Bioconjugate Chem. 2007, 18, 894−902
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The Same Drug but a Different Mechanism of Action: Comparison of Free Doxorubicin with Two Different N-(2-Hydroxypropyl)methacrylamide Copolymer-Bound Doxorubicin Conjugates in EL-4 Cancer Cell Line Lubomı´r Kova´rˇ,*,† Jirˇ´ı Strohalm,‡ Petr Chytil,‡ Toma´sˇ Mrkvan,† Marek Kova´rˇ,† Ondrˇej Hovorka,† Karel Ulbrich,‡ and Blanka R ˇ ´ıhova´† Department of Immunology and Gnotobiology, Institute of Microbiology ASCR, v.v.i., Vı´denˇska´ 1083, 142 20 Prague 4, Czech Republic, and Department of Biomedical Polymers, Institute of Macromolecular Chemistry ASCR, v.v.i., Heyrovsky´ Square 2, 162 06 Prague 6, Czech Republic. Received August 8, 2006; Revised Manuscript Received February 8, 2007
Doxorubicin is one of the most potent anti-tumor drugs with a broad spectrum of use. To reduce its toxic effect and improve its pharmacokinetics, we conjugated it to an HPMA copolymer carrier that enhances its passive accumulation within solid tumors via the EPR effect and decreases its cytotoxicity to normal, noncancer cells. In this study, we compared the antiproliferative, pro-survival, and death signals triggered in EL-4 cancer cells exposed to free doxorubicin and doxorubicin conjugated to a HPMA copolymer carrier via either enzymatically (PK1) or hydrolytically (HYD) degradable bonds. We have previously shown that the intracellular distribution of free doxorubicin, HYD, and PK1 is markedly different. Here, we demonstrated that these three agents greatly differ also in the antiproliferative effect and cell death signals they trigger. JNK phosphorylation sharply increased in cells treated with HYD, while treatment with free doxorubicin moderately decreased and treatment with PK1 even strongly decreased it. On the other hand, treatment with free doxorubicin greatly increased p38 phosphorylation, while PK1 and HYD increased it slightly. PK1 also significantly increased ERK phosphorylation, while both the free doxorubicin and HYD conjugate slightly decreased it. Long-term inhibition of JNK significantly increased both proliferation and viability of EL-4 cells treated with free doxorubicin, showing that the JNK signaling pathway could be critical for mediating cell death in EL-4 cells exposed to free doxorubicin. Both activation of caspase 3 and decreased binding activity of the p50 subunit of NFκB were observed in cells treated with free doxorubicin and HYD, while no such effects were seen in cells incubated with PK1. Analysis of the expression of genes involved in apoptosis and regulation of the cell cycle demonstrated that free doxorubicin and HYD have very similar mechanisms of action, while PK1 has very different characteristics.
INTRODUCTION Doxorubicin is one of the most effective anthracycline antibiotics with a broad anti-tumor spectrum. Unfortunately, numerous side effects such as severe cardiotoxicity, bone marrow suppression, and alopecia limit its use. Development of tumor-targeted formulations is one of the main lines of research for improving the safety and efficacy of anthracyclines. For this purpose, we have used a water-soluble, nontoxic copolymerbasedonN-(2-hydroxypropyl)methacrylamide(HPMA) as a carrier for anti-cancer drugs. Covalent conjugation of doxorubicin to HPMA copolymer eliminates its adverse cytotoxicity, as this conjugate is biologically inactive when circulating in the bloodstream (1). Enhanced and tumor-selective accumulation of HPMA conjugate within the solid tumor is achieved either passively due to the enhanced permeability and retention (EPR) effect (2) or actively by addition of a targeting moiety (e.g., antibodies, lectins, carbohydrates) (3). Anthracyclines cause apoptosis of cancer cells by a complex network of events which include intercalation into the DNA, generation of reactive oxygen species, topoisomerase II inhibition, and DNA damage (4). HPMA conjugate that contains doxorubicin bound via a pH-sensitive bond (HYD) possesses similar biological properties as free doxorubicin (5), because * Corresponding author. Tel.: +420241062158. Fax: +420241721143. E-mail address:
[email protected]. † Institute of Microbiology ASCR. ‡ Institute of Macromolecular Chemistry ASCR.
doxorubicin is released inside the cell and then acts analogously to free doxorubicin. Controlled release of doxorubicin within cancer cells is achieved by hydrolysis of hydrazone conjugates having doxorubicin bound via pH-sensitive bonds (6). Those bonds are stable at physiological pH 7.4, but are effectively degraded at pH 5. Inside the cell, doxorubicin is released quickly in endosomes or lysosomes. Conjugates with doxorubicin bound to the HPMA carrier through a hydrazone bond are highly cytotoxic and cytostatic in vitro and in vivo (7). On the other hand, intracellular events triggered by HPMA copolymer-conjugated doxorubicin via an enzymatically degradable bond (PK1) remain unclear. These conjugates were originally designed to release the drug from the oligopeptide side chain by the activity of lysosomal proteases (8). Some authors initially demonstrated that HPMA copolymer conjugates with enzymatically degradable bonds can be stronger proapoptotic inducers than free doxorubicin (9). However, later it was shown that both apoptosis and necrosis are induced in cells exposed to proteolytically cleavable HPMA copolymer conjugates (10-12). Our recent data do not support the idea that the enzymatic degradation and doxorubicin release from PK1 are the most important mechanisms responsible for target cell death. They rather suggest that immediately after penetrating the plasma membrane the whole conjugate intercalates into the membrane system of the cell, causing its collapse and death (10, 11). PK1 with enzymatically degradable bond between the oligopeptide spacer GFLG and doxorubicin was tested in phase I
10.1021/bc060246e CCC: $37.00 © 2007 American Chemical Society Published on Web 04/03/2007
Doxorubicin and HPMA Conjugates in Cancer Cells
and was recommended for phase II of clinical study (13). A conjugate similar to PK1 but containing human nonspecific antibody was used for the treatment of patients with advanced cancer (14). In this study, we used the EL-4 T-cell lymphoma to determine the differences in intracellular action of free doxorubicin, PK1, and HYD. We determined their impact on intracellular signaling pathways (mitogen-activated protein kinases, NFκB, and apoptosis) and expression of genes involved in the regulation of cell cycle, and showed differences in their intracellular distribution.
EXPERIMENTAL PROCEDURES Materials. Hydrazine monohydrate, methacryloyl chloride, 1-aminopropan-2-ol, 4-nitrophenol, 6-aminohexanoic acid, glycyl-L-phenylalanine, L-leucylglycine, 2,2′-azobis(isobutyronitrile) (AIBN), 2-(dimethylamino)ethyl methacrylate, dimethylformamide (DMF), N,N′-dicyclohexylcarbodiimide (DCC), 1-hydroxybenzotriazole (HOBT), dimethyl sulfoxide (DMSO), tert-butylcarbazate, and doxorubicin hydrochloride (Dox.HCl) were purchased from Fluka Chemie AG. 2,4,6-Trinitrobenzene1-sulfonic acid was purchased from SERVA Feinbiochemica Heidelberg. All other reagents and solvents were of analytical grade. Synthesis and Characterization of Monomers. N-(2-Hydroxypropyl)methacrylamide (HPMA) was synthesized as described in (15) using Na2CO3 as a base in the methacryloylation reaction (mp 70 °C; elemental analysis (calcd/found): 58.80/ 58.98% C; 9.16/9.18% H; 9.79/9.82% N). N-(tert-Butoxycarbonyl)-N′-(6-methacrylamidohexanoyl)hydrazine (Ma-AH-NHNHBoc) was prepared by a two-step synthesis consisting of methacryloylation of 6-aminohexanoic acid followed by reaction of the resulting N-methacryloyl-6-aminohexanoic acid with tertbutylcarbazate as described in (7). Yield: 6.06 g (46%). Mp 110-114 °C. Elemental analysis: calcd C 57.70%, H 8.33%, N 13.46%; found C 58.66%, H 8.84%, N 13.16%. N-Methacryloylglycyl-DL-phenylalanyl-L-leucylglycine and N-methacryloylglycyl-DL-phenylalanyl-L-leucylglycine 4-nitrophenyl ester (Ma-GFLG-ONp) were synthesized as described earlier (1). Mp 134-136 °C. Amino acid analysis: Gly/L-Phe/ D-Phe/L-Leu ) 2.05/0.54/0.47/1.00. Elemental analysis (calcd/ found): C 59.89/59.21%; H 6.07/6.25%; N 12.04/12.32%. HPLC showed two peaks of equal areas at 14.41 min (L-Phe peptide) and 14.71 min (D-Phe peptide). Purity of all monomers was examined by HPLC (LDC Analytical, U.S.A.) using a reversed-phase column Tessek SGX C18 (15 × 33 mm) with UV detection at 230 nm, eluent watermethanol with gradient 50-100 vol % methanol, flow rate 0.5 mL/min. Synthesis of Polymer Precursors. Copolymer poly(HPMAco-MA-AH-NHNH2). Copolymer poly(HPMA-co-MA-AHNHNH-Boc) was prepared by radical solution polymerization (AIBN, 1 wt %; monomer concentration, 14 wt % in methanol solution; molar ratio HPMA/MA-AH-NHNH-Boc 93/7; 60 °C; 23 h) in a sealed ampule under nitrogen. The copolymer was isolated by precipitation into a mixture of acetone/diethyl ether (2:1). The protecting Boc group was removed by dissolution of the polymer in concd trifluoroacetic acid (TFE), and a final polymer was isolated after dilution with methanol by precipitation into ethyl acetate and purified by gel filtration using a column filled with Sephadex LH-20 and methanol as a solvent. The poly(HPMA-co-MA-AH-NHNH2) was isolated by precipitation into ethyl acetate, separated by filtration, and dried in vacuum to constant weight. Poly(HPMA-co- Ma-GFLG-ONp) was prepared by radical precipitation polymerization as described in (15).
Bioconjugate Chem., Vol. 18, No. 3, 2007 895
HPMA homopolymer poly(HPMA) was prepared by radical solution polymerization using AIBN as initiator (0.8 wt %) and monomer concentration 16 wt % in methanol. Polymerization was carried out at 60 °C for 22 h in a sealed ampule under nitrogen; the polymer was isolated by precipitation into acetone and reprecipitated from methanol into acetone. Synthesis and Characterization of Polymer-Dox Conjugates. The conjugate bearing Dox attached via a pH-sensitive hydrazone bond poly(HPMA-co-MA-AH-NHN ) Dox) (HYD) and the conjugate bearing Dox attached via the enzymatically degradable oligopeptide sequence poly(HPMA-co- Ma-GFLGDox) (PK1) were prepared as described earlier, HYD in (6) and PK1 in (15). The polymer-drug conjugates were freed of low-molecularweight impurities (such as Dox, 4-nitrophenol) by gel filtration using a Sephadex LH-20 column with methanol elution and tested for the content of the free drug using a Pharmacia FPLC equipped with a Superose 6 column and by HPLC after extraction of free Dox from an aqueous polymer solution into chloroform. HPLC analysis of monomers was conducted on an HPLC analyzer (LDC Analytical, U.S.A.) using a reversed-phase column Tessek SGXC18 (125 × 4 mm) with UV detection at 230 nm, solvent methanol-water, gradient 50-100% methanol, and flow rate 0.5 mL/min. Content of Dox in the conjugates was measured spectrophotometrically in water using ) 11 500 L mol-1 cm-1 at λ ) 488 nm). Amino acid analysis was performed using an amino acid analyzer (LDC Analytical, U.S.A.) (precolumn OPA derivatization, reversed-phase column Tessek SGX C18, 250 × 4 mm, gradient sodium acetate buffer-methanol, fluorescence detector Fluoromonitor 4100). Determination of the molecular weight of all polymers was carried out with a FPLC Pharmacia system equipped with RI, UV, and multiangle light scattering DAWN DSP-F (Wyatt Co., U.S.A.) detectors using 0.3 M acetate buffer pH 6.5 and a Superose 6 column. The content of hydrazide groups in a precursor and HYD conjugate was determined by a modified TNBSA assay as described (16). The following polymers were prepared: HPMA copolymer bearing Dox attached via enzymatically degradable spacer (PK1), Mw ) 24.8 kDa, Mw/Mn ) 1.5, total Dox content 5.2 wt %, free Dox content
