Cytotoxic effect of calcein acetoxymethyl ester on human tumor cell lines: drug delivery by intracellular trapping

Calcein acetoxymethyl ester (calcein/AM) and some related cellular dyes with a cytoplasmic distribution were investigated with respect to cellular hydrolysis, accumulation, efflux and cytotoxicity in a panel of established human cell lines, including multidrug resistant (MDR) phenotypes. At 0.1-1 micrograms/ml, calcein/AM was highly cytotoxic against several cell lines, even after short-term exposure (30 min). Calcein/AM induced no immediate loss (3 h) of membrane integrity and the drug was more active against low compared with high density plated cells. In cell lines with the MDR phenotype and in the renal carcinoma cell line ACHN, the drug was considerably less active. Non-esterified calcein had no effect and calcein/AM was significantly more potent than other structurally related fluorescein analogs and AM esters tested. Although MDR cell lines showed a decreased cellular hydrolysis and accumulation of the dye, there was no strict relationship between cytoplasmic calcein exposure and cytotoxic activity. The rate of efflux was low in the two most sensitive cell lines, the human lymphoma U-937-GTB and its vincristine (vcr) resistant subline U-937/vcr10, while the remaining cell lines showed similar biphasic efflux patterns, including cell lines of the MDR phenotype. The results show that calcein/AM has cytotoxic activity against human tumor cell lines at low concentrations. The effect appears dependent on the intracellular trapping of the drug, although the specific cellular target remains unknown. Due to its cytotoxic efficacy and unique principle of cellular drug delivery, further investigation of calcein/AM and related compounds as potentially new Anticancer agents seems warranted.