Identification of anti‐cancer chemical compounds using Xenopus embryos

Cancer tissues have biological characteristics similar to those observed in embryos during development. Many types of cancer cells acquire pro‐invasive ability through epithelial–mesenchymal transition (EMT). Similar processes (gastrulation and migration of cranial neural crest cells [CNCC]) are observed in the early stages of embryonic development in Xenopus during which cells that originate from epithelial sheets through EMT migrate to their final destinations. The present study examined Xenopus embryonic tissues to identify anti‐cancer compounds that prevent cancer invasion. From the initial test of known anti‐cancer drugs, AMD3100 (an inhibitor of CXCR4) and paclitaxel (a cytoskeletal drug targeting microtubules) effectively prevented migration during gastrulation or CNCC development. Blind‐screening of 100 synthesized chemical compounds was performed, and nine candidates that inhibited migration of these embryonic tissues without embryonic lethality were selected. Of these, C‐157 (an analog of podophyllotoxin) and D‐572 (which is an indole alkaroid) prevented cancer cell invasion through disruption of interphase microtubules. In addition, these compounds affected progression of mitotic phase and induced apoptosis of SAS oral cancer cells. SAS tumors were reduced in size after intratumoral injection of C‐157, and peritoneal dissemination of melanoma cells and intracranial invasion of glioma cells were inhibited by C‐157 and D‐572. When the other analogues of these chemicals were compared, those with subtle effect on embryos were not tumor suppressive. These results suggest that a novel chemical‐screening approach based on Xenopus embryos is an effective method for isolating anti‐cancer drugs and, in particular, targeting cancer cell invasion and proliferation.     

Rapid detection of germline mutations for hereditary gastrointestinal polyposis/cancers using HaloPlex target enrichment and high-throughput sequencing technologies

Genetic testing for hereditary colorectal polyposis/cancers has become increasingly important. Therefore, the development of a timesaving diagnostic platform is indispensable for clinical practice. We designed and validated target enrichment sequencing for 20 genes implicated in familial gastrointestinal polyposis/cancers in 32 cases with previously confirmed mutations using the HaloPlex enrichment system and MiSeq. We demonstrated that HaloPlex captured the targeted regions with a high efficiency (99.66 % for covered target regions, and 99.998 % for breadth of coverage), and MiSeq achieved a high sequencing accuracy (98.6 % for the concordant rate with SNP arrays). Using this approach, we correctly identified 33/33 (100 %) confirmed alterations including SNV, small INDELs and large deletions, and insertions in APC, BMPR1A, EPCAM, MLH1, MSH2, MSH6, PMS2, and SKT11. Our approach yielded the sequences of 20 target genes in a single experiment, and correctly identified all previously known mutations. Our results indicate that our approach successfully detected a wide range of genetic variations in a short turnaround time and with a small sample size for the rapid screening of known causative gene mutations of inherited colon cancer, such as familial adenomatous polyposis, Lynch syndrome, Peutz–Jeghers syndrome, and Juvenile polyposis syndrome.     

Identification of novel immunohistochemical tumor markers for primary hepatocellular carcinoma; clathrin heavy chain and formiminotransferase cyclodeaminase

Early diagnosis of hepatocellular carcinoma (HCC) greatly improves its prognosis. However, the distinction between benign and malignant tumors is often difficult, and novel immunohistochemical markers are necessary. Using agarose two-dimensional fluorescence difference gel electrophoresis, we analyzed HCC tissues from 10 patients. The fluorescence volumes of 48 spots increased and 79 spots decreased in tumor tissues compared with adjacent nontumor tissue, and 83 proteins were identified by mass spectrometry. Immunoblot confirmed that the expression of clathrin heavy chain (CHC) and Ku86 significantly increased, whereas formiminotransferase cyclodeaminase (FTCD), rhodanese, and vinculin decreased in tumor. The protein expression in tumor and nontumor tissues was further evaluated by immunostaining. Interestingly, CHC and FTCD expression was strikingly different between tumor and nontumor tissues. The sensitivity and specificity of individual markers or a combination for the detection of HCC were 51.8% and 95.6% for CHC, 61.4% and 98.5% for FTCD, and 80.7% and 94.1% for CHC+FTCD, respectively. Strikingly, the sensitivity and specificity increased to 86.7% and 95.6% when glypican-3, another potential biomarker for HCC, was used with FTCD. Moreover, CHC and FTCD were useful to distinguish early HCC from benign tumors such as regenerative nodule or focal nodular hyperplasia, because the sensitivity and specificity of the markers are 41.2% and 77.8% for CHC, 44.4% and 80.0% for FTCD, which is comparable with those of glypican-3 (33.3% and 100%). The sensitivity significantly increased by combination of these markers, 72.2% for CHC+FTCD, and 61.1% for CHC+glypican-3 and FTCD+glypican-3, as 44.4% of glypican-3 negative early HCC were able to be detected by either CHC or FTCD staining. Conclusion: Immunostaining of CHC and FTCD could make substantial contributions to the early diagnosis of HCC.     

Fungal metabolite, epoxyquinol B, crosslinks proteins by epoxy-thiol conjugation

Epoxyquinol B (EPQB) is a fungal metabolite, containing two alpha,beta-epoxy ketones. We previously showed that EPQB inhibited the signal transduction involved in angiogenesis through the binding to cysteine residues of receptor kinases. However, the inhibitory mechanism was undefined. In this report, we found that one EPQB molecule is covalently bound to two L-cysteine molecules through two epoxide residues on EPQB. Furthermore, EPQB crosslinked binding proteins through the cysteine residues. These results suggest that EPQB inhibits receptor kinases by crosslinking with other protein or by intramolecular crosslinking.     

Endovascular Management of Intractable Postpartum Hemorrhage Caused by Vaginal Laceration

Purpose

We evaluated the management of transcatheter arterial embolization for postpartum hemorrhage caused by vaginal laceration.

Materials and Methods

We reviewed seven cases of patients (mean age 30.9 years; range 27–35) with intractable hemorrhages and pelvic hematomas caused by vaginal lacerations, who underwent superselective transcatheter arterial embolization from January 2008 to July 2014. Postpartum hemorrhage was evaluated by angiographic vascular mapping to determine the vaginal artery’s architecture, technical and clinical success rates, and complications.

Results

The vaginal artery was confirmed as the source of bleeding in all cases. The artery was found to originate from the uterine artery in three cases, the uterine and obturator arteries in two, or the internal pudendal artery in two. After vaginal artery embolization, persistent contrast extravasation from the inferior mesenteric artery as an anastomotic branch was noted in one patient. Nontarget vessels (the inferior vesical artery and nonbleeding vaginal arterial branches) were embolized in one patient. Effective control of hemostasis and no post-procedural complications were confirmed for all cases.

Conclusion

Postpartum hemorrhages caused by vaginal lacerations involve the vaginal artery arising from the anterior trunk of the internal iliac artery with various branching patterns. Superselective vaginal artery embolization is clinically acceptable for the successful treatment of vaginal laceration hemorrhages, with no complications. After vaginal artery embolization, it is suggested to check for the presence of other possible bleeding vessels by pelvic aortography with a catheter tip at the L3 vertebral level, and to perform a follow-up assessment.

     

Murine Cyp3a knockout chimeric mice with humanized liver: prediction of the metabolic profile of nefazodone in humans

Chimeric mice with humanized livers (PXB mice) are used to investigate the metabolism and pharmacokinetics of drugs in humans. However, residual murine enzymatic activities derived from the liver and the presence of mouse small intestinal metabolism can hamper the prediction of human drug metabolism. Recently murine Cytochrome P450 3a gene knockout chimeric mice with humanized livers (Cyp3a KO CM) were developed. To evaluate the prediction of drug metabolism, nefazodone (NEF) was administered orally at 10 mg/kg to the following mouse strains: Cyp3a KO CM, murine Cyp3a gene knockout (Cyp3a KO), PXB and severe combined immunodeficiency (SCID) mice. Liquid chromatography‐mass spectrometry was used for metabolic profiling of plasma, urine and bile. The prediction of human metabolite levels such as hydroxy nefazodone (OH‐NEF), triazoledione form (TD), m‐chlorophenylpiperazine and dealkyl metabolites in Cyp3a KO CM was superior to that in Cyp3a KO, PXB or SCID mice. Further, clinical exposure levels of NEF, OH‐NEF and TD were reproduced in Cyp3a KO CM. In contrast, NEF was rapidly metabolized to TD in both PXB and SCID mice but not in Cyp3a KO mice, suggesting that murine CYP3A is involved in the elimination of NEF in these mice. These findings demonstrate that the metabolic profile of NEF in Cyp3a KO CM differs qualitatively and quantitatively from that in PXB mice due to the higher metabolic rate of NEF and its metabolites via murine CYP3A. Therefore Cyp3a KO CM might be useful in predicting the metabolic profiles of drug candidates in humans. Copyright © 2016 John Wiley & Sons, Ltd.     

Effects of the combination of SGLT2 selective inhibitor ipragliflozin and various antidiabetic drugs in type 2 diabetic mice

The sodium-glucose cotransporter 2 (SGLT2) is responsible for most glucose reabsorption in the kidney and has been proposed as a novel therapeutic target for the treatment of type 2 diabetes. In the present study, the combinatory effects of SGLT2 selective inhibitor ipragliflozin and various antidiabetic drugs in high-fat diet and streptozotocin-nicotinamide-induced type 2 diabetic mice were investigated. Ipragliflozin dose-dependently increased urinary glucose excretion and improved glucose tolerance. In addition, each antidiabetic drug (mitiglinide, glibenclamide, sitagliptin, insulin, metformin, voglibose, or rosiglitazone) also significantly improved glucose tolerance without affecting urinary glucose excretion. Combination treatment of ipragliflozin with each antidiabetic drug additively improved glucose tolerance. In these experiments, ipragliflozin-induced increases in urinary glucose excretion were not influenced by combination treatment with antidiabetic drugs. Further, ipragliflozin did not affect antidiabetic drug-induced insulinotropic action (mitiglinide and glibenclamide), increases in plasma glucagon-like peptide-1 and insulin levels via inhibition of dipeptidyl peptidase 4 activity (sitagliptin), increases in plasma insulin level (insulin), decreases in hepatic phosphoenolpyruvate carboxykinase activity (metformin), inhibition of small intestinal disaccharidase activity (voglibose), or improvement of impaired insulin secretion (rosiglitazone). These results suggest that combination treatment of ipragliflozin with various antidiabetic drugs additively enhances the improvement in glucose tolerance without affecting each drug’s unique pharmacological effects. Ipragliflozin may therefore be expected to be effective when administered as part of a combination regimen in the treatment of type 2 diabetes.     

In silico analysis‐based identification of the target residue of integrin α6 for metastasis inhibition of basal‐like breast cancer

Metastasis causes death in breast cancer patients. To inhibit breast cancer metastasis, we focused on integrin α6, a membrane protein that contributes to cell migration and metastasis. According to in silico analysis, we identified Asp‐358 as an integrin α6‐specific vertebrate‐conserved residue and consequently as a potential therapeutic target. Because Asp‐358 is located on the surface of the β propeller domain that interacts with other molecules for integrin α6 function, we hypothesized that a peptide with the sequence around Asp‐358 competitively inhibits integrin α6 complex formation. We treated basal‐like breast cancer cells with the peptide and observed reductions in cell migration and metastasis. The result of the immunoprecipitation assay showed that the peptide inhibited integrin α6 complex formation. Our immunofluorescence for phosphorylated paxillin, a marker of integrin‐regulated focal adhesion, showed that the peptide reduced the number of focal adhesions. These results indicate that the peptide inhibits integrin α6 function. This study identified the functional residue of integrin α6 and designed the inhibitory peptide. For breast cancer patients, metastasis inhibition therapy may be developed in the future based on this study.