Functional studies of calcein acetoxymethyl ester in human tumor cells

Sammanfattning: A fluorometric assay for functional measurement of P-glycoprotein 170 (Pgp) mediated transport in cell lines, using the acetoxymethyl ester of calcein (calcein/AM) as a probe was established. The myeloma cell line RPMI 8226/S and the two Pgp-expressing doxorubicin resistant subcell lines 8226/dox6 (low) and 8226/dox40 (high) were used. There was significantly lower accumulation of fluorescent calcein in both dox6 and dox40 cells compared to 8226/S. The calcein/AM uptake assay also detected membrane transport related to themultidrug resistance associated protein, MRP1.Furthermore, the in vitro cytotoxic activity of calcein/AM was investigated in cell lines and in primary cultures of patient tumor cells derived from both solid and hematological tumors. Calcein/AM was cytotoxic in the µg/ml (µM) range to both cell lines and patient samples. At 2.5 µg/ml, calcein/AM produced a cell kill greater than 50% in 94% of the hematological and in 63% of the solid tumor samples.In the histiocytic lymphoma cell line, U-937 GTB, calcein/AM at 2.5 µg/ml, induced an almost complete inhibition of DNA synthesis and a partial disruption of the mitochondrial membrane potential within one hour of incubation. This was followed by an increase of caspase-3 activity and an increasing cell fraction judged as apoptotic by morphological and TUNEL evaluation at 3 and 6 h of incubation, respectively.High coefficients of correlation were observed between cytotoxicity of calcein/AM, topoisomerase II active/intercalating agents and mitochondria active compounds, using a cell line panel consisting of 10 cell lines expressing defined mechanisms of resistance. Kinetic studies of extracellular metabolism and confocal microscopy evaluation further supported the notion that calcein/AM interacts with nuclear and/or mitochondrial DNA to induce cytotoxicity and apoptosis.The rapid and potent induction of tumor cell apoptosis in combination with the esterase dependent delivery mechanism makes calcein/AM a candidate for further in vivo evaluation.