Fangling Ji

Biocompatible cationic pullulan-g-desoxycholic acid-g-PEI micelles used to co-deliver drug and gene for cancer therapy

The greatest crux in the combination of chemotherapy and gene therapy is the construction of a feasible and biocompatible carrier for loading the therapeutic drug and gene simultaneously. Here, a new amphiphilic bifunctional pullulan derivative (named as PDP) synthesized by grafting lipophilic desoxycholic acid and low-molecular weight (1kDa) branched polyethylenimine onto the backbone of pullulan was evaluated as a nano-carrier for the co-delivery of drug and gene for potential cancer therapy. PDP exhibited good blood compatibility and low cytotoxicity in the hemolysis and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, respectively. By self-assembly process, the amphiphilic PDP polymer formed cationic core-shell nanomicelles in aqueous solution with an average diameter of 160.8nm and a zeta potential of approximate 28mV. The PDP micelles had relative higher drug encapsulation efficiency (84.05%) and loading capacity (7.64%) for doxorubicin (DOX), an effective anti-tumor drug, demonstrating sustained drug release profile and good DNA-binding ability. The flow cytometry and confocal laser scanning microscopy showed that PDP/DOX micelles could be successfully internalized by MCF-7 cells, and presenting higher cytotoxicity against MCF-7 cells than that of free DOX. Furthermore, PDP micelles could efficiently transport tumor suppressor gene p53 into MCF-7 cells, and the expressed exogenous p53 protein induced MCF-7 cells to die. More excitedly, in comparison with single DOX or p53 delivery, the co-delivery of DOX and gene p53 using PDP micelles displayed higher cytotoxicity, induced higher apoptosis rate of tumor cells and blocked more effectively the migration of cancer cells in vitro. In tumor-bearing mice, co-delivery of DOX and p53 also exhibited enhanced antitumor efficacy as compared with single delivery of DOX or p53 alone. In summary, these results demonstrated that it is highly promising to use cationic PDP micelles for effectively co-delivering functional gene and chemotherapeutic agent, and thus improving antitumor efficacy and systemic toxicity.

Efficient mono-acylation of fructose by lipase-catalyzed esterification in ionic liquid co-solvents

Fructose monoesters are eco-friendly nonionic surfactants in various applications. Selective preparation of mono-acylated fructose is challenging due to the multiple hydroxyl sites available for acylation both chemically and enzymatically. Ionic liquids (ILs) have profound impacts not only on the reaction media but also on the catalytic properties of enzymes in the acylation process. In this study, utilizing an IL co-solvent system, selective synthesis of mono-acylated fructose with lauric acid catalyzed by immobilized Candida antarctica lipase B (CALB) was investigated. The imidazolium-based ILs selected as co-solvents with 2-methyl-2-butanol (2M2B) markedly improved the ratios of monolauroyl fructose in the presence of 60% [BMIM][TfO] (v/v) and 20% [BMIM][BF4] (v/v), in which the mono-acylated fructose was 85% and 78% respectively. Based on a Ping-Pong Bi-Bi model, a kinetic equation was fitted, by which the kinetic parameters revealed that the affinity between fructose and acyl-enzyme intermediate was enhanced. The inhibition effect of fructose on free enzyme was weakened in the presence of IL co-solvents. The conformation of CALB binding substrates also changed in the co-solvent system as demonstrated by Fourier transform infrared spectra. These results demonstrated that the variation of CALB kinetic characteristics was a crucial factor for the selectivity of mono-acylation in ILs/2M2B co-solvents.

Quercetin exerts synergetic anti-cancer activity with 10-hydroxy camptothecin

&NA; Quercetin (Qu) is known as a dietary antioxidant with numerous bioactivities, but its function in anti‐cancer has not been fully investigated. Here, we show that Qu at low doses (≤10 &mgr;M) significantly enhances the inhibition of 10‐hydroxy camptothecin (HCPT) on the proliferation of MCF7, BGC823 and HepG2 cells. A plasmid DNA relaxation assay indicates that the inhibition of HCPT on the catalytic activity of topoisomerase I (Topo I) is increased by Qu at 10 &mgr;M. Compared to the treatment by Qu or HCPT alone, phosphorylation at Ser139 of &ggr;H2A.X in MCF7 cells starts to increase significantly (P < 0.05) at 6 h when treated by the combination of 10 &mgr;M Qu and 0.62 &mgr;M HCPT. Moreover, the combinational group successively arrests MCF7 cells at G1, S and G2/M phases from 12 h to 48 h via up‐regulation of p21 and induces apoptosis at 24 h by triggering intrinsic cell death pathways. In addition, the inhibition effects of the combinational group on the proliferation of MCF7 cells are eliminated by pretreatment with 100 &mgr;M Z‐VAD‐FMK (a caspase inhibitor). Finally, by using nude mice xenografting assay of MCF7 cells, we demonstrate that tumor inhibition rates of combinational group are significantly higher than single‐drug group. In summary, the synergic anti‐cancer mechanism of Qu and HCPT in MCF7 cells is through the combined inhibitory effects of Qu and HCPT on Topo I, which synergistically induce cell cycle arrest and apoptosis by triggering DNA damage. Graphical abstract Figure. No caption available.

Combination of baicalein and 10-hydroxy camptothecin exerts remarkable synergetic anti-cancer effects.

BACKGROUND 10-Hydroxy camptothecin (HCPT), a naturally occurring alkaloid, is a clinical drug for cancer chemotherapy. Baicalein (BA) is a flavonoid extracted from the root of Scutellaria baicalensis. The synergistic anti-cancer effect of BA and HCPT has not been reported. PURPOSE To explore whether and how BA enhances the anti-cancer effect of HCPT in BGC823 cells. METHODS Cell viability was measured by MTT assay. Apoptosis and cell cycle were analyzed through flow cytometry and western blotting analysis. DNA damage was determined by a comet assay. The activity of topoisomerase I (Topo I) was detected by the plasmid DNA relaxation assay. The synergistic anti-cancer effect of BA and HCPT in vivo was tested by BGC823 xenografted tumor model. RESULTS BA at non-toxic doses prominently enhanced the anti-cancer activities of HCPT in BGC823, MCF7 and SMMC7721 cells. Combination treatment of BA and HCPT induced BGC823 cells apoptosis mainly via intrinsic rather than extrinsic pathways, and preferentially arresting cell cycle in G1 and G2 phases with the aid of p21. Of note, p53, the upstream regulator of cell apoptosis and cycle, was increased by 5 folds in combination group. It helped to further trigger DNA damage and inhibit Topo I catalytic activity after combination treatment of BA and HCPT. Moreover, the BGC823 xenografted tumor growth rate in nude mice was repressed in a greater degree (P< 0.01) in the combinational group than the single-drug group. CONCLUSION HCPT and BA, a new and effective combination therapy, synergistically target Topo I and up-regulate p53 to induce cell apoptosis and cell cycle arrest.

LotS/LotR/Clp, a novel signal pathway responding to temperature, modulating protease expression via c-di-GMP mediated manner in Stenotrophomonas maltophilia FF11

Stenotrophomonas maltophilia as one of increasing food spoilage bacteria and fish pathogens has become a threat to aquiculture industry. A major factor contributing to the success of bacterium is its outstanding ability to secrete protease at low temperatures. Here, a cAMP receptor like protein (Clp) shows a positive regulation on this protease, named S. maltophilia temperature-response protease (SmtP). Interestingly, a two-component system, comprising of LotS sensor and LotR regulator, for low-temperature response is also confirmed to modulate SmtP expression with similar effect to Clp. Evidence is presented that LotS/LotR modulates smtP (coding SmtP) expression via Clp: clp promoter activity was reduced significantly at low temperatures and protease activity was partially restored by Clp overexpressed in lotS or lotR deletion strain. Furthermore, as a Clp negative effector, the binding ability of c-di-GMP with Clp is not impacted by temperature. c-di-GMP level was increased in S. maltophilia growing at high temperature, but not exhibited significantly in lotR deleted strain, these indicate that LotR is required for temperature modulating c-di-GMP level, although the synthesis or degradation activity of c-di-GMP by LotR was not detected. These findings suggest that LotS/LotR/Clp play an important role in responding to temperature stimuli via c-di-GMP mediated manner.

Rational design of Meso-2,3-butanediol Dehydrogenase by Molecular Dynamics Simulation and Experimental Evaluations

Meso‐2,3‐butanediol dehydrogenase (meso‐2,3‐BDH) catalyzes NAD+‐dependent conversion of meso‐2,3‐butanediol to acetoin, a crucial external energy storage molecule in fermentive bacteria. In this study, the active tunnel of meso‐2,3‐BDH was identified. The two short α helixes positioned away from the α4‐helix possibly expose the hydrophobic ligand‐binding cavity, gating the exit of product and cofactor from the activity pocket. Further MM/GBSA‐binding free energy analysis shows that Phe212 and Asn146 function as the key product‐release sites. Site‐directed mutagenesis experiments targeted to the sites show that the kcat of Phe212Tyr is enhanced up to (4–8)‐fold. The original activity of Asn146Gln is retained, but the activity of Asn146Ala mutation is lost. These results could provide helpful guidance on rational design of short‐chain dehydrogenases/reductases.