The influence of tumor cell density on cellular accumulation of doxorubicin or cisplatin in vitro

Abstract

The effect of tumor cell density on the cellular pharmacokinetics of doxorubicin (DXR) and cisplatin (CDDP) was studied using MOLT-3 human acute lymphoblastic leukemia cells. As determined by the MTT assay, the growth-inhibitory effect of DXR was approx. 40 times lower when cell density was increased from 10(6) to 10(8) cells/ml (positive inoculum effect), whereas little or no influence of cell density was observed in CDDP-induced cell-growth inhibition. As measured by high-performance liquid chromatography using a fluorescence detector, the cellular accumulation of DXR showed 6- and 18-fold decreases after 1 h incubation when the cells were concentrated from 10(6) to 10(7) and 10(8) cells/ml, respectively. Only at low cell density (10(6) cells/ml) did the amount of DXR in the cells increase with increasing exposure times of up to 6 h. The DXR concentration in the supernatant that was separated from a cell suspension showing a density of 10(8) cells/ml fell to 20% of that obtained at 10(6) cells/ml. The metabolites of DXR, including Adriamycinol and Adriamycinone, were not detectable in the cell extracts or supernatants at any cell density examined. In contrast, the cellular accumulation of CDDP calculated from the platinum concentration, which was measured with a flameless atomic absorption spectrophotometer, was essentially identical at all cell densities examined; moreover, extension of the exposure period resulted in a linear increase in the amount of CDDP in the cells. CDDP concentrations in the supernatants were equally retained, irrespective of cell densities. These observations indicate that the positive inoculum effect shown in DXR-induced cell-growth inhibition results from the decreased cellular accumulation of the drug at high cell densities. We found no influence for cell density on the cellular accumulation of CDDP that might be relevant to the therapeutic potentiation of this drug at high tumor-cell density.

Copper ions regulate cytotoxicity of disulfiram to myeloid leukemia cells

“…Similar effect was described also for vincristine (5). In contrast, cisplatin and carboplatin do not exhibit the inoculum effect, since cell density does not affect cytotoxic doseresponse curves and cellular accumulation of these drugs (4,25).…”

“…6), nor extended time of treatment (24 h) (not shown) elevated the intracellular content of copper in HD U937 cells. DSF-treated HD U937 cells reached similar level of copper as LD U937 cells only when growth medium was supplemented with external CuSO 4 (Fig. 6).…”

“…For cell cycle analyses, the cells were cultured for 24 and 48 h, washed twice with PBS, resuspended in 0.5 ml of PBS, fixed in 4 ml of 70% ethanol and stored at 4˚C for 24 h. Fixed cells were centrifuged, washed with PBS and stained in 0.5 ml of Vindelov solution (22) for 30 min at 37˚C. DNA content in at least 1.5x10 4 cells was determined by a Cytomics FC 500 CXP flow cytometry system (Beckman Coulter, Fullerton, CA, USA).…”

“…Interindividual variations in WBC count at diagnosis are common, ranging from leukopenia to more than 400 million cells/ml blood. The WBC count can significantly affect efficacy of chemotherapy because cytotoxicity of chemotherapeutics, such as daunorubicin, doxorubicin, vincristine, tamoxifen and paclitaxel, rapidly decreases in cultures of high density (3)(4)(5)(6)(7). Several explanations of this phenomenon have been suggested.…”

Antitumor Efficacy of Doxorubicin in Combination with Cisplatin on Human Lymphoma Cells at Various Cell Densities in vitro.

“…1 The positive inoculum effect shown in DXR-induced tumor cell kill resulted from lessened cellular accumulation of the drug at high cell densities rather than drug inactivation. 5,6 This phenomenon (posi tive inoculum effect) was originally demonstrated in certain antimicrobial antibiotics of which the inhibitory concentrations in vitro were influenced by the inoculum size of some microbial organisms. 7,8 In experimental animal tumor systems, a remarkable therapeutic synergism was observed in the administration of DXR in combination with CDDP against advanced P388 leukemia and Ridgway osteogenic sarcoma9 as well as Sarcoma 180.10 Combination chemotherapy of these two drugs had marked therapeutic potentiation against ovarian carcinoma and other cancer in man.11 -13 We hypothesized that if DXR is less active at high tumor cell densities and the antitumor effect of CDDP is not influenced by cell density, the proper sequence of administration should be CDDP first followed by DXR, rather than simultaneous exposure or reversed order of the combination.…”