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Purpose: To evaluate the local tissue concentrations of the antineoplastic agent doxorubicin and the amount of drug still present inside drug delivery embolization beads at different time points after embolization and to compare doxorubicin levels with histologic modifications around the beads in a pig liver model. It was hypothesized that doxorubicin-eluting beads maintain cytotoxic concentrations of drug locally over a period of several weeks, as suggested by in vitro elution tests., Materials and Methods: Left lobe hepatic artery embolization was performed in 10 pigs with 100-300-microm or 700-900-microm beads loaded with 37.5 mg doxorubicin/mL. Control unloaded 100-300-microm beads were injected in five pigs. Livers were sampled 28 days or 90 days after embolization. The amount of drug retained inside the beads was assessed with infrared microspectroscopy. Doxorubicin concentration and distribution in the tissue around the beads were determined with microspectrofluorimetry and compared with tissue modifications on hematein eosin saffron-stained sections., Results: Doxorubicin-eluting beads eluted 43% of their initial drug load after 28 days and 89% after 90 days. Doxorubicin was present in tissues around the beads at both time points, with a significant decrease over time (P = .0004). The drug was detected at distances as far as 600 microm from the bead edge. Doxorubicin tissue concentrations ranged from 0.55 microM to 6.80 microM, [corrected] which are cytotoxic levels in hepatocyte cell cultures. High concentrations of drug were associated with coagulative necrosis of liver parenchyma. Doxorubicin-eluting beads 100-300 microm in size induced more necrosis than 700-900-microm beads (P = .0036)., Conclusions: Doxorubicin-eluting beads deliver high concentrations of the drug over a period of at least 3 months at several hundred micrometers from the bead, leading to significant cytotoxic effects., (Copyright (c) 2010 SIR. Published by Elsevier Inc. All rights reserved.)

In solid tumors, the cell microenvironment appears to be a key determinant in the emergence of drug resistance, a major obstacle to the successful use of antitumor drugs. Our aim was to determine whether type I collagen and fibronectin, proteins of the extracellular matrix, were able to influence the antimigratory properties induced by the antitumor drug doxorubicin. These properties were investigated at doxorubicin concentrations of 10 and 20 nM, which do not affect cell proliferation on a 24 h drug exposure. Using videomicroscopy, we found that these subtoxic doses of doxorubicin were sufficient to inhibit individual tumor cell motion on two-dimensional plastic surfaces. Such a drug treatment induced a dramatic disturbance of actin stress fiber formation and of vinculin distribution in 80% of cells. In contrast, on extracellular matrix proteins, cell speed was unaffected by drug and perturbation of both actin network and vinculin distribution was detected in only 50% of cells, suggesting a protective effect of the microenvironment. In addition, the phosphorylation of focal adhesion kinase and GTPase RhoA was less affected by doxorubicin with cells cultured on extracellular matrix proteins. In conclusion, our findings indicate that the cell microenvironment prevents drug-dependent inhibition of cell migration in vitro. They reveal cell locomotion as a key factor of microenvironment-mediated drug resistance. This new concept needs to be exploited in in vitro models to optimize the screening of new antimigratory drugs.

1alpha,25-dihydroxyvitamin D3 (VD3) and the EB1089 analog are well known for their roles in the modulation of proliferation and the differentiation of several malignant cells. In addition, VD3 or EB1089 displayed a high disposal of oxidant features and the ability to cause release of reactive oxygen species (ROS). We attempted to enhance HL60 cell differentiation and to limit ROS generation, by the association of deltanoids with doxorubicin and the antioxidants catalase (CAT), superoxide dismutase (SOD) and N-acetyl cystein (NAC). Differentiation of HL60 cells into monocytic lineage was studied by expression of mRNA, protein CD14 and functional differentiation by the nitroblue tetrazolium assay. The 2',7'-dichlorodihydrofluorescein diacetate (H2-DCFDA) dye allowed to evaluate in situ ROS generation. When associated with 0.1 nM EB1089, 15 nM doxorubicin induced an increase of differentiated cell percentage from 29% to 87% and did not affect VD3-treated cells. The association with doxorubicin also induced a significant increase of ROS release (p<0.05) versus VD3 and EB1089-treated cells. These results correspond to additivity of individual effects of doxorubicin and deltanoids. Antioxidant agents (10 nM NAC, 50 U/ml SOD or 2000 U/ml CAT) were associated with 10 nM VD3 or 1 nM EB1089 for 72 h. Compared to VD3 and EB1089 treatments, associations with antioxidants induced a slight increase of differentiated cells and a significant increase of CD14 mRNA. The highest differentiation effect occurred in the case of the EB1089-NAC association. Antioxidants induced a decrease (p<0.05) in ROS release generated by VD3 or EB1089 near the level of untreated cells. Thus, antioxidant agents demonstrated a protective effect against VD3 and EB1089 oxidative cytotoxicity and an enhancement of the monocyte differentiation. Combinations of antioxidants with deltanoids could dissociate the oxidative stress and differentiation.

In solid tumors, chemotherapeutics must adequately diffuse through the extracellular compartment to achieve their cytotoxic effect. Using quantitative microspectrofluorometry, both Doxorubicin penetration through three-dimensional (3D) collagen I matrices and its subsequent intranuclear accumulation into HT-1080 cells cultured in this microenvironment were directly assessed. Evidence that collagen delayed the Doxorubicin penetration for 1 h is presented. During that period, drug concentrations were lower in the nuclei in 3D compared to 2D matrices. Anthracyclines were also found to exhibit similar cytotoxicity in 2D and 3D after long term incubation. In conclusion, in this 3D culture model, collagen type I matrices delayed the early distribution of low molecular weight therapeutics and failed to affect their long-term cytotoxic effects, as previously reported. This model may provide a rationale for avoiding the emergence of intrinsic chemoresistance in tissue.

We used confocal microspectrofluorometry to investigate intracellular distribution of pirarubicin or THP-DOX in parental K562, CEM, and LR73 tumor cells and their corresponding multidrug-resistant (MDR) strains. Each spectrum of a recorded image was considered as a combination of cell autofluorescence and fluorescence of the drug. In the cytoplasm of parental K562, CEM, and LR73 cells, THP-DOX fluorescence emission profile was similar to that of free drug in aqueous buffer. The (I550nm/I600nm) ratio was 0. 50 +/- 0.1. However, in the cytoplasm of resistant cells the 550-nm band profile was modified. The I550nm/I600nm ratio was 0.85 +/- 0.2 in MDR K562 cells, which is significantly different from the ratio in sensitive cells (p<0.01). This appeared first to correspond to accumulation and self-oligomerization of THP-DOX in cytoplasmic organelles of MDR cells. Transfection of LR73 cells with the mdr1 gene conferred this characteristic on the resistant LR73R cells. Bodipy-ceramide, a trans-Golgi probe, was co-localized with the typical fluorescence emission peak at 550 nm observed in the cytoplasm of MDR cells. This organelle has been shown to be more acidic in MDR cells. Moreover, this specific pattern was similar to that observed when anthracycline is complexed with sphingomyelin. The typical fluorescence emission peak at 550 nm decreased in MDR cells incubated simultaneously in the presence of the drug and quinine, verapamil, or S9788. Growth inhibitory effect and nuclear accumulation of THP-DOX data obtained on LR73R and LR73D cell lines showed that only during reversion of resistance by verapamil and S9788 was an increase of nuclear THP-DOX accumulation observed. Our data suggest that characteristics of molecular environment, such as higher pH gradient or lipid structures, would be potential mechanisms of multidrug-resistance via the sequestration of anthracyclines.

Microspectrofluorometry allows the analysis of fluorescent molecules such as anthracyclines in the nucleus of isolated living cells. Using this technique, we confirmed that the amount of doxorubicin or THP-doxorubicin incorporated into the nucleus was related to the resistant or sensitive character of K562 cells. It was then extended to the study of fresh leukemic cells and kinetic studies were performed allowing the calculation of the retention rate (RR) of anthracycline (THP-doxorubicin) into the cell nucleus. A reproducibility study confirmed the accuracy of the method. Blast cells collected in patients with acute myeloid (n = 22) or lymphoid (n = 8) leukemia, at diagnosis (n = 26), or in relapse (n = 4) have been studied. RR varied from 8 to 98% independently of the type of leukemia or the clinical status. RR did not correlate either with P-glycoprotein or with CD34 expression although this latter result should be confirmed on a higher number of subjects. Among 18 patients presenting with AML at diagnosis, 14 have been treated with intensive chemotherapy including anthracyclines; the only one who had resistant disease had the lowest RR value. In conclusion, the results obtained here show that microspectrofluorometry allows the performance of kinetic studies on fresh leukemic cells in order to quantify chemo-resistance phenomena related to drug transport.

We have demonstrated that in vitro resistance of tumor cells to doxorubicin (Dox) can be fully circumvented by using doxorubicin-loaded nanospheres (Dox-NS), consisting of biodegradable polyisohexylcyanoacrylate polymers of 300 nm diameter and containing 2.83 mg of Dox per 31.5 mg of polymer. Five different multidrug-resistant cell lines, characterized by mdr1 amplification, were used in this study: Dox-R-MCF7, a human breast adenocarcinoma; SKVBL1, a human ovarian adenocarcinoma; K562-R, a human erythroleukemia; and two murine lines: P388-Adr-R, a monocytic leukemia of DBA2 mouse, and LR73MDR, a Chinese hamster ovarian cell line. These lines were 38.7, 210, 232, 143 and 20 times more resistant than their corresponding sensitive counterparts, respectively. Using Dox-NS, we obtained complete reversion of drug resistance in vitro, i.e. cell growth inhibition comparable with that obtained with sensitive cells exposed to free Dox. In vivo, we significantly prolonged the survival of DBA2 mice which had previously received P388-Adr-R cells by i.p. injections of Dox-NS, while free Dox injection was ineffective toward this rapidly growing tumor. (Prolongation of survival time: 115% vs 167% after Dox vs Dox-NS treatment, respectively.) Using the MCF7 cell line and its resistant variant, we studied the intracellular concentration and the cytoplasmic and nuclear distribution of Dox by laser microspectrofluorometry (LMSF). In sensitive cells, we observed a similar accumulation and distribution of Dox whatever the form of Dox delivery, i.e. whether free or carried by nanospheres. Analysis by LMSF showed that 99% of intranuclear Dox was bound to DNA after treatment with both forms of Dox. Of Dox, 81 and 83% were found in the intranuclear compartment of sensitive cells incubated with free Dox and Dox-NS, respectively. Resistant cells incubated with Dox-NS accumulated the same amount of Dox as sensitive cells incubated with free Dox or with Dox-NS. Dox, when loaded in nanospheres, bypasses the efflux mechanism responsible for multidrug resistance. LMSF analysis showed that Dox, transported and released by nanospheres, interacts with DNA identically in sensitive and resistant cells.

The accumulation of doxorubicin (DOX) in white blood cells of treated patients has been studied by quantitative microspectrofluorometry. From blood samples of treated patients, leucocyte subpopulations were separated by the gradient method. Emission fluorescence spectra from a microvolume of a single living cell nucleus were analysed in terms of spectral shape and fluorescence yield between free and DNA-bound doxorubicin. With this non-destructive analysis technique, intranuclear doxorubicin concentrations were determined within +/- 10%. Doxorubicin concentrations were measured in patients treated with bolus injection. After an accumulation of DOX in leucocytes during the first 30 min, intranuclear doxorubicin concentration did not vary significantly for 24 h, whereas its concentration in plasma decreased. Despite large differences between patients, monocytes accumulated significantly more doxorubicin than granulocytes or lymphocytes did.

Intranuclear drug concentration in cells treated with doxorubicin (DXR) or with 4'-deoxy-4'-iododoxorubicin (IDX) was measured by means of a quantitative microspectrofluorometric technique recently developed by us. Resolution of free and bound drug contributions in fluorescence emission spectra, as collected from a microvolume of single living cell nuclei, provided concentration data with about 10% indetermination. Uptake of DXR and IDX into the nucleus of K562 cells and DXR-resistant K562/DXR cells could then be studied with a sensitive, nondestructive technique. Growth inhibitory concentrations of K562 and K562/DXR cells, when measured with respect to drug content in the medium, differed by a factor of 25 in the case of DXR and by a factor of three in the case of IDX. By contrast, intranuclear drug concentrations measured at corresponding growth inhibitory concentrations are found to be nearly constant, i.e., independent of cellular-resistant phenotype and of anthracycline structure. This result supports an identical mechanism of action for the two drugs, most probably targeted to the nucleus, and ascribes to intracellular transport the different potency of the two drugs in the two cell lines.

Doxorubicin-DNA association has been studied by quantitative microspectrofluorometry. Fluorescence emission spectra from a microvolume of single living cell nuclei treated with doxorubicin have been analyzed in terms of difference in spectral shape and fluorescence yield between free and DNA-bound drug. Contribution of each spectral component to the total signal was calculated by least-squares linear regression. With this method of analysis, total drug concentration has been determined with an error of less than 10%. Moreover, the uptake into the nucleus has been studied in a non destructive way, avoiding use of 14C-labelled drug. Kinetic studies of drug accumulation into the nuclei were conducted on sensitive and resistant cells.