Biomedical subjects

Qixin Leng

Publications and source records attributed to Qixin Leng.

7 recordsLinked to original sources

PubMed2006

A branched histidine/lysine peptide, H2K4b, in complex with plasmids encoding antitumor proteins inhibits tumor xenografts.

BACKGROUND: In this study we investigated whether a particular branched HK polymer, H2K4b, was an effective in vivo carrier of plasmids expressing the antiangiogenic kringle 1-5 or the tumor suppressor p53. METHODS: H2K4b was synthesized on a solid-phase peptide synthesizer. Distribution, optimization and time course studies were done in tumor-bearing nude mice by systemically administering H2K4b in complex with a luciferase-expressing plasmid. We examined the amount of tumor angiogenesis in C6 with MDA-MB-435 xenografts utilizing the carmine dye. The ability of H2K4b to carry luciferase plasmids to different tissues was compared with several liposomal carriers. Medium from cells transfected with mKr1-5 was tested for its capacity to inhibit angiogenesis with an in vivo Matrigel assay. We then determined if systemically delivered H2K4b in complex with plasmid encoding mKr1-5 inhibited tumor growth; we also compared the antitumor activity of HK polyplexes containing hKr1-5, mKr1-5, and p53 plasmids. RESULTS: H2K4b carried the luciferase-expressing plasmid in order of descending efficacy to these tissues: lung, spleen, tumor, and liver. Compared to DOTAP-containing liposomes, H2K4b was a more effective carrier of a luciferase-containing plasmid to extrapulmonary tissues. We then determined that mKr1-5 in complex with H2K4b reduced MDA-MB-435 tumor growth by approximately 50% compared to the control group (P < 0.01). Similarly, H2K4b/mKr1-5 polyplexes reduced the growth of C6 xenografts. In MDA-MB-435 xenografts, p53- and Kr1-5-expressing plasmids in complex with H2K4b had comparable antitumor activity. CONCLUSION: H2K4b demonstrates potential as a carrier of plasmids encoding antiangiogenic and/or tumor suppressor proteins in a tumor-bearing mouse model.

Animals↗

PubMed2006

Synthetic histidine-rich peptides inhibit Candida species and other fungi in vitro: role of endocytosis and treatment implications.

A family of histidine-rich peptides, histatins, is secreted by the parotid gland in mammals and exhibits marked inhibitory activity against a number of Candida species. We were particularly interested in the mechanism by which histidine-rich peptides inhibit fungal growth, because our laboratory has synthesized a variety of such peptides for drug and nucleic acid delivery. In contrast to naturally occurring peptides that are linear, peptides made on synthesizers can be varied with respect to their degrees of branching. Using this technology, we explored whether histidine-lysine (HK) polymers of different complexities and degrees of branching affect the growth of several species of Candida. Polymers with higher degrees of branching were progressively more effective against Candida albicans, with the four-branched polymer, H2K4b, most effective. Furthermore, H2K4b accumulated efficiently in C. albicans, which may indicate its ability to transport other antifungal agents intracellularly. Although H2K4b had greater antifungal activity than histatin 5, their mechanisms were similar. Toxicity in C. albicans induced by histatin 5 or branched HK peptides was markedly reduced by 4,4'-diisothiocyanato-stilbene-2,2'-disulfonate, an inhibitor of anion channels. We also determined that bafilomycin A1, an inhibitor of endosomal acidification, significantly decreased the antifungal activity of H2K4b. This suggests that the pH-buffering and subsequent endosomal-disrupting properties of histidine-rich peptides have a role in their antifungal activity. Moreover, the ability of the histidine component of these peptides to disrupt endosomes, which allows their escape from the lysosomal pathway, may explain why these peptides are both effective antifungal agents and nucleic acid delivery carriers.

Adenosine Triphosphate↗

PubMed2005

Modified branched peptides with a histidine-rich tail enhance in vitro gene transfection.

Successful gene therapy depends on the development of efficient, non-toxic gene delivery systems. To accomplish this objective, our laboratory has focused on solid-phase synthesized peptide carriers, in which the amino acid sequence can be varied precisely to augment intracellular DNA transport. We previously determined that linear and branched co-polymers of histidine and lysine in combination with liposomes enhanced the efficiency of gene transfection. In this study, we have modified two branched histidine-lysine (HK) peptides by adding a histidine-rich tail. In a variety of cell lines, this histidine-rich tail markedly improved transfection efficiency, presumably by increasing the buffering capacity of the polymer. One polymer with a histidine-rich tail, H2K4bT, compared favorably with the commonly used transfection agents. Together with modification of our transfection protocol, these improved HK peptides alone, without liposomes, are the effective carriers of plasmids into a variety of cells. We anticipate that branched HK peptides will continue to be developed as carriers of nucleic acids for in vitro and in vivo applications.

Amino Acid Sequence↗

PubMed2005

Alteration in the IL-2 signal peptide affects secretion of proteins in vitro and in vivo.

BACKGROUND: Although hundreds of different signal peptides have now been identified, few studies have examined the factors enabling signal peptides to augment secretion of mature proteins. Signal peptides, located at the N-terminus of nascent secreted proteins, characteristically have three domains: (1) a basic domain at the N-terminus, (2) a central hydrophobic core, and (3) a carboxy-terminal cleavage region. In this study, we investigated whether alterations in the basic and/or the hydrophobic domains of a commonly used signal peptide from interleukin-2 (IL-2) affected secretion of two proteins: placental alkaline phosphatase (AP) and endostatin. METHODS: A series of modifications in the basic and/or hydrophobic domains of the IL-2 signal peptide were made by polymerase chain reaction with endostatin or AP plasmids as templates. Transfection of wild-type or modified IL-2 signal peptides fused in-frame with endostatin or AP were done with Superfect in vitro or by the hydrodynamic method in vivo. RESULTS: Increasing both the basicity and hydrophobicity of the signal peptide augmented the secretion of AP and endostatin by approximately 2.5- and 3.5-fold, respectively, from MDA-MB-435 cells in vitro. Over a range of DNA concentrations and times, the most effective IL-2 signal peptide increased AP levels in the medium compared to the wild-type IL-2 signal peptide. Comparable results from these modified IL-2 signal peptides were found to increase AP levels in the medium from bovine aortic endothelial cells. Moreover, the combined changes in basic and hydrophobic domains of the IL-2 signal peptide augmented serum levels of endostatin and placental AP by 3-fold when the optimal plasmid constructs were injected in vivo. CONCLUSIONS: Modification of the IL-2 signal peptide augments protein secretion both in vitro and in vivo. As a result, optimizing the signal peptide should be considered for increasing the therapeutic levels of secreted proteins.

Alkaline Phosphatase↗

PubMed2005

Highly branched HK peptides are effective carriers of siRNA.

BACKGROUND: Both viral and nonviral carriers have been used to carry small interfering RNA molecules (siRNA) to their cytosolic mRNA target. To date, few peptide carriers have been developed that have proved effective for siRNA delivery. Our previous branched carriers composed of histidine and lysine were useful for transfection of plasmids. In this study, we determined if these and more highly branched HK polymers were effective carriers of siRNA. METHODS: Several branched polymers were synthesized on a Ranin Voyager synthesizer. These polymers were then screened for their ability to transfer siRNA into SVR-bag4 cells, MDA-MB-435 cells, and C6 cells. After one polymer, H3K8b, was identified as an effective carrier of siRNA, additional polymers were synthesized to determine the essential domains for siRNA transport. The size/zeta-potential of HK : siRNA complexes were measured with the N4 submicron particle size analyzer and the Delsa 440 SX zeta-potential analyzer, respectively. Toxicity of the highly branched polymers in complex with siRNA was investigated by flow cytometry. RESULTS: In an endothelial cell line (SVR-bag4) that stably expressed beta-galactosidase (beta-gal), an siRNA in complex with the H3K8b polymer inhibited beta-gal expression by more than 80%. In contrast, the polymer H2K4b, which was an effective carrier of plasmids, was not an efficient carrier of siRNA. The size and surface charge did not distinguish effective from ineffective HK carriers of siRNA. By modifying H3K8b, we then determined what properties of H3K8b augmented siRNA delivery. The histidine-rich domain and the length of the terminal arms of H3K8 were important for siRNA delivery. The modestly more effective analog of H3K8b containing an integrin ligand, H3K8b(+RGD), was able to inhibit markedly intracellular beta-gal expression. Furthermore, we determined that H3K8b(+RGD) in complex with a luciferase-targeting siRNA inhibited luciferase expression in MDA-MB-435 cells. At its optimal concentration for inhibiting its target, H3K8b(+RGD) : siRNA complex had minimal toxicity. In contrast, carriers of siRNA such as Oligofectamine and Lipofectamine 2000 were significantly more toxic. CONCLUSIONS: Both the degree of complexity and the sequence specificity are important factors to be considered for developing the HK carrier of siRNA. In particular, we found that certain branched HK polymers (H3K8b, H3K8b(+RGD), and similar structural analogs) with eight terminal branches and a histidine-rich domain were effective carriers of siRNA.

Amino Acid Sequence↗

PubMed2005

Small interfering RNA targeting Raf-1 inhibits tumor growth in vitro and in vivo.

Raf-1 is a cytosolic serine-threonine kinase that plays an important role in tumor cell growth, proliferation, and apoptosis. Upregulated Raf-1 activity has also been implicated in tumor angiogenesis and metastasis. In this study, we used a promising new RNA interfering technology that targets Raf-1 mRNA both in vitro and in vivo. We initially found that Raf-1 siRNA markedly reduced Raf-1 mRNA in MDA-MB-435 cells in vitro by approximately 75% compared to control siRNA treatment groups. Raf-1 siRNA also reduced cell number by inducing apoptosis in a number of cell lines including HUVEC, MDA-MB-435, and C6 cells. After screening several histidine-lysine polymers in complex with Raf-1 siRNA to reduce tumor growth, we further evaluated the efficacy of this siRNA in complex with the optimal histidine-lysine carrier to reduce the tumor growth in vivo. MDA-MB-435 xenografts treated by intratumoral injections of Raf-1 siRNA were significantly reduced compared with the control groups. By the fourth measurement, tumor growth was reduced by nearly 60% in the Raf-1 siRNA treatment group compared with the untreated group (P < .02). Taken together, our data provide evidence that Raf-1 siRNA may be an effective strategy for reducing tumor growth.

Animals↗

PubMed2003

EAPII interacts with ETS1 and modulates its transcriptional function.

Ets proteins constitute a family of conserved sequence-specific DNA-binding proteins and function as transcription factors. ETS1 plays important roles in differentiation, lymphoid cell development, invasiveness and angiogenesis. Such diverse roles of ETS1 are likely to be dependent on its associated proteins. A yeast two-hybrid screen was conducted and here we describe a novel ETS1 interacting protein designated as ETS1-associated protein II (EAPII). EAPII protein interacts with ETS1 and other Ets proteins (ETS2 and FLI1) both in vitro and in vivo. Indirect immunofluorescence demonstrated that EAPII is predominately localized to the nucleus of mammalian cells. EAPII negatively modulates ETS1 transcriptional activity and attenuates synergistic transactivation by ETS1 and AP-1. Significantly, re-expression of EAPII inhibits the migration of epithelial cancer cells, but does not affect cell viability. Therefore, EAPII is a novel ETS1 modulator that regulates specific aspects of the ETS1 functions.

Base Sequence↗