| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2005: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2004: ¥2,500,000 (Direct Cost: ¥2,500,000)
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| Research Abstract |
First, we have finished establishing the growth-inhibition system by our novel transporter-mediated intracellularly delivering of the p16 functional peptide for highly aggressive lymphomas, which was the project supported by the grant-in-aid from 2002-2004. Its achievement was published in the journal "Molecular Cancer Therapeutics", 3(12),2004. As a next step, we examined the range of molecular weight of proteins as a cargo for this highly efficient protein/peptide transporter. It was finally revealed that the transporter could intracellularly target various kinds of proteins with 2kDa to 500kDa in molecular weight efficiently. Then, we tried thetransporter-mediated delivery of monoclonal antibodies into various kinds of human tumor cells using the anti-actin mAb as an representative example, so as to check if the antibody was well incorporated into these cells and if it showed specific intracellular localization in response to the actin filament as a target-antigen. In the case of th
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e anti-actin mAb, it was incorporated quite efficiently into these cells such as hepatocellular carcinoma cells, osteosarcoma cells, and melanoma cells in addition to lymphoma cells. It was also revealed that the fluorescein-labeled antibody showed the specific intracellular localization of the actin filamentous pattern after 24 hours of antibody introduction. However, other monoclonal antibodies, especially those recognizing nuclear antigens such as p53, PCNA, and c-myc proteins were not able to show the specific nuclear localization even those could be well incorporated into the cells. It was considered that this might be caused by the form of immunoglobulin which comprise the antibody, we tried several kinds of immunoglobulins of an anti-nuclear protein mAbs, including whole form, F(ab)2, and also IgG1, IgG2a, and IgG2b. The result was the same, whcich all of these showed endosomal incorporation at the cytoplasm of the tumor cells, so this difference was considered not to be critical for intracellular localization of delivered-antibody. At this step, it was considered that the antibody delivery system via our novel transporter needs more improvement, so we are continuing the research to solve this problem.
Taking together these experimental results, we moved to the another research that the establishing double molecular targeting sytem using the transporter. We choose human glioblastoma cells as a model for this study, because glioblastomas are well known as a intractable human malignanciy even under the present therapeutic situations. These neoplastics call often lack the endogenous expression of tumor suppressor genes such as p14,p16 encoded by CDKN2A locus. Therefore, we tried to inhibit the growth of the tumor cells by efficiently restoring the lost function of these tumor suppressor gene using our transporter-mediated double peptide delivery of the p14 and p16 functional peptides. The result showed drastic growth inhibition on U87deltaEGFR human glioblastoma cells, upto 97% growth inhibition. Moreover, in vivo double delivery of these peptide via the transporter induced significant growth retardation of the gliomas implanted in the nude mice brains, which suggests this double targeting system using the transporter-fuctional peptide complex is quite effective against in vivo tumors, and is considered that this reseach is deserve to be developed further for the development of a novel therapeutic approach. Less
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