Redounding of Cuscuta chinensis Lam. on BxPC-3, HepG2, and U2OS Human Cancer Cell Lines

Objective: The present study was designed to investigate in vitro cytotoxic effect of aqueous extract from whole Cuscuta chinensis on human hepatocellular carcinoma (HepG2), biopsy xenograft of pancreatic carcinoma line-3 (BxPC-3), and children human bone osteosarcoma cell line (U2OS). Materials and Methods: The anticancer effectiveness of the methanol-watery extract of C. chinensis Lam. was determined by using methyl tetrazolium bromide test (MTT) assay against cancer cells by using suspensions of BxPC-3, HepG2, and U2OS cell lines. The inhibitory concentration (IC50) was tested for each cancer cell line. BxPC-3, HepG2, and U2OS cell line death percent after incubation with extract for 24, 48, and 72-hours interval was compared with cisplatin death percent. Results: The results showed that the IC50 of Cuscuta extract for BxPC-3, HepG2, and U2OS cell lines was 13, 6.5, and 0.73 μg/mL, respectively. The HepG2 cell line death%, when treated with 50 μg/mL Cuscuta extract at 24, 48, and 72-hour time interval, was 90.41, 91.45, and 92.93%, while cells were treated with 15 μg/mL cisplatin, the death percent was 88.8, 93.7, and 96.61%, respectively. The BxPC-3 cell line death%, when treated with 50 μg/mL Cuscuta extract, was 51.46, 83.37, and 91.28%, respectively, and when treated with 15 μg/mL cisplatin was 81.64, 88.02, and 96.67%, respectively. The U2OS cell line death%, when treated with 50 μg/mL Cuscuta extract, was 69.43, 69.75, and 88.89%, and was 74.1, 84.61, and 93.39%, respectively, when treated with 15 μg/mL cisplatin. Conclusion: The methanol-watery extract of C. chinensis Lam. may have a potential role as an adjunct therapy for cancers in the future.

Potential Anticancer Activity of Crude Ethanol, Ethyl Acetate, and Water Extracts of Ephedra foeminea on Human Osteosarcoma U2OS Cell Viability and Migration

Medicinal plants are potential sources for a wide range of complex compounds with probable anticancer activity. Ephedra foeminea Forssk. (E. foeminea), a medicinal plant found in the Eastern Mediterranean, has recently been gaining popularity as a cancer remedy; there is, however, a paucity of empirical evidence supporting this claim. In this study, the effect of E. foeminea ethyl acetate, ethanol, and water crude extracts on viability, migratory ability, and the steady-state mRNA levels of genes involved in these processes was, respectively, examined using MTT assay, wound healing assay, and reverse transcriptase PCR (RT-PCR). The study concludes that all extracts significantly reduce human osteosarcoma U2OS percentage viability in a dose- and time-dependent manner, with varying potencies. The least half-maximal inhibitory concentration (IC50) was observed in the water extract after 48 h incubation (30.761±1.4 μg/mL) followed by the ethyl acetate extract after 72 h incubation (80.35±1.233 μg/mL) and finally the ethanol extract after 48 h incubation (97.499±1.188 μg/mL). Ethanol extract significantly reduced U2OS percentage wound closure. On the other hand, both ethanol and water extracts considerably reduced the steady-state mRNA expression of beta-catenin, promoting both cell proliferation and migration in osteosarcoma by regulating target genes. Additionally, E. foeminea showed no hemolytic activity. These effects suggest that E. foeminea decreases U2OS cell viability and migratory ability by modulating the expression of critical genes involved in regulating these processes and is likely cytocompatible with human erythrocytes.

Parallel siRNA screens to identify kinase and phosphatase modulators of NF-κB activity following DNA damage

DNA damage, such as that experienced by people undergoing chemotherapy, can directly activate NF-κB signalling which in turn can lead to resistance to genotoxic stress. NF-κB signalling is highly regulated by phosphorylation, but the enzymes required for these processes remain largely unknown. Identifying those enzymes responsible for regulating NF-κB activity may yield attractive targets for new clinical therapies, as well as provide the basis for better understanding of signalling network crosstalk. Here we present datasets from two independent RNAi screens using a stable NF-κB reporter U2OS cell line with the aim of identifying enzymes that alter NF-κB activity in response to DNA damage following etoposide and ionising radiation treatments. Although we observed high internal validity and specificity to NF-κB modulation within the screens, there was a striking dissimilarity between the results of the two different screens. These data therefore provide a cautionary lesson regarding the use of RNAi screening but also provide new candidates for kinase and phosphatase regulation of NF-κB activity in response to genotoxic stress.

Screening GLP-1 Receptor Ligands from Natural Products in Herbs through High-Content Technique

Aim and Objective: Screening of active components from a natural product, especially from a crude extract, is a great challenge. To avoid potential activity interference of the N-terminus modification in the most common constructs based on GCPRs labeled with GFP technology, a Cterminus tGFP-labeled hGLP-1 receptor containing recombinant cell line hGLP-1R-tGFP was constructed and tried to be used in the screening of natural products from Chinese herb. Materials and Methods: The GLP1 receptor gene was amplified and the inserts pCMV6-AC-tGFP and tGFP were fused at the C-terminus of GLP1 receptor to construct a recombinant plasmid. The recombinant was transfected into U2OS cell and selected with antibiotics and flow cytometry. The constructed cell line was named as hGLP-1R-tGFP cell line. The expression levels of GLP-1R-tGFP protein were confirmed by western-blot. The fluorescence imaging of re-distribution from diffusing to aggregate spots inside the cells was quantitated and analyzed by High Content Screening (HCS) assay. Meanwhile, the specificity, stability and C-terminus function of hGLP-1R-tGFP cell line were characterized. In order to allow the recombinant cell line of hGLP-1R-tGFP to be suitable in highcontent system of Arrayscan-infinity-700 in screening mode, several conditions have also been optimized. In the end, a total of 100 crude extract samples provided by the Yunnan Institute of Materia Medica have been screened with this method. Results: Upon the activation of GLP-1 receptors by Exendin 4, fluorescent patches appeared on the cell membrane and subsequently internalized to form fluorescent aggregates inside the cells under fluorescent microscopy examination. The agonistic activity, sensitivity and specificity of the formation of fluorescent aggregate spot in hGLP-1R-tGFP cells have been confirmed by the activation of GLP-1R using the GLP-1analogues. The agonistic effects of GLP-1 analogues are blocked by a GLP-1R antagonist, Exendin9-39. The downstream of GLP-1 pathway, the activation of adenylate cyclase and the raising of cellular cAMP levels, remained intact in these tGFP modified C-terminus GLP-1 receptor cells. Meanwhile, a total of 100 crude extract samples from Chinese herbs have been screened by this method to find new active ingredients. Conclusion: Combined with High Content Screening image and data automatic acquisition processing, a new screening assay based on a recombinant U2OS cell line which GFP labeled at the C terminus of GLP1 receptor has been developed. GLP-1R agonist activity in extracts of Astragalus propinquus and Panax notoginseng from Chinese herbs has been determined by this method.

Isoliquiritigenin Suppresses Osteosarcoma U2OS Cell Proliferation and Invasion by Regulating the PI3K/Akt Signalling Pathway

Aims: Isoliquiritigenin (ISL) is a flavonoid, that has been shown to have antioxidant, vasorelaxant, anti-inflammatory, and antitumor activities. This study aimed to explore the antitumor effect of ISL on human osteosarcoma U2OS cells and investigate the mechanism of this effect. Methods: The effect of ISL on osteosarcoma U2OS cell proliferation, invasion, migration, and apoptosis were determined by a CCK8 assay, a transwell invasion assay, a transwell migration assay, and fluorescence-activated cell sorting, respectively. In addition, the protein expression levels of Bcl2, Bax, active Caspase-3, Akt, mTOR, p70, and Cyclin D1 were detected by western blotting. Results: ISL suppressed cell proliferation, inhibited invasion and migration, and promoted apoptosis in U2OS cells. After treatment with ISL, the protein expression levels of Bax and active Caspase-3 increased, while the level of Bcl-2 declined significantly. Furthermore, the phosphorylation levels of Akt and mTOR declined significantly compared with that of the control. Conclusion: ISL could retard proliferation and promote apoptosis of U2OS cells possibly by suppressing the PI3K/Akt signalling pathway, indicating that it might be a potential therapeutic agent for osteosarcoma treatment.

Chimaphilin inhibits human osteosarcoma cell invasion and metastasis through suppressing the TGF-β1-induced epithelial-to-mesenchymal transition markers via PI-3K/Akt, ERK1/2, and Smad signaling pathways

Epithelial-to-mesenchymal transition is a cellular process associated with cancer invasion and metastasis. However, the antimetastatic effects of chimaphilin remain elusive. In this study, we attempted to investigate the potential use of chimaphilin as an inhibitor of TGF-β1-induced epithelial-to-mesenchymal transition in U2OS cells. We found that TGF-β1 induced epithelial-to-mesenchymal transition to promote U2OS cell invasion and metastasis. Western blotting demonstrated that chimaphilin inhibited U2OS cell invasion and migration, increased the expression of the epithelial phenotype marker E-cadherin, repressed the expression of the mesenchymal phenotype marker vimentin, as well as decreased the level of epithelial-to-mesenchymal-inducing transcription factors Snail1 and Slug during the initiation of TGF-β1-induced epithelial-to-mesenchymal transition. In this study, we revealed that chimaphilin up-regulated the E-cadherin expression level and inhibited the production of vimentin, Snail1, and Slug in TGF-β1-induced U2OS cells by blocking PI-3K/Akt and ERK 1/2 signaling pathway. Additionally, the TGF-β1-mediated phosphorylated levels of Smad2/3 were inhibited by chimaphilin pretreatment. Above all, we conclude that chimaphilin represents an effective inhibitor of the metastatic potential of U2OS cells through suppression of TGF-β1-induced epithelial-to-mesenchymal transition.