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Cancer-cell-specific cytostatic agent

a cytostatic agent and cancer-cell technology, applied in the field of compounds, can solve the problem that compounds cannot be expected to be useful anticancer agents, and achieve the effect of suppressing the expression of recq1 gen

Inactive Publication Date: 2013-05-23
GENECARE RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides a new type of anticancer agent that targets the RecQ1 gene expression in cancer cells. These siRNA molecules have the effect of suppressing cancer cell proliferation while not affecting normal cells. The invention also includes a double-stranded RNA, a DNA vector, and a cancer cell-specific cell proliferation inhibitor that can all be used to target the RecQ1 gene expression. These new agents offer a more effective and safe way to treat cancer.

Problems solved by technology

However, even if a compound can suppress the proliferation of cancer cells, if it has similar proliferation-suppressing effects on normal cells, that compound cannot be expected to be a useful anticancer agent.

Method used

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Examples

Experimental program
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Effect test

example 1

Cell Cultures

[0090]HeLa cells (human cervical cancer cells) were used as human cancer cells, and TIG3 cells (normal diploid fibroblast cells) were used as normal human cells. HeLa cells and TIG3 cells were cultured at 37° C. under 5% CO2 using Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 50 μg / mL gentamicin.

example 2

siRNA Design

[0091]Thirty-two siRNAs against RecQ1 gene were designed according to the method of Elbasher et al. (Elbasher, M. S. et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494-498 (2001)) and the method of Reynolds et al. (Reynolds A. et al., Rational siRNA design for RNA interference. Nat. Biotechnol. 3, 326-30 (2004)). FIG. 1 shows each of the siRNA sequences. The siRNAs were synthesized at Qiagen.

example 3

Cancer Cell-Specific Cell Proliferation-Suppressing Effects Due to the Suppression of RecQ1 Gene Expression

[0092](1) Suppression of RecQ1 Gene Expression by siRNAs

[0093]Cells were plated onto 24-well plates at a density of 0.8-1.5×104 cells / well 24 hours before transfection, and siRNAs were transfected under the condition of 20-50% confluency. 10 pmol of siRNA was transfected per well using Oligofectamine (Invitrogen) or Lipofectamine 2000 (Invitrogen) following the manufacturer's protocol. Expression of the RecQ1 gene mRNA 24 hours after introduction of siRNA was quantified using Taqman PCR. Specifically, total RNA was extracted from cells at 24 hours after siRNA transfection using an RNeasy Mini Kit (Qiagen). ABI PRISM 7000 Sequence Detection System (Applied Biosystems) was used for quantitative PCR. RT-PCR primers for the RecQ1 gene and β-actin gene, and TaqMan probes were purchased from Applied Biosystems. RT-PCR reactions were performed using a QuantiTect Probe RT-PCR Kit (Qiag...

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Abstract

The present inventors discovered that although suppressing expression of the RecQ1 gene, a RecQ helicase family gene, shows suppressive effects on cell proliferation in cancer cells, such effects are not observed in human TIG3 cells (a normal diploid fibroblast cell line), which are normal cells. Hence, the present inventors discovered that siRNAs against RecQ1 gene have cancer cell-specific cell proliferation-suppressing effects that are mediated by suppression of the expression of said gene.

Description

TECHNICAL FIELD[0001]The present invention relates to compounds that suppress expression of RecQ1 genes, and particularly relates to cell proliferation inhibitors comprising siRNAs that exhibit the effect of suppressing expression of these genes.BACKGROUND ART[0002]Genes belonging to the RecQ DNA helicase family are widely present in organisms ranging from prokaryotes such as Escherichia coli (E. coli) to higher eukaryotes including humans. Conserved in the evolution process, these genes diversified along with the multicellularization of organisms. The E. coli RecQ gene was the first of the RecQ family genes to be discovered. This gene was identified as a gene participating in zygotic recombination and in the RecF pathway for UV damage repair (see Non-Patent Document 1). The E. coli RecQ gene has been revealed to have the function of suppressing incorrect recombinations (see Non-Patent Document 2). The budding yeast SGS1 gene and the fission yeast Rqh1 gene are the only known RecQ h...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/113
CPCC12N2310/14C12N15/1137A61P35/00A61P35/02A61P43/00C12N15/09A61K31/7088C12N15/11
Inventor TAKAGI, MOTOKISHIMAMOTO, AKIRAFURUICHI, YASUHIROSATO, AYUMI
Owner GENECARE RES INST
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