Role of the aclacinomycin A – doxorubicin association in reversal of doxorubicin resistance in K562 tumour cells

Abstract

Summary Acquired resistance to anthracyclines is characterised by a lower sensitivity to these agents, associated with impaired accumulation of drug. We have examined the ability of aclacinomycin A (ACM) associated with doxorubicin (DOX), to increase intranuclear DOX concentrations and, consequently, to enhance cytotoxic effects against drug resistant cells in vitro. A recently developed microspectrofluorometric technique is used to measure intranuclear DOX concentrations in sensitive and DOX-resistant K562 cells treated with DOX and ACM. Fluorescence emission spectra are collected from a microvolume of single living cell nuclei. From both DOX and ACM model fluorescence spectra (free, DNA-bound and metabolites), the intranuclear spectral profile is analysed according to the amount of each component. This quantitative analysis determines intranuclear DOX concentrations with an error of 10%. Non-cytotoxic doses of ACM, in combination with DOX, increase cytotoxic activity of DOX against K562 resistant cells. When DOX-resistant cells are exposed simultaneously to ACM and DOX, significant increases in intranuclear DOX concentrations are found compared with the case of exposure to DOX alone. The measure of the intranuclear retention of DOX shows that ACM partly blocks the DOX efflux in resistant cell nuclei, resulting in enhanced accumulation of DOX. These data lead us to conclude that ACM-DOX association partly reverses the DOX resistance at clinically achievable concentrations.A major obstacle to successful use of anthracyclines and other cytotoxic drugs in cancer chemotherapy is the development of clinical drug resistance. Tumour cell modifications (resulting in multidrug resistance) are characterised by a complex phenotype of cross-resistance to antineoplasic agents Bradley et al., 1988). These are accompanied by gene amplification, a deficient accumulation of drug, an enhanced drug efflux function and a build up of an integral membrane glycoprotein with a molecular weight of 170,000-180,000 (P-glycoprotein) . A study with DNA transfectants (Riordan et al., 1985) has demonstrated that the P-glycoprotein was intimately involved in resistance and might be serving as an active efflux pump to remove the drug from the cell. One way to overcome in vitro resistance to anti-cancer drugs has been to use simultaneously calcium channel blockers (Tsuruo et al., 1983) or calmodulin inhibitors (Ganapathi et al., 1984) together with the anti-cancer drug. The mechanism of the resistance reversal appears to be related to the inhibition of outward drug transport which subsequently leads to increased intracellular drug levels.With the recent development of microspectrofluorometry, we have studied fluorescence signals from microvolumes within a single living cell (Ginot et al., 1984;Manfait et al., 1987). DOX concentrations in nuclei of sensitive and DOXresistant K562 human leukaemia cell lines may thus be determined (Tsuruo et al., 1986;Sugimoto & Tsuruo, 1987). Cell growth and viability were determined by phase contrast microscop...

Human hepatoma cells rich in P-glycoprotein are sensitive to aclarubicin and resistant to three other anthracyclines

“…However, the intracellular anthracycline fluorescence does not accurately reflect the drug content because binding to DNA may cause quenching of the fluorescence, which is particularly pronounced for ACL (Skovsgaard, 1987; al., 1989). The fluorescence intensity of free ACL has been measured to be 200 times that of nucleus-bound drug (Millot et al, 1989). In the cytoplasm of the HB8065/S cells, and to a much lesser extent in the resistant HB8065/R cells, the anthracycline fluorescence appeared in numerous cytoplasmic vesicles.…”

Characterization of Acidic Vesicles in Multidrug-resistant and Sensitive Cancer Cells by Acridine Orange Staining and Confocal Microspectrofluorometry

“…We suggest that the intracellular sequestration of weak bases and the enhanced outward transport should be distinct mechanisms. Indeed, it has been shown previously that the resistant K562-R cells present an enhanced doxorubicin efflux out of the nucleus compared with K562 cells (Millot et al 1989). However, we have determined that, for resistant cells, the efflux of AO was not enhanced (data not shown), although this molecule was intracellularly redistributed.…”

Anthracycline subcellular distribution in human leukemic cells by microspectrofluorometry: factors contributing to drug-induced cell death and reversal of multidrug resistance

“…39 This affinity has also been reported on tumor cells using resonance Raman and surface-enhanced Raman scatescence of aclacinomycin A located in the nucleus is quenched 200 times upon binding to nuclear DNA. 31 Thus, tering microspectroscopies. 40,41 Knowing the potential target of DOX (Figure 1) in the cell by measuring absolute concentrations of nuclear anthracyclines, we were able to study the real drug transport.…”

“…46 DOX-resistant K562 cells overexstudy the relationship between the nuclear accumulation of DOX and the observed biological effects, the influence of press the P-glycoprotein responsible for active drug efflux. 31 For our purposes the comparison with DOX was very interestincubation temperatures on these processes was studied. Differentiation was assessed by the count of recruited hemoglobiing, because this analogue is taken up faster and to a higher extent by the cells, and shows reduced cross-resistance with nized cells after exposure to DOX at 37°C and 4°C.…”

“…48 We have extended these inhibition growth studies with nontoxic doses of ACM against DOX-resistant K562 cells in order to determine which kind of exposure with ACM and DOX, simultaneously or sequentially, produced the most cytotoxicity. 31 Cells were incubated for 4 h in the presence of DOX with and without nontoxic ACM concentrations (growth inhibition Ͻ5%): 10 nM for K562, 50 nM for K562-DOX. For the K562 cell line (Figure 6a), simultaneous incubation with DOX and nontoxic ACM dose (10 nM) did not produce any enhancement of DOX cytotoxicity.…”