Comparative Evaluation of Cellular Uptake of Free and Liposomal Doxorubicin Following Short Term Exposure.

BACKGROUND/AIM: Liposomal Doxorubicin (lipDOX) and free Doxorubicin (DOX) are reported to exhibit similar antitumor efficacy. However, cellular internalization mechanisms of lipDOX are still a subject of controversy. MATERIALS AND METHODS: Intact and permeabilized cells were exposed for short time to lipDOX and free DOX and drug intracellular content was evaluated by flow cytometry. Then, the antiproliferative capacities of lipDOX and free DOX were compared by the leukocyte nadir test in mice in vivo. RESULTS: The fluorescence increase was 11.2-fold higher in intact cells and 19.7-fold higher in permeabilized cells after exposure to free DOX as compared to lipDOX. Mice injected with DOX showed pronounced antiproliferative activity with a leukocyte count decrease to 2.8±0.65 k/µl (p<0.01) - an effect significantly stronger than that in the lipDOX group. CONCLUSION: Intact and permeabilized cells internalize free DOX manifold faster than lipDOX. The LipDOX formulation does not induce a remarkable leukocyte nadir effect in vivo.

Doxorubicin uptake in ascitic lymphoma model: resistance or curability is governed by tumor cell density and prolonged drug retention.

Background/Aims: Chemotherapy resistance of malignancies is a universal phenomenon which unfavorably affects therapeutic results. Genetic adaptations as well as epigenetic factors can play an important role in the development of multidrug resistance. Cytotoxic drug content in plasma of cancer patients is known to variate up to one hundred-fold regardless of the same dose injected per m2 body surface. The relationship between plasma concentrations, tissue uptake, and chemotherapy response is not completely understood. The main objective of this study was to investigate how the identical dose of Doxorubicin (Dox) can result in a different therapeutic response pattern depending on tumor size. Study Design: The study was performed on ascitic EL4 lymphoma in an exponential growth phase focusing on the rapidly changing tumor susceptibility to the Dox treatment. Well distinguishable tumor response patterns (curability, remission-relapse, resistance) were selected to unveil Dox intratumoral uptake and drug tissue persistence. Intratumoral Dox content within peritoneal cavity (PerC) in conjunction with systemic toxicity and plasma pharmacokinetics, were monitored at several time points following Dox injection in tumor bearing mice (TBM) with differing patterns of response. Results: Following intraperitoneal (i.p.) transplantation of 5x104 EL4 lymphoma cells rapid exponential proliferation with ascites volume and animal mass increase resulted in median survival of 14.5 days. The increase in tumor cell mass in PerC between day 3 and day 9 was 112.5-fold (0.2±0.03 mg vs 22.5±0.31 mg respectively). However, tumors at this time interval (day 3 to day 9 post-transplantation) were relatively small and constituted less than 0.05% of animal weight. An identical dose of Dox (15 mg/kg) injected intravenously (i.v.) on Day 3 lead to a cure whereas a TBM injected on day 9 exhibited resistance with a median survival time no different from the untreated TBM control. Injection of Dox resulted in noticeable differences of cellular uptake in PerC between all three groups of TBM ("cure", relapse", "resistance"). Larger tumors were consistently taking up less Dox 60 min after the 15 mg/kg i.v. bolus injection. Higher initial uptake resulted also in longer retention of drug in PerC cells. The area under the concentration curve in PerC cells AUC0-10d was 8.2±0.57 µg/g x h, 4.6±0.27 µg/g x h and 1.6±0.02 µg/g x h in "cure", "relapse" and "resistance" TBM respectively (p<0.05 "relapse" vs "cure" and p<0.001 "resistance" vs "cure"). No differences in plasma Dox pharmacokinetics or systemic hematological effects were observed in TBM following a single i.v. Dox push. Hematologic nadir was tested on day 2 and subsequent hematologic recovery was evaluated on day 10 following Dox administration. Hematologic recovery on day 10 coincided with complete drug efflux from PerC and rising tumor cell numbers in PerC of "relapse" TBM. Myelosuppression and hematological recovery patterns were identical in all surviving animal groups regardless of the tumor size on the day of Dox injection. Conclusions: Within a few days of exponential tumor growth, an identical dose of Dox produced dramatically different responses in the TBM with increasing resistance. Systemic toxicity and plasma pharmacokinetics were indistinguishable between all TBM groups. Initial uptake in tumor cells was found to be consistently lower in larger tumors. Drug uptake in tumor cells was regulated locally - a phenomenon known as inoculum effect in vitro. The duration of drug retention in cells was directly related to initial cellular uptake. The magnitude of Dox cellular retention could potentially play a role in determining tumor remission and relapse.

Amyloid ß oligomers inhibit growth of human cancer cells.

Effects of amyloid beta (Aß) oligomers on viability and function of cell lines such as NB4 (human acute promyelocytic leukemia), A549 (human lung cancer (adenocarcinomic alveolar basal epithelial tumor)) and MCF-7 (human breast cancer (invasive breast ductal carcinoma)) were investigated. Two types of Aß oligomers were used in the study. The first type was produced in the presence of oligomerization inhibitor, hexafluoroisopropanol (HFIP). The second type of amyloids was assembled in the absence of the inhibitor. The first type preparation was predominantly populated with dimers and trimers, while the second type contained mostly pentadecamers. These amyloid species exhibited different secondary protein structure with considerable amount of antiparallel ß sheet structural elements in HFIP oligomerized Aß mixtures. The effect of the cell growth inhibition, which was stronger in the case of HFIP Aß oligomers, was observed for all cell lines. Tests aiming at elucidating the effects of the amyloid species on cell cycles showed little differences between amyloid preparations. This prompts us to conclude that the effect on the cancer cell proliferation rate is less specific to the biological processes developing inside the cells during the proliferation. Therefore, cell growth inhibition may involve interactions with the peripheral parts of the cancer cells, such as a phospholipid membrane, and only in case of the NB4 cells, where accumulation of amyloid species inside the cells was detected, one may imply the opposite. In general, cancer cells were much less susceptible to the damaging effects of amyloid oligomers compared to earlier observations in mixed neuronal cell cultures.