Anti-cancer effects of naturally occurring compounds through modulation of signal transduction and miRNA expression in human colon cancer cells

Much evidence indicates that various naturally occurring compounds have an anti-cancer effect, but the detailed mechanisms are not well understood. In this study, we selected anti-cancer phytochemicals such as epigallocatechin-3-gallate (EGCG), resveratrol (RES) and α-mangostin (α-M), all of which are well-characterized chemopreventive agents. We sought to elucidate the mechanism of their anti-cancer effects and the synergistic effects obtained by combined treatment with the anti-cancer drug 5-fluorouracil (5-FU) in three human colon cancer cell lines. The numbers of viable cells were consistently decreased by the treatment with EGCG, RES or α-M at more than 10 μM in all three cell lines tested. All compounds mainly induced apoptosis and suppressed the PI3K/Akt signaling pathway. Additionally, α-M, which had the greatest PI3K/Akt-suppressing activity, also suppressed MAP kinase (MAPK)/Erk1/2 signaling. Importantly, the combination treatment with RES and 5-FU induced a remarkably synergistic enhancement of growth inhibition and apoptosis through the additional suppression of the MAPK/Erk1/2 signaling pathway in colon cancer DLD-1 cells. Interestingly, RES increased the intracellular expression level of miR-34a, which down-regulated the target gene E2F3 and its downstream Sirt1, resulting in growth inhibition. These findings indicate that these compounds functioned as chemosensitizers when combined with anti-cancer drugs through the modulation of apoptotic and growth-related signaling pathways. Also, RES exerted its anti-cancer activity in part through a newly defined mechanism, i.e., the miR-34a/E2F3/Sirt1 cascade.     

Genetic and phenotypic characterization of a Japanese wild-derived DOB/Oda rat strain

Wild-derived rat strains can provide novel genome resources that are not available in standard laboratory strains. Genetic backgrounds of wild-derived strains can facilitate effective genetic linkage analyses and often modulate the expression of mutant phenotypes. Here we describe the development and characterization of a new inbred rat strain, DOB/Oda, from wild rats (Rattus norvegicus) captured in Shitara, Aichi, Japan. Phenotype analysis of 109 parameters revealed that the DOB/Oda rats had small body weight, preference for darkness, and high locomotor activity compared with the rat strains in the National BioResource Project for the Rat (NBRP-Rat) database. Genome analysis with 357 SSLP markers identified DOB/Oda-specific alleles in 70 markers. The percentage of SSLP markers that showed polymorphism between the DOB/Oda strain and any of 132 laboratory strains from NBRP-Rat varied from 89 to 95 %. The polymorphic rate (average of the values of the percentage) for the DOB/Oda strain was 91.6 %, much higher than the rates for available wild-derived strains such as the Brown Norway rat. A phylogenic tree constructed with DOB/Oda and all the strains in NBRP-Rat showed that the DOB/Oda strain localized within the wild rat groups, apparently separate from the laboratory strains. Together, these findings indicated that the DOB/Oda rat has a unique genome that is not available in the laboratory strains. Therefore, the new DOB/Oda strain will provide an important genome resource that will be useful for designing genetic experiments and for the discovery of genes that modulate mutant phenotypes.     

Development of a Host-vector System for Gluconobacter suboxydans

A shuttle vector for Gluconobacter suboxydans and Escherichia coli was constructed by ligation of a cryptic plasmid, pMV201, found in G. suboxydans IFO 3130 to E. coli plasmid pACYC177. The chimeric plasmid named pMGlOl carries the ampicillin resistance gene derived from pACYC177 and transforms G. suboxydans var. α IFO 3254 as well as E. coli. The transformation conditions for G. suboxydansvar. α IFO 3254 were examined using pMGlOl DNA. Competent cells were induced efficiently by treatment with LiCl or RbCl CaCl₂ which induced the competency of Acetobacter was much less effective. Addition of polyethylene glycol enhanced the transformation efficiency significantly. An efficiency of approximately 10² transformants per μg DNA was finally obtained.     

Construction of New Shuttle Vectors for Acetbacter

Shuttle vector pMV301 was constructed by ligation of pMV102 found in A. aceti subsp. xylinum NBI 1002 to E. coli plasmid pACYC177. It is 6.0 kb in size, has unique restriction sites suitable for insertion of a foreign DNA fragment and confers ampicillin resistance to the Acetobacter host. This vector transforms A. aceti subsp. aceti 10-80S1 and industrial vinegar producer A. aceti subsp. xylinum NBI 1002 as well as E. coli. Various chimeric plasmids were also constructed by ligation of pMV102 to E. coli plasmids to examine the expression of drug resistance genes. In addition to the ampicillin resistance gene, resistance genes for kanamycin, chloramphenicol and tetracycline derived from E. coli plasmids were expressed in Acetobacter. Most of the constructed shuttle vectors were stably maintained in Acetobacter.     

Improved Transformation Method for Acetobacter with Plasmid DNA

An improved method for transformation of derivative strains of A. aceti subsp. aceti No. 1023 with plasmid DNA has been developed. Addition of polyethylene glycol or dimethylsulfoxide increased the transformation efficiency by a factor of about ten. In the presence of PEG 4,000, various transformation conditions were examined. Cells were also made transformation competent by treatment with other divalent cations than Ca²⁺ . The pH of the buffer did not affect the efficiency significantly. The growth phase influenced the efficiency. Mutants showing high competence were derived by treatment with N-methyl-N′-nitro-N-nitrosoguanidine. By the improved method using the highly transformable mutants, a transformation efficiency of approximately 105 transformants per γg plasmid DNA was achieved.     

Production of Acidic Actinomycin Congeners by a Mutant Strain of Streptomyces antibioticus,No. B-1625

Streptomyces sp. No. B-1625, which was identified as a strain of Streptomyces antibioticus, is a typical producer of actinomycin, but also produces minor acidic antibiotic components (FA), besides actinomycins X₂, D and X_0β. The FA-components, which were obtained with a high-producing mutant, 11M-21, showed antibacterial and antitumor activities, and also similar visible and UV absorption spectra to those characteristic of actinomycin. The FA-components were separated into five components, FA₁ FA_2α, FA_2β, FA_3α and FA_3β, on TLC. Among them, one component, FA_3β, isolated in a purified state as an orange powder, has a composition of C, 52.97: H, 6.34: N, 10.48%, and is active against B. subtilis at a MIC of 5mcg/ml. The FA_3β component showed pK_a′ of 5.4 and 12.0 and λ_max at 443, 427 and 233 nm. From these properties, FA_3β is considered to be an acidic actinomycin congener.     

Separation of Lepidopterous Sex Pheromones by Reversed-phase Thin Layer Chromatography and High Performance Liquid Chromatography

Bis(4-chloro-2-ethylphenyl) phenylphosphonate was metabolically transformed into the cor-responding cyclic ester, i.e., 6-chloro-4-methyl-2-phenyl-4/f-1,3,2-benzodioxaphosphorin 2-oxide, in houseflies in vivo. In a p-unsubstituted analog, hydroxylation at the para-position of an ester linkage occurred preferably to alpha-hydroxylation with subsequent cyclization. The cyclization was diastereomerically selective, giving predominantly the cis ester. The biological activities of synthesized and related cyclic esters were similar to but weaker than saligenin cyclic phosphorus esters lacking a methyl group at the 4-position.     

Administration of a maple syrup extract to mitigate their hepatic inflammation induced by a high-fat diet: a transcriptome analysis

Effects of the administration of maple syrup extract (MSX) on hepatic gene expression were investigated in mice fed a high-fat diet. Gene annotation enrichment analysis based on gene ontology revealed some changes in the expression of genes related to lipid metabolism and the immune response in MSX-fed mice. Detailed analysis of these data indicated that MSX ingestion mitigates hepatic inflammation.