Inhibition of anthracycline semiquinone formation by ICRF-187 (Dexrazoxane) in cells

The formation of semiquinone free radicals of doxorubicin, epirubicin, daunorubicin, and idarubicin was measured by electron paramagnetic resonance (EPR) spectroscopy in hypoxic suspensions of chinese hamster ovary (CHO) cells. The amount of semiquinone produced was in the order idarubicin > doxorubicin > daunorubicin > epirubicin. The idarubicin semiquinone signal was both the fastest to be formed and to decay. Idarubicin, which was the most lipophilic of the anthracyclines studied, also displayed the fastest fluorescence-measured cellular uptake of drug. Thus, it was concluded that semiquinone formation was dependent upon the rate of cellular uptake. Lysed cell suspensions were also shown to be capable of producing the doxorubicin semiquinone in the presence of added NADPH. The cardioprotective agent dexrazoxane (ICRF-187) was observed to decrease the amount of doxorubicin semiquinone observed in cell suspensions. Dexrazoxane also decreased the amount of doxorubicin semiquinone observed in the NADPH-lysed cell suspension mixture. Neither bipyridine nor deferoxamine decreased NADPH-dependent doxorubicin semiquinone formation. These results suggest that dexrazoxane does not decrease doxorubicin semiquinone formation through an iron complex formed from hydrolyzed dexrazoxane. Dexrazoxane may be inhibiting an NADPH-dependent enzyme.     

Lack of effect of glutathione depletion on cytotoxicity, mutagenicity and DNA damage produced by doxorubicin in cultured cells

Since endogenous glutathione (GSH), the main non-protein intracellular thiol compound, is known to provide protection against reactive radical species, its depletion by diethylmaleate (DEM) was used to assess the role of free radical formation mediated by doxorubicin in DNA damage, cytotoxicity and mutagenicity of the anthracycline. Subtoxic concentrations of DEM that produced up to 75% depletion of GSH did not increase doxorubicin cytotoxicity in a variety of cell lines, including Chinese hamster ovary (CHO) and lung (V-79) cells, LoVo human carcinoma cells and P388 murine leukemia cells. Similarly, the number of doxorubicin-induced DNA single strand breaks in CHO cells and the mutation frequency in V-79 cells were not affected by GSH depletion. The results obtained suggest that mechanisms other than free radical formation are responsible for DNA damage, cytotoxicity and mutagenicity of anthracyclines.     

Gadopentatic acid affects in vitro proliferation and doxorubicin response in human breast adenocarcinoma cells

Contrasting agents (CAs) that are administered to patients during magnetic resonance imaging to facilitate tumor identification are generally considered harmless. However, gadolinium (Gd) based contrast agents can be retained in the body, inflicting specific cell line cytotoxicity. We investigate the effect of Gadopentatic acid (Gd-DTPA) on human breast adenocarcinoma MCF-7 cells. These cells exhibit a toggle switch response: exposure to 0.1 and 1 mM concentrations of Gd-DTPA enhances proliferation, which is hindered at a higher 10 mM concentration. Proliferation is enhanced when cells transition to 3D morphologies in post confluent conditions. The proliferation dependence on the concentration of CA is absent for Hs 578T and MDA-MB-231 triple negative cell lines. MCF-7 cells reveal a double toggle switch related to the expression of VEGF, which goes through high–low–high downregulation when cells are exposed to 0.1, 1, and 10 mM Gd-DTPA, respectively. Finally, doxorubicin drug response is assessed, which also reveals a double toggle switch behavior, where drug cytotoxicity exhibits a nonlinear dependence on the CA concentration. A toggle switch in cell characteristics that are exposed to 1 mM of Gd-DTPA amplifies the importance of this threshold, affecting several cell behaviors if surpassed. This work emphasizes the important effects that CAs can have on cells, specifically Gd-DTPA on MCF-7 cells, and the implications for cell growth and drug response during clinical and synthetic biology procedures.     

Mechanisms of induction of apoptosis by anthraquinone anticancer drugs aclarubicin and mitoxantrone in comparison with doxorubicin: Relation to drug cytotoxicity and caspase-3 activation

We examined molecular events and morphological features associated with apoptosis induced by anthraquinone anticancer drugs aclarubicin, mitoxantrone and doxorubicin in two spontaneously immortalized cell lines (NIH 3T3 and B14) in relation to cytotoxicity of these drugs. The investigated cells showed similar sensitivity to aclarubicin but different sensitivity to doxorubicin and mitoxantrone: mitoxantrone was the most cytotoxic drug in both cell lines. All three drugs triggered both apoptosis and necrosis but none of these processes was positively correlated with their cytotoxicity. Apoptosis was the prevalent form of cell kill by aclarubicin, while doxorubicin and mitoxantrone induced mainly the necrotic mode of cell death. The extent and the timing of apoptosis were strongly dependent on the cell line, the type of the drug and its dose, and were mediated by caspase-3 activation. A significant increase in caspase-3 activity and the percentage of apoptotic cells, oligonucleosomal DNA fragmentation, chromatin condensation and formation of apoptotic bodies was observed predominantly in B14 cells. NIH 3T3 cells showed lesser changes and a lack of DNA fragmentation. Aclarubicin was the fastest acting drug, inducing DNA fragmentation 12 h earlier than doxorubicin, and 24 h earlier than mitoxantrone. Caspase-3 inhibitor Ac-DEVD-CHO did not show any significant effect on drug cytotoxicity and DNA nucleosomal fragmentation.     

Effect of gamma irradiation on spleen cell function and cytotoxicity of doxorubicin

The present study was attempted to evaluate the effects of gamma-irradiated doxorubicin (IRD) on spleen cell proliferation, cytokines release (IFN-gamma and IL-2) and lung metastasis in mice. Gamma irradiation induced degradation of doxorubicin molecule and cytotoxicity on melanoma (B16BL6) and myoblast (H9c2) cell lines were determined by high performance liquid chromatography (HPLC) and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tetrazole) assay, respectively. Non-irradiated doxorubicin (NIRD) was used as a control. The mice injected with NIRD (2mg/kg body weight for 5 days, 24h interval) showed a considerable decrease (P<0.05) in the body, spleen weight, proliferation and cytokine release (IL-2 and IFN-gamma) as compared to control. However, a non-significant variation was observed in IRD treated mice compared with normal. Tumor bearing mice treated with NIRD and IRD (2mg/kg body weight, five doses at 48 h interval) showed diverse results on spleen cell cytokine release, proliferation and metastasis. HPLC results revealed the formation of several trace level degradation (P<0.05) products of IRD. IRD displayed a non-significant variation of cytotoxicity on B16BL6 cells, and low percentage (P<0.01) of cardiotoxicity on H9c2 cells as compared to NIRD. Altogether, this present study emphasis that gamma irradiation altered the property of doxorubicin.     

MicroRNA200a enhances antitumor effects in combination with doxorubicin in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is often treated with doxorubicin. MicroRNAs have been shown to have important regulatory roles in cancer and serve as a target in chemoresistance. In this study, we investigated the effects of specific microRNA-200a (miR-200a) on HCC tumor cell growth and effect of doxorubicin-mediated cytotoxicity. Our results show miR-200a is downregulated in human HCC and HCC tumor cell lines. Increasing miR-200a expression inhibited HCC growth and synergized with the antitumor effects of doxorubicin. Inhibiting endogenous miR-200a promoted tumor growth and chemotherapeutic resistance. Increasing miR-200a expression inhibited tumor metabolism (ATP production, mitochondrial respiration, glycolysis), while inhibition of endogenous miR-200a reversed these effects. MiR-200a expression also increased autophagy and synergized with doxorubicin-mediated cytotoxicity. This study identifies a novel role of miR-200a in potentiating doxorubicin-mediated therapeutic effects in HCC.     

Effects of cis-unsaturated fatty acids on doxorubicin sensitivity in P388/DOX resistant and P388 parental cell lines

It has been reported that several cis-unsaturated fatty acids (c-UFAs) could increase doxorubicin (DOX) accumulation in cancer cells and hence elevate its cytotoxicity. However, some researchers showed that c-UFA pretreatment did not affect its cytotoxicity in special cell lines. It is possible that the different results occurred due to different cellular characteristics. We hypothesized that c-UFA treatment might modulate the activities of some antioxidant enzymes to affect the resistance of cells to DOX. In the present study, we examined how c-UFA pretreatment affected DOX cytotoxicity on mouse leukemia cell line, P388, and its resistant subline, P388/DOX, which we found to have significantly higher glutathione peroxidase (GPx) activity as well as P-glycoprotein (p-gp) overexpression. We chose two c-UFAs, gamma-linolenic acid (GLA) (18:3n-6) and docosahexaenoic acid (DHA) (22:6n-3). Cytotoxicity was measured by MTT (3-(4.5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and trypan blue exclusion assays. DOX accumulation and p-gp expression were measured by flow cytometry. The activities of catalase (CAT), superoxide dismutase (SOD), glutathione S-transferase (GST), and GPx were determined for both cell lines with and without treatment with GLA or DHA. Significant DOX accumulation occurred in both cell lines with GLA or DHA pretreatment, but without any change in p-gp expression in either cell line. Sensitivity to DOX cytotoxicity was improved by GLA or DHA pretreatment in P388/DOX in which only SOD activity was significantly increased, but not in the parental cell line P388 in which both SOD and CAT were significantly increased by the pretreatment. However, combined pretreatment of GLA or DHA with antioxidants, pyrrolidinedithiocarbamate (PDTC) or Vitamin C, could sensitize not only P388/DOX but also P388 cells to DOX. We conclude that the effects of c-UFA pretreatment on the sensitivity of cancer cells to DOX not only depend on the change in drug accumulation but also the change in the levels of antioxidant enzyme activities, and suggest that combined administration of c-UFAs, antioxidants, and DOX may be more effective in treating leukemia.     

Arylboronate prodrugs of doxorubicin as promising chemotherapy for pancreatic cancer

This study describes the synthesis of arylboronate-based ROS-responsive prodrugs of doxorubicin and their biological evaluation as anticancer agents. The determination of the most sensitive cancer type toward arylboronate prodrugs is crucial for further consideration of these molecules in clinical phase. To address this goal, an arylboronate-based profluorescent probe was used to compare the capacity of various cancer cell lines to efficiently convert the precursor into the free fluorophore. On the selected MiaPaCa-2 pancreatic cancer cells, a benzeneboronate prodrug exhibited 67% of the cytotoxicity obtained with the free doxorubicin. The prodrug was also able to induce tumor regression on MiaPaCa-2 pancreatic tumor model in ovo. Using this model, the amount of free doxorubicin liberated from this prodrug into the tumor was equivalent to the quantity measured after direct intratumoral injection of the same concentration of doxorubicin.     

Cytotoxicity, intracellular distribution and uptake of doxorubicin and doxorubicin coupled to cell-penetrating peptides in different cell lines: A comparative study

One of the major obstacles which are opposed to the success of anticancer treatment is the cell resistance that generally develops after administration of commonly used drugs. In this study, we try to overcome the tumour cell resistance of doxorubicin (Dox) by developing a cell-penetrating peptide (CPP)-anticancer drug conjugate in aim to enhance its intracellular delivery and that its therapeutic effects. For this purpose, two cell-penetrating peptides, penetratin (pene) and tat, derived from the HIV-1 TAT protein, were chemically conjugated to Dox. The cytotoxicity, intracellular distribution and uptake were accessed in CHO cells (Chinese Hamster Ovarian carcinoma cells), HUVEC (Human Umbilical Vein Endothelial Cells), differentiated NG108.15 neuronal cell and breast cancer cells MCF7drug-sensitive or MDA-MB 231 drug-resistant cell lines. The conjugates showed different cell killing activity and intracellular distribution pattern by comparison to Dox as assessed respectively by MTT-based colorimetric cellular cytotoxicity assay, confocal fluorescence microscopy and FACS analysis. After treatment with 3 μM with Dox-CPPs for 2 h, pene increase the Dox cytotoxicity by 7.19-fold in CHO cells, by 11.53-fold in HUVEC cells and by 4.87-fold in MDA-MB 231 cells. However, cytotoxicity was decreased in NG108.15 cells and MCF7. Our CPPs-Dox conjugate proves the validity of CPPs for the cytoplasmic delivery of therapeutically useful molecules and also a valuable strategy to overcome drug resistance.     

pH and reduction dual-sensitive copolymeric micelles for intracellular doxorubicin delivery

This study develops novel pH and reduction dual-sensitive micelles for the anticancer drug doxorubicin (DOX) delivery owing to the fact that the tumor tissues show low pH and high reduction environment. These sub-100 nm micelles present a core-shell structure under physiological conditions, but quickly release the loaded drugs responding to acidic and reductive stimuli. With disulfide bonds in each repeat unit of poly(β-amino ester)s, the novel copolymer was synthesized via Michael addition polymerization from 2,2'-dithiodiethanol diacrylate, 4,4'-trimethylene dipiperidine, and methoxy-PEG-NH(2). DOX released faster from micelles in a weakly acidic environment (pH 6.5) than at pH 7.4 or in the presence of a higher concentration (5 mM) of reducing agent (DTT). The release is even more effective in a scenario of both stimuli (pH 6.5 and 5 mM DTT). MTT assay showed that the DOX-loaded micelles had a higher cytotoxicity for HepG2 tumor cells than DOX at higher concentrations, and that blank micelles had a very low cytotoxicity to the tumor cells. Confocal microscopy observation showed that the micelles can be quickly internalized, effectively deliver the drugs into nuclei, and inhibit cell growth. These results present the copolymer as a novel and effective pH and reduction dual-responsive nanocarrier to enhance drug efficacy for cancer cells.     

In vitro cytotoxic study of immunoliposomal doxorubicin targeted to human CD34(+) leukemic cells

The expression of CD34 antigen in acute myelogenous leukemias is considered an unfavourable prognosis marker for response to anticancer drugs and duration of remission. This study investigated the applicability of long-circulating immunoliposomes loaded with doxorubicin targeted to CD34 antigen present on MDR(+) human myelogenous leukemia KG-1a cell line. Immunoliposomal doxorubicin showed a higher cytotoxicity against KG-1a cells than non-targeted liposomal doxorubicin, but it did not improve over that of free drug. Although no reversal of doxorubicin resistance was found to occur through its liposomal encapsulation, a therapeutic benefit can be obtained by the selective cytotoxicity observed. Endocytosis studies demonstrated that, after binding to CD34 antigen, the immunoliposomes are not internalized by the KG-1a cells and so the cytotoxic effect might be due to drug released into the space near the cell membrane. Thus, immunotargeting of liposomal doxorubicin to CD34(+) leukemic cells may only provide an ex vivo strategy for site-selective CD34(+) leukemia cell killing.     

Increased level of exogenous zinc induces cytotoxicity and up-regulates the expression of the ZnT-1 zinc transporter gene in pancreatic cancer cells

A balance between zinc uptake by ZIP (SLC39) and efflux of zinc from the cytoplasm into subcellular organelles and out of the cell by ZnT (SLC30) transporters is crucial for zinc homeostasis. It is not clear whether normal and cancerous pancreatic cells respond differently to increased extracellular zinc concentrations. Use of flow cytometry-based methods revealed that treatment with as little as 0.01 mM zinc induced significant cytotoxicity in two human ductal adenocarcinoma cell lines. In contrast, normal human pancreatic islet cells tolerated as high as 0.5 mM zinc. Insulinoma cell lines of mouse and rat origin also succumbed to high concentrations of zinc. Exposure to elevated zinc concentrations enhanced the numbers of carcinoma but not primary islet cells staining with the cell-permeable zinc-specific fluorescent dye, FluoZin-3, indicating increased zinc influx in transformed cells. Mitochondrial membrane depolarization, superoxide generation, decreased antioxidant thiols, intracellular acidosis and activation of intracellular caspases characterized zinc-induced carcinoma cell death. Only the antioxidant glutathione but not inhibitors of enzymes implicated in apoptosis or necrosis prevented zinc-induced cytotoxicity in insulinoma cells. Immunoblotting revealed that zinc treatment increased the ubiquitination of proteins in cancer cells. Importantly, zinc treatment up-regulated the expression of ZnT-1 gene in a rat insulinoma cell line and in two human ductal adenocarcinoma cell lines. These results indicate that the exposure of pancreatic cancer cells to elevated extracellular zinc concentrations can lead to cytotoxic cell death characterized by increased protein ubiquitination and up-regulation of the zinc transporter ZnT-1 gene expression.     

Lack of mechanism-based inactivation of rat hepatic microsomal cytochromes P450 by doxorubicin.

Administration of the antineoplastic doxorubicin to rodents causes depression of hepatic cytochrome P450 (CYP) dependent biotransformation, an effect that has been partially attributed to the ability of doxorubicin to stimulate microsomal lipid peroxidation. Since doxorubicin can be bioactivated by the CYP/NADPH-CYP reductase system to products that bind covalently to microsomal protein, we hypothesized that doxorubicin functions as a mechanism-based inactivator of hepatic microsomal CYPs and (or) NADPH-CYP reductase under conditions in which doxorubicin-stimulated NADPH-dependent lipid peroxidation is minimized. In vitro studies were conducted with hepatic microsomes isolated from untreated and phenobarbital-treated male rats. Unlike the positive control carbon tetrachloride, doxorubicin (10 microM) did not stimulate NADPH-dependent lipid peroxidation in microsomal incubations containing EDTA (1.5 mM). Doxorubicin did not cause NADPH-dependent loss of microsomal CYP, heme, or steroid hydroxylation activities selective for CYP2A, CYP2B, CYP2C11, and CYP3A. The positive control 1-aminobenzotriazole caused marked NADPH-dependent decreases in all of these parameters. Neither doxorubicin nor 1-aminobenzotriazole caused NADPH-dependent loss of NADPH-CYP reductase activity, and neither compound altered the immunoreactive protein levels of CYP2B, CYP2C11, CYP3A, and NADPH-CYP reductase. These results indicate that a pharmacologically relevant concentration of doxorubicin does not cause direct mechanism-based inactivation of hepatic microsomal CYPs or NADPH-CYP reductase, suggesting that the ability of doxorubicin to depress hepatic CYP-mediated biotransformation in vivo is due to lipid peroxidation mediated heme destruction, altered heme metabolism, and (or) decreased expression of selected CYP enzymes.     

Decreased zinc toxicity resulting from doxorubicin without increased GSSG export in three human lung cell lines

Zinc-mediated cytotoxicity is recognized, at least in part, by a decrease of reduced glutathione (GSH) and an increase in the oxidized form of glutathione (GSSG). Doxorubicin is a common inducer of multidrug-resistance-associated proteins and such proteins might, furthermore, be associated by an increased GSSG export rate. Therefore, zinc-mediated toxicity should be abolished after doxorubicin pretreatment. In the present study, zinc toxicity was characterized by methionine incorporation, glutathione content, and the GSSG/GSH ratio. Experiments were performed in three established lung cell lines comparing doxorubicin-pretreated cells with controls. Zinc-mediated toxicity was significantly decreased after pretreatment with doxorubicin as assessed by methionine-incorporation inhibition, GSH depletion, and/or GSSG increase in the two nonmalignant cell lines. Unexpectedly, zinc-associated GSSG export was not increased after doxorubicin pretreatment. This inconsistency might be explained as a result of a decreased zinc content in these cells, probably because of an increased export rate of zinc. The findings are in contradiction to the opinion of metal excretion by multidrug-resistance-associated proteins, matched to GSH conjugate excretion, as it is discussed for cadmium, for example.     

Synergy between enzastaurin doxorubicin in inducing melanoma apoptosis

Melanoma is resistant to most standard chemotherapeutics. We analysed the combined effect of doxorubicin and enzastaurin on cell death of four melanoma cell lines, namely G361, SK‐MEL3, A375 and SAN. Enzastaurin IC50 was calculated by measure of growth inhibition with MTS assay and corresponded to 2 μM; the half maximal cytotoxicity of doxorubicin was obtained at 3 μM dose. Evaluation of combination index showed synergism (CI > 1) or additive effect (CI = 1) with all melanoma cell lines, with enzastaurin doses ≥0.6 μM and doxorubicin doses ≥1 μM. Combination of the two drugs resulted in increase in caspase 3 and 8 activation, in comparison with activation by single agents. Caspase 8 activation was impaired by TNFR‐1 blocking. Our results show doxorubicin‐stimulated production of TNFα, whereas enzastaurin‐stimulated TNFR‐1 expression on plasma membrane. The effect on TNFR‐1 appeared to be mediated by PKCζ inhibition. Taken together, our findings suggest that enzastaurin increases doxorubicin‐induced apoptosis of melanoma by a mechanism involving, at least in part, activation of the TNF‐α signal.     

Oxidative and non-oxidative mechanisms in the inactivation of cardiac mitochondrial electron transport chain components by doxorubicin.

The quinonoid anthracycline, doxorubicin (Adriamycin) is a potent anti-neoplastic agent whose clinical use is limited by severe cardiotoxicity. Mitochondrial damage is a major component of this cardiotoxicity, and rival oxidative and non-oxidative mechanisms for inactivation of the electron transport chain have been proposed. Using bovine heart submitochondrial preparations (SMP) we have now found that both oxidative and non-oxidative mechanisms occur in vitro, depending solely on the concentration of doxorubicin employed. Redox cycling of doxorubicin by Complex I of the respiratory chain (which generates doxorubicin semiquinone radicals, O2-, H2O2, and .OH) caused a 70% decrease in the Vmax. for NADH dehydrogenase during 15 min incubation of SMP, and an 80% decrease in NADH oxidase activity after 2 h incubation. This inactivation required only 25-50 microM-doxorubicin and represents true oxidative damage, since both NADH (for doxorubicin redox cycling) and oxygen were obligatory participants. The damage appears localized between the NADH dehydrogenase flavin (site of doxorubicin reduction) and iron-sulphur centre N-1. Succinate dehydrogenase, succinate oxidase, and cytochrome c oxidase activities were strongly inhibited by higher doxorubicin concentrations, but this phenomenon did not involve doxorubicin redox cycling (no NADH or oxygen requirement). Doxorubicin concentrations of 0.5 mM were required for 50% decreases in these activities, except for cytochrome c oxidase which was only 30% inhibited following incubation with even 1.0 mM-doxorubicin. Our results indicate that low concentrations of doxorubicin (50 microM or less) can catalyse a site-specific oxidative damage to the NADH oxidation pathway. In contrast, ten-fold higher doxorubicin concentrations (or more) are required for non-oxidative inactivation of the electron transport chain; probably via binding to cardiolipin and/or generalized membrane chaotropic effects. The development of agents to block doxorubicin toxicity in vivo will clearly require detailed clinical studies of doxorubicin uptake in the heart.     

Reactions of Adriamycin with haemoglobin. Superoxide dismutase indirectly inhibits reactions of the Adriamycin semiquinone.

The Adriamycin semiquinone produced by the reaction of xanthine oxidase and xanthine with Adriamycin has been shown to reduce both methaemoglobin and cytochrome c. In air, but not N2, both reactions were inhibited by superoxide dismutase. With cytochrome c, superoxide formed by the rapid reaction of the semiquinone with O2, was responsible for the reduction. However, even in air, methaemoglobin was reduced directly by the Adriamycin semiquinone. Superoxide dismutase inhibited this reaction by removing superoxide and hence the semiquinone by displacing the equilibrium: Semiquinone + O2 in equilibrium or formed from quinone + O2-. to the right. This ability to inhibit indirectly reactions of the semiquinone could have wider implications for the protection given by superoxide dismutase against the cytotoxicity of Adriamycin. Oxidation of haemoglobin by Adriamycin has been shown to be initiated by a reversible reaction between the drug and oxyhaemoglobin, producing methaemoglobin and the Adriamycin semiquinone. Reaction of the semiquinone with O2 gives superoxide and H2O2, which can also react with haemoglobin. Catalase, by preventing this reaction of H2O2, inhibits oxidation of oxyhaemoglobin. Superoxide dismutase, however, accelerates oxidation, by inhibiting the reaction of the semiquinone with methaemoglobin by the mechanism described above. Although superoxide dismutase has a detrimental effect on haemoglobin oxidation, it may protect the red cell against more damaging reactions of the Adriamycin semiquinone.     

Quantification by laser scan microscopy of intracellular doxorubicin distribution.

Changes in intracellular drug localization accompany doxorubicin resistance in multidrug resistant tumor cells. The purpose of this study was to develop a method to quantify these changes and so detect different levels of resistance. Tumor cells were incubated with the fluorescent anthracycline doxorubicin (excitation at 480 nm; emission maximum at 560-590 nm) and were quantified using laser scanning microscopy. The fluorescent mode was used to record the intracellular drug distribution, whereas the absorption mode was used to define the nuclear and cytoplasmic boundaries. The cell compartments were delineated interactively on an image processing system and the ratio nuclear fluorescence/cytoplasmic fluorescence (N/C ratio) was determined. N/C ratios were: 1.8 in the Chinese hamster ovarian cell line AUXB1 and 0.1 in its MDR subline CHRC5; 3.8 in the human squamous lung cancer cell line SW-1573 and 1.8 and 0.4 in its MDR sublines SW-1573/2R120 and SW-1573/2R160, respectively; and 3.6 in the human myeloma cell line 8226/S and 2.1 and 1.0 in its MDR sublines 8226/Dox4 and 8226/Dox40, respectively. The doxorubicin distribution was independent of the doxorubicin concentration within a range from 1-32 microM. Furthermore, the progressive mean of the nuclear/cytoplasmic doxorubicin fluorescence ratio showed that a minimal sample size of 30 cells is necessary for reliable results. The results of two independent assessments showed a high reproducibility (r = 0.97). Thus, with the method described in this paper, it is possible to detect relatively low levels of doxorubicin resistance (factor 8).     

A mathematical model of doxorubicin penetration through multicellular layers

Inadequate drug delivery to tumours is now recognised as a key factor that limits the efficacy of anticancer drugs. Extravasation and penetration of therapeutic agents through avascular tissue are critically important processes if sufficient drug is to be delivered to be therapeutic. The purpose of this study is to develop an in silico model that will simulate the transport of the clinically used cytotoxic drug doxorubicin across multicell layers (MCLs) in vitro. Three cell lines were employed: DLD1 (human colon carcinoma), MCF7 (human breast carcinoma) and NCI/ADR-Res (doxorubicin resistant and P-glycoprotein [Pgp] overexpressing ovarian cell line). Cells were cultured on transwell culture inserts to various thicknesses and doxorubicin at various concentrations (100 or 50 μM) was added to the top chamber. The concentration of drug appearing in the bottom chamber was determined as a function of time by HPLC-MS/MS. The rate of drug penetration was inversely proportional to the thickness of the MCL. The rate and extent of doxorubicin penetration was no different in the presence of NCI/ADR-Res cells expressing Pgp compared to MCF7 cells. A mathematical model based upon the premise that the transport of doxorubicin across cell membrane bilayers occurs by a passive “flip-flop” mechanism of the drug between two membrane leaflets was constructed. The mathematical model treats the transwell apparatus as a series of compartments and the MCL is treated as a series of cell layers, separated by small intercellular spaces. This model demonstrates good agreement between predicted and actual drug penetration in vitro and may be applied to the prediction of drug transport in vivo, potentially becoming a useful tool in the study of optimal chemotherapy regimes.     

Comparative accumulation of the Hoechst 33258 fluorescent probe in leukemia P388 cells sensitive and resistant to doxorubicin

The authors studied accumulation of the fluorescent probe Hoechst 33258 in leukemia P 388 sensitive (P 388/0) and resistant to doxorubicin (P 388/DOX) cells. It was shown that intensity of fluorescence of the dye increased after binding with nuclear DNA during 25 min for both lines of the cells. Intensity of fluorescence was 40% greater in sensitive than resistant cells. If Triton X-100 was added no difference between two lines of the cell was observed. When doxorubicin was added to the cells with dye, the intensity of fluorescence decreased. It was suggested to use Hoechst 33258 for assessment extent doxorubicin accumulation in nuclei of the cells.