Wenyun Zheng

Functionalization of single-walled carbon nanotubes enables efficient intracellular delivery of siRNA targeting MDM2 to inhibit breast cancer cells growth

The delivery of DNA or RNA to cells represents the limiting step in the development of cancer gene therapy and RNA interference protocols. Single walled carbon nanotubes (SWNTs) are of interest as carriers of biologically active molecules because of their ability to cross cell membranes. In this study, we developed a novel strategy for chemical functionalization of SWNTs (f-SWCNTs) with DSPE-PEG-Amine to bind small interfering RNA (siRNA) by disulfide bonds applied to siRNA-mediated gene silencing in breast cancer cells. Results indicated the efficiency of f-SWNTs carrying siRNA reached 83.55%, and the new f-SWNTs-siRNA-MDM2 complexes were successfully introduced into the breast carcinoma B-Cap-37 cells at a concentration of 100 nM in mediums, and caused proliferation inhibition of B-Cap-37 cells significantly. The proliferation inhibition ratio of B-Cap-37 cells was detected as 44.53% for 72 h, and the apoptosis ratio was measured as 30.45%. It was obvious that MDM2 can serve as a novel therapeutic target by an effective carrier system of DSPE-PEG-Amine-functionalized SWNTs, which would be very advanced and significant to therapy of breast cancer further.

Molecular cloning and bioinformatics analysis of a novel spliced variant of survivin from human breast cancer cells

Survivin gene and its two alternatively spliced variants, survivin-2 B and survivin- Δ Ex3 gene were cloned from human breast cancer cell lines B-cap37 firstly. A new gene designated as survivin-image (SI) was cloned from above cell lines, which has not been reported yet to clone from any cell lines. It was found that the novel gene 507 bp comprises partial survivin gene (345 bp), partial image gene (155 bp) of eye cancer and other insertion of 7 bp by analyzing with a series of recent bioinformatics software at the level of nucleotide and protein deduced. Predicted 3-D structures of the new molecule showed greatly similar to that of survivin in N-terminal containing BIR by homology modeling. These results suggested SI gene (GenBank accession No.AY830084) might be a novel alternatively spliced isoforms of the survivin gene involved in other functional significances related to tumorigenesis.

Discovery of the cell-penetrating function of A2 domain derived from LTA subunit of Escherichia coli heat-labile enterotoxin

Heat-labile enterotoxin (LT) is a protein toxin produced by enterotoxigenic Escherichia coli (ETEC). As a bacterial toxin, LT holotoxin can enter intestinal epithelial cells and cause diarrhea. In addition, LT is also a powerful mucosal adjuvant capable of enhancing the strong immune responses to co-administered antigens. However, the LT immunological mechanism is still not clear in some aspects, especially with the respect to how the LTA subunit functions alone. Here, we discovered that the A2 domain of LTA could carry a fluorescent protein into cells, whose function is similar to a cell-penetrating peptide. The transmembrane-transporting ability of the A2 domain is non-specific in its cell-penetrating function, which was shown through testing with different cell types. Moreover, the LTA2 fusion protein penetrated a fluorescently labeled cell membrane that identified LTA2 internalization through membrane transport pathways, and showed it finally localized in the endoplasmic reticulum. Furthermore, low-temperature stress and pharmacological agent treatments showed that the LTA2 internalization route is a temperature-dependent process involving the clathrin-mediated endocytosis and the macropinocytosis pathways. These results could explain the internalization of the LTA subunit alone without the LTB pentamer, contributing to a better understanding of LTA working as a mucosal adjuvant; they also suggest that the A2 domain could be used as a novel transport vehicle for research and treatment of disease.

Cascade enzymatic reactions for efficient carbon sequestration.

Thermochemical processes developed for carbon capture and storage (CCS) offer high carbon capture capacities, but are generally hampered by low energy efficiency. Reversible cascade enzyme reactions are examined in this work for energy-efficient carbon sequestration. By integrating the reactions of two key enzymes of RTCA cycle, isocitrate dehydrogenase and aconitase, we demonstrate that intensified carbon capture can be realized through such cascade enzymatic reactions. Experiments show that enhanced thermodynamic driving force for carbon conversion can be attained via pH control under ambient conditions, and that the cascade reactions have the potential to capture 0.5 mol carbon at pH 6 for each mole of substrate applied. Overall it manifests that the carbon capture capacity of biocatalytic reactions, in addition to be energy efficient, can also be ultimately intensified to approach those realized with chemical absorbents such as MEA.

Genetic protein TmSm(T34A) enhances sensitivity of chemotherapy to breast cancer cell lines as a synergistic drug to doxorubicin.

In order to eliminate common side effects to cancer patients and resistance from chemotherapy, a genetic protein TmSm(T34A) was investigated as a sensitizer to doxorubicin. The results indicated TmSm(T34A) enhanced the sensitivity of three breast cancer cell lines to doxorubicin with low dose, and reduced the dose of doxorubicin significantly in contrast to common effective dose. As a synergistic therapy, the TmSm(T34A) also caused strongest apoptotic activity in MCF-7, and the possible molecular mechanisms were explored primarily. The research showed the TmSm(T34A) is promising to be a potential drug in strengthening therapy effects of breast cancer chemotherapy.

Effect on apoptosis and cell cycle of recombinant double negative dominant mutation Survivin (T34/117A) in breast cancer cell B-Cap-37

As a bi-functional molecule in anti-apoptosis and pro-proliferation, Survivin is considered to be an attractive target for anti-cancer drug development all the time. Some studies show that these effects of survivin are mainly from two phosphorylation sites on its different domain, Thr 34 and Thr117, but it remains unclear that how they involve respectively in the apoptosis and the cell cycle of cancer cells, hindering the design and preparation of novel anti-cancer drug target to survivin. In the study, a series of recombinant double negative dominant mutants of survivin were constructed, expressed and purified efficiently, and their effects on cell cycle and apoptosis of breast cancer cell B-Cap-37 were investigated. The results showed that Survivin Thr117 is a key site on regulation of proliferation and cycle by Aurora B kinase phosphorylation, and Survivin Thr34 involves cell apoptosis by decreasing mitochondria membrane potential (MMP) and activating caspase-3. Further studies have also shown that recombinant double negative dominant mutation Survivin (T34/117A) could significantly inhibit the proliferation of B-Cap-37 cells and arrest cell in G0/G1 phase and G2/M phase, indicating the double mutant is a more potential candidate as anti-cancer drug.

Design and screening of a chimeric survivin-specific nanobody and its anticancer activities in vitro.

Survivin is a strong inhibitor of apoptosis protein and a promising target for cancer prevention and treatment. Here, we report the design and preparation of novel chimeric nanobodies (Nbs) that could specifically bind to survivin. We screened the peptides from phage-displayed libraries (7-mer, 12-mer) for nonconserved sequences of complementarity-determining regions (CDRs) in the scaffold of the Nb. By a combination of the nonconserved sequences for CDRs, the corresponding chimeric Nbs (10 Nbs) were prepared with genetic operations. The antisurvivin Nb TAT-Nb4A (a fusion with cellular transduction peptide TAT) was found to be the most efficient antibody on the basis of the results from enzyme-linked immunosorbent assay, MTT, and flow cytometry when these nanobodies were tested with hepatoma carcinoma cell HepG2. TAT-Nb4A could inhibit the growth of HepG2 and promote cancer cell apoptosis significantly in a dose-dependent and time-dependent manner: the apoptosis rate reached 52.5% when the concentration of TAT-Nb4A was 120 &mgr;g/ml. Western blotting with cells expressing survivin showed that the prepared nanobody could efficiently bind to expressed survivin and blocked the signaling pathway in which survivin played a role. This study provided a convenient and feasible method of obtaining a novel specific Nb with the case of survivin as a good example.

Screening and Identification of Highly Specific MAbs for Discovering Novel Biomarkers of Bone Marrow Stromal Cells

Bone marrow stromal cells (BMSCs) are very useful model systems for a better understanding of cell behavior and differential gene expression. Up to now, there have not been specific markers and MAbs for BMSCs that hamper the identification and isolation of BMSCs populations. In this study, chicken BMSCs were isolated from 1-day-old Beijing fatty chickens by adherent culture. After biological characteristics were detected, the chicken BMSCs were used to immunize BALB/c mice to prepare BMSCs-specific monoclonal antibodies (MAbs) by the routine hybridoma technique. These MAbs were characterized by FACS analysis, immunocytochemistry, immunohistochemistry, subtype identification, and Western blotting assay and were used to explore markers of chicken BMSCs. Our data showed that BMSCs expressing antigens CD29, CD44, and CD105, but not expressing antigens CD34, CD45, and CD11b, could be isolated from postnatal chicken bone marrow and hold great potential for multiline age differentiation. Meanwhile, we obtained two hybridoma cell lines secreting chicken BMSCs-specific MAbs (named CHK1 and CHK2), which specifically recognized the surface antigens expressed on chicken BMSCs. According to our subtype identification, heavy chains of CHK1 and CHK2 were typed as IgG1 and IgG2b, respectively; all the light strands were kappa subtype. MAbs CHK1 and CHK2 can be used to develop the detection assay and to discover novel biomarkers of chicken BMSCs.

A Novel Method for Efficient Preparation of Mucosal Adjuvant Escherichia coli Heat-Labile Enterotoxin Mutant (LTm) by Artificially Assisted Self-Assembly In Vitro

As well-known powerful mucosal adjuvant proteins, Escherichia coli heat-labile enterotoxin (LT) and its non-toxic or low-toxic mutants (LTm) are capable of promoting strong mucosal immune responses to co-administered antigens in various types of vaccines. However, due to the complex composition and special structure, the yield of LTm directly from the recombinant genetic engineering strains is quite low. Here, we put forward a novel method to prepare LTm protein which designed, expressed, and purified three kinds of component subunits respectively and assembled them into a hexamer structure in vitro by two combination modes. In addition, by simulated in vivo environment of polymer protein assembly, the factors of the protein solution system which include environment temperature, pH, ionic strength of the solution, and ratio between each subunit were taken into consideration. Finally, we confirmed the optimal conditions of two assembly strategies and prepared the hexamer holotoxin in vitro. These results are not only an important significance in promoting large-scale preparation of the mucosal adjuvant LTm but also an enlightening to produce other multi-subunit proteins.

Preparation of Recombinant HIV-TATm-Survivin (T34A) Protein and Its Proapoptosis Activity to Four Cancer Cell Lines in vitro

Abstract In this study, the cDNA encoding survivin was cloned by RT-PCR obtained from human breast cancer cell lines, B-Cap37. After the mutation of survivin (T34A) by site-directed mutagenesis, an expression vector of pRSET-B-HIV-TATm-Survivin (T34A) was constructed by PCR. Subsequently, the resultant plasmid was transformed into E. coli BL21 (DE3). The recombinant HIV-TATm-Survivin (T34A) protein was expressed efficiently by inducing IPTG at a concentration of 0.5 mmol/L, reaching a yield of 650 mg/L (as inclusion body) in fermentation culture. The inclusion bodies were solubilized, refolded, and purified by ion exchange chromatography and size-exclusion chromatography to a purity of 96 %. Remarkable effects of the purified recombinant HIV- TATm-Survivin (T34A) on the morphology of cell line SW1990 and B-Cap37 were observed after being administrated for 4 h. MTT assay showed that the recombinant HIV-TATm-Survivin (T34A) protein could restrain the cell proliferation of SW1990, B-Cap37, and SSMC-7721, in vitro. Apoptosis rate and cell circle of SW1990 and B-Cap37 that were treated with the target protein (final concentration 60 μg/mL) were detected with flow cytometry. The results revealed that the apoptosis rate of SW1990 and B-Cap37 were 25.6 % and 19.3 %, respectively. More than 65 % of cancer cells were arrested at the G1 phase. The study suggested that TATm-Survivin (T34A) protein was a hopeful protein drug for the treatment of cancer by facilitating apoptosis of cancer cells.