| Project/Area Number |
22KF0337
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| Project/Area Number (Other) |
22F21773 (2022)
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| Research Category |
Grant-in-Aid for JSPS Fellows
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| Allocation Type | Multi-year Fund (2023)
Single-year Grants (2022) |
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| Section | 外国 |
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| Review Section |
Basic Section 54010:Hematology and medical oncology-related
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| Research Institution | Juntendo University |
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Principal Investigator |
ハイジッヒ ベアーテ 順天堂大学, 大学院医学研究科, 特任准教授 (30372931)
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| Co-Investigator(Kenkyū-buntansha) |
YATSENKO TETIANA 順天堂大学, 大学院医学研究科, 外国人特別研究員
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| Project Period (FY) |
2023-03-08 – 2024-03-31
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| Project Status |
Granted (Fiscal Year 2023)
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| Budget Amount *help |
¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 2023: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2022: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Keywords | fibrinolysis / protein fragment / plasmin / multiple myeloma |
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| Outline of Research at the Start |
Multiple myeloma (MM) requires neovascularization. Accumulating evidence suggests that the truncated tPA derivatives kringle 1-2 and kringle 2-protease domain have show antiangiogenic effect. Here, we will examine the role of kringle 2 of tPA during MM progression and it effects as an anti-angiogenic drug. In addition, we will test its anti-proliferative effects on myeloma cell growth and establish its effects on the coagulation system through its potential interactions with platelets.
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| Outline of Annual Research Achievements |
This study aimed to find an efficient method to generate tissue-type plasminogen activator (tPA) fragments, which naturally occur during tumor progression/inflammation of the hematologic cancer called multiple myeloma. To determine whether it will occur. In the first year of this proposal, we used different approaches to generate tPA fragments. Using a plasmid-based expression system in bacteria, we evaluated multiple proteases, including alpha elastase and plasmin, to cleave the purchased protein. We have successfully expressed the fragment in bacteria and are currently evaluating various methods for large-scale isolation. In one of his original articles published this year, we identified p53 as a downstream target of the small molecule and plasmin inhibitor YO2 (Cancers 15, 288; doi 10.3390/cancers15010288, 2023). A published review article summarizes the knowledge of the plasminogen-plasmin system in the oral cavity (Cells, 12(3), 445 https://doi.org/10.3390/cells12030445, 2023)."
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
We have initially used recombinant tPA and performed time course studies to identify proteases that will generate the tPA fragments. Unfortunately, non of the tested proteases was able to generate sufficient hydrolysis of the input full-length protein. Therefore, we shifted the strategy to express the tPA fragment in a plasmid. The generation of the fragment in bacteria turned out to be more difficult than expect, but we identified a strain in which the protein could be expressed. We are currently using various purification methods to obtain the protein in a tissue culture pure form.
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| Strategy for Future Research Activity |
We are planning in the following year to continue our studies on the role of the tPA fragment in tumor progression* 1. Purification of the tPA fragment
We already have expressed the protein in an expression vector and obtained supernatants containing the protein fragments. 2. Identification of the natural occurrence of the tPA fragment(s) in selected hematological and solid cancers using Western blotting in cell lines in vitro and murine disease models in vivo. 3. Identification of fragment properties during cancer progression with focus on cell proliferation and migration. A solid cancer and the hematological disease multiple myeloma will be targeted. An orthotopic murine model for the solid cancer has been established in the lab. Furthermore, the multiple myeloma model as described in Salama et al Blood advances 2020) will be used in the current study. 4. Evaluation of the anti-angiogenic properties of the newly synthesized fragment。
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