Yanfei Cai

MiR-489 regulates chemoresistance in breast cancer via epithelial mesenchymal transition pathway.

To investigate the role of microRNAs in the development of chemoresistance and related epithelial–mesenchymal transition (EMT), we examined the effect of miR‐489 in adriamycin (ADM)‐resistant human breast cancer cells (MCF‐7/ADM). MiR‐489 was significantly suppressed in MCF‐7/ADM cells compared with chemosensitive parental control MCF‐7/WT cells. Forced‐expression of miR‐489 reversed chemoresistance. Furthermore, Smad3 was identified as the target of miR‐489 and is highly expressed in MCF‐7/ADM cells. Forced expression of miR‐489 both inhibited Smad3 expression and Smad3 related EMT properties. Finally, the interactions between Smad3, miR‐489 and EMT were confirmed in chemoresistant tumor xenografts and clinical samples, indicating their potential implication for treatment of chemoresistance.

Apigenin, a plant‐derived flavone, activates transient receptor potential vanilloid 4 cation channel

BACKGROUND AND PURPOSE Transient receptor potential vanilloid 4 (TRPV4) is a Ca2+‐permeable channel with multiple modes of activation. Apigenin is a plant‐derived flavone, which has potential preventive effects on the development of cardiovascular disease. We set out to explore the effects of apigenin on TRPV4 channel activity and its role in vasodilatation.

Luteolin inhibits pyrogallol-induced apoptosis through the extracellular signal-regulated kinase signaling pathway

Luteolin is an antioxidative, antitumor and anti‐inflammatory flavone. It has been shown to reduce endothelial dysfunction, but the mechanism is not clear. We set out to explore the effects of luteolin on apoptosis and its mechanism of action in endothelial cells. The effect of luteolin on pyrogallol‐induced superoxide stress and the subsequent apoptosis was studied in the mouse heart capillary endothelial cell line H5V and human umbilical vein endothelial cells, by the use of flow cytometry, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide, Hoechst staining, and western blot. Pyrogallol (0–400 μm) dose‐dependently induced reactive oxygen species production, cytotoxicity, an annexin V–fluorescein isothiocyanate increase, mitochondrial transmembrane depolarization and DNA condensation in both H5V and human umbilical vein endothelial cells; these actions were reversed by luteolin (0.78–50 μm) in a concentration‐dependent manner. Luteolin suppressed the poly (ADP‐ribose) polymerase activation, caspase‐8 cleavage and p38 mitogen‐activated protein kinase activation triggered by pyrogallol, and stimulated the extracellular signal‐regulated kinase signaling pathway to counteract the pyrogallol‐induced apoptotic signals. Luteolin is an effective agent for the protection of endothelial cells from superoxide stress‐induced apoptosis via the extracellular signal‐regulated kinase signaling pathway.

Enhanced recombinant factor VII expression in Chinese hamster ovary cells by optimizing signal peptides and fed-batch medium

ABSTRACT Signal peptides play an important role in directing and efficiently transporting secretory proteins to their proper locations in the endoplasmic reticulum of mammalian cells. The aim of this study was to enhance the expression of recombinant coagulation factor VII (rFVII) in CHO cells by optimizing the signal peptides and type of fed-batch culture medium used. Five sub-clones (O2, I3, H3, G2 and M3) with different signal peptide were selected by western blot (WB) analysis and used for suspension culture. We compared rFVII expression levels of 5 sub-clones and found that the highest rFVII expression level was obtained with the IgK signal peptide instead of Ori, the native signal peptide of rFVII. The high protein expression of rFVII with signal peptide IgK was mirrored by a high transcription level during suspension culture. After analyzing culture and feed media, the combination of M4 and F4 media yielded the highest rFVII expression of 20 mg/L during a 10-day suspension culture. After analyzing cell density and cell cycle, CHO cells feeding by F4 had a similar percentage of cells in G0/G1 and a higher cell density compared to F2 and F3. This may be the reason for high rFVII expression in M4+F4. In summary, rFVII expression was successfully enhanced by optimizing the signal peptide and fed-batch medium used in CHO suspension culture. Our data may be used to improve the production of other therapeutic proteins in fed-batch culture.

Antitumor and anti‑angiogenic activity of the recombinant human disintegrin domain of A disintegrin and metalloproteinase 15

A disintegrin and metalloproteinases (ADAMs), a family of transmembrane glycoproteins, are expressed in numerous tissues and organs, and have been implicated in a variety of physiological and pathological processes. ADAM15 is unique among the ADAMs in having an Arg-Gly-Asp motif in its disintegrin domain. In the present study, the antitumor and anti-angiogenic effects of the recombinant human disintegrin domain (rhdd) ADAM15, expressed by Escherichia coli, were evaluated. rhddADAM15 inhibited the proliferation and migration of several tumor cells, with a half maximal inhibitory concentration of 1.0-6.0 µM. In addition, rhddADAM15 inhibited the proliferation of Bel-7402 cells via the mitogen-activated protein kinase pathway and reduced the activation of Src. rhddADAM15 (1-10 µM) inhibited the proliferation, migration and tube formation of vascular endothelial EA.hy926 cells. G0/G1 arrest (10.96 ± 1.40%) and apoptotic cells (55.85 ± 1.06%) were observed in the EA.hy926 cells treated with 4 µM and 6 µM rhddADAM15, respectively. In vivo, rhddADAM15 significantly inhibited angiogenesis in zebrafish. rhddADAM15 at concentrations of 20 nmol/fish or 5 nmol/fish inhibited the angiogenesis of subintestinal and intersegmental vessels in the zebrafish by 72 ± 1.26 and 48 ± 2.92%, respectively. In conclusion, the results of the present study identified rhddADAM15 as a potent inhibitor of tumor formation and angiogenesis, rendering it a promising tool for use in anticancer treatment.

Efficient expression of stable recombinant human insulin-like growth factor-1 fusion with human serum albumin in Chinese hamster ovary cells

ABSTRACT Insulin-like growth factor-1 (IGF-1) plays a crucial role in cell development, differentiation, and metabolism, and has been a potential therapeutic agent for many diseases. Chinese hamster ovary (CHO) cells are widely used for production of recombinant therapeutic proteins, but the expression level of IGF-1 in CHO cells is very low (1,500 µg/L) and the half-life of IGF-1 in blood circulation is only 4.5 min according to previous studies. Therefore, IGF-1 was fused to long-circulating serum protein human serum albumin (HSA) and expressed in CHO cells. After 8-day fed-batch culture, the expression level of HSA–IGF-1 reached 100 mg/L. The fusion protein HSA–IGF-1 was purified with a recovery of 35% using a two-step chromatographic procedure. According to bioactivity assay, the purified HSA–IGF-1 could stimulate the proliferation of NIH3T3 cells in a dose-dependent fashion and promote the cell-cycle progression. Besides this, HSA–IGF-1 could bind to IGF-1 receptor on cell membrane and activate the intracellular PI3K/AKT signaling pathway. Our study suggested that HSA fusion technology carried out in CHO cells not only provided bioactivity in HSA–IGF-1 for further research but also offered a beneficial strategy to produce other similar cytokines in CHO cells.

Binding and biologic characterization of recombinant human serum albumin-eTGFBR2 fusion protein expressed in CHO cells

ABSTRACT Transforming growth factor-β1 (TGF-β1) signaling is involved in cell metabolism, growth, differentiation, carcinoma invasion and fibrosis development, which suggests TGF-β1 can be treated as a therapeutic target extensively. Because TGF-β1 receptor type α(TGFBR2) is the directed and essential mediator for TGF-β1 signals, the extracellular domain of TGFBR2 (eTGFBR2), binding partner for TGF-β1, has been produced in a series of expression systems to inhibit TGF-β1 signaling. However, eTGFBR2 is unstable with a short half-life predominantly because of enzymatic degradation and kidney clearance. In this study, a fusion protein consisting of human eTGFBR2 fused at the C-terminal of human serum albumin (HSA) was stably and highly expressed in Chinese Hamster Ovary (CHO) cells. The high and stable expression sub-clones with Ig kappa signal peptide were selected by Western blot analysis and used for suspension culture. After fed-batch culture over 8 d, the expression level of HSA-eTGFBR2 reached 180 mg/L. The fusion protein was then purified from culture medium using a 2-step chromatographic procedure that resulted in 39% recovery rate. The TGF-β1 binding assay revealed that HSA-eTGFBR2 could bind to TGF-β1 with the affinity constant (KD of 1.42 × 10−8 M) as determined by the ForteBio Octet System. In addition, our data suggested that HSA-eTGFBR2 exhibited a TGF-β1 neutralizing activity and maintained a long-term activity more than eTGFBR2. It concluded that the overexpressing CHO cell line supplied sufficient recombinant human HSA-eTGFBR2 for further research and other applications.

Efficient expression of glucagon-like peptide-1 analogue with human serum albumin fusion protein in Pichia pastoris using the glyceraldehyde-3-phosphate dehydrogenase promoter

Glucagon-like peptide-1 (GLP-1) was a potential therapeutic drug for type II diabetes, mainly because of the stimulatory effect on insulin secretion under condition of high blood glucose. We used PCR to obtain a recombination gene, GGH, in which two GLP-1 (GLP-1A2G) mutants were connected in series and then fused to the N terminal of human serum albumin. The fusion gene was inserted into pGAPZaA plasmid with Saccharomyces cerevisiae α-factor secretion signal sequence, and was expressed by the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. The engineered strain was constructed by integrating the recombinant plasmid pGAPZαA/GGH into the genome of Pichia pastoris GS115. Genome PCR and western blot showed that the recombinant P. pastoris successfully expressed the fusion protein GGH. The yield of GGH reached 78 mg/L after 72 h fermentation in a flask, using glucose as the optimal carbon source. Fed-batch fermentation was investigated in a 5 L bioreactor, and the expression level of GGH reached 246 mg/L in 52 h. The fusion protein GGH was purified in four steps, and the final purity was 96.1%. The in vitro bioactivity of GGH was the same as that expressed in P. pastoris by the AOX1 promoter. This study described an efficient way to express GGH fusion protein in P. pastoris using GAP promoter, fermentation was easier to control without carbon source change and fermentation time was 20 h less than AOX1 promotercontrolled GGH fermentation.

A simple, rapid method for evaluation of transfection efficiency based on fluorescent dye

ABSTRACT Enhanced transfection efficiency of transient gene expression (TGE) and electroporation is a useful approach for improvement of recombinant therapeutic proteins in mammalian cells. A novel method is described here in which CHO cells expressing recombinant FVII (rFVII) were labeled with fluorescent dye and analyzed by confocal microscopy. Cells with or without rFVII encoding gene were detectable by flow cytometry. Thus, we were able to distinguish positive cells (with rFVII encoding gene) and quantify their percentages. We evaluated the effects of varying electroporation conditions (voltage, number of repetitions, plasmid amount, carrier DNA) in order to optimize transfection efficiency. The highest transfection efficiency achieved was ∼86%. The method described here allows rapid evaluation of transfection efficiency without co-expression of reporter genes. In combination with appropriate antibodies, the method can be extended to evaluation of transfection efficiency in cells expressing other recombinant proteins.