pegylated epirubicin - The FASEB Journal

©2006 FASEB

The FASEB Journal express article 10.1096/fj.05-4452fje. Published online February 2, 2006.

Nitric oxide modulates proapoptotic and antiapoptotic properties of chemotherapy agents: the case of NOpegylated epirubicin Luca Santucci,* Andrea Mencarelli,* Barbara Renga,* Gianfranco Pasut,† Francesco Veronese,† Antonella Zacheo,‡ Antonia Germani§ and Stefano Fiorucci* *Clinica di Gastroenterologia ed Epatologia, Dept. of Clinical and Experimental Medicine, University of Perugia, Perugia; †Dept. of Pharmaceutical Sciences, University of Padova, Padova; ‡Laboratorio di Patologia Vascolare, Istituto Dermopatico dell'Immacolata, Istituto di Ricovero e Cura a Carattere Scientifico, Rome; and §Laboratorio di Biologia Vascolare e Terapia Genica, Centro Cardiologico Fondazione Monzino, Istituto di Ricovero e Cura a Carattere Scientifico, Milan, Italy Corresponding author: Dr. Luca Santucci, Dept. of Clinical and Experimental Medicine, University of Perugia, Via Enrico dal Pozzo, 06122 Perugia, Italy. E-mail: [email protected] ABSTRACT The use of the anthracycline epirubicin (EPI) is limited by the risk of a dilatory congestive heart failure that develops as a consequence of induction of a mitochondrial-dependent cardiomyocyte and endothelial cell apoptosis. Nitric oxide (NO) increases the antitumoral activity of several chemotherapics, while it provides protection against apoptosis induced by oxidative stress both in endothelial cells and cardiomyocytes. The aim of the present study was to investigate whether the addition of an NO-releasing moiety to a pegylated derivative of EPI (p-EPI-NO) confers to the drug a different cytotoxic profile against tumoral and normal cells. The cytotoxic profile of the drugs was investigated in Caco-2 cell line, in embryonic rat heart-derived myoblasts (H9c2), in adult cardiomyocytes, and in endothelial cells (HUVEC). p-EPI-NO was more efficient than EPI in inducing Caco-2 cell apoptosis, while it spared HUVEC, H9c2 cells and adult cardiomyocytes from EPI-induced toxicity. Exposure of cells to p-EPI-NO resulted in a NOmediated inhibition of cellular respiration followed by mitochondrial membrane depolarization and cell death in Caco-2 cells but not in HUVEC and H9c2 cells in which mitochondrial membrane polarization was maintained at the expense of glycolytically generated ATP. These findings indicate that addition of an NO-releasing moiety to p-EPI increases the anti-neoplastic activity of the drug, while it reduces its cytotoxicity against nonneoplastic cells. Key words: anthracycline-induced miocardiopathy membrane depolarization ● colon cancer cells

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he anthracycline semiquinone doxorubicin is one of the most effective and widely used anti-cancer drugs. Doxorubicin is employed in many chemotherapy regimens for the treatment of breast, liver, and colon cancers; sarcomas; and leukemia. Unfortunately, its

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use is limited by the risk of a delayed, life-threatening, and irreversible form of dilatory congestive heart failure, with a mortality that exceeds 50% within 2 yr (1–3). Current strategies to limit myocardial injury caused by this drug include the encapsulation of doxorubicin in longcirculating pegylated liposomes (4) and the use of anthracycline analogs, such as epirubicin (EPI) (5). EPI differs from doxorubicin only in the orientation of the 4′-hydroxyl group, and it has greater lipid solubility resulting in faster cellular uptake. Although EPI-induced cardiotoxicity occurs at higher cumulative doses (>900 mg/m2) compared with doxorubicin (>500 mg/m2), chronic and irreversible myocardial damage leading to congestive heart failure increases abruptly with higher cumulative doses (5–7). Cellular mechanisms of EPI-induced myocardial damage are similar to that of doxorubicin and involve a mitochondria-dependent apoptosis of cardiomyocytes (8–15). Polyethylene glycol (PEG) is a widely used polymer for drug delivery. Thanks to its unique characteristics, like absence of immunogenicity and toxicity, high solubility either in aqueous and organic solvents, PEG is extensively used for conjugation to peptide and nonpeptide drugs (16). The resulting conjugates are less immunogenic and more hydrosoluble than parent molecules. The derivatives are also more stable toward degradative enzymes and show an increased molecular weight, resulting in a prolonged half-life (16). In addition, in the case of anti-tumor drugs, PEG conjugation increases the ability of the chemotherapy agent to penetrate tumor cell membranes (17). Nitric oxide (NO) is a diffusible messenger that plays an important role in cell growth and differentiation and in apoptosis. NO sensitizes neoplastic cells to ionizing radiation and photodynamic therapy (18–20) and increases the antitumoral activity of several chemotherapic agents, including doxorubicin (21), cisplatin (22), melphalan (23), and fludarabine (24). Interestingly, NO-releasing nonsteroidal antiinflammatory drugs (NSAIDs) inhibit proliferation and cause apoptosis of tumoral cells more efficiently than traditional NSAIDs (25–27). Along with its antitumoral effects, NO participates in a wide range of biological reactions to maintain normal myocardial function and an antithrombotic intravascular milieu (28). In particular, NO protects endothelial cells and cardiomyocytes from apoptosis induced by oxidative stress, proinflammatory cytokines and chemotherapy agents (28–31). Furthermore, NO provides protection against apoptosis induced by doxorubicin in cardiomyocytes (32), suggesting that it may be useful also in the prevention of anthracycline-induced cardiomyopathy. Since the PEG backbone allows addition of multiple molecules of NO, we have taken advantage of this property to generate a series of polyethylene glycol-EPI (p-EPI) derivatives carrying different amounts of NO. Here, we report on the effect of one of such agents, obtained by adding eight molecules of NO to p-EPI (p-EPI-NO). Compared with standard EPI, p-EPI-NO shows enhanced anti-tumor activity, while it spares embryonic rat heart-derived myoblasts, cardiomyocytes isolated from adult mice, and human umbilical endothelial cells (HUVEC) from EPI-induced toxicity. This study provides the ground for development of more potent and safer anticancer drugs based on the combination of NO with pegylated anthracyclines.


MATERIALS AND METHODS Synthesis of the NO derivative of p-EPI The synthesis of the NO derivative of pegylated EPI is based on the hetero-bifunctional HOPEG-COOH; to increase the number of NO releasing groups per PEG chain, a dendrimer structure was built at the carboxylic end of this polymer using the amino adipic acid as branching moiety. One gram of HO-PEG-COOH (MW 3400 Da), dehydrated by toluene azeotropic distillation, was dissolved in 10.0 ml of CH2Cl2. To the solution, 177.8 mg of pnitrophenylchloroformate (MW 201.5 Da) were added and the pH was brought to 8.5 by Et3N. The reaction was left to proceed overnight under stirring. The product, p-nitrophenyl-carbonatePEG-COOH, was recovered by precipitation from diethyl ether and filtered in a funnel. The derivative (970.0 mg) was coupled with 205.1 mg of EPI in 10.0 ml of DMF, and the pH was adjusted to 8.0 by Et3N. The reaction flask was covered by aluminum foil to prevent drug degradation from light. After 10 h, 80.0 ml of CH2Cl2 were added and the excess of EPI was removed by extraction from the organic phase with several acid water treatments until the aqueous phase did not present red color. The organic phase, dried by anhydrous sodium sulfate, was concentrated to small volume and dropped into 250.0 ml of diethyl ether under vigorous stirring to precipitate of the product EPI-PEG-COOH (p-EPI) that was recovered by filtration and dried under vacuum. The carboxylic group of p-EPI (900 mg) was activated by DCC/NHS in CH2Cl2, and, after recovery by precipitation from diethyl ether, the activated EPI-PEG-NHS was added to 8.0 ml of a solution containing 72.4 mg of amino adipic acid (AD) in borate buffer 0.1 M pH 8.0. The coupling reaction was left to proceed for 1 h. Then, the pH was brought to 5.0 by HCl 1N and the product, EPI-PEG-AD (p-EPI-AD), was extracted from the reaction mixture by CH2Cl2 (60.0 ml×6). The organic phase, dried by anhydrous sodium sulfate and concentrated to small volume, was dropped into 250.0 ml of diethyl ether under vigorous stirring to precipitate and to recover p-EPI-AD. The two reaction steps of carboxylic group activation and coupling with AD, reported above, were repeated two times with p-EPI-AD leading to the formation of the dendrimer structure on the PEG chain end, namely EPI-PEG-AD-(AD)2-(AD)4. EPI-PEGAD-(AD)2-(AD)4 (830.0 mg) was dissolved in 10.0 ml of a 15.0% (p/v) solution of butandiol mononitrate in CH2Cl2. To the solution, 533.8 mg of dicyclohexylcarbodiimide (DCC) and 233.0 mg of 1-hydroxybenzotriazole were added and the pH was adjusted to 8.0 by Et3N. The reaction was left to proceed overnight protected from light and under stirring. The product was recovered by precipitation from ethyl ether and purified by precipitation from ethyl acetate at 0°C. The final product p-EPI-NO was collected by filtration on a funnel and dried under vacuum; 797.0 mg were recovered with an overall yield of 45.1%. Cell cultures The embryonic rat heart-derived cell line H9c2 was obtained from the Istituto Zooprofilattico of Brescia, Italy, and was maintained in DMEM supplemented with 10% FBS in 95% air-5% CO2 using standard culture methods. Cells were plated 24 h before treatment at a density of 150 cells/mm2. All studies were conducted in the presence of 10% FBS. Although growth factors and other possible confounding factors are present in the serum, removing the serum from the experiments could also confound interpretation through altering the baseline viability of the cultures and causing apoptotic changes independently of the doxorubicin treatment. Cardiomyocytes were isolated from 2– to 3-mo-old C57Bl6J male mice as described previously


(33, 34). Briefly, hearts were explanted and retrogradely perfused in MEM containing 3 mM HEPES, 2 mM glutamine, 10 mM taurine, 20 U/l insulin, 100 U/ml penicillin, and 100 mg/ml streptomycin at constant pressure (100 cm H2O) at 37°C. The enzymatic digestion was performed in the indicated medium containing 280 U/ml type II collagenase (Worthington, Lakewood, NJ) and 50 mM Ca2+. When the heart became swollen (after 10 min), ventricles were removed, cut, and further digested at 37°C in the same solution. The supernatant containing cardiomyocytes was then filtered and centrifuged at 500 rpm. Cells were resuspended before in 250 and then in 500 mM Ca2+ and then cultured in the same medium containing 1.2 mM Ca2+ with or without p-EPI or p-EPI-NO for 48 h. At the end of the treatment, cardiomyocytes were counted and processed for the apoptosis assay using an ELISA kit accordingly to manufacturer’s instructions (Cell Death Detection ELISA kit, Roche, Milan, Italy). Primary cultures of HUVEC were from Istituto Zooprofilattico of Brescia (Brescia, Italy). HUVEC were grown in endothelial basal medium supplemented with bovine brain extract (12 µg/ml), human epithelial growth factor (10 ng/ml), hydrocortisone (1 µg/ml), penicillin (100 units/ml), streptomycin (100 µg/ml), and gentamycin (5 µg/ml) at 37°C in a humidified atmosphere containing 5% fetal bovine serum. Caco-2 cells, a human colon adenocarcinoma cell line, were obtained from American Type Culture Collection (Rockville, MD). Caco-2 cells were cultured in MEM containing 20% fetal calf serum, glutamine, and antibiotics and supplemented with nonessential amino acids and 1 mM sodium pyruvate. Cells were grown in 75 cm2 culture flasks, and the medium was changed every other day. The cells were aliquoted into 12-well plates (104 cells/well) and allowed to adhere overnight before drug challenge. Detection of nitrite/nitrate generation Cells were exposed to the drugs and cultured in minimum essential medium without fetal bovine serum for 24 h. The concentration of nitrite and nitrate in the medium was measured with a NO2/NO3 detection kit (Cayman Chemical, Ann Arbor, MI). Briefly, an aliquot of the medium was collected and centrifuged at 1000 g for 15 min at room temperature. According to the instructions, 80 µl of the supernatant were treated with nitrate reductase and co-enzyme for 1 h at 37°C and then reacted with 2,3-diaminonaphtalene under acidic conditions at room temperature for 15 min. After neutralization with sodium hydroxide, the fluorescence from naphtalenetriazole at 460 nm (excited at 355 nm) was measured with a fluorescence plate reader. Determination of apoptosis At the end of the incubation period, apoptotic cells were identified at cytofluorimeter (EPICS Elite flow cytometer; Coulter, Miami, FL) using annexin V and propidium iodide (PI) according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN). Cytosolic cytochrome c measurement To measure cytochrome c translocation, cells were harvested, lysed in buffer A [250 mM sucrose, 20 mM HEPES-KOH (pH 7.4), 10 mM KCl, 1.5 mM Na-EGTA, 1.5 mM Na-EDTA, 1 mM MgCl2, 1 mM DTT, and mixture of protease inhibitors], and ground in a glass Dounce homogenizer with a tight pestle (B-type). Homogenates were centrifuged at 800 g for 10 min at 4°C. Supernatants were further centrifuged at 26,000 g for 20 min at 4°C. The concentration of


cytochrome c on the resulting supernatant (cytosolic fraction) was measured by using a specific ELISA kit according to the manufacturer’s instruction (Alexis Italia, Florence, Italy). Caspase 3 activity measurement Cell lysates were evaluated for caspase 3 activity using a specific caspase fluorometric assay according to manufacturer’s instructions (ApoAlert™, Clontech, Palo Alto, CA). Determination of mitochondrial membrane potential Mitochondrial membrane potential (Δψm) was measured by using the probe JC-1, which selectively enters mitochondria. JC-1 exists in a monomeric form emitting at 527 nm after excitation at 490 nm. Depending on Δψm, JC-1 forms J-aggregates that are associated with a large shift in emission (590 nm). Color dye changes reversibly from orange to green as mitochondrial membranes become depolarized. For staining, cell suspensions were adjusted to a density of 0.5 × 106 cells/ml and incubated in complete medium with JC-1 (10 µg/ml) for 10 min at 37°C in the dark. Cells were then washed in PBS, resuspended in a total volume of 400 µl, and immediately analyzed by flow cytometry (EPICS Elite flow cytometer; Coulter) equipped with a single 488 nm argon. A total of 5,000 cells was analyzed for green fluorescence with a 525 nm filter and for orange fluorescence with a 575-nm filter. All data were analyzed with Elite software version 4.02. Western blot Western blots were performed according to standard protocols. Briefly, whole cell lysates, mitochondrial and cytosolic fractions from a fixed quantity of cells (2.5×105) were boiled and reduced and underwent 10−15% sodium dodecyl sulfate-PAGE (SDS-PAGE). After the blots were transferred onto nitrocellulose membranes and were blocked, they were incubated overnight at 4°C with the following polyclonal antibodies: rabbit anti-human Bax, anti-Bcl-2, anti-p53 and anti-phosphorylated p53 (Pp53) (Santa Cruz Biotechnology, Santa Cruz, CA). After samples were washed, specific binding was detected with an appropriate horseradish peroxidaseconjugated second layer. Control blots were probed with isotype-matched primary reagents followed by an appropriate horseradish peroxidase-conjugated second layer. The blots were developed by use of an enhanced chemiluminescent technique (ECL, Amersham Biosciences Europe, Milan, Italy) according to the manufacturer’s instructions. The protein bands were then scanned (Wathman, Biometra), and relative intensities were quantified with the use of a specific software (Delta Sistemi, Roma, Italy). Real-time reverse transcription-PCR Total RNA was processed directly to cDNA by reverse transcription with Superscript III (Invitrogen). All PCR primers were designed using software PRIMER3-NEW using published sequence data from the NCBI database. Primers were synthesized by MWG BIOTECH. For human GAPDH, the sense primer was 5′-gacaacagcctcaagatcatcagc-3′ and antisense 5′gtagaggcagggatgatgttctgg-3′; for human survivin the sense primer was: 5′-ggaccaccgcatctctacat3′ and the antisense 5′-gttcctctatggggtcgtca-3′. All PCR reactions were performed in triplicate in an iCycler iQ system (Bio-Rad, Hercules, CA). We used the expression of human GAPDH to


normalize the expression data of target genes. GAPDH was used to correct for differences in the amount of total RNA added to a reaction and to compensate for different levels of inhibition during reverse transcription of RNA and during PCR. Production of reactive oxygen species The fluorogenic probe DCFH-DA was used to determine the intracellular production of reactive oxygen species (ROS). DCFH-DA can be deacetylated in cells, where it can react quantitatively with intracellular radicals, mainly H2O2, to be converted to its fluorescent product, 2,7dichlorofluorescein (DCF), which is retained within the cells providing an index of cell cytosolic oxidation. At the end of the incubation period, samples of cells were washed, incubated with 20 µM DCFH-DA for 30 min at 37°C, and analyzed using a flow cytometer (EPICS Elite flow cytometer; Coulter). The results were expressed as fluorescence units. Metabolite determinations ATP and lactate concentrations were measured in the same experiments. Aliquots of the cell suspensions were acidified with HClO4 and neutralized with KHCO3. Lactate concentrations were measured in the supernatants by using a specific ELISA kit (Sigma Chemical, Milan, Italy) and ATP/ADP concentrations by chemiluminescence using a commercially available kit (Sigma Chemical) following the manufacturer’s instructions. Cytocrome oxidase activity was measured by using a commercial kit (Sigma Chemical). Cellular concentration of reduced thiols and superoxide dismutase activity Cells were harvested in 200 µl of buffer containing 20 mM Tris (pH 7.4), 150 mM NaCl, 10% glycerol, 1% Triton X-100, and 4 mM EGTA. An aliquot was used to quantify both protein and nonprotein thiols, using a thiol quantification kit (Molecular Probes, Eugene, OR) according to directions provided by the manufacturer. Superoxide dismutase (SOD) activity was measured by using a commercial kit following the manufacturer’s instruction (Cayman Chemical CO, Ann Arbor, MI). Statistical analysis All values in the figures and text are expressed as mean ± SE. The variation between data sets was tested with ANOVA, and the significance was tested with unpaired t tests, with a Bonferroni modification for multicomparison of data. Differences were considered significant when P was < 0.05. RESULTS p-EPI-NO is metabolized by cancer and normal cells to generate NO We have first investigated whether p-EPI-NO undergoes cell metabolization and generates free NO. Exposure of H9c2, HUVEC and Caco-2 cells to p-EPI-NO resulted in a concentration- and time-dependent increase in nitrite/nitrate released in cell supernatants (Fig. 1A and B). On the other hand, no release of NO was observed by incubating p-EPI-NO with medium alone or


exposing cells to EPI and p-EPI (data not shown). Therefore, these data indicate that p-EPI-NO penetrates cell membrane and releases free NO and/or NO-derived compounds. p-EPI-NO exerts a potent cytotoxic activity against colon cancer cells As shown in Fig. 2A, incubation of Caco-2 cell line with p-EPI-NO resulted in a concentrationdependent induction of cancer cell apoptosis that was significantly higher as compared with either EPI and p-EPI (n=8, P