Characterization of metapopulation of Ellobium chinense through Pleistocene expansions and four covariate COI guanine-hotspots linked to G-quadruplex conformation.

The land snail Ellobium chinense (L. Pfeiffer, 1855) (Eupulmonata, Ellobiida, Ellobiidae), which inhabits the salt marshes along the coastal areas of northwestern Pacific, is an endangered species on the IUCN Red List. Over recent decades, the population size of E. chinense has consistently decreased due to environmental interference caused by natural disasters and human activities. Here, we provide the first assessment of the genetic diversity and population genetic structures of northwestern Pacific E. chinense. The results analyzed with COI and microsatellites revealed that E. chinense population exhibit metapopulation characteristics, retaining under the influence of the Kuroshio warm currents through expansion of the Late-Middle and Late Pleistocene. We also found four phylogenetic groups, regardless of geographical distributions, which were easily distinguishable by four unidirectional and stepwise adenine-to-guanine transitions in COI (sites 207-282-354-420: A-A-A-A, A-A-G-A, G-A-G-A, and G-G-G-G). Additionally, the four COI hotspots were robustly connected with a high degree of covariance between them. We discuss the role of these covariate guanines which link to form four consecutive G-quadruplexes, and their possible beneficial effects under positive selection pressure.

Stem Cells Antigen-1 Enriches for a Cancer Stem Cell-Like Subpopulation in Mouse Gastric Cancer.

There is a strong need to identify markers to enrich gastric cancer stem cells (CSCs). However, CSC enrichment markers for mouse gastric cancers have not yet been determined. In our previous study, we generated primary mouse gastric cancer cell line NCC-S1 (S1) established from a Villin-cre;Smad4(F/F) ;Trp53(F/F) ;Cdh1(F/wt) mouse and its metastatic variant cell line NCC-S1M (S1M). Interestingly, S1M cells exhibited CSC-like features, such as increased tumorigenic potential and chemoresistance. By comparing gene expression profiles between S1 and S1M cells, we identified Stem Cells Antigen-1 (Sca-1) as a cell surface marker, which was mostly upregulated in S1M. Sca-1 was upregulated in tumorspheres from S1 cells or after cisplatin treatment in S1 cells. Immunofluorescence (IF) analysis showed that approximately 7% of cancer cells exhibited positivity for Sca-1 in primary mouse gastric cancer tissues. An in vivo-limiting dilution assay showed that Sca-1(high) mouse gastric cancer cells demonstrated increased tumorigenicity compared with Sca-1(negative) cells. The Sca-1 expression was downregulated by TGF-ß pathway activation and Wnt pathway inhibition in mouse gastric cancer cells. Sca-1(high) cells showed relatively low TGF-ß reporter activity and high TCF/LEF1 reporter activity compared with Sca-1(negative) cells. A chromatin immunoprecipitation analysis demonstrated that Sca-1 was a ß-catenin/LEF1 target gene. Sca-1(high) allografts were more resistant to cisplatin/fluorouracil chemotherapy than Sca-1(negative) allografts, and overexpressed Bcl-xL. Eighty-five mouse genes overexpressed in Sca-1(high) S1 cells compared with Sca-1(negative) cells clustered 123 pretreatment gastric cancer patient samples according to survival following chemotherapy. Taken together, Sca-1 is a novel CSC enrichment marker that mediates TGF-ß and Wnt/ß-catenin signaling in mouse gastric cancer. Stem Cells 2016;34:1177-1187.

Establishment and characterization of metastatic gastric cancer cell lines from murine gastric adenocarcinoma lacking Smad4, p53, and E-cadherin.

There is a strong need for murine gastric cancer cell line models recapitulating human gastric cancers. Here, we describe two murine gastric cancer cell lines designated as NCC-S1 and NCC-S3. They were generated from gastric adenocarcinomas that formed in a Villin-cre, Smad4(F/F) , Trp53(F/F) , Cdh1(F/wt) mouse and a Pdx1-cre, Trp53(F/F) , Cdh1(F/F) mouse, respectively. Molecular profiles of both cell lines were very similar to human gastric cancer. NCC-S1M and NCC-S3M subpopulation clones were isolated from pulmonary metastasis of heterotopic allografts of NCC-S1 and NCC-S3 cells, respectively. NCC-S1M and NCC-S3M showed enhanced in vivo growth rates and metastatic potentials and exhibited epithelial-to-mesenchymal transition features. NCC-S1M cells developed orthotopic and heterotopic tumors in immunocompetent mice in predictable manner, and were useful for testing the efficacy of an immunotherapeutic agent, anti-4-1BB antibody. NCC-S1M and NCC-S3M cells demonstrated Wnt/ß-catenin pathway activation, and knockdown of Ctnnb1 reversed the metastatic phenotype of NCC-S1M. These results underscore the role of Wnt/ß-catenin pathway in metastatic phenotype of gastric cancer. Taken together, our novel metastatic gastric cancer cell lines are useful resources for drug development and metastasis research.

Cooperativity of E-cadherin and Smad4 loss to promote diffuse-type gastric adenocarcinoma and metastasis.

UNLABELLED: Loss of E-cadherin (CDH1), Smad4, and p53 has been shown to play an integral role in gastric, intestinal, and breast cancer formation. Compound conditional knockout mice for Smad4, p53, and E-cadherin were generated to define and compare the roles of these genes in gastric, intestinal, and breast cancer development by crossing with Pdx-1-Cre, Villin-Cre, and MMTV-Cre transgenic mice. Interestingly, gastric adenocarcinoma was significantly more frequent in Pdx-1-Cre;Smad4(F/F);Trp53(F/F);Cdh1(F) (/+) mice than in Pdx-1-Cre;Smad4(F/F);Trp53(F/F);Cdh1(+/+) mice, demonstrating that Cdh1 heterozygosity accelerates the development and progression of gastric adenocarcinoma, in combination with loss of Smad4 and p53. Pdx-1-Cre;Smad4(F/F);Trp53(F/F);Cdh1(F) (/+) mice developed gastric adenocarcinomas without E-cadherin expression. However, intestinal and mammary adenocarcinomas with the same genetic background retained E-cadherin expression and were phenotypically similar to mice with both wild-type Cdh1 alleles. Lung metastases were identified in Pdx-1-Cre;Smad4(F/F);Trp53(F/F);Cdh1(F) (/+) mice, but not in the other genotypes. Nuclear ß-catenin accumulation was identified at the invasive tumor front of gastric adenocarcinomas arising in Pdx-1-Cre;Smad4(F/F);Trp53(F/F);Cdh1(F) (/+) mice. This phenotype was less prominent in mice with intact E-cadherin or Smad4, indicating that the inhibition of ß-catenin signaling by E-cadherin or Smad4 downregulates signaling pathways involved in metastases in Pdx-1-Cre;Smad4(F/F);Trp53(F/F);Cdh1(F) (/+) mice. Knockdown of ß-catenin significantly inhibited the migratory activity of Pdx-1-Cre;Smad4(F/F);Trp53(F/F);Cdh1(F) (/+) cell lines. Thus, loss of E-cadherin and Smad4 cooperates with p53 loss to promote the development and metastatic progression of gastric adenocarcinomas, with similarities to human gastric adenocarcinoma. IMPLICATIONS: This study demonstrates that inhibition of ß-catenin is a converging node for the antimetastatic signaling pathways driven by E-cadherin and Smad4 in Pdx-1-Cre;Smad4(F/F);Trp53(F/F);Cdh1(F) (/+) mice, providing novel insights into mechanisms for gastric cancer metastasis.

A rapid, simple and reliable HPLC-triple quadrupole tandem mass spectrometer method for a simultaneous quantification of irinotecan and its active metabolite 7-ethyl-10-hydroxycamptothecin (SN38) in mouse plasma.

A simple, fast and reliable high-performance liquid chromatography-triple quadrupole mass spectrometry method (HPLC-MS/MS method) was developed, validated and used for the simultaneous quantification of irinotecan and 7-ethyl-10-hydroxycamptothecin (SN38) in heparinized mouse plasma. Camptothecin was used as the internal standard. A single-step protein precipitation without evaporation and reconstitution steps was adopted as sample processing method. Our bioanalytical method was validated in compliance with the guidelines from the European Medicines Agency. The lower limit of quantification for both irinotecan and SN38 was 5 ng/mL. The calibration curves for both analytes fitted to a 1/x(2) weighted linear regression model and ranged from 5 to 1000 ng/mL. The intra-run and inter-run precisions were within 8.6%, and the intra-run and inter-run accuracies were within 96.4-103.9%. Our validated bioanalytical method was successfully applied to the pharmacokinetic study in mice, in which 4 mg/kg irinotecan was intraperitoneally injected.

Development and validation of a microfluidic chip-based nano-liquid chromatography-triple quadrupole tandem mass spectrometry method for a sensitive and reliable quantification of 7-ethyl-10-hydroxycamptothecin (SN38) in mouse plasma.

There is an increasing need for more sensitive analytical methods in pharmacokinetic studies, for example, for phase 0 clinical trials. A novel HPLC Chip-triple quadrupole mass spectrometer method (HPLC Chip-MS/MS method) for the quantification of 7-ethyl-10-hydroxycamptothecin (SN38) was developed, validated, and employed to the pharmacokinetic analysis of SN38 in ICR mice. Protein precipitation with a ratio of plasma/acetonitrile of 1:10 was chosen as the sample processing method. The nano-electrospray inserted in the microfluidic chip operated in positive mode, and selected reaction monitoring was used for quantification. Our bioanalytical method met all essential validation parameters-selectivity, accuracy, precision, dilution integrity, calibration curve, matrix effect, recovery, and different stability tests (benchtop, freeze-thaw, autosampler stability). The calibration curves (weight 1/x (2)) were linear for the range 50-10,000 pg/mL. Clogging was not observed until the end of the lifetime of the microfluidic chip (350-400 injections), and carryover was practically eliminated through the introduction of a step gradient elution program. After intraperitoneal injection of 0.1 mg/kg irinotecan, SN38 concentration could be measured up to 6 h with accuracy and precision. Thus, we developed a new, very sensitive HPLC Chip-MS/MS method for the determination of plasma SN38 that has been validated in compliance with guidelines from different regulation authorities.

Development of chitosan-based ondansetron buccal delivery system for the treatment of emesis.

BACKGROUND: For the buccal drug delivery, chitosan (CS) can be used to improve drug absorption and reduce application frequency and drug amount. The aim of this study is to develop and evaluate mucoadhesive ondansetron buccal films for the treatment of emesis using CS as a mucoadhesive polymer. METHODS: The film prepared by solvent casting method was comprised of ondansetron (approximately 65 µg)-loaded mucoadhesive gels containing 1, 2 or 3% CS and impermeable backing layer. Rheological property of the gels, physiochemical properties of the films (weight, thickness, drug content, swelling ratio, adhesion time and mucoadhesive force) and in vitro ondansetron release profile from the films were determined to evaluate the formulation. The films containing 3% CS (diameter: 0.5 cm; thickness: 170 µm) was selected as the novel formulation, and were used for the in vivo study. Comparative pharmacokinetic studies of ondansetron with this film and oral solution were performed at the same dose in hamsters. RESULTS: The mean values of T(max) and C(max) of the film and oral solution were similar. However, the half-life, mean residence time and AUC(0-24 h) of the film were about 1.7, 1.4 and 2.0-fold higher than those of the oral solution, respectively. The film showed enhanced bioavailability and prolonged efficacy compared to the oral solution. CONCLUSIONS: The mucoadhesive ondansetron buccal film may be a potential alternative to the marketed oral formulation, parenterals and solid suppositories with better patient compliance and higher bioavailability for the treatment of emesis.