Evaluation of IC50 levels immediately after treatment with anticancer reagents using a real-time cell monitoring device

Abstract

A real-time cell-monitoring analysis (RTCA) system was previously developed based on the change in impedance when cells attach and spread in a culture dish coated with a gold microelectrode array. However, the potential applications of this system have not yet been fully demonstrated. The purpose of this study was to test the utility of the RTCA system to determine the cytotoxicity of four anticancer agents in carcinoma cells. The results were compared with those of the conventional WST-8 assay at the endpoint to determine the potential of the RTCA system as a new real-time assay method to evaluate cytotoxicity. iCELLigence was used as the RTCA system in this study. Suspensions of oral squamous cell carcinoma (OSCC) cell lines were seeded (2×10⁴ cells/well) onto the E-plate (the culture plate of the iCELLigence system). After 24 h of culture, anticancer agents were added to each well, and changes in electrical impedance (cell index, CI) were recorded for another 72 h of culture. Cell proliferation was detected in real-time by the RTCA device in an automated, high throughput manner. Then, the IC₅₀ profiles of the four anticancer agents were calculated based on the real-time cell index values. The results indicated that the RTCA system was useful in evaluating cytotoxic reactions immediately after the addition of the anticancer agents as it was able to record the data in real-time. Furthermore, the IC₅₀ levels measured by the real-time assay were lower than those measured by the endpoint assay. Thus, RTCA systems can be used to evaluate chemotherapeutic agents in cancer cells as well as their side effects in normal cells.

Keywords: IC50; RTCA system; anticancer reagent; cytotoxicity; squamous cell carcinoma.

Figure 1.

CI measurements of the SQUU-A cell line treated with four anticancer agents. (A) 5-FU, (B) doxifluridine, (C) carboplatin, and (D) docetaxel. Data are represented as the mean ± SD (n=3) though the SD values were too small to see. Black arrows indicate the time of addition of anticancer agents. CI, cell index; 5-FU, 5-fluorouracil.

Figure 2.

CI measurements of the SQUU-B cell line treated with four anticancer agents. (A) 5-FU, (B) doxifluridine, (C) carboplatin, and (D) docetaxel. Data are represented as the mean ± SD (n=3) though the SD values were too small to see. Black arrows indicate the time of addition of the anticancer reagents. CI, cell index; 5-FU, 5-fluorouracil.

Figure 3.

CI measurements of the NA cell line treated with four anticancer agents. (A) 5-FU, (B) doxifluridine, (C) carboplatin and (D) docetaxel. Data are represented as the mean ± SD (n=3) though the SD values were too small to see. Black arrows indicate the time of addition of the anticancer reagents. CI, cell index; 5-FU, 5-fluorouracil.

Figure 4.

CI measurements of the SAS cell line treated with four anticancer reagents. (A) 5-FU, (B) doxifluridine, (C) carboplatin, and (D) docetaxel. Data are represented as the mean ± SD (n=3) though the SD values were too small to see. Black arrows indicate the time of addition of the anticancer reagents. CI, cell index; 5-FU, 5-fluorouracil.

Figure 5.

Real-time IC₅₀ profiles of the SQUU-B cell line after treatment with four anticancer reagents. The results of 5-FU, doxifluridine, carboplatin, and docetaxel are shown in (A-D), respectively. Data are represented as the mean ± SD (n=3) though the SD values were too small to see. CI, cell index; 5-FU, 5-fluorouracil.

Figure 6.

Correlations between real-time measurements of IC₅₀ values using the RTCA system and endpoint measurements of IC₅₀ values using the WST-8 assay in OSCC cells. The average IC₅₀ values at 24, 48, and 72 h after treatment with anticancer agents in the four OSCC cell lines (12 points) were plotted. The results of 5-FU, doxifluridine, carboplatin, and docetaxel are shown in (A-D), respectively. OSCC, oral squamous cell carcinoma; RTCA, real-time cell analysis.