(−)-9′-O-(α-l-Rhamnopyranosyl)lyoniresinol from Lespedeza cuneata suppresses ovarian cancer cell proliferation through induction of apoptosis

Lespedeza cuneata (Dum. Cours.) G. Don. (Fabaceae), known as Chinese bushclover or sericea lespedeza, has been used in traditional medicine to treat diabetes, hematuria, and insomnia, and it has been reported that bioactive compounds from L. cuneata possess various pharmacological properties. However, there has been no study to determine the active compounds from L. cuneata with potential activity against ovarian cancer. This study aimed to isolate cytotoxic compounds from L. cuneata and identify the molecular mechanisms underlying the apoptosis pathway in ovarian cancer cells. Based on cytotoxic activity identified in the screening test, chemical investigation of the active fraction of L. cuneata led to the isolation of nine compounds including four lignanosides (1–4), three flavonoid glycosides (5–7), and two phenolics (8–9). Cytotoxicity and the molecular mechanism were examined by methyl thiazolyl tetrazolium (MTT) assay and Western blot analysis. Of the isolated compounds, (?)-9′-O-(α-l-rhamnopyranosyl)lyoniresinol (3) demonstrated the strongest effect in suppressing A2780 human ovarian carcinoma cell proliferation in a dose-dependent manner, with an IC₅₀ value of 35.40?±?2.78?μM. Control A2780 cells had normal morphology, whereas cell blebbing, shrinkage, and condensation were observed after treatment with compound 3. Western blotting analysis showed that compound 3 inhibited A2780 human ovarian cancer cell viability by activating caspase-8, caspase-3, and PARP, which contributed to apoptotic cell death. These results suggest that (?)-9′-O-(α-l-rhamnopyranosyl)lyoniresinol (3) has potent anticancer activities against A2780 human ovarian carcinoma cells through the extrinsic apoptotic pathway. Therefore, (?)-9′-O-(α-l-rhamnopyranosyl)lyoniresinol is an excellent candidate for the development of novel chemotherapeutics.     

First record of the family Bothrideridae (Coleoptera) in Korea represented by the wood-boring beetle ectoparasite,Dastarcus helophoroides

The ectoparasitic beetle, Dastarcus helophoroides (Fairmaire), was observed for the first time in Korea as a result of a study of the natural enemies of Monochamus alternatus Hope and M. saltuarius Gebler, the vector of the pine wood nematode (Bursaphelenchus xylophilus (Steiner & Buhner)). Diagnostic illustrations are provided and the biology and host range of D. helophoroides (Fairmaire) are reviewed.     

Transgenic Arabidopsis thaliana expressing a barley UDP-glucosyltransferase exhibit resistance to the mycotoxin deoxynivalenol

Fusarium head blight (FHB), caused by Fusarium graminearum, is a devastating disease of small grain cereal crops. FHB causes yield reductions and contamination of grain with trichothecene mycotoxins such as deoxynivalenol (DON). DON inhibits protein synthesis in eukaryotic cells and acts as a virulence factor during fungal pathogenesis, therefore resistance to DON is considered an important component of resistance against FHB. One mechanism of resistance to DON is conversion of DON to DON-3-O-glucoside (D3G). Previous studies showed that expression of the UDP-glucosyltransferase genes HvUGT13248 from barley and AtUGt73C5 (DOGT1) from Arabidopsis thaliana conferred DON resistance to yeast. Over-expression of AtUGt73C5 in Arabidopsis led to increased DON resistance of seedlings but also to dwarfing of transgenic plants due to the formation of brassinosteroid-glucosides. The objectives of this study were to develop transgenic Arabidopsis expressing HvUGT13248, to test for phenotypic changes in growth habit, and the response to DON. Transgenic lines that constitutively expressed the epitope-tagged HvUGT13248 protein exhibited increased resistance to DON in a seed germination assay and converted DON to D3G to a higher extent than the untransformed wild-type. By contrast to the over-expression of DOGT1 in Arabidopsis, which conjugated the brassinosteriod castasterone with a glucoside group resulting in a dwarf phenotype, expression of the barley HvUGT13248 gene did not lead to drastic morphological changes. Consistent with this observation, no castasterone-glucoside formation was detectable in yeast expressing the barley HvUGT13248 gene. This barley UGT is therefore a promising candidate for transgenic approaches aiming to increase DON and Fusarium resistance of crop plants without undesired collateral effects.     

Proteomic analysis of cellular change involved in mitochondria-to-nucleus communication in L6 GLUT4myc myocytes

Genetic or biochemical abnormalities in mitochondria are closely associated with apoptosis, aging, cancer, and other chronic degenerative diseases. Mitochondrial dysfunction resulting from mitochondrial DNA (mtDNA) depletion dispatches retrograde signals to the nucleus to compensate by altering the expression of various genes. In this study, a proteomic approach was used to gain insight into the nuclear gene targets of mitochondrial stress signaling and the pathophysiological mechanisms associated with mitochondrial dysfunction. We have used 2-DE to characterize the nuclear gene responses resulting from mtDNA depletion in L6 GLUT4myc myocytes. Our results showed that 77 polypeptides were differentially expressed in mtDNA-depleted cells; 33 polypeptides were down-regulated and 44 polypeptides were up-regulated. Of these differentially expressed polypeptides, 40 were identified as 36 different proteins by MALDI-TOF MS. These proteins are related to various cellular responses, such as apoptosis, cellular metabolism, signaling and cytoskeleton functions. It is suggested that the insulin resistance developed in mtDNA-depleted myocytes may be associated with disorganization of cytoskeleton assembly, and that cellular mtDNA depletion might promote the ability to evade apoptosis or other death effectors.     

VEGF receptor binding peptide‐linked high mobility box group‐1 box A as a targeting gene carrier for hypoxic endothelial cells

High mobility group box‐1 (HMGB‐1) is a nuclear protein that can bind to and condense plasmid DNA. In this study, we developed a recombinant VEGF receptor binding peptide (VRBP) linked to HMGB‐1 box A (VRBP‐HMGB1A) as a targeting gene carrier to hypoxic endothelial cells. Hypoxic endothelial cells in ischemic tissues of solid tumors are important targets for gene therapy. A recombinant VRBP‐HMGB1A expression vector, pET21a‐VRBP‐HMGB1A was constructed. VRBP‐HMGB1A was over‐expressed in BL21 strain and purified by nickel‐chelate affinity chromatography. Complex formation between VRBP‐HMGB1A and pCMV‐Luc was confirmed by gel retardation assay. pCMV‐Luc was retarded completely at a 2/1 weight ratio (peptide/plasmid). For transfection assays, calf pulmonary artery endothelial (CPAE) cells were incubated under hypoxia for 24 h, prior to transfection to induce the VEGF receptors on the cells. VRBP‐HMGB1A/pCMV‐Luc complexes were transfected to hypoxic CPAE cells. The highest transfection efficiency was at a 30/1 weight ratio (peptide/plasmid). In addition, VRBP‐HMGB1A had higher efficiency than poly‐L ‐lysine (PLL) specifically in hypoxic CPAE cells, However, VRBP‐HMGB1A had lower efficiency than PLL in 293, H9C2, and normoxic CPAE cells. In MTT assay, VRBP‐HMGB1A was less toxic than PLL to cells. In conclusion, VRBP‐HMGB1A is a potential gene carrier for targeting hypoxic endothelial cells and thus, may be useful for cancer gene therapy. J. Cell. Biochem. 110: 1094–1100, 2010. Published 2010 Wiley‐Liss, Inc.     

Potential target gene and protein interaction ofPimpinella brachycarpa HD-Zip protein, Phz4 by yeast two-hybrid system

The yeast two-hybrid system was used to investigate dimerization between proteins ofPhz2 andPhz4 clones of the homeodomain-leucine zipper family which were obtained by screening aPimpinella brachycarpa shoot-tip cDNA library. Assays showed that Phz4 formed a homo rather than a heterodimer with Phz2. In addition, we isolated cDNA clones,Phyb1, Phyb2, andPhyb3, that encode proteins interacting with Phz4. Although Phyb1 is not a HD-Zip protein, the activity of interaction between Phyb1 and Phz4 was, surprisingly, stronger than that of the homodimerization of Phz4. The analysis of interacting parts indicated that from 1 bp to 466 bp of Phyb1, there was no interaction with Phz4, but from 467 bp to 593 bp, interactions were found with the N-terminal and C-terminal regions, except for HD-Zip of Phz4. This region ofPhyb1 contained a nuclear localization signal. DNA-binding analysis showed that the Phz4 HD-Zip domain recognized the [T(C/G)ATTG] core sequence and the region containing the [TCATTG] motif, which is, in itself, a promoter in vitro.