細胞株パネル解析

細胞株パネルを用いた化合物の作用機作の評価
Evaluation of modes of action of compounds using cell line panel

(担当:旦 慎吾)
(Member in charge: Shingo Dan)

【概要】
Summary

本系は、機能未知の生理活性物質のうち細胞増殖阻害活性を有するものの作用機作推定に有用な、実績のある評価系である。さまざまな臓器がん由来の細胞株(肺がん7系、胃がん6系、大腸がん5系、卵巣がん5系、脳腫瘍6系、乳がん5系、腎がん2系、前立腺がん2系およびメラノーマ1系)からなるJFCR39パネルに対して、被験化合物が細胞増殖を50%抑制する濃度(GI50)を各々測定し、その有効濃度の違いをフィンガープリントとして表す。これまでの経験上、作用メカニズムが既知の種々の生理活性物質のフィンガープリントを測定し解析した結果、作用機作が共通な薬剤は互いに類似したフィンガープリントを示すことを明らかにしている。本系では、この性質を利用して、機能未知の化合物のフィンガープリントを測定し、およそ250種類のレファレンス化合物のフィンガープリントとの類似性を検討することで、被験化合物の作用機作の推定を行うことが可能である。また、本系はセル(細胞)ベースのアッセイであり、セルフリーのスクリーニング系で見出された新規分子標的薬の標的特異性評価(オンターゲット・オフターゲット)にも有用である。

This evaluation system has long been used for estimating the modes of action of compounds that cause cell growth inhibition. Using JFCR39, a panel of 39 cancer cell lines derived from various tissues of origin (7 from lung cancers, 6 from gastric cancers, 5 from colon cancers, 5 from ovarian cancers, 6 from central nervous system (CNS) cancers, 5 from breast cancers, 2 from renal cancers, 2 from prostate cancers and 1 from melanoma), the concentration at which the test compound causes half growth inhibition (GI50) in each of the cell lines is measured, and the fingerprint, or the spectrum of GI50 concentrations across the JFCR39 panel, is used to predict its mode of action. It has been clarified that compounds with a common mechanism of action show fingerprints similar to each other. In this system, prediction of the mode of action of a compound is accomplished by comparing its fingerprint with those of 250 reference compounds including anticancer drugs and other physiologically active substances with known modes of action. Since this system is a cell-based assay, it is also useful to evaluate the target specificity of a novel molecule-targeted drug found in a cell-free screening system.

【方法】
Method

JFCR39パネルの各細胞株を96ウェルプレートにまき込み、翌日被験化合物の希釈系列(通常、最終濃度10-8~10-4mol/L)を添加する。2日間培養後、細胞増殖をスルホローダミンBによる比色定量で測定する。各細胞株の用量反応曲線から細胞増殖を50%に抑制する濃度を算出し、39系の平均薬剤有効濃度に対する個々の細胞株の有効濃度偏差を求め、フィンガープリントとして表示する。

Each cell line of the JFCR 39 panel is plated in a 96-well plate, and the dilution series of the test compound (usually at a final concentration of 10-8 to 10-4 mol / L) is added the following day. After 2 days of culture, cell proliferation is estimated colorimetrically by measuring A525 in each well stained with sulforhodamine B. The concentration of the compound that inhibit cell proliferation to 50% (GI50) is calculated from the dose response curve, and the deviation of log GI50 in each cell line to the average log GI50 across the JFCR39 is determined and displayed as a fingerprint.

【化合物の評価】
Evaluation of compounds

JFCR39パネルの細胞株のうち、被験化合物が最も効きやすい細胞株と効きにくい細胞株のlog GI50の差(Range)、および、log GI50の平均値と最も効きやすい細胞株のlog GI50の差(delta)を計算し、それぞれ1以上、0.5以上の場合、薬効の差が大きい(Differential Activity/DA (+))と判定する。また、測定したフィンガープリントを、およそ250種類の標準物質のフィンガープリントと比較することにより、作用機作の推定あるいは作用機作の新規性の評価を行う。

To evaluate the differential activity of a compound across the JFCR39 panel, we calculated the difference between log GI50 in the most sensitive cell line and the least sensitive cell line (Range) and the difference between log GI50 in the most sensitive cell line and the average GI50 across the JFCR39 (Delta). The criteria that the test compound has the differential activity, or DA(+), is “Range≧1 and Delta≧0.5”. To predict the mode of biological action of a test compound, its fingerprint is subjected to “COMPARE analysis”, or comparing it with those of approximately 250 reference compounds in the database. If it is similar to a certain reference compound, its mode of action is supposed to be similar to the reference compound. If it does not exhibit similarity to any of the reference compounds, the test compound is supposed to have a unique mechanism of action.

【欲しい情報】
Desired information (if available)

細胞増殖阻害濃度およびそのときの薬剤接触時間

The concentration of the compounds and time of exposure required for cell growth inhibition.

【参考文献】
References

  1. Yamori T. Panel of human cancer cell lines provides valuable database for drug discovery and bioinformatics. Cancer Chemother Pharmacol 2003;52 Suppl 1:S74-9. https://https://doi.org/10.1007/s00280-003-0649-1
  2. Dan S, Tsunoda T, Kitahara O, et al. An integrated database of chemosensitivity to 55 anticancer drugs and gene expression profiles of 39 human cancer cell lines. Cancer Res 2002;62:1139-47.
  3. Shoemaker RH. The NCI60 human tumour cell line anticancer drug screen. Nat Rev Cancer 2006;6:813-23.  https://doi.org/10.1038/nrc1951
  4. Paull KD, Shoemaker RH, Hodes L, et al. Display and analysis of patterns of differential activity of drugs against human tumor cell lines: development of mean graph and COMPARE algorithm. J Natl Cancer Inst 1989;81:1088-92.  https://doi.org/10.1093/jnci/81.14.1088
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