Discovery of sesquiterpene lactones with anti-inflammatory effect from Youngia japonica.

The preliminary study revealed that the ethyl acetate eluate of Youngia japonica (YJ-E) could inhibit the expression of key proteins of p-p65, p-IκBα, p-IKKα/ß, and p-AKT in LPS stimulated BV2 cell. Further phytochemical study led to the isolation of eight compounds from YJ-E, including one new sesquiterpene lactone. Their structures were elucidated by several spectroscopic data, and comparing the NMR data of known compound. In addition, all of the isolates were evaluated for the anti-inflammatory effect. As a result, compounds 3 and 4 distinctly attenuated the expressions of p-IκBα, p-p65, and p-AKT in LPS stimulated BV2 cell, respectively.

Discovery of Sesquiterpene Lactones with Cytotoxicity from the Herb of Youngia japonica.

In the process of searching for anti-breast cancer agents, five sesquiterpene lactones (1-5), including two previously undescribed ones, yjaponica B-C (1-2), were isolated from the herb of Youngia japonica. Their structures were elucidated by spectroscopic data analyses and Marfey's method. Cytotoxic activities of all compounds against A549, U87, and 4T1 cell lines were tested using the CCK8 assay. The result showed that compound 3 possessed the highest cytotoxic activity against 4T1 cells with an IC50 value of 10.60 µM. Furthermore, compound 3 distinctly induced apoptosis, inhibited immigration, and blocked the cell cycle of 4T1 cells. In addition, compound 3 induced the production of reactive oxygen species. Further anticancer mechanism studies showed that compound 3 significantly upregulated expression of the cleaved caspase 3 and PARP, whereas it downregulated the expression of Bcl-2, cyclin D1, cyclin A2, CDK4, and CDK2. Taken together, our results demonstrate that compound 3 has a high potential of being used as a leading compound for the discovery of new anti-breast cancer agent.

Comprehensive molecular characterisation of the complete mitogenome of Ergasilus tumidus and phylogenetic relationships of Copepoda inferred from mitogenomes.

Although parasitic copepods of the genus Ergasilus von Nordmann, 1832 are globally distributed parasites of fish, their phylogenetic relationships with other Copepoda are not clear, and the characteristics of their mitochondrial genomes (mitogenomes) are not thoroughly understood. The objective of this study was to address these knowledge gaps by sequencing the complete mitogenome of Ergasilus tumidus Markevich, 1940. The complete mitogenome (GenBank Acc. No. OQ596537) was 14,431 bp long and it comprised 13 protein-coding genes (PCGs), 22 tRNAs, two tRNAs, and two control regions (CRs). Phylogenetic analyses, conducted using concatenated nucleotide and amino acid sequences of 13 protein-coding genes, produced two partially incongruent topologies. While the order Calanoida was consistently resolved as the sister lineage to the other three orders, topological instability was observed in the relationships of the orders Cyclopoida, Siphonostomatoida and Harpacticoida. Siphonostomatoida clustered with Cyclopoida in the nucleotide-based phylogeny, but with Harpacticoida in the amino acid-based phylogeny. The latter topology conforms to the widely accepted relationships, but we speculate that the former topology is more likely to be the correct one. Our study provides a complete mitogenome sequence of E. tumidus, which helps us better understand the molecular evolution of the genus Ergasilus. Additionally, we suggest a different perspective on the controversial phylogenetic relationships among Siphonostomatoida, Cyclopoida and Harpacticoida, diverging from previously accepted views.

Discovery of sesquiterpene from Youngia japonica with antitumor effect.

Fourteen sesquiterpenes, including one undescribed sesquiterpene lactone, were isolated from Youngia japonica, and their structures were identified by NMR, HRESIMS, ECD and calculated ECD. Cytotoxic activities of all isolates against A549, HeLa, and 4 T1 cell lines were detected by CCK8 assay. Among them, 2 showed obvious cytotoxic activity against A549 cells. Subsequently, the production of ROS, and apoptosis of A549 cells treated with 2 were evaluated. The result showed that 2 distinctly increased the ROS level, and induced the apoptosis of A549 cells. Further anticancer mechanism studies showed that 2 increased the expression of cleaved caspase 3. Taken together, our results demonstrated that 2 might become potential leading compounds for the treatment of lung cancer.

Four undescribed coumarin derivatives, with ten amides from the roots of Ficus hirta and their cytotoxic activities.

Four undescribed coumarin derivatives, ficusalt A (1) and ficusalt B (2), a pair of racemic coumarins, (±) ficudimer A (3a/3b), along with ten known amides, were isolated from the roots of Ficus hirta. Their structures were elucidated by several spectroscopic data analyses, including HRESIMS, NMR, and X-ray single-crystal diffraction. The cytotoxic activities of all compounds against HeLa, HepG2, MCF-7, and H460 cell lines were detected using the MTT assay. Among these, 5 showed the highest activity against HeLa cells. Subsequently, the apoptotic, anti-invasive, and anti-migration effects of 5 on HeLa cells were determined by flow cytometer, transwell invasion assay, and wound-healing assay, respectively. The result suggested that 5 distinctly induced the apoptosis in HeLa cells and inhibited their invasion and migration. Further studies on anticancer mechanisms were conducted using Western blotting. As a result, 5 increased the cleavage of PARP and the expression of pro-apoptotic protein Bax. Moreover, 5 notably upregulated the phosphorylation of p38 and JNK, whereas inhibited the expression of p-ERK and p-AKT. Our results demonstrated that 5 could be a potential leading compound for further application in the treatment of cervical cancer.

TRIM22 can activate the noncanonical NF-κB pathway by affecting IKKα.

Tripartite motif 22 (TRIM22) is involved in various cellular processes. It has been reported that TRIM22 can activate nuclear factor-κB (NF-κB) pathway, but the precise mechanism remains unclear. In this study, we explored the exact role of TRIM22 in activating the NF-κB pathway. Different to tumor necrosis factor-α (TNF-α) induction, we found that the overexpression of TRIM22 could induce the processing of p100 to p52 in HEK293T cells. Furthermore, based on the results of co-immunoprecipitation and co-localization experiments, we demonstrated that TRIM22 could interact with IκB kinase (IKK)α but not IKKß and could increase the level and phosphorylation of IKKα through its really interesting new gene (RING) and spla-ryanodine receptor (SPRY) domains. These results suggest that TRIM22 is able to activate the noncanonical but not the canonical NF-κB pathway by activating IKKα. This finding will aid our understanding of the biological function of TRIM22.

DNA methyltransferases 1 and 3B are required for hepatitis C virus infection in cell culture.

DNA methyltransferases (DNMTs) are responsible for establishing and maintaining DNA methylation, which are dysregulated in hepatitis C virus (HCV)-associated hepatocellular carcinoma (HCC). In this report, using lentivirus-mediated shRNA interference technology, we identified DNMT1 and DNMT3B as host factors involved in HCV propagation. Our results demonstrated that down-regulation of DNMT1 or DNMT3B expression in Huh7.5.1 cells severely impaired cell culture-produced HCV (HCVcc) infection. Furthermore, knockdown of DNMT1 or DNMT3B did not affect HCV entry and internal ribosome entry site (IRES)-directed translation but did inhibit subgenomic replication. In contrast, knockdown of DNMT3A had no significant effect on HCV infection, entry, translation, or replication, which suggested that DNMT3A did not play a significant role in HCV life cycle. Moreover, we showed that DNMT inhibitors 5-Aza-C and 5-Aza-dC significantly suppressed HCVcc infection, viral RNA replication, and protein expression. These results suggest that DNMTs are critical for HCV replication and may represent potent targets for the treatment of HCV infection.

Characterization of TRIM62 as a RING finger E3 ubiquitin ligase and its subcellular localization.

TRIM62, also named DEAR1, is a member of the TRIM/RBCC family, which includes proteins with conserved RING finger, B-box and coiled-coil domains. Several reports have identified a role for this family in cancer, retroviral infection and innate immunity. In this study, the E3 ubiquitin ligase activity and subcellular localization of TRIM62 were characterized. TRIM62, in association with the E2 enzyme UbcH5b, was found to catalyze self-ubiquitination in vitro, a process that required an intact RING finger domain. A ubiquitination assay performed in HEK293T cells further confirmed the E3 ubiquitin ligase activity and self-ubiquitination activity of TRIM62 and the requirement of the RING finger domain. Importantly, the treatment of HEK293T cells with a proteasome inhibitor stabilized poly-ubiquitinated TRIM62, indicating that self-ubiquitination promoted the proteasomal degradation of TRIM62. Additionally, TRIM62 and its two mutants were distinctly localized in the cytoplasm in both HEK293T and HeLa cells. Collectively, our data indicate that TRIM62, a cytoplasmic protein, is a RING finger domain-dependent E3 ubiquitin ligase that catalyzes self-ubiquitination both in vitro and in vivo.

(-)-Epigallocatechin-3-gallate inhibits the replication cycle of hepatitis C virus.

(-)-Epigallocatechin-3-gallate (EGCG) is the most abundant catechin in green tea. In this study, we found that hepatitis C virus (HCV) infection was significantly suppressed by EGCG in an HCV cell culture (HCVcc) system using a JFH1-GFP chimeric virus, with a 50 % effective concentration (EC(50)) of 17.9 µM. The inhibitory activity of EGCG was confirmed by monitoring HCV RNA and protein expression levels in Huh7.5.1 cells infected with the JFH1 virus. Moreover, we demonstrated that the inhibitory mechanisms of EGCG were attributable to the suppression of both the HCV entry and RNA replication steps, although EGCG had little effect on translation directed by the viral internal ribosome entry site (IRES). Furthermore, HCV could be rapidly eliminated from cell cultures after two and five passages in the presence of 50 and 25 µM EGCG, respectively. These results indicate that EGCG is a potential candidate as a preventive and antiviral drug for HCV infection.