Flow cytometric analysis of early steps in development of adriamycin resistance in a human gastric cancer cell line

We have established a low-level adriamycin (ADM)-resistant human gastric cancer cell line (MKN45R) from the parental cell line (MKN45) by exposure to stepwise increases of ADM concentration (final concentration, 0.026 microgram/ml). The purpose of this study was to identify the early steps in the development of ADM resistance in MKN45R by flow cytometric (FCM) analysis. Comparison of the concentration required for 50% growth inhibition, determined by a tetrazolium-based colorimetric assay, showed that MKN45R was about 2.6-fold more resistant to ADM than MKN45. However, the inhibition index values were 89.5% for MKN45 and 86.4% for MKN45R, respectively, showing that ADM was judged to be "effective" against both cell lines. On the other hand, cell kinetic analysis by FCM revealed that the increase of the ratio of G2M accumulation induced by ADM treatment was significantly lower (P < 0.01) in MKN45R. Moreover, the efflux of ADM estimated by FCM analysis was significantly increased (P < 0.05) in MKN45R even though there was no significant increase of P-glycoprotein expression. These results suggest that although ADM was still effective based on a standard drug sensitivity test, the cancer cells were already acquiring resistance to ADM as judged from FCM analysis. Moreover, the mechanism of this ADM resistance is considered to be independent of P-glycoprotein expression. Thus, FCM analysis is useful for detecting the early steps in the development of drug resistance of cancer cells.