Uptake and effects of doxorubicin-linked poly (l-lysine iso-phthalamide) compounds in resistant and sensitive cancer cells

Anikina E, Pawlyn C, Chiu M, Lim L, Wong E, 1Eccleston M, 1Slater N and Barrand MA. University of Cambridge, Department of Pharmacology, Tennis Court Road, Cambridge CB2 1PD and 1Department Of Chemical Engineering, New Museum Site, Pembroke Street, Cambridge, CB2 3RA

Obstacles to effective anticancer chemotherapy include low therapeutic indices of many of the drugs with toxicity in normal tissues, eg cardiotoxicity of doxorubicin (DOX), and development of drug resistance, an important mechanism being drug efflux by multidrug transporters such as P-glycoprotein (Pgp) and multidrug resistance associated proteins (MRPs). To avoid such efflux, some pseudopeptidic polymers conjugated to DOX (Eccleston et al., 2000) have been designed to enter cells, not by diffusion as with free drug, but by endocytosis with subsequent release of DOX into the cytoplasm so by-passing cell surface transporters.

This study investigates the properties of two of these DOX-conjugates (poly-DOX and the pegylated variant, peg-poly-DOX): their cytotoxic effects, their ability to accumulate within cells, factors affecting their uptake and the intracellular sites to which they localize in cells of the large cell lung cancer line, L23P and its MRP1-overexpressing DOX-resistant variant, L23R and of the colon carcinoma cell line, SW620P and its Pgp-overexpressing DOX-resistant variant, SW620R. As measured by a 4day MTT cell viability assay, cells appeared more sensitive to DOX as the free drug than in polymer-conjugate form (IC 50 values as means ±s.e.m. for DOX, poly-DOX and peg-poly-DOX of 0.2± 0.1, 2.5±1.0 and 10.1±3.7μM with SW620P cells and of 2.5±0.5, 20±4.6 and 12.0±3.3μM respectively with SW620R cells, n=3). To determine the extent of DOX uptake, cells were trypsinised and exposed in suspension for up to 90 mins to free DOX (2μM) or the DOX polymer conjugates (2μM DOX-equivalent) and fluorescence due to DOX subsequently assessed by flow cytometry. Accumulation of free DOX increased over 90 mins with more entering sensitive than resistant cells whilst that of poly-DOX reached equilibrium within 10 min and was similar in both cell types. Intracellular distribution of DOX observed by confocal microscopy in sensitive L23P cells on cover slips was seen to be nuclear following exposure to free DOX but localized to the cytoplasm with poly-DOX and peg-poly-DOX.

As with free DOX, accumulation of the DOX conjugates varied with external pH, being higher at pH 6.9 (1.5 fold, n=6, p<0.05) and lower at pH 7.9 (0.45 fold, n=6, p<0.05) than at pH 7.4. However, uptake of the DOX conjugates appeared independent of energy (ratios for ATP-depleted to ATP-replete cells of 0.94± 0.45 for poly-DOX and 1.00± 0.21 for peg-poly-DOX, n=7), ATP being reduced to 1% of normal values as determined by a luciferase assay by treatment for 1 h with 6mM deoxyglucose and 10mM sodium azide in the absence of glucose. Uptake was also independent of temperature (ratios of uptake at 4°C compared to 37°C of 1.2±0.13 for poly-DOX and 0.97±0.2 for peg-poly-DOX, n=6). Such features hint at entry mechanisms unrelated to endocytosis but more akin to those used by some penetrating peptides (Zorko and Langel, 2005).

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