMPs) have been implicated in tumorigenesis and metastatic progression in various types of cancer cells, but the role and cellular mechanism in the invasive phenotype of gastric cancer cells is not known. Herein, we determined the roles of phosphoinositide 3-kinase (PI3K)/AKT, extracellular signal-regulated protein kinase (ERK), nuclear factor (NF)-κB, and matrix metalloproteinase (MMP) expression in BMP-2-mediated metastatic function in gastric cancer. We found that stimulation of BMP-2 in gastric cancer cells enhanced the phosphorylation of AKT and ERK. Accompanying activation of AKT and ERK kinase, BMP-2 also enhanced phosphorylation/degradation of IκBα and the nuclear translocation/activation of NF-κB. Interestingly, blockade of PI3K/AKT and ERK signaling using LY294002 and PD98059, respectively, significantly inhibited BMP-2-induced motility and invasiveness in association with the activation of NF-κB. Furthermore, BMP-2-induced MMP-9 expression and enzymatic activity was also significantly blocked by treatment with PI3K/AKT, ERK, or NF-κB inhibitors. Immunohistochemistry staining of 178 gastric tumor biopsies indicated that expression of BMP-2 and MMP-9 had a significant positive correlation with lymph node metastasis and a poor prognosis. These results indicate that the BMP-2 signaling pathway enhances tumor metastasis in gastric cancer by sequential activation of the PI3K/AKT or MAPK pathway followed by the induction of NF-κB and MMP-9 activity, indicating that BMP-2 has the potential to be a therapeutic molecular target to decrease metastasis.

A microorganism (strain HJ6) producing extracellular beta-glycosidase was isolated from a hot springs located in Arima-cho, Hyogo, Japan. The cells were long-rods (2-4 microm) about 0.4 microm in diameter, and formed yellow-colored colonies, like most other strains of the genus Thermus. The pH and temperature for optimal growth were 6.5 and 80 degrees C. Thus, the HJ6 strain displayed a higher optimal temperature than other described Thermus sp. The gene encoding beta-glycosidase (TtbetaGly) was cloned, sequenced, and comprised of 1296 nucleotides encoding a protein (431 amino acids) with a predicted molecular mass of 48.7 kDa. TtbetaGly was expressed in Escherichia coli cells, and the recombinant protein was purified to homogeneity. The optimal temperature and pH for beta-glycosidase activity were found to be 90 degrees C and 8.5, respectively. The half-life of heat inactivation was about 30 min at 95 degrees C indicating that TtbetaGly had higher thermostability than beta-glycosidases from other Thermus sp. The results of the kinetics experiment indicated that beta-D-fucoside and beta-D-glucoside were better substrates of TtbetaGly than beta-D-galactoside. The catalytic efficiency (k(cat)/K(m)) of TtbetaGly at 80 degrees C increased 70-fold to that at 40 degrees C, indicating that this enzyme was activated at high temperatures. Thin layer chromatography showed that the enzyme had transglycosylation activity at high temperature and that various transfer products were formed in the reaction with lactose or cellobiose.

The proper homeostasis of the liquid lining the surface of the middle ear cavity is vitally important for maintaining a fluid-free middle ear cavity. Disruption of this homeostasis leads to fluid collection in the middle ear cavity and results in otitis media with effusion. We demonstrated the molecular and functional expression of the Na+/H+ exchanger (NHE)s in normal human middle ear epithelial (NHMEE) cells. We also evaluated the role of NHEs in fluid absorption and the effect of dexamethasone on NHE function and NHE-dependent fluid absorption in NHMEE cells. Western blot analysis was performed for NHE1, -2, and -3 in NHMEE cells. The fluid absorption rate was measured after liquid application on the luminal surface of the cells. Intracellular pH (pHi) was measured using the pH-sensitive fluorescent probe bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-AM. NHE activity was determined as Na+-induced pHi recovery from an acid load achieved by luminal exposure to 40 mmol/l NH4Cl. NHE1, -2 and -3 were all expressed in the NHMEE cells. The pHi recovery rate was suppressed by inhibition of NHE2 and -3 with HOE694 at concentrations greater than 50 microM. Inhibition of NHE3 with 650 microM of HOE694 or S3226 significantly decreased the fluid absorption rate. Dexamethasone increased the Na+-induced pHi recovery rate which was reversed by the inhibition of NHE3 with 650 microM of HOE694. Dexamethasone treatment up-regulated NHE3 expression in a dose-dependent manner. The fluid absorption rate was increased by treatment with dexamethasone (10(-7) M) and reversed by the inhibition of NHE3. In summary, we have shown that NHE3 are involved in the regulation of both pHi and fluid absorption on the luminal surface of NHMEE cells. Dexamethasone stimulates NHE3 expression and NHE3-dependent fluid absorption in NHMEE cells. These findings provide a new insight into mechanisms that regulate periciliary fluid and the therapeutic mechanisms behind steroid treatment of otitis media with effusion.

Ion channels, such as the epithelial sodium channel (ENaC), are essential for maintaining a fluid-free middle ear cavity by controlling periciliary fluid. Deviations from the normal volume or compositions of periciliary fluid are probably responsible for otitis media with effusion. To elucidate the physiologic roles of the ENaC and cystic fibrosis transmembrane conductance regulator (CFTR) in the middle ear mucosa, we compared the electrophysiological activity and protein expressions of ENaC and CFTR in normal human middle ear epithelial (NHMEE) cells with those in normal human nasal epithelial (NHNE) cells. We also evaluated the role of ENaC and CFTR in fluid transport by NHMEE cells. Short-circuit current (Isc) was measured in cell monolayers by modified Ussing chambers. Immunoblotting was performed for ENaC and CFTR. In addition, transepithelial fluid transport was measured after loading 100 microl of fluid onto the luminal cell surface. The amiloride-sensitive Isc in NHMEE cells was much larger than in NHNE cells, whereas the forskolin-induced Isc, presumably mediated by CFTR, was significantly smaller in NHMEE cells. ENaC subunits alpha, beta, and gamma were all detected in NHMEE cells, and their expressions were stronger than those in NHNE cells. In comparison, CFTR was also detected in the middle ear mucosa, but at a lower expression level than in NHNE cells. NHMEE cells showed more amiloride-sensitive fluid absorption than NHNE cells. In contrast, fluid absorption was less sensitive to forskolin/IBMX in NHMEE cells than in NHNE cells. The ATP induced Cl- efflux and the amplitude of ATP-induced current in NHMEE cells was much larger than in NHNE cells. In the present study, we have demonstrated an enhanced amiloride-sensitive Isc and fluid absorption in NHMEE cells, where the role of CFTR is limited. Our data also suggest that the ATP-induced Cl- channel could be an alternative Cl- channel to CFTR in NHMEE cells.

Purinergic agonists regulate mucin secretion in the airway epithelial cells. This study examined the effects of the apical application of purinergic agonists on Ca 2+ influx ([ Ca 2+ ] i ), and mucin secretion along with their underlying signaling pathway in normal human middle ear epithelial (NHMEE) cells. The apical membrane of NHMEE cells were stimulated with various purinergic agonists, including UTP, and the [ Ca 2+ ] i was measured using a miniature Ussing double perfusion chamber. P2Y 2 receptor in NHMEE cells was also localized by immunohistochemistry. UTP-induced mucin secretion was quantified by an immunoblotting assay. The order of the purinergic agonist potency with respect to [ Ca 2+ ] i determined in this study was ATP = UTP > 2-MeSATP > UDP ≫ adenosine which is consistent with that obtained from P2Y 2 receptor activation. The P2Y 2 receptor is expressed in the apical membrane of monolayered cultured NHMEE cells. Apical UTP-induced [ Ca 2+ ] i was inhibited by 2-aminoethoxydiphenyl borate (2-APB) but not by ryanodine. UTP-induced mucin secretion was inhibited by a Ca 2+ chelating agent, BAPTA-AM, and was stimulated by ionomycin. UTP-induced mucin secretion was also suppressed by U73122 and 2-APB, while Calphostin C suppressed it to a small extent and PD98059 was ineffective. Caffeine also inhibited the UTP-induced [ Ca 2+ ] i and mucin secretion. These results suggest that the P2Y 2 receptor is expressed in NHMEE cells, and plays a major role in modulating the [ Ca 2+ ] i from the IP 3 -sensitive intracellular Ca 2+ store. UTP-induced mucin secretion in NHMEE cells is strongly dependent on Ca 2+ - and IP 3 .