ABSTRACT
Ultrasound (US) is an effective tool for local delivery of genes into target tumors or organs. In combination with microbubbles, US can temporarily change the permeability of cell membranes by cavitation and facilitate entry of plasmid DNA into cells. Here, we demonstrate that repeated US-mediated delivery of anti-angiogenic genes, endostatin or calreticulin, into muscle significantly inhibits the growth of orthotopic tumors in the liver, brain or lung. US-mediated anti-angiogenic gene therapy also seems to function as an adjuvant therapy that significantly enhances the antitumor effects of the chemotherapeutic drug doxorubicin and adenovirus-mediated cytokine gene therapy. Significantly higher levels of tumor apoptosis or tumor-infiltrating lymphocytes were observed after combined therapy consisting of either anti-angiogenic therapy and chemotherapy, or anti-angiogenic therapy and immunotherapy. Taken together, our experiments demonstrate that intramuscular delivery of anti-angiogenic genes by US exposure can effectively treat distant orthotopic tumors, and thus has great therapeutic potential in terms of clinical treatment.
Subject(s)
Calreticulin/genetics , Endostatins/genetics , Gene Transfer Techniques , Neoplasms/blood supply , Neoplasms/therapy , Ultrasonics/methods , Amino Acid Sequence , Animals , Antibiotics, Antineoplastic/pharmacology , Calreticulin/biosynthesis , Cell Line, Tumor , Combined Modality Therapy , Doxorubicin/pharmacology , Endostatins/biosynthesis , Genetic Therapy , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Molecular Sequence Data , Neoplasms/genetics , Neovascularization, Pathologic/genetics , Neovascularization, Pathologic/therapy , Random Allocation , Rats , Rats, Inbred F344 , Sonication/methodsABSTRACT
Radiosensitization of cancer cells to irradiation could improve the efficacy of radiotherapy. The early transcriptional factor (Egr-1) promoter induced expression of downstream genes after irradiation. TNF-related apoptosis-inducing ligand (TRAIL) is known to induce apoptosis in malignant cells, but displayed little or no toxicity on normal cells. In this study, we constructed pcDNA3.1-Egr-1-TRAIL (pEgr.1-TRAIL) recombinant plasmid and evaluated its effect on human colon cancer cell line SW480. pEgr.1-TRAIL transfection combined with radiotherapy caused dramatically elevation of TRAIL expression both in mRNA and protein levels, much lower radiobiological parameters in clonogenic assays, accompanied by remarkably increase in apoptosis ratio. Furthermore, pEgr.1-TRAIL transfected cells displayed higher proportion in G0/G1 phase. Our results suggested that pEgr.1-TRAIL can sensitize SW480 cells to radiation, and the radiosensitization is related to cell cycle changes and apoptosis mediated by up-regulation of TRAIL expression. These findings support the potential future application of genetic radiotherapy against carcinoma.
Subject(s)
Colonic Neoplasms/genetics , Early Growth Response Protein 1/genetics , Genetic Therapy/methods , Radiation Tolerance/genetics , TNF-Related Apoptosis-Inducing Ligand/genetics , Apoptosis/genetics , Blotting, Western , Cell Line, Tumor , Colonic Neoplasms/metabolism , Early Growth Response Protein 1/metabolism , Gene Expression Regulation, Neoplastic , Humans , Plasmids , Promoter Regions, Genetic , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , TNF-Related Apoptosis-Inducing Ligand/metabolism , Transfection , Up-RegulationABSTRACT
TRAIL induces apoptosis in a variety of tumorigenic and transformed cell lines, but not in many normal cells. Recent studies have demonstrated that death receptor 5 (DR5), one of the two death receptors bound by TRAIL, showed expression in most malignantly transformed cells. This study evaluated effects of a monoclonal antibody (TRA-8) to human death receptor 5, combined with ionizing radiation, on radioresistant human larynx squamous carcinoma cell line (Hep-2R). Cells were treated with TRA-8 alone or in combination with radiation, cell viability inhibition was measured by MTT assay, and the induction of apoptosis was determined by Annexin V staining. Radionsensitivity of Hep-2R cells treated with TRA-8 were investigated with long-term clonogenic assays. Regulation of DR5 expression in cells after radiation was analyzed by indirect immunofluorescence using murine TRA-8 in combination with flow cytometry. The results suggested that TRA-8 enhanced radionsensitivity of Hep-2R cells, and that TRA-8 regulated Hep-2R cell cycle arrest at G2/M phase. Irradiation up-regulated the expression of DR5, and when combined with TRA-8 yielded optimal survival benefit. Therefore, TRA-8 can be used in combination with irradiation in radioresistant human larynx squamous carcinoma cells. Monoclonal antibodies such as TRA-8 may play an important role in the development of an effective treatment strategy for patients with radioresistant cancers.
Subject(s)
Antibodies, Monoclonal/pharmacology , Carcinoma, Squamous Cell/therapy , Laryngeal Neoplasms/therapy , Radiation-Sensitizing Agents/pharmacology , Receptors, TNF-Related Apoptosis-Inducing Ligand/antagonists & inhibitors , Apoptosis/drug effects , Apoptosis/radiation effects , Cell Cycle/drug effects , Cell Cycle/radiation effects , Cell Line, Tumor , Combined Modality Therapy , Flow Cytometry , Fluorescent Antibody Technique , Humans , Radiation Tolerance , Radiotherapy , Receptors, TNF-Related Apoptosis-Inducing Ligand/immunologyABSTRACT
Selectively substituted hydantoins 1 (15 examples), 4-hydroxy-2-imidazolidinones 2 (13 examples), 2-imidazolones 3 (10 examples), 2-imidazolidinones 4 (four examples), vicinal diamines 5 (two examples), and simple amino acid derivatives 6 (four examples) have been prepared and evaluated in the maximal electroshock seizure (MES), subcutaneous pentylenetetrazole seizure threshold (sc Met), and rotorod (Tox) tests. The medium effective doses (ED50) and the medium toxic dose (TD50) for the most active compounds are reported. In general, the most pronounced activity was observed for hydantoins 1 and protected amino acids 6. Within each series of compounds, enhanced anticonvulsant activity was often noted for compounds containing an aromatic group one carbon removed from a nitrogen atom. Among the most active compounds observed were the amino acid derivative N-acetyl-D,L-alanine benzylamide (6d) and the two 2-imidazolones 4-methyl-1-(phenylmethyl)-1,3-dihydro-2H-imidazol-2-one (3e) and 1-phenyl-1,3-dihydro-2H-imidazol-2-one (3g). Compound 6d proved to be slightly more potent in the MES test than phenacemide.
