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A rapid fluorescent screening method for cellular sensitivity to anti-cancer compound.

ABSTRACT: The tetrazolium salts (MTT, XTT, MTS, WST) based colorimetric assay or resazurin based fluorimetric assay are currently typical methods for cell sensitivity determination to anticancer compounds. We presented here a new rapid method for this purpose. This method uses a fluorescent dye named DCFH-DA which is previously taken as a intracellular probe for measurement of H(2)O(2) levels within a cell. The application basis for this method lies in two facts: the membrane permeable feature of the final metabolite of DCFH-DA inside a cell, and the linearity relationship between cell number and H(2)O(2) level. The results showed that there was a perfect association between cell number and fluorescent intensity determined by the DCFH-DA method, no matter whether using resuspended or adherent cells, and further 50% concentration of inhibition (IC(50)) comparison between data obtained by DCFH-DA method or MTT method using a positive known anticancer compound Baicalin showed that there were no significant differences in cellular sensitivity determination to compound Baicalin though there existed a relatively higher coefficient of variation of IC(50) by the DCFH-DA method than that by the MTT method. Thus our data indicate that DCFH-DA might not only be a fine reagent for determination of H(2)O(2) levels in cells but also an ideal fluorescent dye for cellular sensitivity test of anti-cancer compounds, and may be suitable for primary high-throughput drugs screening.

SUBMITTER: Mao W

PROVIDER: S-EPMC3397105 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature

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A rapid fluorescent screening method for cellular sensitivity to anti-cancer compound.

Mao Wenwei W Chen Xinlin X Yang Tian T Yin Yu Y Ge Mei M Luo Minyu M Chen Daijie D Qian Xiuping X

Cytotechnology 20120404 4

The tetrazolium salts (MTT, XTT, MTS, WST) based colorimetric assay or resazurin based fluorimetric assay are currently typical methods for cell sensitivity determination to anticancer compounds. We presented here a new rapid method for this purpose. This method uses a fluorescent dye named DCFH-DA which is previously taken as a intracellular probe for measurement of H(2)O(2) levels within a cell. The application basis for this method lies in two facts: the membrane permeable feature of the fina ...[more]

PMID: 22476562

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Project description:BACKGROUND: Chemosensitivity and tumor metastasis are two primary issues in cancer management. Cancer cells often exhibit a wide range of sensitivity to anti-cancer compounds. To gain insight on the genetic mechanism of drug sensitivity, one powerful approach is to employ the panel of 60 human cancer cell lines developed by the National Cancer Institute (NCI). Cancer cells also show a broad range of invasion ability. However, a genome-wide portrait on the contributing molecular factors to invasion heterogeneity is lacking. METHODS: Our lab performed an invasion assay on the NCI-60 panel. We identified invasion-associated (IA) genes by correlating our invasion profiling data with the Affymetrix gene expression data on NCI-60. We then employed the recently released chemosensitivity data of 99 anti-cancer drugs of known mechanism to investigate the gene-drug correlation, focusing on the IA genes. Afterwards, we collected data from four independent drug-testing experiments to validate our findings on compound response prediction. Finally, we obtained published clinical and molecular data from two recent adjuvant chemotherapy cohorts, one on lung cancer and one on breast cancer, to test the performance of our gene signature for patient outcome prediction. RESULTS: First, we found 633 IA genes from the invasion-gene expression correlation study. Then, for each of the 99 drugs, we obtained a subset of IA genes whose expression levels correlated with drug-sensitivity profiles. We identified a set of eight genes (EGFR, ITGA3, MYLK, RAI14, AHNAK, GLS, IL32 and NNMT) showing significant gene-drug correlation with paclitaxel, docetaxel, erlotinib, everolimus and dasatinib. This eight-gene signature (derived from NCI-60) for chemosensitivity prediction was validated by a total of 107 independent drug tests on 78 tumor cell lines, most of which were outside of the NCI-60 panel. The eight-gene signature predicted relapse-free survival for the lung and breast cancer patients (log-rank P = 0.0263; 0.00021). Multivariate Cox regression yielded a hazard ratio of our signature of 5.33 (95% CI = 1.76 to 16.1) and 1.81 (95% CI = 1.19 to 2.76) respectively. The eight-gene signature features the cancer hallmark epidermal growth factor receptor (EGFR) and genes involved in cell adhesion, migration, invasion, tumor growth and progression. CONCLUSIONS: Our study sheds light on the intricate three-way interplay among gene expression, invasion and compound-sensitivity. We report the finding of a unique signature that predicts chemotherapy survival for both lung and breast cancer. Augmenting the NCI-60 model with in vitro characterization of important phenotype-like invasion potential is a cost-effective approach to power the genomic chemosensitivity analysis.

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A rapid fluorescent screening method for cellular sensitivity to anti-cancer compound.

Publications

A rapid fluorescent screening method for cellular sensitivity to anti-cancer compound.

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