3EZ,20Ac-ingenol, a catalytic inhibitor of topoisomerases, downregulates p-Akt and induces DSBs and apoptosis of DT40 cells.

We have previously reported that many ingenol compounds derived from Euphorbia kansui exhibit topoisomerase (topo) II inhibitory activity. Of these compounds, 3EZ,20Ac-ingenol inhibited topo I activity. Camptothecin, which inhibits the religation activity of topo I without interfering with the binding of topo I to DNA and induces topo I-mediated DNA cleavage, was used as a positive control. In this study, we found that 3EZ,20Ac-ingenol did not hamper the binding of topo I to DNA in the same manner as camptothecin but affected the inhibition of cleavage of one DNA strand. 3EZ,20Ac-ingenol inhibited cell proliferation by blocking cell cycle progression in the G2/M phase. To define the mechanism of inhibition of DT40 cell proliferation, the change in Akt activity was observed because Akt activity is regulated in response to DNA damage. Western blot analysis revealed that 3EZ,20Ac-ingenol downregulated the expression of p-Akt, and apoptosis was detected by the presence of DNA double-strand breaks and caspase 3 activation.

Accelerated biodegradation of nitrophenols in the rhizosphere of Spirodela polyrrhiza.

We investigated the biodegradation of 2-nitrophenol (2-NP), 4-nitrophenol (4-NP), and 2,4-dinitrophenol (2,4-DNP) in the rhizosphere of Spirodela polyrrhiza plants by conducting degradation experiments with three river water samples supplemented with each nitrophenol (NP). We then isolated NP-degrading bacteria both from the S. polyrrhiza roots and from the river water. In the river water samples, removal of the three NP was accelerated in the presence of S. polyrrhiza plants. The three NPs persisted in an autoclaved solution with sterile plants suggests that NP removal was accelerated largely by bacterial NP biodegradation rather than by adsorption and uptake by the plants. We isolated 8 strains of NP-degrading bacteria: 6 strains from the S. polyrrhiza roots and 2 strains from river water without the plants. The 2-NP- and 2,4-DNP-degrading bacteria were isolated only from the S. polyrrhiza roots. The 4-NP-degrading bacteria different from those isolated from the river water samples were also found on S. polyrrhiza roots. The 2-NP- and 4-NP-degrading strains isolated from the roots utilized the corresponding NP (0.5 mmol/L) as the sole carbon and energy source. The 2,4-DNP-degrading strains isolated from the roots showed substantial 2,4-DNP-degrading activity, but the presence of other carbon and energy sources was required for their growth. The isolated NP-degrading bacteria from the roots must have contributed to the accelerated degradation of the three NPs in the rhizosphere of S. polyrrhiza. Our results suggested that rhizoremediation with S. polyrrhiza may be effective for NP-contaminated surface water.

Isolation and characterization of 3-nitrophenol-degrading bacteria associated with rhizosphere of Spirodela polyrrhiza.

INTRODUCTION: The accelerated biodegradation of 3-nitrophenol (3-NP) in the rhizosphere of giant duckweed (Spirodela polyrrhiza) was investigated. MATERIALS AND METHODS: Biodegradation of 3-nitrophenol in the rhizosphere of a floating aquatic plant, S. polyrrhiza, was investigated by using three river water samples supplemented with 10 mg l(-1) of 3-NP. Isolation and enrichment culture of 3-NP-degrading bacteria were performed in basal salts medium containing 3-NP (50 mg l(-1)). The isolated strains were physiologically and phylogenetically characterized by using an API20NE kit and 16S rRNA gene sequencing. RESULTS AND DISCUSSION: Accelerated removal of 3-NP (100%) was observed in river water samples with S. polyrrhiza compared with their removal in plant-free river water. Also, 3-NP persisted in an autoclaved solution with aseptic plants, suggesting that the accelerated 3-NP removal resulted largely from degradation by bacteria inhabiting the plant rather than from adsorption and uptake by the plant. We successfully isolated six and four strains of 3-NP-degrading bacteria from the roots of S. polyrrhiza and plant-free river water, respectively. Phylogenetic analysis based on 16S rRNA gene divided the 3-NP-degrading bacteria into two taxonomic groups: the genera Pseudomonas and Cupriavidus. The strains belonging to the genus Cupriavidus were only isolated from the roots of duckweed. All strains isolated from the roots utilized 3-NP (0.5 mM) as a sole carbon and energy source, indicating that they could have contributed to the accelerated degradation of 3-NP in the rhizosphere of S. polyrrhiza. CONCLUSIONS: The rhizoremediation using S. polyrrhiza and its rhizosphere bacteria can be an effective strategy for cleaning up the 3-NP-contaminated surface waters.

Analysis of inhibition of topoisomerase IIalpha and cancer cell proliferation by ingenolEZ.

We previously reported that many ingenol compounds derived from Euphorbia kansui exhibit topoisomerase inhibitory activity and/or inhibitory activity of cell proliferation. The inhibitory effects of 20-O-(2'E,4'Z-decadienoyl) ingenol and 3-O-(2'E,4'Z-decadienoyl)-ingenol among these compounds on topoisomerase II activity and on the cell proliferative activity and arrest phase of the cell cycle were studied using a mouse breast cancer (MMT) cell line. Although 20-O-ingenolEZ exerted inhibitory effects on both topoisomerase II activity and cell proliferative activity, 3-O-ingenolEZ exerted inhibitory activity on neither. The 20-O-ingenolEZ-induced cell arrest of MMT-cell proliferation led to a cell cycle arrest in the G2/M phase. Topoisomerase II inhibition can be divided into the poison and catalytic inhibitor types. A checkpoint mechanism is activated when cells are treated with these topoisomerase II inhibitors. Poison-type inhibition occurs via induction of the DNA damage checkpoint and the catalytic-type inhibition occurs via induction of the DNA-decatenation checkpoint, suggestive of distinct checkpoint reactions. 20-O-ingenolEZ inhibited topoisomerase IIalpha activity through inhibition of ATPase, and induced DNA-decatenation checkpoint without signaling for phosphorylation of H2AX.