Cytotoxicity of the traditional chinese medicine (TCM) plumbagin in its copper chemistry

Abstract

The anticancer traditional Chinese medicine (TCM), plumbagin (PLN), was isolated from Plumbago Zeylanica. Reaction of plumbagin with Cu^II salt, afforded [Cu(PLN)₂]·2H₂O (1). With 2,2′-bipyridine (bipy) as a co-ligand, PLN reacts with Cu^II to give [Cu(PLN)(bipy)(H₂O)]₂(NO₃)₂·4H₂O (2). 1 and 2 were characterized by elemental analysis, IR, ESI-MS spectra. Their crystal structures were determined by single crystal X-ray diffraction methods. The in vitro cytotoxicity of PLN, 1 and 2 against seven human tumour cell lines was assayed. The metal-based compounds exhibit enhanced cytotoxicity vs. that of free PLN, suggesting that these compounds display synergy in the combination of metal ions with PLN. The binding properties of PLN, 1 and 2 to DNA were investigated through UV-vis, fluorescence, CD spectra, and gel mobility shift assay, which indicated that 1 and 2 were non-covalent binding and mainly intercalated the neighboring base pairs of DNA. PLN, 1 and 2 exhibit inhibition activity to topoisomerase I (TOPO I), but 1 and 2 were more effective than PLN.

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Introduction

The success of cisplatin and its related platinum complexes as anticancer agents has sparked an ever increasing search for other active transition metal anticancer complexes.^1-5 Copper, as a bioessential element plays a key role in the endogenous oxidative DNA damage associated with aging and cancer, and its potential antitumour properties have attracted attention recently because it is thought to be less toxic than nonessential metals such as platinum.^6-13 A large number of Cu^II complexes have been synthesized, such as synthetic bleomycin models Cu(3-Clip-Phen) and derivatives.¹⁴ Most of them exhibit high antitumour activity towards a variety of tumour cell lines, including copper(II) complexes with thiosemicarbazone, quinoline derivatives and nitrogen-containing heterocyclic ligands.^15,16 For instance, Ruiz-Azuara and co-workers reported and patented a series of new antineoplastic agents based on mixed chelate copper(II) complexes named Casionpeínas^® with general formulae [Cu(N–N)(α-L-aminoacidato)]NO₃ and [Cu(N–N)(O–O)]NO₃ (N–N = 1,10-phenanthroline or 2,2′-bipyridine, O–O = acac or sala, acac is the acetylacetonate anion), showing satisfied cytotoxicity with a significantly lower IC₅₀ value.^17,18

On the other hand, over long-term folk practice, a large number of TCMs have been screened and used for treating and preventing various chronic conditions, such as cancer, atherosclerosis, aging, diabetes, and other degenerative diseases.¹⁹ In recent years, there has been a global surge in the popularity of traditional medicine, and the strongly increasing interest in developing new pharmaceutical products from such resources has been aroused. Some research results indicate that new coordination compounds based on traditional Chinese medicines (TCMs) provide a novel approach to a potential (pro-) drug.^20,21

In this study, our objective was to exploit the benefits of TCM in the synthesis of a new TCM-copper compound to create a therapeutic agent with antitumour activity. A potential candidate was identified as plumbagin, 5-hydroxy-2-methyl-1,4-naphthoquinone (PLN, Scheme 1). Plumbagin is a potent toxic natural product extracted from Plumbago Zeylanica L. (Plumbaginaceae), which has been used in China as well as other Asian countries for the treatment of rheumatoid arthritis, dysmenorrhea, bruising, and even cancer. The anticancer property of PLN against HeLa, P388 lymphocytic, leukemia, colon cancer and hepatoma has been reported.^22,23 PLN is structurally derived from naphthoquinone, as many quinone chemotherapeutic drugs used today, such as daunorubicin and adriamycin. The principal drawback from which these drugs suffer is that they are cardiotoxic and cannot be administered to patients over prolonged periods of time. Earlier studies have shown that formation of metal complexes with daunorubicin and adriamycin greatly influence the toxicity imparted by them, in fact, complexes of adriamycin and carminomycin with Cu^II and Fe^III are less cardiotoxic than the parent drugs, but are effective against P388 leukemia.²⁴ The coordination of copper(II) ion by NSAIDs improves the pharmaceutical activity of the drugs themselves and reduces their undesired toxicity effects in human and veterinary medicine.²⁵ Therefore, we selected plumbagin to react with a copper(II) salt to form new TCM-copper(II) compounds with high cytotoxicity potency and non-covalent DNA binding modes. In addition, the combination of Cu^II, plumbagin, and the N-donor heterocyclic ligand 2,2′-bipyridine (bipy) has also been studied in an attempt to examine the possible synergetic effects. This paper describes the isolation of PLN, synthesis and crystal structures of its two Cu^II complexes, their in vitro cytotoxicity and binding properties to DNA along with inhibition to topoisomerase I (TOPO I).

Scheme 1 Plumbagin (PLN).

Results and discussion

Plumbagin (PLN)

Plumbagin (Scheme 1) is a naphthoquinone and was isolated from the ethanol extract of roots of P. zeylanica. The structure of PLN was confirmed by ¹H NMR and ¹³C NMR, as well as single crystal X-ray diffraction analysis. PLN is a planar molecule with phenol O and carbonyl O atoms as potential donors, comparable with the previous determined structure (Fig. S1, ESI†).²⁶

[Cu(PLN)₂]·2H₂O(1)

PLN reacted with CuCl₂·2H₂O in the presence of MeONa giving rise to 1, which was characterized by elemental analysis, IR, and ESI-MS as well as the X-ray single crystal diffraction method. As shown in Fig. 1, 1 is the monomeric trans-form of the planar CuO4 core with regard to the methyl groups of two PLN ligands (the sum of four angles around the Cu(1) is 360°). The bite angle of O(1)–Cu(1)–O(2) is 93.21(14)°, indicating a slightly distorted square plane around Cu(1). The Cu–O bond lengths (Cu(1)–O(1) = 1.907(3), Cu(1) –O(2) = 1.883(3) Å) are in agreement with other Cu(II) complexes, e.g. [Cu(hino)₂]^27,28 (Hhino: 2-hydoxy-4-isopropylcyclohepta-2,4,6-trienone) (Cu–O: 1.900(2), 1.904(3) Å) and [Cu(ONQ)₂(H₂O)₂]_n (NQ: 2-hydroxy-1,4-naphthoquinone, Cu–O: 1.954(3), 2.336(3) Å).²⁹ The whole molecule is planar (Fig. S2a, ESI†). There are no classical hydrogen bonds in the packing view, but a two-dimensional network is generated via a nonclassical hydrogen bond (C(7)–H(7)⋯O(3), 3.309(6) Å, symmetry code: 1.5 −x, y −0.5, 0.5 −z; Fig. S2b, ESI†). In the ESI-MS of 1, a major peak at m/z 436.8 was observed, which could be attributed to a negatively charged species [Cu(PLN)₂]⁻ (C₂₂H₁₃CuO₆).

Fig. 1 ORTEP view of [Cu(PLN)₂]·2H₂O (1) showing atom labeling, thermal ellipsoids are drawn at the 30% probability and hydrogen atoms as well as two lattice water molecules have been omitted for clarity. Selected bond distances (Å) and angles (°): Cu(1)–O(1) 1.907(3), Cu(1)–O(2) 1.883(3); O(1)–Cu(1)–O(2) 93.21(14).

[Cu(PLN)(bipy)(H₂O)]NO₃·2H₂O (2)

PLN reacted with Cu(NO₃)₂·3H₂O in the presence of MeONa, with bipy as a co-ligand, giving rise to 2, which was characterized by elemental analysis, IR, and ESI-MS as well as X-ray single crystal diffraction method. As shown in Fig. 2, the crystal structure of 2 consists of dicationic [Cu(PLN)(bipy)(H₂O)]₂²⁺, two uncoordinated nitrate anions and four water solvent molecules. The Cu^II centre is in a distorted octahedral N₂O₄ coordination sphere, in which the equatorial plane is formed by two nitrogen atoms from bipy and two oxygen atoms from PLN, and the two apical sites are occupied by one water oxygen with Cu(1)–O(4) of 2.357(5) Å and one carbonyl oxygen (O3A) from another PLN with Cu(1)–O(3A) of 2.7539(2) Å. Thus, a dimeric species is formed. The dihedral angle between the plane defined by Cu(1), O(1) and O(2) and that defined by Cu(1), N(1) and O(2) is 7.989(24)°, which indicates a distortion of the N2O2 equatorial plane. In addition, a one-dimensional infinite chain is generated via H-bonding (C(11)–H(11)⋯O(3), 3.294(7) Å, symmetry code: x, y, 1 + z; Fig. S3, ESI†). The bidentate chelating coordination mode of PLN is similar to that in complex 1. The bond parameters associated with the Cu^II centres are unexceptional and similar to those of naphthoquinone complexes [Cu(ONQ)₂(H₂O)₂]_n²⁹ (NQ: 2-hydroxy-1,4-naphthoquinone, Cu–O: 1.954(3), 2.336(3) Å), [Fe(lawsonato)₂(H₂O)₂]³⁰ (lawsonato is the anion of 2-hydroxy-1,4-naphthoquinone, Fe–O: 2.045(4), 2.143(4) Å). In the ESI-MS of 2, a major peak at m/z 423.9 was observed, which could be attributed to a positively charged species [Cu(bipy)(PLN)(H₂O)]⁺ (C₂₁H₁₇CuN₂O₄).

Fig. 2 ORTEP view of [Cu(PLN)(bpy)(H₂O)]₂(NO₃)₂·4H₂O (2) showing atom labeling, thermal ellipsoids are drawn at 30% probability and hydrogen atoms as well as two NO₃⁻ anions have been omitted for clarity. Selected bond distances (Å) and angles (°): Cu(1)–O(1) 1.895(3), Cu(1)–O(2) 1.920(3), Cu(1)–N(1) 1.999(4), Cu(1)–N(2) 1.978(4), Cu(1)–O(4) 2.357(5), Cu(1)⋯O(3A) 2.7539(2); O(1)–Cu(1)–O(2) 91.63(15), O(1)–Cu(1)–N(1) 171.72(14), O(1)–Cu(1)–O(4) 96.26(16), N(1)–Cu(1)–N(2) 80.91(17), N(1)–Cu(1)–O(4) 90.38(16) (A: 2 − x, −y, 1 − z).

In summary, PLN retains its planarity after deprotonation and subsequent coordination to metal ions. The metal complexes themselves are close to planar because 1 is planar; and in solution, 2, the dimeric species easily breaks to afford the mono-copper(II) complex cation, and the aqua ligand also easily disassociates to give [Cu(bipy)(PLN)]⁺. These structural features may be important in their enhanced antitumour activity because they can intercalate the neighboring base pairs of targeted DNA.

Cytotoxicity assay in vitro

The in vitro cytotoxicity of plumbagin and its two Cu^II complexes were estimated by MTT assay³¹ against seven typical human tumour cell lines (with cisplatin, [Cu(bpy)₂(H₂O)₂](NO₃)₂ and [Cu(acac)(bpy)]NO₃ as the positive control) including Renal 786-O, MCF-7, HepG2, CNE2, HCT116, BEL7404, NCI-H460. As shown in Table 1, against Renal 786-O, 1 is 5 times stronger than PLN, while 2 is 8 times stronger than PLN. Against MCF-7, 2 is 4 times stronger than PLN, and against HCT116, 2 is about 6 times stronger than PLN. Against CNE2, 1 is 6 times stronger than PLN. The two copper(II) complexes exhibit significant cytotoxicity against the selected tumour cell lines, 2 to 27 times stronger than that of cisplatin. Against NCI-H460 and BEL7404, 1 and PLN are inactive, but 2 shows satisfied cytotoxicity. In contrast to PLN and cisplatin, against the selected tumour cell lines, 2 against NCI-H460 is the most sensitive, 27 times stronger than cisplatin. Besides, against MCF-7, the cytotoxicity of 1 and 2 are comparable to copper(II) mixed chelate [Cu(N–N)(acac)]NO₃ and [Cu(N–N)(glycinato)]NO₃ complexes (IC₅₀ in 103.7 ∼ 2.2 μM),¹⁷ also comparable to platinated copper(3-clip-phen) complexes (IC₅₀ in 5.8 ∼ 0.9 μM).^14a Against HepG2, 1 and 2 are less effective than [Cu(4-Mecdoa)(phen)₂] (IC₅₀ = 1.3 μM);¹³ but more effective than Cu–Ad (Cu₂(adenine)₄Cl₄·2EtOH)(IC₅₀ = 40 μM).³² These results suggest that the effectiveness of TCM like PLN as an antitumour agent was improved by coordination to the metal ions.

Table 1 IC₅₀ values for PLN and complexes 1 and 2 against seven tumour cell lines (μmol L⁻¹)

Compounds	786-O	MCF-7	HCT116	HepG2	CNE2	NCI-H460	BEL7404
— represents inactive; ND represents no data available toward CNE2. Cisplatin, [Cu(bpy)2(H2O)2](NO3)2 and [Cu(acac)(bpy)]NO3 as the positive control references.
1	3.4 ± 1.3	7.0 ± 0.9	8.9 ± 1.0	5.0 ± 1.0	11.8 ± 5.9	—	—
2	2.5 ± 0.9	3.2 ± 1.1	5.9 ± 1.4	9.0 ± 0.7	—	2.0 ± 1.2	12.9 ± 3.6
PLN	17.9 ± 4.5	12.9 ± 2.8	34.7 ± 10.4	—	67.6 ± 2.6	—	—
[Cu(bpy)2(H2O)2](NO3)2	5239.3 ± 1411.8	61.2 ± 7.9	267.4 ± 13.3	113.2 ± 17.3	ND	71.7 ± 0.6	266.6 ± 31.7
[Cu(acac)(bpy)]NO3	398.7 ± 39.9	71.4 ± 7.5	254.1 ± 19.7	162.2 ± 13.8	ND	90.5 ± 2.7	160.5 ± 23.8
Cisplatin	70.1 ± 15.0	74.9 ± 10.0	62.0 ± 14.0	98.6 ± 23.0	96.3 ± 7.4	55.5 ± 4.7	36.4 ± 7.6

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In order to investigate the effect of metal ion or the ternary structure on the cytotoxicity, the controlled experiments using the complexes [Cu(bpy)₂(H₂O)₂](NO₃)₂ and [Cu(acac)(bpy)]NO₃ under similar experimental conditions were carried out. The data are tabulated in Table 1. From Table 1, it was found that against the selected tumour cell lines, 1 and 2 are at least 18 times effective as [Cu(bpy)₂(H₂O)₂](NO₃)₂ and [Cu(acac)(bpy)]NO₃. The IC₅₀ values of the two controlled copper(II) complexes against the tested tumour cell lines (except for CNE2 unavailable) studied here, are 1–3 orders of magnitude lower than that of complexes 1 and 2. Additionally, the combination of PLN and Cu^II with bpy, against 786-O, MCF-7 and HCT116, 2 is more effective than 1; moreover, except for CNE2 (inactive), towards the other 6 tested tumour cell lines, 2 also exhibits more effective cytotoxicity than the controlled copper(II) complexes. Interpretation of these results leads to the conclusion that the enhanced effect of 1 and 2 on the viability of the selected tumour cell lines is due to the synergistic contribution of their components.^16c,33

To determine the dose- and time-dependent relationship, 786-O and MCF-7 cell lines were exposed to increasing doses of PLN, 1 and 2 for increasing periods of time (24, 48 and 72 h), and cell viability was measured by the MTT assay. As shown in Fig. 3 and 4, both 786-O and MCF-7 cells exhibit increased sensitivity to the complexes, compared to PLN, in a dose- and time-dependent manner. More specifically, towards 786-O cells, 1 and 2 exhibit more effective cytotoxicity than PLN does, displaying an enhanced antiproliferative effect compared to PLN. Towards MCF-7, with increasing concentrations of 2, 2 causes increased cytotoxicity higher than either PLN or 1. As shown in Fig. 4, against 786-O cell lines, 1 and 2 display a higher cytotoxicity than PLN with increased periods of time. However, towards MCF-7 cells, 2 exhibits a higher cytotoxicity than that of PLN and 1. In the light of the above results, 1 and 2 may be potential metal-based anticancer agents.

Fig. 3 Dose-dependent cytotoxic effects of PLN, 1 and 2 against 786-O and MCF-7 cell lines treated with various doses of PLN (-■-), 1 (-●-) and 2 (-▲-) for periods of time [(a) = 24 h, (b) = 48 h, (c) = 72 h]. Each data point represents the mean for three separate experiments (mean ±SD).

Fig. 4 Time-dependent cytotoxic effects of PLN (-■-), 1 (-●-), 2 (-▲-) against 786-O and MCF-7 cell lines treated with various doses [(a) = 5 μg mL⁻¹, (b) = 10 μg mL⁻¹, (c) =20 μg mL⁻¹] of PLN, 1 and 2 for 24, 48, 72 h. Each data point represents the mean for three separate experiments (mean ±SD).

DNA binding studies

Although there is some evidence to suggest that other biological targets, including RNA or proteins, may be important in the cisplatin action mechanism, it is generally accepted that DNA is the primary target.³⁴ Similarly, interactions between small molecules and DNA rank among the primary action mechanisms of antitumour activity. DNA replication in tumour cells will be blocked by the small molecule intercalation of the neighbouring base pairs of DNA.³⁵ Generally, active compounds have an approximately planar structure, with a medium-sized planar area and some hydrophobic character. In order to investigate the binding properties of PLN and its copper(II) complexes to DNA, we carried out a series of spectroscopic studies including UV-vis, fluorescence and CD spectra in addition to the gel mobility shift assay.

UV–vis absorption titration

It is well documented that intercalative π–π stacking of the aromatic rings of metal complexes with DNA bases affects the transition dipoles of the molecules and usually leads to a hypochromicity in its absorbance.³⁶ The absorption titrations were carried out to determine the DNA binding constants of PLN and its complexes 1 and 2 in Tris-NaCl buffer.

The UV-vis absorption spectra of PLN and complexes 1 and 2 in the absence and presence of ct-DNA are shown in Fig. 5. Comparison with the absorption spectrum of PLN (Fig. S4, ESI†) shows that 1 has similar absorption peaks to those of PLN with an intense absorption band around 269 nm and a weak absorption band at 415 nm, whereas the absorption spectrum of 2 contains three bands at 246, 273 and 298 nm. After the addition of ct-DNA, the absorption bands of PLN and 1 at 269 nm exhibit hypochromism of about 24% for PLN, 25% for 1, and bathochromism about 9 nm for PLN, 2 nm for 1, while the absorption band of 2 at 298 nm showed hypochromism of about 27%, without significant bathochromism. Notably, the absorption band at 298 nm in 2 shows hyperchromism of about 25%; thus an isosbestic point at 283 nm can be observed. The hypochromism and the isosbestic point are regarded as characteristics of intercalative binding.³⁷

Fig. 5 UV–vis spectra in the absence ([dash dash, graph caption]) and presence (––) of increasing amounts of DNA (2.1 × 10⁻³ M), 1 (1.0 × 10⁻⁴ M, 40 μL DNA per scan); 2 (1.0 × 10⁻⁴ M, 100 μL DNA per scan, pH = 7.35).

The intrinsic binding constant, K_b, of the metal complex to DNA can be determined by the classic eqn (1).³⁸

[DNA]/(ε_a−ε_f) = [DNA]/(ε_b−ε_f) + 1/[K_b (ε_b−ε_f)] (1)

[DNA] is the concentration of DNA, ε_a = Abs/[complex], ε_f and ε_b represent the extinction coefficient for the free complex and its fully DNA-bound combination, respectively. In the plot of [DNA]/(ε_f−ε_a) versus [DNA], K_b can be given by the ratio of the slope to the intercept.

From the absorption spectra of PLN and 1 at 269 nm and 2 at 298 nm, in the presence of increasing amounts of ct-DNA, the plots of [DNA]/(ε_f−ε_a) versus [DNA] (Fig. 5 inset) were found to be linear, and K_b values could be determined from the ratio of the slope to the intercept. The binding constants obtained for PLN and 1 and 2 are 5.07 × 10³ M⁻¹, 6.70 × 10³ M⁻¹ and 1.84 × 10⁴ M⁻¹, respectively. The K_b values for PLN and 1 to ct-DNA are nearly the same (taking into account the error limits of the values), since in both copper(II) complexes PLN is the intercalating moiety. However, the K_b value for 2 is slightly larger than those of PLN and 1, which may be ascribed to the co-ligand bipy playing an important role;³⁹ because in solution, 2 can generate near planar [Cu(bipy)(PLN)]⁺ cationic species, other binding modes of 2 should be considered. In the control experiment with sodium dodecyl sulfate (SDS) instead of ct-DNA, with the addition of SDS, the hypochromism of 2 can be observed obviously (Fig. S5, ESI†), but no significant differences were observed in the absorption spectra of PLN and 1 in the absence and presence of SDS. These results indicate that the electrostatic binding of the cationic copper(II) 2 to the polyanionic DNA phosphate back-bone may exist,⁴⁰ which perhaps results from the positive charge on complex 2.⁴¹

The results agree well with their cytotoxicity, suggesting that the cytotoxicity of these complexes is correlated to their binding ability to DNA.⁴²

Fluorescence emission titration

In order to investigate the interaction mode of PLN and 1 and 2 with ct-DNA, fluorescence emission titration analyses were undertaken. The fluorescence emission spectra of PLN, 1 and 2 in the absence and presence of ct-DNA are shown in Fig. 6. PLN ligand shows a weak emission band around 317 nm (Fig. S6, ESI†), while 1 and 2 exhibit strong emission bands around 420 and 413 nm, respectively, due to the reinforcement of the rigid structure by coordination, which could be attributed to an intra-ligand charge transfer.⁴³ Generally, the enhancement of the emission intensity of small molecules can be ascribed to the protection by the hydrophobic environment among the base pairs of DNA from quenching by polar solvent molecules, when the molecules intercalate into DNA base pairs.⁴⁴ In the experiments, with the addition of ct-DNA, PLN exhibits significant enhancement of emission intensity, which suggests an intercalative binding mode to DNA.⁴⁵ While 1 and 2 both exhibit less enhancement compared with that of PLN, they have a much stronger emission intensity than PLN at the same concentration. This result is not consistent with that observed in the UV-vis absorption spectra, other factors which impact on the emission may exist, such as hydrogen bonding and electrostatic interaction (in the case of 2).

Fig. 6 Emission spectra in the absence (----) and presence (––) of increasing amounts of DNA (2.1 × 10⁻³ M, 50 μL per scan). 1 (1.0 × 10⁻⁴ M, excited at 380 nm), 2 (1.0 × 10⁻⁴ M, excited at 310 nm), pH = 7.35.

Binding of 1 and 2 to ct-DNA was further investigated by EthBr-competitive binding experiments. The emission spectra of EthBr bound to ct-DNA in the absence and presence of PLN, 1 and 2 are shown in Fig. 7 (and Fig. S7, ESI†). The addition of free PLN, 1 and 2 to the DNA-bound EthBr solutions caused an obvious reduction in emission intensity, indicating that 1 and 2 compete with EthBr for binding to DNA.⁴⁶ The relative binding propensity of 1 and 2 to ct-DNA was determined by the classical Stern–Volmer equation:⁴⁷I₀/I = 1 + K_sqr, where I₀ and I represent the fluorescence intensities in the absence and presence of the compound, respectively, r is the concentration ratio of the compound to DNA. K_sq is the linear Stern–Volmer quenching constant. The K_sq values, obtained from the ratio of the slope to the intercept from the plot of I₀/I versus [complex]/[DNA] (insets in Fig. 7), for 1 and 2 are 1.16 and 1.82, respectively. These results demonstrate that 1 and 2 have a stronger affinity for DNA than PLN does (K_sq value is 0.16, Fig. S7†). Moreover, 2 possesses a higher DNA affinity than 1. Such a trend is consistent with the previous absorption spectra results.

Fig. 7 Emission spectra of DNA–EB system (3 mL solution, [DNA]/[EB] ratio of 6 : 1, pH = 7.35, excited at 350 nm) in the absence (----) and presence (––) of increasing amounts of 1 and 2, 50 μL of 1.0 × 10⁻³ M stock solution per scan.

From the plot of intensity against complex concentration, the values of the apparent DNA binding constant (K_app) were calculated using the equation K_EthBr× [EthBr] = K_app× [complex],^48a,b in which the complex concentration is the value at a 50% reduction of the fluorescence intensity of EthBr and K_EthBr = 1.0 × 10⁷ M⁻¹ ([EthBr] = 1.3 μM). The K_app values are 2.07 × 10⁴ M⁻¹ for PLN, 1.43 × 10⁵ M⁻¹ for 1 and 1.69 × 10⁵ M⁻¹ for 2. These results suggest that DNA binding by PLN, 1 and 2 might be by the intercalation mode.⁴⁸

Circular dichroism spectra

Circular dichroism (CD) is a useful technique to assess whether nucleic acids undergo conformational changes as a result of complex formation or changes in the environment.⁴⁹

As shown in Fig. 8, the CD spectra of ct-DNA show a positive band at 277 nm and a negative band at 250 nm, due to π–π base stacking and right-hand helicity, respectively, consistent with the characteristic B conformation of DNA.⁵⁰ The CD spectrum of PLN exhibits little alteration on both negative and positive absorptions, only a weak decrease in the positive band and a weak increase in the negative band are observed, indicating a relatively weak intercalation of PLN to ct-DNA. However, on the addition of 1 and 2, the intensities of the peak positive bands both decrease by 15% and 22%, respectively, with the same red shift of 2 nm, which indicates that 1 and 2 can intercalate the neighbouring base pairs of ct-DNA to reduce the energy of the π–π electronic transition, and lead to a decrease in the positive absorption of DNA. Meanwhile, significant decreases in the negative absorption bands of ct-DNA can also be observed, suggesting that a decline of the right-hand helicity character of ct-DNA, by which the intercalation modes of 1 and 2 can be further confirmed.⁵¹

Fig. 8 CD spectra of ct-DNA (3 mL solution, 2.0 × 10⁻⁴ M) in the absence and presence of PLN, 1 and 2 at the same concentration of 5.0 × 10⁻⁵ M.

Gel mobility shift assay

The binding mode to DNA was further examined by a gel mobility shift assay. Incubation with PLN or 1 and 2 for 1 h in the absence of any external reagent or light, resulted in the supercoiled form of pBR322 plasmid DNA being lengthened without any obvious unwinding, but the DNA mobility for PLN was slightly reduced (Fig. 9), as was that for 1, while that for 2 is significantly reduced. These results are comparable to those found when the DNA solution was treated with the classical intercalator — ethidium bromide,⁵² which is probably related to the weak interaction of these complexes with DNA.⁵³

Fig. 9 Gel mobility shift of plasmid pBR322 DNA treated with PLN, 1 and 2 after incubation for 1 h at 37 °C in 20 μL TBE at pH 7.2. Lane 0: Control DNA, lanes 1–5: DNA+ 10, 20, 30, 40, 50 μM compounds respectively.

In summary, the interactions of PLN, 1 and 2 with DNA have been investigated by means of UV-vis, fluorescence, CD spectra, and gel mobility shift assay. The results show that PLN, 1 and 2 bind DNA mainly by intercalation. The binding extent differs following the sequence 2 > 1 > PLN. Based on these results, it can be concluded that the DNA binding ability of the copper(II) complexes with PLN correlates with their respective cytotoxicity, and the Cu^II ions play a key role in its mechanism of action.⁵⁴

Inhibition of topoisomerase I

Topoisomerases are ubiquitous molecules that relieve the torsional stress in the DNA helix generated as a result of replication, transcription, and other nuclear processes; they are also specific targets for a number of anticancer agents,⁵³ including the camptothecins, indolocarbazoles, and indenoisoquinolines. These compounds bind to a transient topoisomerase I (TOPO I)-DNA covalent complex and inhibit the resealing of a single-strand nick that the enzyme creates to relieve superhelical tension in duplex DNA.⁵⁵ From the results of gel mobility shift assays of PLN, 1 and 2 (Fig. 9), it can be presumed that these compounds do not cleave DNA. Therefore, similar to 10-hydroxycamptothecin (HCPT), as shown in Fig. 10, PLN, 1 and 2 inhibit topoisomerase I (TOPO I). Enzyme activity is characterized by conversion of the DNA substrate from the supercoiled conformation (SC) to the fully relaxed conformation (RLX). PLN inhibits the activity of TOPO I at 200 μM. Both 1 and 2 inhibit the TOPO I-mediated relaxation of supercoiled pBR322 plasmid DNA at very low concentrations (both 1.56 μM), indicating that the activity of 1 and 2 are 128 times stronger than that of PLN. Because PLN, 1 and 2 inhibit the growth of the tested tumour cell lines (except for inactive compounds on the tumour cell lines), and effect the activity of TOPO I at very low concentrations, it is likely that inhibition of TOPO I is responsible for their anticancer activity. In addition, the three compounds can intercalate the neighbouring base pairs of DNA. Therefore, their cytotoxic action may be via a dual-targeted mechanism.⁵⁶

Fig. 10 Inhibition of TOPO I-mediated DNA relaxation by PLN, 1 and 2, respectively. For PLN: lane 1–7: 1 U TOPO I + 0.2 μg pBR322 DNA+ PLN (200, 100, 50, 25, 6.25, 1.56, 0.39 μM), lane 8: 1 U TOPO I + 0.2 μg pBR322 DNA, lane 9: 0.2μg pBR322 DNA alone, lane 10: 1 U TOPO I + 0.2 μg pBR322 DNA + 100 μM HCPT; For Complexes 1 and 2: lane 1–8: 1 U TOPO I + 0.2 μg pBR322 DNA + 1 or 2 (200, 100, 50, 25, 6.25, 1.56, 0.39, 0.1 μM), lane 9: 1 U TOPO I + 0.2 μg pBR322 DNA, lane 10: 1U TOPO I + 0.2 μg pBR322 DNA + 100 μM HCPT; lane 11: 0.2 μg pBR322 DNA alone.

Conclusions

The neutral mononuclear Cu^II complex 1 with the anticancer constituent plumbagin (PLN) and the dimeric Cu^II complex 2 with PLN and 2,2′-bipyridine have been synthesized and characterized by elemental analysis, IR, ESI-MS and single crystal X-ray diffraction analysis. 1 contains a square planar Cu^II coordination environment formed by four O atoms from two trans PLN bidentate ligands, while 2 is in a distorted octahedral N₂O₄ coordination sphere from one bipy, two PLN and one aqua ligand. The results of in vitro cytotoxicity assays against a series of selected human tumour cell lines show that the copper(II) complexes 1 and 2 exhibit enhanced cytotoxicity, compared to PLN, cisplatin, [Cu(bpy)₂(H₂O)₂](NO₃)₂ and [Cu(acac)(bpy)]NO₃, suggesting a synergistic effect upon PLN coordination with the Cu^II ion. 2 is the most cytotoxic, perhaps due to the synergistic effect of the N-donor heterocyclic ligand 2,2′-bipyridine. PLN, 1 and 2 exhibit an increased dose- and time-dependent cytotoxic effect. The interactions of PLN, 1 and 2 with ct-DNA reveal that they are bound to ct-DNA by intercalation, for 2 electrostatic interaction also exists. 1 and 2 have a higher affinity than PLN itself, suggesting that the metal ion coordinates to planar PLN to enhance the binding ability, displaying a good coherence with their cytotoxicity. Our findings have shown that 1 and 2 present a high activity of TOPO I inhibition at 1.56 μM, which implies TOPO I may be another target. Interpretation of these results leads to the conclusion that the enhanced effects of 1 and 2 on the viability of the selected tumour cell lines is due to the synergistic contribution of their components. PLN complexation to copper(II) improved the effectiveness of TCM like PLN as an antitumour agent, which afforded a new effective strategy to achieve promising potential metal-based anticancer drugs via formation of TCM-metal complexes with a dual-targeted mechanism and non-covalent binding mode.

Experimental

Materials

All the metallic salts and solvents used were analytical grade. All the materials were used as received without further purification unless noted specifically. Tris-HCl-NaCl buffer solution (5 mM Tris, 50 mM NaCl, pH was digitally adjusted to 7.35 by titration with hydrochloric acid with a Sartorius professional meter, Tris = tri(hydroxymethyl)aminomethane) was prepared using double distilled water. Calf thymus DNA (ct-DNA) was purchased from Sino-American Biotech. Co. Ltd., Beijing and plasmid pBR322 DNA was purchased from Generay Biotech. Co. Ltd., Shanghai. They were both used without further purification. A Tris-buffer solution of ct-DNA gave ratios of UV absorbance at 260 nm and 280 nm of ca. 1.8–1.9 : 1, indicating that the DNA was sufficiently free of protein. The DNA concentration per nucleotide in base pairs was determined spectrophotometrically by employing a molar absorption coefficient (6600 M⁻¹cm⁻¹) at 260 nm. Stock solutions were stored at 4 °C and used for no more than 4 days. HCPT and ethidium bromide were purchased from Sigma Chemical Co. Topoisomerase I was purchased from TakaRa Biotechnology (Dalian of China) Co. Ltd. Gel image formation analytical system performed on the Bio-Rad system.

Measurements

IR spectra were obtained on a Perkin-Elmer FTIR Spectrometer. ¹H-NMR and ¹³C-NMR spectra were recorded on a Bruker AV-500 NMR spectrometer using CD₃Cl as solvent. ESI-MS spectra were recorded on a Thermo-Finnigan LCQ/AD Quadrupole Ion Trap ES-MS. Elemental analyses (C, H, N) were carried out on a Perkin-Elmer Series II CHNS/O 2400 elemental analyzer. UV-vis absorption spectra were performed on a Varian Cary100 UV-visible spectrophotometer. Fluorescence measurements were performed on a Shimadzu RF-5301/PC spectrofluorophotometer. The circular dichroism spectra of DNA were obtained by using a JASCO J-810 automatic recording spectropolarimeter operating at 25 °C. The region between 220 and 320 nm was scanned for each sample.

Plant material

Roots of P. zeylanica were collected in Guangxi province of China, in September, 2004 and identified by Prof. S. Q. Tang (College of Life Science, Guangxi Normal University). A voucher specimen is deposited at the Institute of Chemistry & Chemical Engineering, Guangxi Normal University.

Isolation and structure identification of PLN

The roots of P. zeylanica (15 kg) were extracted with 95% EtOH. The extracted solution was concentrated in vacuo and the residue was successively partitioned between H₂O and n-hexane followed by EtOAc. The EtOAc extract (252 g) was subjected to silica gel column chromatography with a gradient of EtOAc in n-hexane, eluting with n-hexane–EtOAc (9 : 1) to yield orange crystals, plumbagin (13.3 g). Yield, 0.1%. A crystal for X-ray diffraction analysis was grown by slow evaporation of a solution in n-hexane–EtOAc (9 : 1). IR (KBr): 3445, 1663, 1645, 1567, 1456, 1365, 1260, 752 cm⁻¹. ¹H NMR(CDCl₃) (δ ppm): δ 2.22 (3H, s, -CH₃), δ 6.83 (1H, s, H-3), δ 7.28 (1H, d, J = 8.5 Hz, H-6), δ 7.60 (1H, d, J = 6.2 Hz, H-8), δ 7.66 (1H, d J = 7.5 Hz, H-7), δ 11.99(1H, s, -OH); ¹³C NMR (CDCl₃) (δ ppm) δ 16.5 (-CH3), δ 115.2 (C-10), δ 119.3 (C-8), δ 124.2 (C-6), δ 132.0 (C-9), δ 135.5 (C-7), δ 136.1 (C-3), δ 149.6 (C-2), δ 161.2 (C-5), δ 184.8 (C-1), δ 190.3 (C-4).

[Cu(PLN)₂]·2H₂O (1)

A methanolic solution (10 cm³) of PLN (37.6 mg, 0.2 mmol) containing CH₃ONa (11.8 mg, 0.22 mmol) was added to a solution of CuCl₂·2H₂O (17.1 mg, 0.1 mmol) in methanol (15 cm³), the mixture was refluxed with stirring for 2 h, then cooled to room temperature. Slow evaporation of the dark-red solution yielded a dark-red block crystalline product. Calc. for C₂₂H₁₈CuO₈: C, 55.76; H, 3.83. Found: C, 55.65; H, 3.88%. IR (KBr): 3524, 1632, 1589, 1562, 1427, 1195, 1257, 638 cm⁻¹. ESI-MS: 436.8 [M⁻].

[Cu(PLN)(bipy)(H₂O)]₂(NO₃)₂·4H₂O (2)

A mixture of PLN (37.6 mg, 0.2 mmol) containing CH₃ONa (11.8 mg, 0.22 mmol) and Cu(NO₃)₂·3H₂O (96.7 mg, 0.4 mmol) in methanolic solution (20 cm³) was refluxed for 2 h, followed by addition of 2,2′-bipyridine (30.4 mg, 0.2 mmol) in methanol (15 cm³).The resulting mixture was refluxed for another 2 h. Slow evaporation of the dark-red solution yielded a dark-red block crystalline product. Calc. for C₂₁H₂₁CuN₃O₉: C, 48.23; H, 4.15; N, 8.04. Found: C, 48.53; H, 3.97; N, 7.98%. IR (KBr): 3434, 1632, 1593, 1566, 1531, 1423, 1384, 1195, 1112, 775 cm⁻¹. ESI-MS: 423.9 [M⁺].

Cytotoxicity assay in vitro

Cell lines: Human cancer cell lines 786-O (renal carcinoma), MCF-7 (breast carcinoma), CNE-2 (nasopharyngeal cancer), HepG2 (hepatoma), HCT116 (colon cancer), BEL7404 (liver cancer) and NCI-H460 (lung carcinoma) were obtained from the Shanghai Cell Bank in the Chinese Academy of Sciences. Cell lines BEL-7404, NCI-H460, CNE-2, 786-O and MCF-7 were grown in DMEN (Gibco, Scotland, UK), HCT-116 and HepG2 in RPMI 1640 (Gibco, Scotland, UK) at 37 °C in a humidified atmosphere of 5% CO₂/95% air.

Assays of cytotoxicity were conducted in 96-well, flat-bottomed microtitre plates. The supplemented culture medium with seven cell lines was added to the wells. 1, 2, PLN, cisplatin, [Cu(bpy)₂(H₂O)₂](NO₃)₂ and [Cu(acac)(bpy)]NO₃ (positive control) were dissolved in the culture medium with 1% DMSO to various concentrations, and the solutions were subsequently added to a set of wells. Control wells contained supplemented media with 1% DMSO. The microtitre plates were incubated at 37 °C in a humidified atmosphere of 5% CO₂/95% air for a further 3 d. Assessment of cytotoxicity was carried out by using a modified method of the Mosmann-based 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. At the end of each incubation period, MTT solution (10 μL, 5 mg mL⁻¹) was added into each well and the cultures were incubated for a further 4 h at 37 °C in a humidified atmosphere of 5% CO₂/95% air. After removal of the supernatant, DMSO (150 μL) was added to dissolve the formazan crystals. The absorbance was read by an enzyme labelling instrument with a 570/630 nm double wavelength measurement. The cytotoxicity was evaluated based on the percentage cell survival in a dose-dependent manner relative to the negative control.

UV–vis absorption titration

Absorption titrations were performed using a fixed compound concentration (1.0 × 10⁻⁴ M complex 1, 1.0 × 10⁻⁴ M complex 2, 6.0 × 10⁻⁴ M PLN) and varying the concentration of DNA (40 μL DNA per scan for complex 1, 100 μL DNA per scan for complex 2, 10 μL DNA per scan for PLN). While measuring the absorption spectra, the solutions were allowed to incubate for 10 min at room temperature before the absorption spectra were recorded, and an equal amount of ct-DNA was added to both the compound solution and the reference solution to eliminate the absorbance of ct-DNA itself.

Fluorescence emission titration

Fluorescence emission spectra of compounds were performed by using a fixed compound concentration (1.0 × 10⁻⁴ M of 1, 1.0 × 10⁻⁴ M of 2, 1.0 × 10⁻⁴ M of PLN) and increasing the concentration of DNA (2.1 × 10⁻³ M, 50 μL DNA per scan). Samples were excited at 380 nm for 1, 310 nm for 2 and 282 nm for PLN, respectively. Fluorescence emission spectra of the DNA–EB system were carried out with DNA pretreated with ethidium bromide (EB) at a ratio of [DNA]/[EB] = 6 : 1 for 30 min. To the DNA–EB solution, increasing amounts of compounds (1.0 × 10⁻³ M of 1, 1.0 × 10⁻³ M of 2 and 1.0 × 10⁻³ M of PLN, 50 μL per scan, respectively) were added and their effect on the emission intensity was measured. Samples were excited at 350 nm and the emission was observed between 500 and 700 nm.

Circular dichroism spectra

The CD absorption spectra of ct-DNA (2.0 × 10⁻⁴ M) were recorded in the absence and presence of compounds (5.0 × 10⁻⁵ M of 1, 2 and PLN). The sample solution was mixed and incubated at room temperature. Each sample solution was scanned in the range 220–320 nm with a screening rate of 100 nm min⁻¹ at room temperature.

Gel mobility shift assay⁵⁷

For the gel mobility shift experiment, supercoiled pBR322 DNA was treated with all the compounds in 5 mM Tris-HCl, 50 mM NaCl buffer, pH 7.2 and the solutions were incubated for 1 h in the dark. The samples were electrophoresed for 1 h at 110 V on a 0.8% agarose gel in tris-boric acid-EDTA buffer (TBE buffer). The gel was stained with 0.5 μg mL⁻¹ ethidium bromide and then photographed under UV light and visualized by a Bio-Rad Gel Imaging System.

Inhibition of topoisomerase I⁵⁸

Inhibition of the catalytic activity of human DNA Topo I was evaluated by monitoring the enzyme-catalyzed relaxation of a supercoiled DNA substrate by 1% agarose gel electrophoresis. Standard assays were performed in 50 mM Tris-HCl (pH 7.9), 120 mM KCl, 10 mM MgC1₂, 50 mM NaCl, 0.5 mM EDTA, 50 μg mL⁻¹ of bovine serum albumin. Typically, the reactions contained 1 unit of the enzyme and 200 ng of supercoiled pBR322, in the absence or presence of the test compounds at different concentrations as indicated in Fig. 10. HCPT was used as a positive control. The samples were incubated at 37 °C for 30 min, and then quenched by the addition of 5 μL of a concentrated gel loading solution containing 10% SDS, 100 mM EDTA, and 0.125% bromophenol blue. The samples were subjected to electrophoresis in 1% agarose and then stained with 0.5 mg L⁻¹ of ethidium bromide.

X-ray crystallography

The data collections on single crystals were carried out on a Bruker SMART APEX2 CCD equipped with a graphite monochromated Mo-Kα radiation. The structures were solved with direct methods and refined using SHELX-97 programs.⁵⁹ The non-hydrogen atoms were located in successive difference Fourier synthesis. The final refinement was performed by full-matrix least-squares methods with anisotropic thermal parameters for non-hydrogen atoms on F². The hydrogen atoms were added theoretically, riding on the concerned atoms, except for O(4) for complex 1, O(5) and disorder O(6) for complex 2. The crystallographic data and refinement details of the structure analyses are summarized in Table 2.

Table 2 Crystallographic data and refinements of complexes 1 and 2

	1	2
Formula	C22H18CuO8	C21H21CuN3O9
Mr	473.90	522.95
Crystal size/mm	0.28 × 0.17 × 0.12	0.18 × 0.12 × 0.10
Crystal system	Monoclinic	Triclinic
Space group	C2/c	P1̄
a/Å	21.576(4)	8.684(8)
b/Å	10.0549(18)	10.357(9)
c/Å	9.3266(17)	13.036(11)
α (°)	90	72.472(13)
β (°)	109.319(3)	83.105(13)
γ (°)	90	88.979(13)
V/Å^3	1909.4(6)	1109.7(17)
T/K	296(2)	296(2)
Z	4	2
Dc/g cm^−3	1.649	1.565
2θ/°	4.00 to 50.10	4.44 to 50.10
F(000)	972	538
μ(Mo-Kα)/mm^−1	1.195	1.043
Total no. reflns	4662	5332
No. indep. reflns	1684	3794
Rint	0.0385	0.0325
R1 [I > 2σ(I)]	0.0499	0.0670
wR2 (all data)	0.1594	0.1960
Gof (F^2)	1.036	1.002

---

Acknowledgements

This work was supported by National Basic Research Program of China (No. 2007CB516805, 2009CB526503), the National Natural Science Foundation of China (No. 20861002), and the Natural Science Foundation of Guangxi Province (No. 0991012Z), as well as the Project of Ten, Hundred, Thousand Distinguished Talents in New Century of Guangxi (No. 2003223). We also thank Prof. Dr. Zijian Guo for valuable discussions, and Prof. Dr Huai-Ming Hu for crystal structure determination.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: UV-vis absorption spectrum of PLN; Fluorescence emission spectrum of PLN; Emission quenching spectrum of the EB-DNA system by titration with PLN; UV-vis absorption of complex 2 by titration of SDS with increasing concentration. Crystal structure and space filling drawing of PLN and space filling drawing and 2D network of 1; 1D chain structure of 2. CCDC reference numbers 725715 & 725716. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/b910133k

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