| Budget Amount *help |
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 2005: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2004: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Research Abstract |
We have constructed short hairpin RNA (shRNA)-expressing HIV lentiviral vectors specific to the mutated BRAF^<V600E>, which was found in about half of human melanoma cases. Melanoma cell lines with the BRAF^<V600E> mutation were infected with the shRNA vectors, and were evaluated for the effects of BRAF RNAi on the MAPK activity, cellular proliferation, and invasive ability. BRAF^<V600E> -specific RNAi resulted in the significant inhibition of cellular proliferation both in vitro and in vivo, which was accompanied with the reduced activity of MAPK activity in the melanoma cells with BRAF^<V600E>, but not in the melanoma cells without it. Furthermore, BRAF^<V600E> -specific RNAi also resulted in the suppression of matrigel invasive ability, which was associated with the decrease of MMP-2 activity and integrin β1 expression level in the melanoma cells with BRAF^<V600E>. Collectively, our results suggest that the mutated BRAF^<V600E> is closely related to the malignant phenotype of melano
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mas, and is expected to be a promising candidate molecular target of human melanoma therapy.
On the other hand, Skp2 acts as a substrate-specific recognition subunit of an SCF ubiquitin ligase for a cdk inhibitor, p27^<Kip1>, and induces the proteasome-dependent degradation of p27^<Kip1>. In many human cancers, over-expression of Skp2, which is associated with the inverse decrease of p27^<Kip1>, suggesting the causal relationship of the over-expressed Skp2 and the decrease of p27^<Kip1>. We induced Skp2-specific RNAi in human small cell lung carcinoma cell (SCLC) line with the over-expressed Skp2, and found that the inhibition of Skp2 protein leaded to the decrease of cellular proliferation, which was associated with the increase of cdk inhibitors, p27^<Kip1> and p21 with G1 arrest. We also evaluated in vivo tumor model with NOD/SCID mice. The NOD/SCID mice implanted with the human SCLC cells with the over-expressed Skp2, were treated by intra-tumoral injection of an adenovirus vector expressing shRNA for Skp2 after the tumor formation, and the significant inhibition of tumor growth was observed following the Skp2 RNAi treatment.
Furthermore, the melanoma cells with both the mutated BRAF^<V600E> and over-expressed Skp2, were infected with an shRNA lentiviral vector expressing shRNAs for either BRAF^<V600E> or Skp2, and were evaluated for the cellular proliferation and invasive ability. The simultaneous suppression of both BRAF^<V600E> and Skp2 resulted in the significant suppression of in vitro cellular growth compared to the cells with single inhibition of either BRAF^<V600E> or Skp2, which was accompanied with more increase of p27^<Kip1> protein. Our results suggest the usefulness of the simultaneous suppression of both BRAF^<V600E> and Skp2 in melanoma cells with the abnormalities of both genes. Less
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