Synthesis of New Cu Complex Based on Natural 5Z,9Z-Eicosadienoic Acid: Effective Topoisomerase I Inhibitor and Cytotoxin against the Cisplatin-Resistant Cell Line.

The complex (bipy)2Cu(5,9-eicd) was prepared by the reaction of Cu(OAc)2 with 5Z,9Z-eicosadienoic acid and 2,2'-bipyridine in methanol. The new copper complex showed high antitumor activity in vitro toward A2780cis, A2780, Hek293, K562, HL60, Jurkat, and U937 cell lines and efficiently inhibited human topoisomerase I. Using flow cytofluorometry, (bipy)2Cu(5,9-eicd) was studied for the effect on the cell cycle and apoptosis-inducing activity in tumor cells.

Introduction

In 1969, Rosenberg and co-workers reported the first results concerning the cytotoxicity of platinum compounds against murine tumors,[1] which initiated a new area of medicinal chemistry dealing with metal-containing antitumor agents. Currently, platinum-based compounds such as cisplatin, carboplatin, and oxaliplatin are widely used in medical practice.[2] Although cisplatin and its derivatives have been successfully included into the armory of anticancer chemotherapeutical agents, there are problems related to their high toxicity and low selectivity to malignant tumors and, in some cases, drug resistance of cancer cells. This stimulates extensive studies aimed at the development of new metal derivatives that would be free from the indicated drawbacks.[3] Among non-platinum compounds, researchers are focused on less toxic copper-based compounds.[4] The properties exhibited by complexes are substantially dependent on the nature of ligands and the donor atoms that are coordinated to the metal.[5]

The relationship between the structure and biological activity of copper complexes has been investigated for as long as more than 5 decades. The biological activities of many copper compounds is governed by the chelating properties of the ligands towards transition-metal ions.[6] It is also known that metal-ion chelates show a much better uptake by living organisms than free metal ions. However, many issues concerning the behavior of copper ions in the intracellular space remain unsolved: it is absolutely unknown whether or not the structure of sophisticated complexes is retained in the cell as the copper ion is released and what happens when the copper valence changes from Cu(II) to Cu(I).[7] An increase in the copper-ion concentration inside the cells is known to induce general intoxication to inhibit the DNA synthesis and oxidative phosphorylation, and also to result in total thiol oxidation to disulfides.[8] The biological effects of copper complexes are highly diversified and selective, and all this complicates the search for and systematization of literature sources. All of the listed issues can apparently attest to the high diversity of biological effects of these compounds.

A fairly important factor for the design of modern antitumor compounds is the selection of the molecular target, most often, an enzyme that performs a key function in tumor cells. It is known that many copper complexes with bipyridine ligands that exhibit antitumor activity are effective inhibitors of topoisomerases, that is, key cell cycle enzymes.[4,5] Recently, it was also shown in a number of works that 5Z,9Z-dienoic acids are efficient nonspecific inhibitors of human topoisomerase.[9]

In view of the foregoing, we hypothesized that copper carboxylates with pyridine ligands based on 5Z,9Z-dienoic acids could also exhibit high cytotoxicity via targeted action on topoisomerases.

This communication presents preliminary results on the synthesis of copper bipyridine complexes based on 5Z,9Z-eicosadienoic acid, which exhibited the highest inhibitory activity toward topoisomerase I, and study of its antitumor properties in vitro on several tumor cell lines of various etiology by means of flow cytofluorometry.

Results and Discussion

The complex Cu(bpy)2(5,9-eicd) (bpy is 2,2′-bipyridyl, 5,9-eicd is 5Z,9Z-eicosadienoic acid) 1 was synthesized by the reaction of Cu(OAc)2·H2O with 2,2′-bipyridine (2 equiv) and 5Z,9Z-eicosadienoic acid (1 equiv) in methanol by a modified procedure (Figure ).[10] A similar copper complex based on arachidic acid, Cu(bpy)2(arach) (bpy is 2,2′-bipyridyl, arach is arachidic acid) 2, was prepared as a reference compound (Figure ).

Figure 1

Structures of complexes Cu(bpy)2(5,9-eicd) (1) and Cu(bpy)2(arach) (2).

We studied the ability of the compounds Cu(bpy)2(5,9-eicd) (bpy: 2,2′-bipyridyl, 5,9-eicd: 5Z,9Z-eicosadienoic acid) 1 and Cu(bpy)2(arach) (bpy: 2,2′-bipyridyl, arach: arachidic acid) 2 synthesized in this work to inhibit topoisomerase I.

The results presented in Figure A,B indicate that the relaxation of supercoiled plasmid DNA with inhibition of topoisomerase I (Topogen) by the complex Cu(bpy)2(5,9-eicd) (in this case, 3 enzyme units of topoisomerase I are inhibited by 2 nM the test compound) results in decrease in the residual amount of the supercoiled plasmid DNA and increase in the number of formed topoisomers (lanes 2–10) as the concentration of the test compound is increased from 1 to 100 nM. When the compound concentration is in the range from 1 to 20 nM, the supercoiled plasmid is predominantly accumulated upon addition of ethidium bromide to the gel (Figure B, samples 8–12), while at concentrations above 20 nM (Figure B, samples 3–5), the level of the open circular form increases. These results lead to the conclusion that Cu(bpy)2(5,9-eicd) suppresses the topo I catalytic activity; however, its action is dose-dependent, and in concentrations of 20 nM and higher, it can also influence the formation of covalent complexes of DNA with topo I. Compound 2 did not show an inhibitory effect on the enzyme topoisomerase I. Thus, the synthesized copper complex has more than 10 times higher human topoisomerase inhibitory activity than the initial unsaturated acid.

Figure 2

Electropherogram of the products of relaxation of supercoiled plasmid DNA in vitro under the action of topoisomerase I (Topogen) and Cu(bpy)2(5,9-eicd) (A) without ethidium bromide and (B) with ethidium bromide. Lane: (1). Relaxed plasmid DNA (pHOT1) (2). Supercoiled plasmid DNA (pHOT1). (3–12). Supercoiled plasmid DNA + topoisomerase I (1 unit) + compound 1 at various concentrations (3: 100 nM, 4: 80 nM, 5: 20 nM, 6: 5 nM, 7: 4 nM, 8: 3 nM, 9: 2 nM, 10: 1 nM). (11). Supercoiled plasmid DNA + topoisomerase I (1 unit) + camptothecin (10 μM). (12). Supercoiled plasmid DNA + topoisomerase I + dimethyl sulfoxide (DMSO) (3%).

The quantitative and qualitative analyses of cell viability, cell cycle, and apoptosis-inducing activity of Cu(bpy)2(5,9-eicd) (bpy is 2,2′-bipyridyl, 5,9-eicd is 5Z,9Z-eicosadienoic acid) were performed by means of Guava Nexin Reagent, Guava Cell Cycle, and Guava ViaCount kits (Millipore).

The cytotoxic activity of the complex Cu(bpy)2(5,9-eicd) (bpy is 2,2′-bipyridyl, 5,9-eicd is 5Z,9Z-eicosadienoic acid) in vitro against the Jurkat, HL-60, K562, and U937 human leukemia cells and HEK293 kidney cancer cells was tested using the Guava ViaCount kit (Millipore) (Table ).

Table 1

Inhibition of Jurkat, HL-60, K562, U937, A2780cis, A2780, and HEK293 Cell Viability by the Complexes Cu(bpy)2(5,9-eicd) (1) and Cu(bpy)2(arach) (2), CC50 (μM) ± SE (μM)

	Jurkat	HL-60	K562	U937
CC50 (1)	0.05 ± 0.001	0.04 ± 0.001	0.10 ± 0.007	0.08 ± 0.006
CC50 (2)	11.26 ± 1.09	9.38 ± 0.91	15.28 ± 1.43	13.17 ± 1.29
CC50 (cisplatin)	0.12 ± 0.004	0.10 ± 0.003	0.21 ± 0.006	0.18 ± 0.006

The complex Cu(bpy)2(5,9-eicd) in concentrations from 0.05 to 0.22 μM exhibited a clearly pronounced cytotoxic effect against all types of cancer cells used in the tests. However, the highest CC50 value was found for the A2780cis cell line (0.47 μM), whereas the CC50 values for Jurkat, K562, HL-60, and U937 cells were 0.05, 0.1, and 0.08 μM, respectively. It is noteworthy that the arachidic acid-based copper complex 2 and cisplatin taken as reference compounds showed substantially lower antitumor activities in vitro than complex 1 (Table ).

It should be noted that the synthesized compounds exhibit good selectivity index (SI) (SI = CC50 fibroblasts/CC50 cancer cells); in particular for compound 1, the selectivity index varies from 2 to 18 with respect to all tumor cells.

The Cu(bpy)2(5,9-eicd)-induced apoptosis in the Jurkat, HL-60, K562, U937, and HEK293 cell cultures was estimated by the detection of phosphatidylserine externalization on the plasmatic membrane after treatment of cell cultures with the test compound. It is worth noting that the effect of the complex Cu(bpy)2(5,9-eicd) on the induction of apoptosis in Jurkat cells is more pronounced than in other types of cells, which is in line with the higher cytotoxicity of this compound against this cell line. As can be seen from Figure , the action of Cu(bpy)2(5,9-eicd) on the HEK293 tumor cell culture induces a substantial dose-dependent increase in the number of apoptotic cells occurring at early and late apoptosis stages. The highest percentage of early and late apoptosis (∼91%) was observed at a compound concentration of 0.1 μM. In particular, as shown in Figure , both early and late apoptosis stages for the Jurkat cells increase as compared with HL60 (p ≤ 0.0003), U937 (p ≤ 0.0005), HEK293 (p ≤ 0.0007), and K562 (p ≤ 0.00019).

Figure 3

HEK293 cells treated with different concentrations of complex Cu(bpy)2(5,9-eicd) were double-stained with annexine V/PI and analyzed by flow cytometry. (A) Control; (B) 1 (0.025 μM); (C) 1 (0.05 μM); and (D) 1 (0.1 μM).

Under conventional light microscopic examination, HEK293 cells treated with the complex Cu(bpy)2(5,9-eicd) differed from the control untreated sample by the presence of clear-cut morphological changes. The complex Cu(bpy)2(5,9-eicd) induced nuclear condensation and apoptotic body formation. These morphological changes, characteristic of early apoptosis, were visible as soon as 3 h after treatment. Figure shows cells with different concentrations of the test compound after 24 h of exposure.

Figure 4

Conventional light microscopy of HEK293 cells treated with different concentrations of the test compound after 24 h of exposure. (A) Control; (B) 1 (0.025 μM); (C) 1 (0.05 μM); and (D) 1 (0.1 μM).

To find out whether the retarding effect exerted by complex Cu(bpy)2(5,9-eicd) is caused by the cell cycle arrest, we studied the distribution of cell cycle phases for five cell lines, Jurkat, HL60, K562, and U937, and the adhesion cell culture HEK293 after the appropriate treatment with the test compound by flow cytometry. According to cell cycle studies by the Guava Cell Cycle Reagent, the complex Cu(bpy)2(5,9-eicd) proved to be a potent inducer of hypodiploid cell population (sub-G1 phase) in all five cell lines. As shown in Figure , incubation of Jurkat cells for 24, 48, or 72 h after treatment with the complex resulted in 14.37 ± 1.02, 19.35 ± 1.33, or 35.99 ± 2.38% of hypodiploid cells, respectively. The percentage of cells in the G1 phase decreased compared with the control from 67.57 to 16.39%, whereas the percentage of cells in the S-phase substantially increased (from 27.50 to 47.69%) in the samples incubated for 72 and 48 h. These results indicate that the complex Cu(bpy)2(5,9-eicd) arrests the cell cycle in the S phase depending on the time of incubation.

Figure 5

Results of cell-cycle analysis by flow cytometry and representative of the profiles of cell cycle distribution in three independent experiments is shown. Jurkat cells treated with complex Cu(bpy)2(5,9-eicd) for (A) absence of complex, (B) 24 h, (C) 48 h, and (D) 72 h. The percentage of Jurkat cells after staining with propidium iodide. Data are presented as mean ± standard deviation of three independent experiments.

It was shown that the complex Cu(bpy)2(5,9-eicd) is absorbed rather rapidly by cancer cells and induces apoptosis phenomena as soon as after a 3 h incubation. The presence of the 5Z,9Z-eicosadienoic acid moiety in this complex markedly increases the stability and lipophilicity of the molecule, which may be significant for penetration through the tumor cell membrane. The complex Cu(bpy)2(5,9-eicd) showed an exceptionally high cytotoxicity on a panel of five tumor cell lines, the IC 50 values being in the range from 0.05 to 0.22 μM; this is an order of magnitude lower than this value for cisplatin.

It was found that compound 1 significantly reduces the proliferative activity and viability of cisplatin-resistant A2780cis cells, as shown in a study of the cytotoxicity of the compounds under study (Table ). According to real-time cell analysis (RTCA), Cu(bpy)2(5,9-eicd) is a substance that exhibits a strong inhibitory effect even at the lowest concentration on A2870cis-resistant ovarian cancer cells on cisplatin cells (Figure ). At the same time, cisplatin itself, added at a concentration of 1 μM (CC50 = 0.94 ± 0.028 for A2780cis cells), also slightly reduces the proliferative ability of the A2870cis line in the first two days; however, by the third day, the cells regain their ability to grow, which is confirmed by an increase in cell index (CI).

Figure 6

Effect of different sample concentrations of cisplatin and Cu(bpy)2(5,9-eicd) on real-time cell analyzer curves xCELLigence RTCA system generated with A2870cis cells. Effect of cisplatin 0.5 μM/mL (blue line), cisplatin 1.0 μM (gray line), Cu(bpy)2(5,9-eicd) 0.3 μM (turquoise line), Cu(bpy)2(5,9-eicd) 0.5 μM (green line), Cu(bpy)2(5,9-eicd) 1.0 μM (purple line), and control (red line, control group: untreated cells).

Thus, it can be argued that the introduction of 1 into the culture medium reduced the proliferation and viability of cisplatin-resistant ovarian cancer for four days, indicating a potential antiproliferative effect of 1 for cisplatin-resistant ovarian cancer.

The ability of the complex 1 to inhibit topoisomerase I results in disruption of DNA synthesis, inhibition of transcription, and initiation of apoptosis by the extrinsic pathway. These results shed light on the key molecular mechanisms that underlie the anticancer activity of Cu(bpy)2(5,9-eicd) and are important for the development of DNA-specific copper complexes as antitumor drug candidates.

Conclusions

Thus, the newly synthesized copper complex (bipy)2Cu(5,9-eicd) based on 5Z,9Z-eicosadienoic acid and 2,2′-bipyridine shows a high antitumor activity in vitro toward Hek293, K562, HL60, Jurkat, and U937 cell lines, being more efficient than the known anticancer drug cisplatin. Furthermore, (bipy)2Cu(5,9-eicd) was found to arrest the cell cycle in the S phase, depending on the incubation time, and to act as an efficient human topoisomerase I inhibitor, which can help to make assumptions on the possible mechanism of its antitumor activity.

Experimental Section

General (Instruments)

Chromatographic analysis was performed on a Shimadzu GC-9A instrument using a 2000 × 2 mm column, the SE-30 (5%) stationary phase on Chromaton N-AW-HMDS (0.125–0.160 mm), helium carrier gas (30 mL/min), and temperature programming from 50 to 300 °C at a 8 °C/min rate. IR spectra were recorded on a Bruker VERTEX 70V FTIR spectrometer using KBr disks over the range of 400–4000 cm–1. Melting points were recorded on a Stuart SMP3 advanced digital melting point apparatus. The 1H- and 13C NMR spectra were recorded in CDCl3 on a Bruker Avance-400 spectrometer (100.62 MHz for 13C, 400.00 MHz for 1H). High-resolution mass spectra (HRMS) were measured on a Bruker maXis instrument using electrospray ionization.[11] In experiments on selective collisional activation (CAD), activation energy was set at the maximum abundance of fragment peaks (see figures legend). A syringe injection was used for solutions in MeCN–H2O, 50/50 vol % (flow rate 3 mL/min). Nitrogen was applied as a dry gas; interface temperature was set at 180 °C. Elemental analysis of the samples was determined by elemental analyzer firm KarloErba, model 1106. Reactions with organometallic compounds were performed in a dry argon flow. The solvents were dried and distilled immediately prior to use. Commercially available Cu(OAc)2, 2,2′-bipyridine, and arachidic acid (Aldrich) were used. 5Z,9Z-Eicosadienoic acid was synthesized according previously published technique.[9g]

Chemistry

Synthesis of Copper Bipyridine Complexes 1, 2 Based on 5Z,9Z-Eicosadienoic and Arachidic Acids (General Procedure)

A methanol solution (7.5 mL) of 2,2′-bipyridine (0.3 mmol) was added to a solution of Cu(OAc)2·H2O (0.3 mmol) in CH3OH–H2O (15 mL, 3:1) with vigorous stirring. A methanol solution (10 mL) of acid (0.15 mmol) was then added and the mixture stirred for 30 min. The reaction solution was filtered and left to stand at room temperature the precipitate evaluated after partial evaporation of solvent was collected by filtration.

Complex Cu(bpy)2(5,9-eicd) (1)

Yield 30%; Dark blue amorphous powder. IR (KBr pellet, ν, cm–1): 3428, 3118, 3077, 3057, 3030, 2920, 2851, 1654, 1605, 1577, 1562, 1469, 1377, 1159, 1108, 1022, 972, 931, 758, 731, 662. HMRS: [M + H]+ found = 683.3347; [M + Na]+ found = 705.3961; [M + Na]+ found = 721.2904, C40H51CuN4O2 anal. calcd 682.3308.

Complex Cu(bpy)2(arach) (2)

Yield 48%; Blue powder, mp = 126–128 °C. IR (KBr pellet, ν, cm–1): 3433, 3114, 3079, 3058, 3039, 2920, 2851, 1601, 1576, 1562, 1467, 1377, 1162, 1108, 1018, 972, 930, 754, 734, 660. HMRS: [M + H]+ found = 687.3694; [M + Na]+ found = 709.3513; [M + Na]+ found = 725.3253, C40H55CuN4O2 anal. calcd 686.3621.

Biology

Cell Culture

Cells (Jurkat, K562, U937) were purchased from Russian Cell Culture Collection (Institute of Cytology of the Russian Academy of Sciences) and cultured according to standard mammalian tissue culture protocols and sterile technique. Human cancer cell lines HL60, HEK293, A2780cis, and A2780 were obtained from the HPA Culture Collections (U.K.). All cell lines used in the study were tested and shown to be free of mycoplasma and viral contamination.[12]

HEK293 and fibroblasts cell line was cultured as monolayers and maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco BRL) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution at 37 °C in a humidified incubator under a 5% CO2 atmosphere.

Cells were maintained in the DMEM medium (adherent cell culture HEK293, A2780cis, A2780, and fibroblasts) or RPMI 1640 (Jurkat, K562, and U937) (Gibco) supplemented with 4 mM glutamine, 10% FBS (Sigma), and 100 units/mL penicillin-streptomycin (Sigma). HL60 cells were cultured in RPMI 1640 with 20% FBS. All types of cells were grown in an atmosphere of 5% CO2 at 37 °C. The cells were subcultured at 2–3 days intervals. Adherent cells HEK293, fibroblasts, and A2870cis were suspended using trypsin/ethylenediaminetetraacetic acid and counted after they have reached 80% confluency. Cells were then seeded in 24-well plates at 5 × 104 cells per well and incubated overnight. Jurkat, HL-60, K562, and U937 cells were subcultured at 2 day intervals with a seeding density of 1 × 105 cells per 24-well plates in RPMI with 10% FBS or 20% FBS for HL60 cells.[13]

Cytotoxicity Assay

Viability (live/dead) assessment was performed by staining cells with 7-aminoactinomycin D (Biolegend). After treatment, the cells were harvested, washed 1–2 times with phosphate-buffered saline (PBS), and centrifuged at 400g for 5 min. Cell pellets were resuspended in 200 μL of flow cytometry staining buffer (PBS without Ca2+ and Mg2+, 2, 5% FBS) and stained with 5 μL of 7-AAD staining solution for 15 min at room temperature in the dark. The samples were acquired on NovoCyte 2000 FlowCytometry System (ACEA) equipped with 488 nm argon laser. Detection of 7-AAD emission was collected through a 675/30 nm filter in the FL4 channel.[14]

Viability and Apoptosis

Apoptosis was determined by flow cytometric analysis of annexin V and 7-aminoactinomycin D staining. Briefly, 200 μL of Guava Nexin reagent (Millipore, Bedford, MA) was added to 5 × 105 cells in 200 μL, and the cells were incubated with the reagent for 20 min at room temperature in the dark. At the end of incubation, the cells were analyzed on NovoCyte 2000 flow cytometry system (ACEA).[15]

Cell Cycle Analysis

Cell cycle was analyzed using the method of propidium iodide staining. Briefly, the cells were plated in 24-well round-bottom plates at a density 10 × 105 cells per well, centrifuged at 450g for 5 min, and fixed with ice-cold 70% ethanol for 24 h at 0 °C. Cells were then washed with PBS and incubated with 250 μL of Guava Cell Cycle Reagent (Millipore) for 30 min at room temperature in the dark. Samples were analyzed on NovoCyte 2000 flow cytometry system (ACEA).[15]

DNA Topoisomerase I Assay

The inhibitory activity of Cu complexes 1, 2 were determined using the Topoisomerase I Drug Screening Kit TG-1018-2 using protocol by Topogen.

Real-Time Cell Analysis (RTCA)

Eight thousand A2780cis culture Cells were seeded in two 8-well plates with an integrated microelectronic sensor array in 600 μL of culture medium (iCELLigence real-time cell analyzer; ACEA Biosciences, San Diego, CA). After 24 h, the drugs were added for a total volume of 100 μL. The cell proliferation and survival were monitored in real-time by measuring the cell to electrode responses of the seeded cells. In each individual E-well, the cell impedance was measured and converted to cell index (CI) values by the RTCA software version 1.2 (Roche Diagnostics GmbH). The graphs were generated in real time by the iCELLigence system. Untreated and DMSO treated cells served as controls.[16]