Lipoplex-based targeted gene therapy for the suppression of tumours with VEGFR expression by producing anti-angiogenic molecules.

BACKGROUND: The anti-angiogenic fusion protein RBDV-IgG1 Fc (RBDV), which comprises the receptor-binding domain of vascular endothelial growth factor-A (VEGF-A), has shown antitumour effects by reducing angiogenesis in vivo. This study used the cationic lipoplex lipo-PEG-PEI-complex (LPPC) to simultaneously encapsulate both the RBDV targeting protein and the RBDV plasmid (pRBDV) without covalent bonds to assess VEGFR targeting gene therapy in mice with melanoma in vivo. RESULTS: LPPC protected the therapeutic transgene from degradation by DNase, and the LPPC/RBDV complexes could specifically target VEGFR-positive B16-F10 cells both in vitro and in vivo. With or without RBDV protein-targeting direction, the pRBDV-expressing RBDV proteins were expressed and reached a maximal concentration on the 7th day in the sera after transfection in vivo and significantly elicited growth suppression against B16-F10 melanoma but not IgG1 control proteins. In particular, LPPC/pRBDV/RBDV treatment with the targeting molecules dramatically inhibited B16-F10 tumour growth in vivo to provide better therapeutic efficacy than the treatments with gene therapy with IgG1 protein targeting or administration of a protein drug with RBDV. CONCLUSIONS: The simultaneous combination of the LPPC complex with pRBDV gene therapy and RBDV protein targeting might be a potential tool to conveniently administer targeted gene therapy for cancer therapy.

Lipo-PEG-PEI complex as an intracellular transporter for protein therapeutics.

BACKGROUND: Protein or peptide drugs are emerging therapeutics for treating human diseases. However, current protein drugs are typically limited to acting on extracellular/cell membrane components associated with the diseases, while intracellular delivery of recombinant proteins replaces or replenishes faulty/missing proteins and remains inadequate. In this study, we developed a convenient and efficient intracellular protein delivery vehicle. MATERIALS AND METHODS: A cationic liposomal polyethylenimine and polyethylene glycol complex (LPPC) was developed to noncovalently capture proteins for protein transfer into cells via endocytosis. ß-glucuronidase (ßG) was used in vitro and in vivo as a model enzyme to demonstrate the enzymatic activity of the intracellular transport of a protein. RESULTS: The endocytosed protein/LPPC complexes escaped from lysosomes, and the bound protein dissociated from LPPC in the cytosol. The enzymatic activity of ßG was well preserved after intracellular delivery in vitro and in vivo. CONCLUSION: Using LPPC as an intracellular protein transporter for protein therapeutics, we illustrated that LPPC may be an effective and convenient tool for studying diseases and developing therapeutics.

Specific drug delivery efficiently induced human breast tumor regression using a lipoplex by non-covalent association with anti-tumor antibodies.

BACKGROUND: A cationic liposome-PEG-PEI complex (LPPC) was employed as a carrier for achieving targeted delivery of drug to human epidermal growth factor receptor-2 (HER2/neu)-expressing breast cancer cells. LPPC can be easily loaded with an anti-tumor drug and non-covalently associated with an anti-tumor antibody such as Herceptin that is clinically used to rapidly form immunoparticles within 1 h. RESULTS: Drug-loaded LPPC have an average size about 250 nm and a zeta potential of about 40 mV. Herceptin was complexed onto surface of the LPPC to form the drug/LPPC/Herceptin complexes. The size of curcumin/LPPC/Herceptin complexes were 280 nm and the zeta potentials were about 23 mV. Targeting ability of this delivery system was demonstrated through specific binding on surface of cells and IVIS images in vivo, which showed specific binding in HER2-positive SKBR3 cells as compared to HER2-negative Hs578T cells. Only the drug/LPPC/Herceptin complexes displayed dramatically increased the cytotoxic activity in cancer cells. Both in vitro and in vivo results indicated that Herceptin adsorbed on LPPC directed the immunocomplex towards HER2/neu-positive cells but not HER2/neu-negative cells. The complexes with either component (curcumin or doxorubicin) used in the LPPC-delivery system provided a better therapeutic efficacy compared to the drug treatment alone and other treatment groups, including clinical dosages of Herceptin and LipoDox, in a xenografted model. CONCLUSIONS: LPPC displays important clinical implications by easily introducing a specific targeting characteristic to drugs utilized for breast cancer therapy.

Development of a computational promoter with highly efficient expression in tumors.

BACKGROUND: Gene therapy is a potent method to increase the therapeutic efficacy against cancer. However, a gene that is specifically expressed in the tumor area has not been identified. In addition, nonspecific expression of therapeutic genes in normal tissues may cause side effects that can harm the patients' health. Certain promoters have been reported to drive therapeutic gene expression specifically in cancer cells; however, low expression levels of the target gene are a problem for providing good therapeutic efficacy. Therefore, a specific and highly expressive promoter is needed for cancer gene therapy. METHODS: Bioinformatics approaches were utilized to analyze transcription factors (TFs) from high-throughput data. Reverse transcription polymerase chain reaction, western blotting and cell transfection were applied for the measurement of mRNA, protein expression and activity. C57BL/6JNarl mice were injected with pD5-hrGFP to evaluate the expression of TFs. RESULTS: We analyzed bioinformatics data and identified three TFs, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), cyclic AMP response element binding protein (CREB), and hypoxia-inducible factor-1α (HIF-1α), that are highly active in tumor cells. Here, we constructed a novel mini-promoter, D5, that is composed of the binding sites of the three TFs. The results show that the D5 promoter specifically drives therapeutic gene expression in tumor tissues and that the strength of the D5 promoter is directly proportional to tumor size. CONCLUSIONS: Our results show that bioinformatics may be a good tool for the selection of appropriate TFs and for the design of specific mini-promoters to improve cancer gene therapy.

A rapid and convenient method to enhance transgenic expression in target cells.

Gene therapy provides a novel strategy and a new hope for patients with cancer. Unfortunately, the specifics of the delivery systems or the promoters have not achieved the specified efficacy so far, and the perfection of either system will be extremely difficult. In this study, we introduce a simple concept that a combination of a partially specific delivery system and a partially specific promoter activity may achieve a more specific effect on transgenic expression in target cells. The first section describes tumor-related transcription factors that were assayed in tumors or rapidly proliferating cells to determine their activities. The activities of nuclear factor (NF)-κB, CREB, and HIF-1 were higher, and three copies of each response element were used to construct a transcription factor-based synthetic promoter (TSP). The results showed that the expression of the TSP was active and partially specific to cell types. As described in the second section, the multifunctional peptide RGD-4C-HA was designed to absorb polyethyleneimine (PEI) molecules, and this complex was targeted to integrin αvß3 on B16F10 cells. The results indicated that RGD-4C-HA could associate with PEI to mediate specific targeting in vitro. Finally, the combination of the PEI-peptide complex and TSP could enhance the specifically transgenic expression in B16F10 cells. This strategy has been proven to work in vitro and might potentially be used for specific gene therapy in vivo.

Inhibitory effects of chloroform extracts derived from Corbicula fluminea on the release of pro-inflammatory cytokines.

Corbicula fluminea, the primary freshwater bivalve cultivated in Taiwan, was formerly used as a remedy for hepatitis. Recent reports indicate that C. fluminea has many bioactivities, but it remains unknown whether C. fluminea affects inflammation. This study explored the anti-inflammatory activity of C. fluminea. C. fluminea was first treated with chloroform to obtain clam chloroform extracts (CCEs). On the basis of the assay for the release of pro-inflammatory cytokines in vitro and in vivo, the results show that the CCEs significantly lowered the release of lipopolysaccharide (LPS)-induced pro-inflammatory cytokines. Additionally, the CCEs reduced LPS-induced organ damage. Real-time polymerase chain reaction analysis suggested that CCEs inhibit the LPS-induced mRNA expression of interleukin-1ß and tumor necrosis factor-α. Western blot analysis indicated that the CCEs increased expression of IκB and attenuated the phosphorylation of IκB. Gas chromatography-mass spectrometry suggests that phytosterols and fatty acids are responsible for the anti-inflammatory properties of CCEs. Taken together, CCEs have the potential to be developed as an anti-inflammatory functional food.

Liposome-based polymer complex as a novel adjuvant: enhancement of specific antibody production and isotype switch.

The aim of vaccination is to induce appropriate immunity against pathogens. Antibody-mediated immunity is critical for protection against many virus diseases, although it is becoming more evident that coordinated, multifunctional immune responses lead to the most effective defense. Specific antibody (Ab) isotypes are more efficient at protecting against pathogen invasion in different locations in the body. For example, compared to other Ab isotypes, immunoglobulin (Ig) A provides more protection at mucosal areas. In this study, we developed a cationic lipopolymer (liposome-polyethylene glycol-polyethyleneimine complex [LPPC]) adjuvant that strongly adsorbs antigens or immunomodulators onto its surface to enhance or switch immune responses. The results demonstrate that LPPC enhances uptake ability, surface marker expression, proinflammatory cytokine release, and antigen presentation in mouse phagocytes. In contrast to Freund's adjuvant, LPPC preferentially activates Th1-immunity against antigens in vivo. With lipopolysaccharides or CpG oligodeoxynucleotides, LPPC dramatically enhances the IgA or IgG2A proportion of total Ig, even in hosts that have developed Th2 immunities and high IgG1 serum titers. Taken together, the results demonstrate that the LPPC adjuvant not only increases the immunogenicity of antigens but also modulates host immunity to produce an appropriate Ab isotype by combining with immunomodulators.

7,7''-Dimethoxyagastisflavone-induced apoptotic or autophagic cell death in different cancer cells.

7,7''-Dimethoxyagastisflavone (DMGF), a biflavonoid isolated from the needles of Taxus × media cv. Hicksii, was evaluated for its antiproliferative and antineoplastic effects in three human cancer cell lines. Interestingly, DMGF caused cell death via different pathways in different cancer cells. DMGF induced apoptosis, activated caspase-3 activity and changed the mitochondrial membrane potential in HT-29 human colon cancer cells. However, the apoptotic pathway is not the major pathway involved in DMGF-induced cell death in A549 human lung cancer cells and HepG2 human hepatoma cells. Treatment with 3-MA, an inhibitor of autophagy, significantly decreased DMGF-induced cell death in HepG2 and A549 cells, but did not affect DMGF-induced cell death in HT-29 cells. Following DMGF treatment, the HepG2 cells increased expression of LC3B-II, a marker used to monitor autophagy in cells. Thus, DMGF induced apoptotic cell death in HT-29 cells, triggered both apoptotic and autophagic death in A549 cells and induced autophagic cell death in HepG2 cells.

A Lipo-PEG-PEI complex for encapsulating curcumin that enhances its antitumor effects on curcumin-sensitive and curcumin-resistance cells.

A cationic liposome-PEG-PEI complex (LPPC) was used as a carrier for the encapsulation of hydrophobic curcumin to give curcumin/LPPC. Curcumin/LPPC had an average size less than 270 nm and a zeta potential of approximately 40 mV. The LPPC encapsulation efficiency for curcumin was about 45%. The authors found it surprising that the cytotoxic activity of the curcumin/LPPC was fivefold higher than curcumin when tested on curcumin-sensitive cells and 20-fold more active against curcumin-resistant cells. Curcumin/LPPC treatment caused a cell cycle arrest at G2/M phase, which rapidly resulted in apoptosis. The increased cytotoxic activity of curcumin/LPPC is likely attributable to its rapid accumulation in the cell. In vivo, administration of curcumin/LPPC inhibited about 60 - 90% of tumor growth in mice bearing CT-26 or B16F10 cells. These results demonstrate LPPC encapsulation technology is able to enhance the effects of antitumor drugs. Use of this technology may provide a new tool for cancer therapy, especially for drug-resistant cancer. From the Clinical Editor: This team of investigators used a cationic liposome-PEG-PEI complex (LPPC) to encapsulate curcumin. The different delivery method resulted in the five-fold increase of cytotoxic activity against curcumin-sensitive cells and twenty-fold against curcumin-resistant cells.

A fusion protein composed of receptor binding domain of vascular endothelial growth factor-A and constant region fragment of antibody: angiogenesis antagonistic activity.

Vascular endothelial growth factor (VEGF) promotes the growth of solid tumor mainly via VEGF receptor-1 and receptor-2, which are expressed preferentially in proliferating endothelial cells. Therefore, a strategy for simultaneous blockage of both VEGF receptors may have a useful therapeutic effect in tumor growth. In this study, we utilized a fusion protein which is composed of receptor binding domain of VEGF-A (RBDV) and the constant region fragment (Fc) of a human immunoglobulin G1 (IgG1), to interfere with the growth of human umbilical vein endothelial cells (HUVECs) via VEGF receptors. The results showed that RBDV-IgG1 Fc was able to bind with both VEGF receptor-1 and receptor-2. In addition, RBDV-IgG1 Fc could decrease VEGF-induced proliferation and tube formation among HUVECs. Moreover, the cytotoxic test showed RBDV-IgG1 Fc could also enhance the cytotoxic activity of human natural killing cells. The data are suggesting that the fusion protein, RBDV-IgG1 Fc, may have potential as an angiogenesis antagonist for future tumor therapy.

A unique and potent protein binding nature of liposome containing polyethylenimine and polyethylene glycol: a nondisplaceable property.

Most of the currently available targeting vectors are produced via the linkage of targeting molecules. However, the coupling process is complicated, and the covalent linkage may attenuate the activity of certain targeting molecules. In this study, we have developed a cationic liposome complexed with polyethylenimine and polyethylene glycol polymers (LPPC) that can capture various proteins without covalent conjugation. Characterizations of prepared LPPC revealed that the maximal-binding capacity was about 170 µg of bovine serum albumin to 40 µg of sphere-shaped LPPC (180 nm). The proteins were essentially located at or near the surface when analyzed by atomic force or transmission electron microscopy. We demonstrate that polyethylenimine was an essential component to bind the proteins. Upon the saturation of captured proteins, a given protein could not be displaced by other additional proteins and still retained its biological activity. Using a variety of functional proteins, we show some typical examples of the utility of incorporated beta-glucuronidase and antibodies onto the LPPC. The beta-glucuronidase can be used for the study of antigen-antibody interactions, whereas in studies with the antibody complex, we used anti-CD3 as an agonist to stimulate the proliferation of peripheral blood mononuclear cells via a receptor-mediated mechanism and anti-VEGFR for cell staining. In conclusion, the prepared LPPC can provide a platform to capture biologically and biochemically functional proteins on its surface for various applications, such as cell signaling, cell profiling, noncovalent enzyme-linked immunoassays, and others not mentioned.

A fusion protein with the receptor-binding domain of vascular endothelial growth factor-A (VEGF-A) is an antagonist of angiogenesis in cancer treatment: Simultaneous blocking of VEGF receptor-1 and 2.

Vascular endothelial growth factor (VEGF) is an angiogenic factor that signals through VEGFR-1 and VEGFR-2, which are expressed preferentially in proliferating endothelial cells. Thus, simultaneous blockage of both VEGF receptors may provide a more efficient therapeutic response in cancer treatment. We created a recombinant fusion protein (RBDV-IgG1 Fc), which is composed of the receptor binding domain of human VEGF-A (residues 8-109) and the Fc region of human IgG1 immunoglobulin. The recombinant protein can bind to both mouse VEGFR-1 and VEGFR-2 to decrease VEGF-induced proliferation and tube formation of endothelial cells in vitro. In this study, the RBDV-IgG1 Fc fusion protein reduced the effects of proliferation, migration and tube formation induced by VEGF in murine endothelial cells in vitro. In vivo tumor therapy with RBDV-IgG1 Fc resulted in tumor inhibition by reducing angiogenesis. Pathological evidence also shows that RBDV-IgG1 Fc can seriously damage vessels, causing the death of tumor cells. These findings suggest that this chimeric protein has potential as an angiogenesis antagonist in tumor therapy.

A new microsphere-based immunoassay for measuring the activity of transcription factors.

There are several traditional and well-developed methods for analyzing the activity of transcription factors, such as EMSA, enzyme-linked immunosorbent assay, and reporter gene activity assays. All of these methods have their own distinct disadvantages, but none can analyze the changes in transcription factors in the few cells that are cultured in the wells of 96-well titer plates. Thus, a new microsphere-based immunoassay to measure the activity of transcription factors (MIA-TF) was developed. In MIA-TF, NeutrAvidin-labeled microspheres were used as the solid phase to capture biotin-labeled double-strand DNA fragments which contain certain transcription factor binding elements. The activity of transcription factors was detected by immunoassay using a transcription factor-specific antibody to monitor the binding with the DNA probe. Next, analysis was performed by flow cytometry. The targets hypoxia-inducible factor-1α (HIF-1α) and nuclear factor-kappa B (NF-κB) were applied and detected in this MIA-TF method; the results that we obtained demonstrated that this method could be used to monitor the changes of NF-κB or HIF within 50 or 100 ng of nuclear extract. Furthermore, MIA-TF could detect the changes in NF-κB or HIF in cells that were cultured in wells of a 96-well plate without purification of the nuclear protein, an important consideration for applying this method to high-throughput assays in the future. The development of MIA-TF would support further progress in clinical analysis and drug screening systems. Overall, MIA-TF is a method with high potential to detect the activity of transcription factors.