2011 Fiscal Year Final Research Report
Development of a new hypoxia targeting chrono-gene therapy in cancer.
| Project/Area Number |
22790158
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| Research Category |
Grant-in-Aid for Young Scientists (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Medical pharmacy
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| Research Institution | Kyushu University |
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Principal Investigator |
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| Project Period (FY) |
2010 – 2011
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| Keywords | c, 医療薬剤学 |
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| Research Abstract |
Tissue specific gene expression occurs throughout specific transcriptional factor and corresponding response element. CMV promoter is one promoter of type II RNA polymerase which endogenously transcript mRNA tissue-specifically. Then, CMV promoter was selected as a basal promoter. In this study, hypoxia response HRE-CMV promoter was constructed and HRE-CMV driven anti-tumor plasmid vectors were evaluated its anti-tumor effect in tumor bearing mice.
In the first, HRE-CMV promoter was constructed and evaluated the response under hypoxic condition. HRE-CMV promoter contained four HREs derived from glycolytic related genes. The mRNA expression of glycolytic related genes were promoted in 1% 02 condition regardless cell types. HRE-CMV promoter had a high transcriptional activity hypoxic conditional-dependently throughout inserting HRE oligonucleotide. HRE-CMV promoter constructed in this study could express downstream genes in wide range of cell types.
In the second, HRE-CMV promoter driven N
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K4 expression vector was constructed and evaluated its anti-tumor effect in U2sl bearing mice. HRE-CMV-NK4 vector suppressed tumor growth strongly compared to CMV-NK4 vector in U2sl bearing mice. HRE-CMV-NK4 vector suppressed HGF/c-Met signaling strongly for a long-term compared to CMV-NK4 vector, which was correlated with NK4 protein expression. The high expression of NK4 from HRE-CMV promoter was also confirmed in cultured cells.
In the third, HRE-CMV promoter driven bcl-2 shRNA expression vector was constructed and evaluated its anti-tumor effect in Colon-26 bearing mice. HRE-CMV shbcl-2 vector induced apoptosis throughout bcl-2 mRNA knockdown and suppressed tumor growth strongly compared to CMV-shbcl-2 vector in Colon-26 bearing mice. HRE-CMV promoter expressed shRNA efficiently compared to CMV promoter in CoCl2treated Colon-26 cells. Moreover, the shRNA expression activity from HRE-CMV promoter was comparable levels compared to CMV promoter in non-CoCl2-treated Colon-26 cells. These results suggest that inserting HRE oligonucleotide activates shRNA expression in hypoxic tumor and affect little gene expression under normoxic condition.
In conclusion, the constructed HRE-CMV promoter could apply to express several anti-tumor genes downstream of HRE-CMV promoter because almost plasmid vectors enable to insert thousands bases pairs of optional genes. HIF-l/HRE transcriptional factor is correlated with tumor grade and participates in tumor growth and aggravation in 70% of human cancers, especially solid tumor. Therefore, HRE-CMV promoter could widely apply to the treatment for many cancers. In gene therapy, the enhancement of tissue specific gene expression could not only obtain high therapeutic results but also avoid adverse effects. These results may contribute to investment of more efficiency and safety for gene therapy. Less
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