Chu-yan Chan

Synthesis and characterization of folate-PEG-grafted-hyperbranched-PEI for tumor-targeted gene delivery

A great challenge for gene therapy is to develop a high efficient gene delivery system with low toxicity. Nonviral vectors are still attractive although the current agents displayed some disadvantages (i.e., low transfection efficiency, high toxicity). To overcome the high toxicity of poly(ethylene imine) (PEI) and low transfection efficiency of PEGylated PEI (PEG-PEI), we linked a cell specific target molecule folate (FA) on poly(ethylene glycol) (PEG) and then grafted the FA-PEG onto hyperbranched PEI 25kDa. The FA-PEG- grafted-hyperbranched-PEI (FA-PEG-PEI) effectively condensed plasmid DNA (pDNA) into nanoparticles with positive surface charge under a suitable N/P ratio. Tested in deferent cell lines (i.e., HEK 293T, glioma C6 and hepatoma HepG2 cells), no significant cytotoxicity of FA-PEG-PEI was added to PEG-PEI. More importantly, significant transfection efficiency was exhibited in FA-targeted cells. Reporter assay showed that FA-PEG-PEI/pDNA complexes had significantly higher transgene activity than that of PEI/pDNA in folate-receptor (FR) positive (HEK 293T and C6) cells but not FR-negative (HepG2) cells. These results indicated that FA-PEG-PEI might be a promising candidate for gene delivery with the characteristics of good biocompatibility, potential biodegradability, and relatively high gene transfection efficiency.

Comparative proteomic analysis of mesenchymal stem cells derived from human bone marrow, umbilical cord, and placenta: Implication in the migration

Umbilical cord (UC) and placenta (P) have been suggested as alternatives to bone marrow (BM) as sources of mesenchymal stem cells (MSC) for cell therapy, with both UC‐ and P‐MSC possess immunophenotypic and functional characteristics similar to BM‐MSC. However, their migration capacity, which is indispensable during tissue regeneration process, is unclear. Under defined conditions, the migration capacity of BM‐ and P‐MSC was found 5.9‐ and 3.2‐folds higher than that of UC‐MSC, respectively. By the use of 2‐DE and combined MS and MS/MS analysis, six differentially expressed proteins were identified among these MSC samples, with five of them known to be involved in cell migration as migration enhancing or inhibiting proteins. Consistent with their migration capacity, the levels of migration enhancing proteins including cathepsin B, cathepsin D and prohibitin,were significantly lower in UC‐MSC when compared with those in BM‐ and P‐MSC. For the migration inhibiting proteins such as plasminogen activator inhibitor‐1 (PAI‐1) and manganese superoxide dismutase, higher expression was found in the UC‐MSC. We also showed that the overexpression of the PAI‐1 impaired the migration capacity of BM‐ and P‐MSC while silencing of PAI‐1 enhanced the migration capacity of UC‐MSC. Our study indicates that PAI‐1 and other migration‐related proteins are pivotal in governing the migration capacity of MSC.

The use of folate-PEG-grafted-hybranched-PEI nonviral vector for the inhibition of glioma growth in the rat.

Combined treatment using nonviral agent-mediated enzyme/prodrug therapy and immunotherapy had been proposed as a powerful alternative method of cancer therapy. The present study was aimed to evaluate the cytotoxicity in vitro and the therapeutic efficacy in vivo when the cytosine deaminase/5-fluorocytosine (CD/5-FC) and TNF-related apoptosis-inducing ligand (TRAIL) genes were jointly used against rat C6 glioma cells. The potency of the FA-PEG-PEI used as a nonviral vector was tested in the FR-expressed C6 glioma cells and Wistar rats. The C6 glioma cells and animal model were treated by the combined application of FA-PEG-PEI/pCD/5-FC and FA-PEG-PEI/pTRAIL. The antitumor effect was evaluated by survival assays and tumor volume. This study revealed a significant increase of cytotoxicity in vitro following the combined application of FA-PEG-PEI/pCD/5-FC and FA-PEG-PEI/pTRAIL treatments in C6 glioma cells. Animal studies showed a significant growth inhibition of the C6 glioma xenografts using the combined treatment. These results demonstrated that the combined treatment generated additive cytotoxic effect in C6 glioma cells in both in vitro and in vivo conditions, and indicated that such treatment method using both enzyme/prodrug therapy and TRAIL immunotherapy might be a promising therapeutic strategy in treating glioma.

Flavonoids of Herba Epimedii regulate osteogenesis of human mesenchymal stem cells through BMP and Wnt/β-catenin signaling pathway.

Herba Epimedii is one of the most commonly used Chinese herbs for treating osteoporosis. In the present study, the flavonoids of Herba Epimedii (HEF) have shown to promote the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells. They were noted to enhance the mRNA expression of BMP-2, BMP-4, Runx2, beta-catenin and cyclinD1, all of which are BMP or Wnt-signaling pathway related regulators. The osteogenic effect was inhibited by the introduction of noggin and DKK-1, which is classical inhibitor of BMP and Wnt/beta-catenin signaling, respectively. These results suggest that HEF exerts promoting effect on osteogenic differentiation, which plausibly functions via the BMP and Wnt/beta-catenin signaling pathways. Considering the therapeutic efficiency and economical issues, HEF may be a potential candidate for promoting bone regeneration. On the other hand, osteogenic differentiation of MSCs may also be a promising and attractive tool to apply in bone repair.

Seabream growth hormone receptor : Molecular cloning and functional studies of the full-length cDNA, and tissue expression of two alternatively spliced forms

A full-length clone of the growth hormone receptor (GHR) was isolated from a cDNA library constructed from the liver of black seabream (Acanthopagrus schlegeli). The seabream GHR (sbGHR) cDNA sequence encodes a transmembrane protein of 640 amino acids (aa) possessing the characteristic motifs and architectural design of GHRs of other species. When compared to the other fish GHRs, it is most homologous to another marine fish species, the turbot, where the aa identity is 79.3%. But the sbGHR sequence is more remotely related to the goldfish GHR (51.6% aa identity) and the salmonid GHRs (approximately 46-48% aa identities). Phylogenetic comparison with other known GHRs indicates that the fish GHRs constitute a distinct group among the different vertebrate classes. The aa identities between sbGHR and other GHRs are low, being around 40% with mammalian GHRs, around 45% with avian and reptilian GHRs, and less than 35% with Xenopus GHR. CHO cells transfected with the sbGHR cDNA can be stimulated to proliferate by recombinant seabream growth hormone (sbGH). In addition, the transfected cells can transactivate a co-expressed mammalian serine protease inhibitor (Spi) 2.1 promoter upon stimulation by sbGH. These functional assays indicated that the fish receptor can interact with its homologous ligand to evoke the downstream post-receptor events. Reverse transcription-polymerase chain reaction (RT-PCR) and genomic PCR using a pair of gene-specific primers revealed the expression of two alternatively spliced forms of sbGHR in various tissues of the fish. A 93-bp intron, unique to the sbGHR gene and not found in any other known GHR genes, is alternatively spliced to give rise to two forms of receptor mRNA transcripts. The two forms of the receptor are differentially expressed among the different tissues of the fish.

Proteomic identification of molecular targets of gambogic acid: Role of stathmin in hepatocellular carcinoma

Gamboge has been developed as an injectable drug for cancer treatment in China. In this study, the inhibition ratio and their IC50 values of two derivatives from Gamboge in hepatocellular carcinoma (HCC) were determined. Proteomic approach was employed to reveal the target proteins of these two derivatives, gambogic acid (GA), and gambogenic acid (GEA). HCC cells were cultured under varied conditions with the addition of either GA or GEA. Twenty differentially expressed proteins were identified and the four most distinctly expressed proteins were further validated by Western blotting. GA and GEA revealed inhibitory effects on HCC cell proliferation. The expression of cyclin‐dependent kinase 4 inhibitor A and guanine nucleotide‐binding protein β subunit 1 were upregulated by both xanthones, whilst the expression of 14‐3‐3 protein sigma and stathmin 1 (STMN1) were downregulated. Furthermore, overexpression of STMN1 in HCC cells decreased their sensitivity, whilst small interfering RNAs targeting STMN1 enhanced their sensitivity to GA and GEA. In conclusion, our study suggested for the first time that STMN1 might be a major target for GA and GEA in combating HCC. Further investigation may lead to a new generation of anticancer drugs exerting synergistic effect with conventional therapy, thus to promote treatment efficacy.

Molecular cloning and expression studies of a prolactin receptor in goldfish (Carassius auratus).

A full-length cDNA clone, of a size of 4.6 kb, for the goldfish prolactin receptor has been isolated. This cDNA clone encodes a protein of 600 amino acids homologous to prolactin receptors of other species. A Kyte-Doolittle hydropathy analysis of the receptor indicates that the translated protein consists of a signal peptide of 22 amino acids, an extracellular domain of 228 amino acids, a single transmembrane domain of 24 amino acids, and an intracellular domain of 346 amino acids. Several characteristic landmarks of prolactin receptor could be identified in this clone. These include the four conserved cysteine residues and the WS motif within the extracellular domain, and the box 1 and box 2 regions of the intracellular domain. Among all the prolactin receptor sequences known to date, this clone bears the closest resemblance to the tilapia prolactin receptor, although homology between these two fish prolactin receptors is rather low. There are only 57.4% of nucleotide and 48.3% of amino acid sequence identities between these two fish receptors. This receptor cDNA was transfected into CHO-K1 cells for functional analysis. RT-PCR analysis with a pair of gene specific primers indicate that the receptor was transcribed in the transfected cells. Using a cell proliferation assay based on the reduction of the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, the receptor transfected CHO-K1 cells can be stimulated to proliferate upon the addition of ovine prolactin in the culture medium. The tissue distribution of the prolactin receptor in goldfish was studied by RT-PCR/Southern analysis and by Northern analysis. The results indicated that the receptor is expressed mostly in the kidney, the gill and the intestine of goldfish, corroborating with the osmoregulatory role of prolactin in fish. In addition, an appreciable level of the receptor is also found in the brain and gonads of goldfish. Northern analysis showed that there are two transcript sizes, a major 4.6 kb and a minor 3.5 kb mRNAs, in the kidney, gill and intestine.

Total flavonoids of Herba Epimedii improves osteogenesis and inhibits osteoclastogenesis of human mesenchymal stem cells.

INTRODUCTION In China Herba Epimedii is one of the most common herbs that could be prescribed for treating osteoporosis. It is known to increase the overall mineral content, therefore, to promote bone formation and to increase lumbar bone mineral density (BMD). The present study was aimed at investigating the effect of flavonoids of Herba Epimedii (HEF) on osteogenesis in human MSCs. METHODS The human bone marrow-derived MSCs (BM-MSCs) were isolated and their osteogenic differentiation was evaluated by their alkaline phosphatase (ALP) activities and level of mineralization. After treating with total flavonoids during osteogenic differentiation process, differential mRNA expression was examined by RT-PCR. RESULTS The total time needed for osteogenic differentiation of BM-MSCs was significantly shortened by adding HEF. Up-regulation of mRNA expression by HEF was observed for several marker genes and osteogenic regulators. HEF was also found to inhibit osteoclastogenesis of MSCs by enhancing the ratio OPG/RANKL. CONCLUSIONS Our study demonstrated that the HEF could improve osteogenic differentiation and inhibit the osteoclast differentiation of BM-MSCs concurrently.

Two-dimensional differential gel electrophoresis/analysis of diethylnitrosamine induced rat hepatocellular carcinoma

Diethylnitrosamine (DEN) is a known carcinogen that can alkylate DNA molecules. In rats, DEN‐induced hepatocellular carcinoma (HCC) model is well established. In this study, we used a two‐dimensional differential gel electrophoresis (2D‐DIGE) system and liquid chromatography/mass spectrometry/mass spectrometry to identify the differential expression protein profiles between the DEN‐induced HCC and healthy liver cells. Western blotting and semiquantitative RT‐PCR were used to further confirm the results. Seventeen differentially expressed spots were identified in DEN‐induced HCC cells. Among all, the most prominent upregulated proteins include the members of the glutathione S‐transferase super family, aldo‐keto reductase superfamily and proteins involved in the response to oxidative stress. Downregulation was observed in 2 proteins that were known to contribute to hepatic dysfunction. This study provides the first comprehensive protein profiling of the DEN‐induced HCC in rats. This model simulates the differential protein expression of human HCC and may be useful for further understanding the mechanism of HCC tumorigenesis.

Glucose-regulated protein 78 is an intracellular antiviral factor against hepatitis B virus.

Hepatitis B virus (HBV) infection is a global public health problem that plays a crucial role in the pathogenesis of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. However, the pathogenesis of HBV infection and the mechanisms of host-virus interactions are still elusive. In this study, two-dimensional gel electrophoresis and mass spectrometry-based comparative proteomics were applied to analyze the host response to HBV using an inducible HBV-producing cell line, HepAD38. Twenty-three proteins were identified as differentially expressed with glucose-regulated protein 78 (GRP78) as one of the most significantly up-regulated proteins induced by HBV replication. This induction was further confirmed in both HepAD38 and HepG2 cells transfected with HBV-producing plasmids by real time RT-PCR and Western blotting as well as in HBV-infected human liver biopsies by immunohistochemistry. Knockdown of GRP78 expression by RNA interference resulted in a significant increase of both intracellular and extracellular HBV virions in the transient HBV-producing HepG2 cells concomitant with enhanced levels of hepatitis B surface antigen and e antigen in the culture medium. Conversely overexpression of GRP78 in HepG2 cells led to HBV suppression concomitant with induction of the positive regulatory circuit of GRP78 and interferon-β1 (IFN-β1). In this connection, the IFN-β1-mediated 2′,5′-oligoadenylate synthetase and RNase L signaling pathway was noted to be activated in GRP78-overexpressing HepG2 cells. Moreover GRP78 was significantly down-regulated in the livers of chronic hepatitis B patients after effective anti-HBV treatment (p = 0.019) as compared with their counterpart pretreatment liver biopsies. In conclusion, the present study demonstrates for the first time that GRP78 functions as an endogenous anti-HBV factor via the IFN-β1-2′,5′-oligoadenylate synthetase-RNase L pathway in hepatocytes. Induction of hepatic GRP78 may provide a novel therapeutic approach in treating HBV infection.