Involvement of cardiac glycosides targeting Na/K-ATPase in their inhibitory effects on c-Myc expression via its transcription, translation and proteasomal degradation.

Cardiac glycosides (CGs) have been used for decades to treat heart failure and arrhythmic diseases. Recent non-clinical and epidemiological findings have suggested that CGs exhibit anti-tumor activities. Therefore, CGs may be repositioned as drugs for the treatment of cancer. A detailed understanding of the anti-cancer mechanisms of CGs is essential for their application to the treatment of targetable cancer types. To elucidate the factors associated with the anti-tumor effects of CGs, we performed transcriptome profiling on human multiple myeloma AMO1 cells treated with periplocin, one of the CGs. Periplocin significantly down-regulated the transcription of MYC (c-Myc), a well-established oncogene. Periplocin also suppressed c-Myc expression at the protein levels. This repression of c-Myc was also observed in several cell lines. To identify target proteins for the inhibition of c-Myc, we generated CG-resistant (C9) cells using a sustained treatment with digoxin. We confirmed that C9 cells acquired resistance to the inhibition of c-Myc expression and cell proliferation by CGs. Moreover, the sequencing of genomic DNA in C9 cells revealed the mutation of D128N in α1-Na/K-ATPase, indicating the target protein. These results suggest that CGs suppress c-Myc expression in cancer cells via α1-Na/K-ATPase, which provides further support for the anti-tumor activities of CGs.

Prenylated-coumarins from Gmelina arborea and evaluation for neurotrophic activity.

A MeOH extract of the stem of Gmelina arborea Roxb. ex Sm. (Lamiaceae) exhibited neurite outgrowth-promoting activity in NGF-mediated PC12 cells. Bioassay-guided fractionation resulted in the isolation of eight previously undescribed prenylated coumarin compounds along with nine known compounds. Structural elucidation of these compounds was accomplished by analysis of extensive spectroscopic data, comparison with the literature, and chemical reactions. It was the first time to find prenylated coumarin compounds from G. arborea. Among the isolated compounds, N-methylflindersine and artanin showed neurite outgrowth-promoting activity in NGF-mediated PC12 cells.

A case of breast cancer with extensive colon metastasis.

Breast cancer is one of the most common malignancies in women worldwide. Although most breast cancers are curable, in cases of metastasis, many are often found in the lungs, bones, liver, and central nervous system; however, metastasis to the gastrointestinal tract is rare. Invasive lobular carcinoma, which represents only 5%-10% of breast cancers, has a higher risk of metastasis to the gastrointestinal tract than invasive ductal carcinoma. Here, we report a rare case of gastrointestinal metastasis of invasive lobular carcinoma that spread extensively to the colonic mucosa. Given the improved survival rates of breast cancer patients with current treatments, many rarer metastatic diseases, including gastrointestinal metastases, are likely to be increased in the future.

Characterization of Triterpene Saponin Glycyrrhizin Transport by Glycyrrhiza glabra.

Glycyrrhizin (GL), a triterpene compound produced by Glycyrrhiza species, is a crucial pharmacologically active component of crude drugs. In contrast to the biosynthesis of GL in plants, little is known about GL transport and accumulation in plants. The transport mechanism of GL was characterized using cultured cells of Glycyrrhiza glabra. Cultured cells of G. glabra efficiently incorporated exogenously supplied GL. Proton pump inhibitors, such as probenecid and niflumic acid, as well as a protonophore (carbonylcyanide m-chlorophenylhydrazone), markedly inhibited GL uptake by cultured cells, whereas vanadate exhibited a moderate inhibition. Furthermore, GL transport by G. glabra tonoplast vesicles is dependent not on a H+-electrochemical gradient but MgATP and is markedly inhibited by vanadate. These results suggest that GL uptake by cultured cells is mediated by a H+-symporter in the plasma membrane and an ATP-binding cassette transporter, which has high specificity for the aglycone structure of GL on the tonoplast.

Chukranoids A-I, isopimarane diterpenoids from Chukrasia velutina.

Bioactivity guided separation of Chukrasia velutina root methanolic extract led to the isolation of nine new isopimarane diterpenoids, chukranoids A-I (1-9). The absolute configuration was then assigned by comparing the experimental CD spectra and the calculated CD spectra. Chukranoids A-I (1-9) showed moderate antimalarial activity against Plasmodium falciparum 3D7 strain. It seems that conjugate system in the isopimarane skeleton may influence their antimalarial activity.

Periplocin and cardiac glycosides suppress the unfolded protein response.

The unfolded protein response (UPR) controls protein homeostasis through transcriptional and translational regulation. However, dysregulated UPR signaling has been associated with the pathogenesis of many human diseases. Therefore, the compounds modulating UPR may provide molecular insights for these pathologies in the context of UPR. Here, we screened small-molecule compounds that suppress UPR, using a library of Myanmar wild plant extracts. The screening system to track X-box binding protein 1 (XBP1) splicing activity revealed that the ethanol extract of the Periploca calophylla stem inhibited the inositol-requiring enzyme 1 (IRE1)-XBP1 pathway. We isolated and identified periplocin as a potent inhibitor of the IRE1-XBP1 axis. Periplocin also suppressed other UPR axes, protein kinase R-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF6). Examining the structure-activity relationship of periplocin revealed that cardiac glycosides also inhibited UPR. Moreover, periplocin suppressed the constitutive activation of XBP1 and exerted cytotoxic effects in the human multiple myeloma cell lines, AMO1 and RPMI8226. These results reveal a novel suppressive effect of periplocin or the other cardiac glycosides on UPR regulation, suggesting that these compounds will contribute to our understanding of the pathological or physiological importance of UPR.

Mitochondrial toxins potentiate hydroxyl radical production in rat striatum during carbon monoxide poisoning.

Hydroxyl radical (•OH) production in the rat striatum during carbon monoxide (CO) poisoning, which inhibits complex IV, was enhanced synergistically by malonate, a mitochondrial complex II inhibitor, but not N-methyl-4-phenylpyridinium or NaCN, complex I and IV inhibitors, respectively. No such enhancement appeared in the case of NaCN combined with malonate. Intrastriatal dopamine, which is involved in •OH production by malonate, did not synergistically enhance CO-induced •OH production. Diphenyleneiodonium, a nonselective NADPH oxidase inhibitor, partly suppressed the potentiation of CO-induced •OH production by malonate. Impairment of mitochondrial functions might potentiate oxidative stress and intensify CO toxicity in the brain.

Three Cases of Esophageal Cancer Related to Fanconi Anemia.

The risk of carcinogenesis increases after 20 years old in patients with Fanconi anemia (FA). We herein report three rare cases of FA combined with esophageal cancer in women; all patients were diagnosed with FA in early childhood. Patients 1 and 2 were diagnosed with advanced and superficial esophageal cancer, respectively, at 21 and 30 years old, respectively. Patient 3 was diagnosed with superficial esophageal cancer, underwent curative surgery at 26 years old, and survived for over 5 years without recurrence. Therefore, establishing a protocol for the early detection of esophageal cancer in FA patients over 20 years old is important.

Identification of pheophorbide a as an inhibitor of receptor for advanced glycation end products in Mallotus japonicus.

Accumulation of advanced glycation end products (AGEs) plays an important role in diabetes, immunoinflammation, and cardiovascular and neurodegenerative diseases. Since AGEs mediate their pathological effects through interaction with receptor for AGEs (RAGE), RAGE antagonists would provide a useful therapeutic option for various health disorders. Therefore, in this study, we aimed to identify phytochemicals that would inhibit binding of AGEs to RAGE, which may help develop new drug leads and/or nutraceuticals for AGE-RAGE-related diseases. On screening ethanol extracts obtained from 700 plant materials collected in Myanmar, we found that the ethanol extract from the leaves of Mallotus philippensis inhibited the binding of AGEs to RAGE. We also found that the leaves of M. japonicus, which belongs to the same genera and distributes abundantly in Japan, exhibited the inhibitory activity similar to M. philippensis. Activity-guided fractionation and LC/MS analysis of the ethanol extract of M. japonicus helped identify pheophorbide a (PPBa) as a major component in the active fraction, along with some other pheophorbide derivatives. PPBa exhibited potent inhibitory activity against AGE-RAGE binding, with an IC50 value (0.102 µM) comparable to that of dalteparin (0.084 µM). PPBa may be a valuable natural product for use as a therapeutic agent and/or a nutraceutical against various health complications arising from activation of the AGE-RAGE axis.

Triterpenoids from Walsura trichostemon.

Bioactivity guided separation of Walsura trichostemon stem methanolic extract led to the isolation of four new dammarane (1-4) and two new apotirucallane triterpenoids (5-6), together with one limonoid (7), 11,25-dideacetyltrichostemonate, 12ß, 20S, 24R-trihydroxydammar-25-en-3-one and 12ß, 20S, 25-trihydroxydammar-23-en-3-one. Compounds 1-7 showed in vitro inhibitory activity on the proliferation of A549, human lung adenocarcinoma cell line.

Celiac Disease Complicated by Rhabdomyolysis.

At 37 years old, a patient developed chronic watery diarrhea, generalized pain, severe hypokalemia and elevated creatine kinase levels. She was thought to have rhabdomyolysis due to hypokalemia from chronic diarrhea. No organic cause was found. Her symptoms subsided with potassium correction, but hypokalemia persisted; she visited our hospital at 44 years old. Endoscopy detected prominent atrophy of the intestinal villi. Histology indicated Marsh-Oberhuber type-3b disease. Anti-gliadin and anti-tissue transglutaminase IgA antibody tests were positive. She was diagnosed with celiac disease and started on a gluten-free diet, which improved her symptoms. This report is only the tenth of its kind worldwide.

Increase in the Lipopolysaccharide Activity and Accumulation of Gram-Negative Bacteria in the Stomach With Low Acidity.

INTRODUCTION: Lipopolysaccharides (LPSs) of Gram-negative bacteria (GNB) are highly toxic and induce inflammation. Therefore, we investigated both the LPS activity and composition of GNB in the gastric fluid (GF) to assess the potential toxicity of them accumulated in the stomach. METHODS: GF and saliva samples were obtained from 158 outpatients who were undergoing upper gastrointestinal endoscopy and 36 volunteers using a nasogastric tube. The LPS activity was measured by assay kits including recombinant Factor C or Limulus amebocyte lysate. To assess the bacterial composition in the samples, a 16S ribosomal DNA-based operational taxonomic unit analysis was performed. We focused on the genera representing >0.1% of the whole microbiota. RESULTS: We found a high LPS activity in the GF samples with weak acidity (approximately > pH 4), whereas little/no activity in those with strong acidity (approximately < pH 2). Spearman test also demonstrated a close correlation between pH and LPS in those samples (r = 0.872). The relative abundance of GNB in the saliva showed no significant difference between the subject groups with weak- and strong-acidity GF. In addition, in the subjects whose GF acidity was weak, the GNB abundance in the GF was almost the same as that in the saliva. By contrast, in the subjects whose GF acidity was strong, the GNB abundance in the GF was significantly lower than that in the saliva. DISCUSSION: GNB that have recently moved from the oral cavity might account for the prominent LPS activity in a stomach with weak acidity.

Blockade of the renin-angiotensin system suppresses hydroxyl radical production in the rat striatum during carbon monoxide poisoning.

Oxidative stress has been suggested to play a role in brain damage during carbon monoxide (CO) poisoning. Severe poisoning induced by CO at 3000 ppm, but not 1000 ppm, enhances hydroxyl radical (˙OH) production in the rat striatum, which might be mediated by NADPH oxidase (NOX) activation associated with Ras-related C3 botulinum toxin substrate (Rac) via cAMP signaling pathway activation. CO-induced ˙OH production was suppressed by antagonists of angiotensin II (AngII) type 1 receptor (AT1R) and type 2 receptor (AT2R) but not an antagonist of the Mas receptor. Suppression by an AT1R antagonist was unrelated to peroxisome proliferator-activated receptor γ. Angiotensin-converting enzyme inhibitors also suppressed CO-induced ˙OH production. Intrastriatal AngII at high concentrations enhanced ˙OH production. However, the enhancement of ˙OH production was resistant to inhibitors selective for NOX and Rac and to AT1R and AT2R antagonists. This indicates a different mechanism for ˙OH production induced by AngII than for that induced by CO poisoning. AT1R and AT2R antagonists had no significant effects on CO-induced cAMP production or ˙OH production induced by forskolin, which stimulates cAMP production. These findings suggest that the renin-angiotensin system might be involved in CO-induced ˙OH production in a manner independent of cAMP signaling pathways.

Kurarinone from Sophora Flavescens Roots Triggers ATF4 Activation and Cytostatic Effects Through PERK Phosphorylation.

In response to cellular stresses, activating transcriptional factor 4 (ATF4) regulates the expression of both stress-relieving genes and apoptosis-inducing genes, eliciting cell fate determination. Since pharmacological activation of ATF4 exerts potent anti-tumor effects, modulators of ATF4 activation may have potential in cancer therapy. We herein attempted to identify small molecules that activate ATF4. A cell-based screening to monitor TRB3 promoter activation was performed using crude drugs used in traditional Japanese Kampo medicine. We found that an extract from Sophora flavescens roots exhibited potent TRB3 promoter activation. The activity-guided fractionation revealed that kurarinone was identified as the active ingredient. Intriguingly, ATF4 activation in response to kurarinone required PKR-like endoplasmic reticulum kinase (PERK). Moreover, kurarinone induced the cyclin-dependent kinase inhibitor p21 as well as cytostasis in cancer cells. Importantly, the cytostatic effect of kurarinone was reduced by pharmacological inhibition of PERK. These results indicate that kurarinone triggers ATF4 activation through PERK and exerts cytostatic effects on cancer cells. Taken together, our results suggest that modulation of the PERK-ATF4 pathway with kurarinone has potential as a cancer treatment.

A Case of Mesentero-axial Type of Acute Gastric Volvulus Successfully Reduced by a Balloon-assisted Endoscopy.

A 95-year-old man was admitted to the hospital due to a sudden onset of nausea and abdominal pain. Physical examination revealed abdominal distension with mild epigastric tenderness. Computed tomography showed a grossly distended stomach with displacement of the antrum above the esophago-gastric junction, and he was diagnosed with acute mesentero-axial gastric volvulus. We attempted urgent reduction using conventional endoscopy, but failed. He and his family did not want surgery because of his extreme advanced age, and a nasogastric tube was inserted to his stomach for decompression expecting a natural reduction. On the next day, however, it was not improved, so endoscopic reduction was tried again by a balloon-assisted endoscope without an overtube under X-ray fluoroscopy. When the scope reached the descending portion of the duodenum, the balloon on the scope tip was inflated, and the stomach position was reduced by pulling back the scope with twisting to the right. He was discharged from the hospital without any complication, and no recurrence has been observed for 12 months thereafter. We suggest a balloon-assisted endoscope as a useful tool for reduction of gastric volvulus especially in cases of reduction failure by a conventional one.

Two autopsy cases of rupture of the aorta by fistula formation after thoracic endovascular aortic repair and open stent-grafting on aortic arch aneurysm.

The mortality rate of aortic aneurysm/dissection is low in Japan. Two surgical procedures, the thoracic endovascular aortic repair (TEVAR) and the open stent-grafting have contributed much in survival of such aneurysmal patients. We encountered with two autopsy cases of death by aortic rupture with fistula formation after these procedures. Case 1 is an 85-year-old male who had the history of TEVAR for thoracic aorta aneurysm one and a half year before his death. His endovascular stent-graft was composed of a steel endoskeleton consisting of six Z-shape elements while at autopsy, one of the elements locating at the distal part was found inserted deep into the wall of descending aorta, causing aorto-esophageal fistula. Case 2 is an 88-year-old male who had the history of open stent-grafting for aortic aneurysm eight years ago. At autopsy, the stent-graft was found detached from aorta at its lesser curvature, causing gap formation between the aorta and the stent. Six Z-shaped stent elements, the parts of stent-graft, were found separated from descending aorta and located in the aneurism. Furthermore, three of the separated elements were found inserted deep in the aortic wall, causing aorto-pulmonary fistula. Since aorto-esophageal fistula formation after surgery for aortic aneurysm is very rare in TEVAR and there are no reported cases of death by aorto-pulmonary fistula in the open stent-grafting, these cases are reported here.

A glucosyltransferase specific for 4-hydroxy-2,5-dimethyl-3(2H)-furanone in strawberry.

4-Hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) is a key aroma compound in Fragaria × ananassa (strawberry). A considerable amount of HDMF is converted into HDMF ß-D-glucoside and accumulated in mature strawberry fruits. Here we isolated a novel UDP-glucose: HDMF glucosyltransferase, UGT85K16 from Fragaria × ananassa. UGT85K16 preferentially glucosylated the hydroxyl group of HDMF and its structural analogs. Although UGT85K16 also catalyzed the glucosylation of vanillin, its affinity and efficiency toward HDMF was higher. The expression of UGT85K16 mRNA correlated with the accumulation of HDMF and its glucoside in Fragaria × ananassa plants. These results suggest that UGT85K16 might be UDP-glucose: HDMF glucosyltransferase in strawberries. ABBREVIATIONS: DMMF: 2,5-dimethyl-4-methoxy-3(2H)-furanone; EHMF: 2(5)-ethyl-4-hydroxy-5(2)-methyl-3(2H)-furanone; GBV: glycosidically bound volatile; HDMF: 4-hydroxy-2,5-dimethyl-3(2H)-furanone; HMF: 4-hydroxy-5-methyl-3(2H)-furanone; HMMF: 4-hydroxy-5-methyl-2-methylene-3(2H)-furanone; PSPG: Plant secondary product glycosyltransferase; RT-PCR: reverse transcription-PCR; OMT: O-methyltransferase; UGT: UDP-glycosyltransferase.

A Case-control Study on the Risk Factors for Ischemic Colitis.

OBJECTIVE: Ischemic colitis (IC) is a relatively common acute inflammation disorder of the intestine. It was considered to be a disorder of elderly people with risk factors for arteriosclerosis; however, a considerable number of young people with IC have been reported recently. We performed a case-control study to determine the risk factors for IC and compare the risk factors between elderly and non-elderly people. METHODS: The study included 209 consecutive patients diagnosed with IC between December 2004 and March 2017 at Tokai University Hospital. The study also included 209 randomly selected controls in the same calendar year so as to match age and sex. Possible risk factors for IC were identified and compared between age groups. RESULTS: The mean age of IC group was 64.9 with 60 males and 115 elderly patients aged 65 or more in each group. On multivariable conditional logistic regression analysis, drinking, abdominal surgery, hypertension, and malignant diseases were risk factors for IC in all ages. In non-elderly patients, only hypertension and laxative/enema use were significant factors, while in elderly, abdominal surgery, hypertension, COPD, malignant disease and antiplatelet drugs were significant. CONCLUSION: The risk factors in elderly people might be quite different from younger ones, while hypertension seemed to be a common risk in all ages.

Anti-Tumorigenic Activity of Chrysin from Oroxylum indicum via Non-Genotoxic p53 Activation through the ATM-Chk2 Pathway.

The p53 tumor suppressor plays critical roles in cell cycle regulation and apoptotic cell death in response to various cellular stresses, thereby preventing cancer development. Therefore, the activation of p53 through small molecules is an attractive therapeutic strategy for the treatment of cancers retaining wild-type p53. We used a library of 700 Myanmar wild plant extracts to identify small molecules that induce p53 transcriptional activity. A cell-based screening method with a p53-responsive luciferase-reporter assay system revealed that an ethanol extract of Oroxylum indicum bark increased p53 transcriptional activity. Chrysin was isolated and identified as the active ingredient in the O. indicum bark extract. A treatment with chrysin increased p53 protein expression and the p53-mediated expression of downstream target genes, and decreased cell viability in MCF7 cells, but not in p53-knockdown MCF7 cells. We also found that chrysin activated the ATM-Chk2 pathway in the absence of DNA damage. Hence, the inactivation of the ATM-Chk2 pathway suppressed p53 activation induced by chrysin. These results suggest the potential of chrysin as an anti-cancer drug through the activation of p53 without DNA damage.

Hydroxyl radical production via NADPH oxidase in rat striatum due to carbon monoxide poisoning.

Severe poisoning induced by carbon monoxide (CO) at 3000 ppm, but not 1000 ppm, enhances hydroxyl radical (OH) production in rat striatum, which is greatly susceptible to inhibitors of NADPH oxidase (NOX), including diphenyleneiodonium (DPI), but not xanthine oxidase. The quantitative real-time PCR confirmed the previous microarray finding that CO at 3000 ppm, but not 1000 ppm, enhanced mRNA expression of dual oxidase 2 (DUOX2), but not DUOX1, in rat striatum, both of which are NOX family members producing reactive oxygen species. However, the protein levels of DUOX2 and DUOX1 were decreased by 3000 ppm CO. The CO-induced OH production was resistant to chelerythrine and SB230580, inhibitors of protein kinase C and p38MAPK, respectively, which are reported to mediate activation of DUOX1 and DUOX2, respectively. Deprivation of Ca2+, which is required for activation of both DUOXs, failed to suppress the CO-induced OH production. The CO-induced OH production was strongly suppressed by EHT1864, an inhibitor of Rac (Ras-related C3 botulinum toxin substrate), which is a factor for activation of NOX1, NOX2 and NOX3 (the role of Rac on Nox3 activation is controversial) as much as that was suppressed by DPI. In addition, EHT1864 in combination with DPI further suppressed the CO-induced OH production. There were no significant changes in the protein levels of NOX1 through NOX4 and Rac1. It is likely that the CO-induced OH production is mediated through the activation of Rac-dependent NOX enzymes, such as Nox1, Nox2, and Nox3.