Two hydrazone copper(II) complexes: synthesis, crystal structure, cytotoxicity, and action mechanism

Abstract

Two copper(II) complexes with 8-quinolinecarbaldehyde o-vanilloylhydrazone (H-L1) and 8-quinolinecarbaldehyde salicylhydrazone (H-L2), [Cu(L1)NO₃] (1) and [Cu(L2)NO₃] (2), were synthesized and structurally characterized, respectively. Complexes 1 and 2 exhibited enhanced cytotoxicity against BEL-7402, Hep-G2, NCI-H460, MGC80-3, HeLa tumor cells compared with free ligands and copper(II) salt and slightly lower than that shown by cisplatin. And MGC80-3 cells are more sensitive to these two complexes relative to the normal liver cells. Cytotoxicity and action mechanism studies suggest 1 and 2 could cause MGC80-3 cell cycle arrest at G1 phase, which is induced by limiting the supply of cyclins D1 and E1 and inhibiting the activity of G1-phase-promoting cyclin–Cdk complexes. And complexes 1 and 2 led to cell apoptosis via the activation of Bcl-2 protein. Moreover, mitochondrial dysfunction was induced by both of complexes.

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Introduction

The discovery and success of cisplatin as an anticancer agent ushered in an era of tumor chemotherapy, which has placed coordination chemistry of metal-based drugs in the frontline in the fight against cancer.^1–3 Although cisplatin as a broad-spectrum agent exhibits high efficiency towards a variety of cancer cell types, it is still limited because it causes severe side effects and intrinsic or acquired resistance.^3–5 These problems have stimulated researchers to develop alternative strategies with improved pharmacological properties and to aimed at different targets based on different metals and ligands.^6,7

Copper is an essential trace element for most aerobic organisms which plays important roles in a wide range of physiological cellular processes.⁸ Numerous experimental evidences indicated that copper metabolism is severely altered in neoplastic diseases.⁹ Therefore, the copper complexes have been designed and synthesized to research its role in the development of medicines for the treatment of cancer and other diseases.^7,10–14 For example, homoleptic phosphino copper(I) complexes were prepared and their dual cytotoxic and anti-angiogenic activities were studied in vitro and in vivo.⁹ Bojja Sreedhar et al. have synthesized and characterized a series of new copper(II) polypyridyl complexes, and two of them are found to have significant anticancer and antiangiogenic potential as observed by several in vitro and in vivo assays.¹⁵ Copper complexes based on nitroimidazole and glucosamine conjugated heteroscorpionate have been designed and found to display a similar growth inhibitory potency in the micromolar range, and both copper(II) complexes overcame acquired resistance to cisplatin, although the activities are slightly lower than that shown by cisplatin.¹⁶ These findings have demonstrated that copper complexes as potential anticancer agents are especially promising and represent good alternatives to platinum drugs.^7,9

Quinolines and their derivatives are very important in medicinal chemistry because of their wide occurrence in natural products¹⁷ and drugs.¹⁸ Recently, a large number of quinolines derivatives, such as 8-hydroxyquinoline galactosides,^19,20 arylethylenequinolinic²¹ and other quinoline derivatives,^19,20,22,23 were utilized as ligands to prepare copper complexes with good anti-cancer and antineurodegenerative activities. In particular, the copper complex of arylethylenequinolinic exhibited highest cytotoxicity.²¹

On the other hand, hydrazones are versatile ligands in the field of analytical chemistry and medicines.^24,25 Hydrazone moieties are the most important pharmacophoric cores of several anticancer, anti-inflammatory, antinociceptive, and antiplatelet drugs.^26,27 Recently, copper-bound aroylhydrazone derivatives of pyridoxal isonicotinoyl hydrazone show great potential as effective antiproliferative/anticancer agents.^7,28 And the researchers found that change of the substitution in the hydrazone moiety effected the bio-activity.²⁹

Taking into account of the above factors, in this present study, two new tetragonal pyramid copper(II) hydrazones complexes with change of substitution (H or CH₃O) in salicylhydrazone moieties were designed and synthesised. The ligand can interact with copper ions by virtue of the N, O donor of hydrazone resulting in two stable copper complexes, [Cu(L1)NO₃] (1) and [Cu(L2)NO₃] (2), respectively. The structures of them were characterized by FT-IR, HRMS (ESI), elemental analysis and single crystal X-ray diffraction analysis. H-L1, H-L2, complex 1 and 2 were tested for their in vitro antitumor activities on BEL-7402, Hep-G2, NCI-H460, MGC80-3, HeLa cancer cells. Moreover, the mechanism of their cytotoxicity was also investigated.

Experimental methods

Materials

Commercially available reagents were used directly without further purification unless otherwise noted. Quinoline-8-carboxaldehyde, salicylhydrazide and 3-methoxy salicyhydrazide were purchased from Sigma-Aldrich. BEL-7402, Hep-G2, NCI-H460, MGC80-3 and HeLa were obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences. Cell lines were grown in DMEN (Gibco, Scotland, UK) at 37 °C in a humidified atmosphere of 5% CO2/95% air.

Cell cycle analysis results were recorded on FACS Aria IIflow cytometer (BD Biosciences, San Jose, CA, USA). MTT assay were performed on M1000 microplate reader (Tecan Trading Co. Ltd., Shanghai, China). The liquid chromatography (LC) and high resolution electrospray ionization-mass spectroscopy (HRMS-ESI) were recorded on Exactive LC-MS mass spectrometer (Thermofisher Scientific, USA). Elemental analyses (C, H, N) were performed on an Elementar Vario Micro Cube elemental analyzer. FT-IR spectra were recorded as KBr pellets using a Perkin-Elmer Spectrum One FTIR spectrometer in the 400–4000 cm⁻¹ region. NMR spectra were measured on a BRUKER AVANCE AV500 spectrometer.

Synthesis of 8-quinolinecarbaldehyde o-vanilloylhydrazone (H-L1) and 8-quinolinecarbaldehyde salicylhydrazone (H-L2)

A mixture of 8-quinolinecarbaldehyde (1.57 g) and 3-methoxy salicyhydrazide (1.82 g) or salicylhydrazide (1.52 g) in anhydrous methanol (20 mL) was refluxed for 24 h. After cooling to room temperature, the resulting yellow for H-L1 and light yellow for H-L2 product suitable for structural characterization were isolated, washed with methanol and ether, and air-dried, respectively. The chemical composition of these ligands was characterized by elemental analyses, FT-IR, HRMS (ESI) and NMR spectra (Fig. S1–S6†).

Data for H-L1: the yellow color product suitable for structural characterization. Yield (3.05 g, 95%). FT-IR (KBr): 3277, 2931, 2835, 1649, 1542, 1471, 1286, 1244, 1153, 1064, 940, 801, 761, 606 cm⁻¹. HRMS (ESI): calcd for C₁₈H₁₆N₃O₃ [M + H]⁺ 322.11917, found 322.11743. Elemental analysis calcd (%) for C₁₈H₁₅N₃O₃: C 67.28, H 4.71, N 13.08; found: C 67.25, H 4.70, N 13.11. ¹H NMR (500 MHz, DMSO) δ 12.17 (s, 1H), 12.07 (s, 1H), 9.78 (s, 1H), 9.05–8.97 (m, 1H), 8.44 (dd, J = 22.3, 7.7 Hz, 2H), 8.11 (d, J = 8.0 Hz, 1H), 7.74 (t, J = 7.7 Hz, 1H), 7.66–7.57 (m, 2H), 7.17 (d, J = 7.9 Hz, 1H), 6.91 (t, J = 8.0 Hz, 1H), 3.83 (s, 3H). ¹³C NMR (125 MHz, DMSO) δ 166.27, 150.95, 150.78, 148.92, 146.42, 145.89, 137.19, 131.40, 130.86, 128.53, 127.06, 126.34, 122.44, 119.63, 118.75, 116.18, 115.63, 56.37, 49.08.

Data for H-L2: the light yellow product suitable for structural characterization. Yield (2.62 g, 90%). FT-IR (KBr): 3254, 3050, 2843, 2707, 2566, 1633, 1544, 1305, 1217, 1155, 1084, 1033, 793, 754, 638 cm⁻¹. HRMS (ESI): calcd for C₁₇H₁₄N₃O₂ [M + H]⁺ 292.10860, found 292.10674. Elemental analysis calcd (%) for C₁₇H₁₃N₃O₂: C 70.09, H 4.50, N 14.42; found: C 69.94, H 4.52, N 14.40. ¹H NMR (500 MHz, DMSO) δ 12.17 (s, 1H), 12.08 (s, 1H), 9.77 (s, 1H), 9.05–8.95 (m, 1H), 8.44 (dd, J = 19.0, 7.1 Hz, 2H), 8.11 (d, J = 7.9 Hz, 1H), 8.01 (d, J = 7.2 Hz, 1H), 7.73 (t, J = 7.7 Hz, 1H), 7.63 (dd, J = 8.3, 4.1 Hz, 1H), 7.46 (t, J = 7.2 Hz, 1H), 6.98 (dd, J = 14.6, 7.7 Hz, 2H). ¹³C NMR (125 MHz, DMSO) δ 165.88, 160.21, 150.93, 146.28, 145.89, 137.19, 134.47, 131.43, 130.82, 128.75, 128.53, 127.05, 126.34, 122.42, 119.35, 117.87, 115.83.

Synthesis of [Cu(L1)NO₃] (1) and [Cu(L2)NO₃] (2)

Cu(NO₃)·3H₂O (0.15 mmol, 0.0361 g), H-L1 (0.15 mmol, 0.0481 g) or H-L2 (0.15 mmol, 0.0436 g), 1.0 mL methanol were placed into a thick Pyrex tube (ca. 25 cm long). And then the tube was quenched in liquid N₂, evacuated and sealed. The mixture was heated at 80 °C for four days, then slowly cooled (5°C h⁻¹) to room temperature. Green block (1) and dark green block (2) crystals suitable for X-ray structural characterization were collected, respectively. Yield (0.0601 g, 90%). Their structures were characterized by X-ray diffraction analysis, elemental analysis, FT-IR, and HRMS (ESI) spectroscopy (Fig. S7–S10†).

Complex 1: FT-IR (KBr): 3477, 2934, 1630, 1514, 1478, 1387, 1293, 1247, 1069, 1013, 916, 776 cm⁻¹. HRMS (ESI): calcd for C₁₈H₁₄N₃O₃Cu [M − NO₃]⁺ 383.03312, found 383.03246. Elemental analysis calcd (%) for C₁₈H₁₄CuN₄O₆: C 48.49, H 3.16, N 12.57; found: C 48.45, H 3.20, N 12.61.

Complex 2: FT-IR (KBr): 1592, 1514, 1478, 1380, 1293, 1251, 1062, 1010, 835, 764 cm⁻¹. HRMS (ESI): calcd for C₁₇H₁₂N₃O₂Cu [M − NO₃]⁺ 353.02255, found 353.02136. Elemental analysis calcd (%) for C₁₇H₁₂CuN₄O₅: C 49.10, H 2.91, N 13.47; found: C 49.05, H 2.94, N 13.50.

Cell lines, culture conditions and cytotoxicity assay

Assays of cytotoxicity were conducted in 96-well microtitre plates. The supplemented culture medium with cell lines was added to the wells. Complexes 1 and 2, H-L1, H-L2, Cu(NO₃)·3H₂O, DMSO and cisplatin (as a reference metallodrug in order to investigate the potency of these synthetic complexes) were dissolved in the culture medium with 1% DMSO at various concentrations 1.25, 2.5, 5, 10, 20 μmol L⁻¹, respectively. The resultant 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 48 d.

Cytotoxic screening by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was carried out. At the end of each incubation period, the MTT solution (10 μL, 5 mg mL⁻¹) was added into each well and the cultures were incubated further for 4 h. After removal of the supernatant, DMSO (150 μL) was added to dissolve the formazan crystals. The absorbance was read by enzyme labelling instrument with 570/630 nm double wavelength measurement. The cytotoxicity was evaluated based on the percentage of cell survival in a dose-dependent manner relative to the negative control. The final IC₅₀ values were calculated by the Bliss method (n = 5). All tests were repeated in at least three independent trials.

Cell cycle analysis

MGC80-3 cells were incubated in 10% FBS-supplemented culture medium with complexes 1 (5 and 10 μM) and 2 (3.5 and 7 μM) for 48 h at 37 °C and 5% CO₂. After treatment, cells were collected and fixed with ice-cold 70% ethanol at −20 °C overnight. Fixed cells were resuspended in 0.5 mL of PBS containing 50 μg mL⁻¹ propidium iodine, 100 μg mL⁻¹ RNase A. The cell cycle distribution was analyzed by FACS Calibur flow cytometer (BD) and calculated using ModFIT LT software (BD).

Apoptosis assay by flow cytometry

MGC80-3 cells were plated at 1 × 10⁵ cells per mL in 6-well plates. Cells were then incubated with complete medium only (control), medium with the complexes 1 (5 and 10 μM) and 2 (3.5 and 7 μM). After incubation for 12 h, the cell was trypsinized and collected. Induced apoptotic was assayed by the BD Pharmingen FITC Annexin V Apoptosis Detection kit, according to the manufacture's instructions.

Western blotting

MGC80-3 cells (5 × 10⁵) were cultured on 60 mm dish and incubated overnight before experiments, which were treated with complexes 1 (5 and 10 μM) and 2 (3.5 and 7 μM). After incubation for 24 h, cells were harvested and lysed using the lysis buffer with protease inhibitor. Total protein extracts (50 μg) were loaded onto suitable concentration SDS-polyacrylamide gel, and then transferred to polyvinylidene fluoride (PVDF) membranes. The membrane was blocked with 5% BSA in TBST buffer and incubated with corresponding primary antibodies at 4 °C overnight. After washing, the membrane was incubated with secondary antibody conjugated with horseradish peroxidase for 120 min. The immuno reactive signals were detected using enhanced chemoluminance kit (Pierce ECL Western Blotting Substrate).

Mitochondrial membrane potential detection

MGC80-3 cells incubated with complexes 1 (5 and 10 μM) and 2 (3.5 and 7 μM) for 16 h in poly-HEMA coated 6-wells were collected and resuspended in a fresh medium. The cells were incubated in a 5% CO₂ incubator for 20 min at 37 °C after the addition of 0.5 mL JC-1 working solution. The staining solution was removed by centrifugation and cells were washed with JC-1 staining buffer twice, and were analyzed immediately by flow cytometry.

X-Ray crystallography

The data collection of single crystals of complexes 1 and 2 were performed on a SuperNova CCD Area Detector equipped with graphite monochromated Mo-Kα radiation (λ = 0.71073 Å) at room temperature. Absorption correction was applied to the raw intensities with the SADABS program.³⁰ The structures were solved with direct methods and refined using SHELX-97 programs.^31,32 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 all no-hydrogen atoms on F². The hydrogen atoms were added theoretically and riding on the concerned atoms with Uiso constrained to be 1.2 or 1.5 times U_eq of the carrier atoms. Details of crystal data, intensity collection and refinement parameters are summarized in Table 1. Selected bond lengths and bond angles are tabulated in Table 2.

Table 1 Crystal data and structure refinement for complex 1 and 2

Formula	C18H14CuN4O6	C17H12CuN4O5
Fw	445.88	415.85
T/K	293(2)	293(2)
Crystal system	Triclinic	Triclinic
Space group	P1̄	P1̄
a/Å	6.9484(11)	7.052(4)
b/Å	15.176(2)	10.386(6)
c/Å	16.475(2)	11.404(7)
α/°	90.222(11)	85.415(9)
β/°	90.699(12)	77.055(10)
γ/°	91.171(12)	77.165(9)
V/Å^3	1736.8(4)	793.2(8)
Z	4	2
Dc, g cm^−3	1.705	1.741
μ, mm^−1	1.306	1.418
F(000)	908.0	422.0
Reflns (collected/unique)	17 362	9213
Rint	0.0818	0.0373
R [I ≥ 2σ(I)]	0.0861	0.0461
Final R indexes [all data]	0.1507	0.0719

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Table 2 Selected bond lengths (Å) and bond angles (°) for complex 1 and 2

1
Cu1–O3	1.933(5)	O4–Cu1–N3	97.1(2)
Cu1–O4	1.989(5)	O6–Cu1–N2	120.35(18)
Cu1–O6	2.548(5)	O6–Cu1–N3	97.76(18)
Cu1–N2	1.931(5)	N2–Cu1–N3	94.6(2)
Cu1–N3	1.965(5)	O10–Cu2–N6	156.1(3)
Cu2–N5	1.979(5)	O11–Cu2–N5	96.8(2)
Cu2–N6	1.908(5)	O11–Cu2–N6	142.1(2)
Cu2–O9	1.922(5)	N5–Cu2–N6	93.4(2)
Cu2–O10	2.169(6)	O9–Cu2–N5	174.4(2)
Cu2–O11	2.405(6)	O9–Cu2–N6	81.5(2)
O3–Cu1–O4	86.8(2)	O10–Cu2–O11	54.9(2)
O3–Cu1–O6	86.88(17)	O10–Cu2–N5	101.3(2)
O3–Cu1–N2	81.7(2)	O9 –Cu2–O10	84.3(2)
O4–Cu1–O6	55.02(18)	O9–Cu2–O11	86.0(2)
O4–Cu1–N2	167.9(2)

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2
Cu1–N1	2.001(3)	N1–Cu1–O3	97.17(13)
Cu1–O1	1.940(3)	O1–Cu1–N1	173.99(11)
Cu1–O3	2.013(3)	O1–Cu1–O3	88.72(12)
Cu1–N2	1.924(3)	N2–Cu1–N1	93.04(12)
Cu1–O5	2.586(3)	N2–Cu1–O1	81.29(12)
		N2–Cu1–O3	166.95(12)

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Results and discussion

Synthesis of ligands and their copper(II) complexes

Two new ligands, H-L1 and H-L2 were first synthesized via the synthetic routes shown in Scheme 1, starting from quinoline-8-carboxaldehyde.

Scheme 1 Synthetic routes for the preparation of H-L1, H-L2 and theirs copper(II) complexes 1 and 2. Reagents are the following: (a) methanol (reflux); (b) Cu(NO₃)·3H₂O, 80 °C.

Complexes 1 and 2 were prepared by the reactions of Cu(NO₃)·3H₂O with H-L1 and H-L2 in the solvent of methanol under solvothermal conditions, as shown in Scheme. 1. The characteristic results are well consistent with the compositions and structures of the compounds.

Crystal structure

The single crystal X-ray diffraction analysis demonstrated that complex 1 belongs to monoclinic crystal system with space group of P1̄. As shown in Fig. 1, the asymmetric unit of 1 contains two similar Cu(L1) (NO₃) molecules. And the geometry of the copper(II) centre in complex 1 is a distorted N₂O₃ tetragonal pyramid completed by one quinoline nitrogen atom, one iminic nitrogen atom and one carbonyl oxygen atom derived from a tridentate chelating planar ligand L1⁻(μ₁-η¹:η¹:η¹-L₁), as well as two oxygen atoms provided by a bidentate chelating NO₃⁻ ligand. Except the long distance of Cu1–O6 (2.546 (2) Å) and Cu2–O11 (2.402 (8) Å) in the axial positions, indicating weak coordination interactions between them,³³ all other Cu–O (range from 1.925 to 2.167 Å) and Cu–N (range from 1.930 to 1.979 Å) bond lengths in the equatorial plane are within the normal range. Intramolecular hydrogen bonds could be observed between the non-coordinating phenol hydroxyl group and the deprotonated amide group of the acylhydrazone linker (O2–H2⋯N1 2.604(7) Å and O8–H8⋯N7 2.613 (7) Å).

Fig. 1 ORTEP drawing of the complex 1 with atom numbering scheme. Thermal ellipsoids for non-hydrogen atoms are drawn at the 30% probability level. Hydrogen atoms are omitted for clarity. Color scheme: Cu, green; O, red; N, light blue; C, gray.

Complex 2 also crystallizes in the monoclinic crystal system with space group of P1̄. And the molecule structure of 2 is analogue to that of 1, with the L1 ligand replaced by L2 (Fig. 2). Different from complex 1, the asymmetric unit of 2 contains only one Cu(L2)(NO₃) molecule. Except the long distance of Cu1–O5 bond length (2.586 (3) Å) in the axial position, indicating a weak coordination interaction between them,³³ all the Cu–O (range from 1.940 to 2.013 Å) and Cu–N (range from 1.924 to 2.001 Å) bond distances are much shorter than the axial Cu1–O5 bond distance (2.586 (3) Å). An intramolecular hydrogen bond is formed between the phenol hydroxyl group and acylhydrazone amide group (O2–H2⋯N3 2.595(3) Å).

Fig. 2 ORTEP drawing of the complex 2 with atom numbering scheme. Thermal ellipsoids for non-hydrogen atoms are drawn at the 30% probability level. Hydrogen atoms are omitted for clarity. Color scheme: Cu, green; O, red; N, light blue; C, gray.

Stability of 1 and 2 in solution

The stabilities of complexes 1 and 2 were tested in 10 mM Tris–HCl buffer (Tris buffer solution, TBS), pH 7.4, 0.1% DMSO by UV-Vis spectroscopy. As shown in Fig. 3, the time-dependent (0, 24 and 48 h) UV-Vis spectra of the complexes indicated that 1 and 2 were stable in TBS for 48 h at room temperature. The stabilities of complexes 1 and 2 were further confirmed by LC-MS experiments. As shown in Fig. S11,† the peak area of complexes 1 and 2 keeps basically constant in the TBS (0.1% DMSO). These results demonstrated that complexes 1 and 2 exhibit good stability in the TBS (0.1% DMSO).

Fig. 3 UV-Vis absorption spectra of 1 (A) and 2 (B) (1.0 × 10–6 M) in TBS (0.1% DMSO) with time course 0, 24 and 48 h, respectively.

In vitro cytotoxicity

The in vitro cytotoxicity of H-L1, H-L2, complexes 1 and 2 against BEL-7402, Hep-G2, NCI–H460, MGC80-3, HeLa and one normal liver cell line (HL-7702 cells) were assessed by MTT method. Cisplatin, Cu(NO₃)·3H₂O, and 1% DMOS were used as the positive control groups. The IC₅₀ values of these compounds were shown in Table 3, toward all the tested human tumor cells, complexes 1 and 2 exhibited significantly enhanced cytotoxicity compared with the corresponding ligands, copper(II) salt and solvent, suggesting a synergistic effect between ligands and copper(II) ion. The chelation of the ligand with the Cu(II) ion may be a responsible factor for the observed cytotoxic properties of these complexes.³⁴ Interestingly, the cytotoxicity of 2 is higher than that of 1, which may be that 3-methoxy group reduced the antiproliferative effects.^35,36 The antiproliferative efficiency of complexes 1 and 2 on the human normal liver cells (HL-7702) is lower than that on the other five tumor cell lines. Complexes 1 and 2 exhibited lower IC₅₀ values (10.35 and 7.24 μM for 1 and 2, respectively) for MGC80-3, and compared with the case of normal liver cell HL-7702, the cytotoxicity of toward the MGC80-3 tumor cells is enhanced by 9.6 and 3.9 times, respectively. All these results indicate that the complexes have a certain degree of selective cytotoxicity.

Table 3 IC₅₀^a (μM) values of H-L1, H-L2, 1, 2 and cisplatin on the selected cells for 48 h

a IC50 values are presented as the mean. SD (standard error of the mean) from five independent experiments.
Compounds	BEL-7402	Hep-G2	NCI–H460	MGC80-3	HeLa	HL-7702
H-L1	>100	72.33 ± 1.18	90.83 ± 1.52	86.56 ± 1.35	>100	>100
H-L2	>100	>100	28.23 ± 0.71	58.83 ± 0.65	100	>100
1	12.52 ± 0.28	9.69 ± 0.14	12.48 ± 0.63	10.35 ± 0.29	14.56 ± 0.53	>100
2	10.30 ± 0.28	9.70 ± 0.43	10.29 ± 0.42	7.24 ± 0.63	7.72 ± 0.19	28.24 ± 0.58
Cisplatin	60.35 ± 0.93	27.31 ± 2.30	47.68 ± 0.54	21.28 ± 2.17	35.25 ± 1.88	23.02 ± 0.46
Cu(NO3)·3H2O	86.21 ± 4.52	42.18 ± 0.26	>100	>100	72.58 ± 4.46	>100
1% DMSO	>100	>100	>100	>100	>100	>100

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Cell cycle

The high in vitro cytotoxicity of the copper(II) complexes 1 and 2 were attractive enough for us to explore the antitumor mechanism in detail at the cellular and molecular levels, respectively. There are many researchers have shown that cell cycle arrest and cell apoptosis induction closely correlated to tumor cells death.³⁷

The cell cycle in MGC80-3 cells induced by 1 (5 and 10 μM) and 2 (3.5 and 7 μM) were examined by flow cytometry. As shown in Fig. 4, with the increase of concentrations of complexes, the population of cells in S and G2/M phases decreased profoundly, while a dramatic increase in G1 phase was observed in the complexes 1 and 2 treated groups from 62.19% to 72.02%, 80.21%, respectively. The results indicated that complexes 1 and 2 could arrest the MGC80-3 cell cycle in G1 phase. In addition, the ability of cell cycle arrest of complex 1 and 2 was in accordance with their in vitro cytotoxicity.

Fig. 4 Effect of cell cycle of MGC80-3 cells treated with complexes 1 (5 and 10 μM) and 2 (3.5 and 7 μM) for 24 h comparing with the control cells.

Effect of 1 and 2 on the levels of cell cycle regulators

It is known that cyclin-dependent kinases 2/4 (CDK2, CDK4), cyclin E and cyclin D1 cooperate to promote G1 and G1/S phase progression in cell cycle checkpoints. Therefore, western blotting was utilized to determine whether corresponding concentration of complexes 1 and 2 treatment affected the expressions of these proteins in MGC80-3 cells. As shown in Fig. 5, cyclin E, and cyclin D1 decreased significantly in a done-dependent manner. In contrast, the expression of CDK2 and CDK4 changed slightly. As expected, in higher concentration, complexes 1 (10 μM) and 2 (7 μM) could lead to up-regulation of inhibitors of cyclin-dependent kinase (CKIs) p27, which is associated with the activity of cyclin–Cdks complexes.^38,39 These results revealed that the activity of G1-phase-promoting cyclin–Cdk complexes can be inhibited by limiting the supply of cyclins D1 and E1. Furthermore, the up-regulation of cyclin-dependent kinase (CKIs) p27 protein can bind to the G1-phase -promoting cyclin–Cdk complexes, ultimately lead to cell cycle arrest in G1 phase.

Fig. 5 Western blot was used to determine the expression of EGFR, CDK2, CDK4, cyclin E, cyclin D1 or p27 in MGC80-3 cells treated with complexes 1 (5 and 10 μM) and 2 (3.5 and 7 μM) for 24 h, respectively.

Cell apoptosis

Since the cell cycle arrest induction is mostly correlated with the cell apoptosis induction,⁴⁰ thus, the ability of complexes 1 (5 and 10 μM) and 2 (3.5 and 7 μM) to promote apoptosis in MGC80-3 cells were determined by flow cytometry after staining the cells with propidium iodide (PI) and annexin-V-FITC. It was found that the populations of the apoptotic MGC80-3 cells (both at the early stage and the late stage of apoptosis, Q2 + Q3) significantly increased in a dose-dependent manner when incubating with complexes 1 (5 and 10 μM) and 2 (3.5 and 7 μM) for 24 h, respectively, as shown in Fig. 6. For complexes 1 (10 μM) and 2 (7 μM), the populations of cell in late apoptosis were 10.1% and 13.4%, respectively, which suggested that cell apoptosis in MGC80-3 cells can be effectively induced by complexes 1 (10 μM) and 2 (7 μM).

Fig. 6 Apoptosis of MGC80-3 cells treated with complexes 1 (5 and 10 μM) and 2 (3.5 and 7 μM) for 24 h, comparing with the control group cells.

Expressions of bcl-2 and bax proteins

It was reported that bcl-2 and related cytoplasmic proteins (including bax protein) are key regulators of apoptosis, the cell suicide program critical for development, tissue homeostasis, and protection against pathogens.^41,42 Thus, to detect whether the release of bcl-2 and bax occurred in complexes treated MGC80-3 cells, western blot was carried out. As shown in Fig. 7, complexes 1 (5 and 10 μM) and 2 (3.5 and 7 μM) induced significantly up-regulation of Bax protein expression and down-regulation of Bcl-2 protein expression. This observation suggested that complexes could activate the Bcl-2 and Bax proteins which closely associated with the mitochondria-involved apoptotic cell death, ultimately induced cells apoptosis. By comparing the expression level of Bax proteins, we can see that complex 2 induces higher protein expression than that of complex 1 (7 and 10 μM, respectively), which is consistent with staining by cell apoptosis assay.

Fig. 7 Western blot was used to determine the expression of bcl-2 and bax in MGC80-3 cells treated with complexes 1 (5 and 10 μM) and 2 (3.5 and 7 μM) for 24 h, respectively.

Detection of mitochondrial membrane potential

Mitochondrial dysfunction and the release of apoptogenic factors are critical events in triggering various apoptotic pathways, which is demonstrated by several key events such as the reduction of mitochondrial membrane potential. To confirm whether apoptosis induced by complexes 1 and 2 is related to mitochondrial dysfunction, the mitochondrial transmembrane potential (MMP) was investigated by flow cytometry using JC-1. As shown in Fig. 8, the MGC80-3 cells treated with complexes 1 and 2 cause a significant decrease in mitochondrial membrane potential compared with the control cells, which indicating the typical property of cell apoptosis induced by complexes 1 and 2 in a dose-dependent manner.

Fig. 8 Effects of complex 1 and 2 on MMP analyzed by JC-1 staining and flow cytometry. MGC80-3 were treated with complexes at the indicated concentrations for 4 h.

Conclusions

Two copper(II) complexes [Cu(L1)NO₃] (1) and [Cu(L2)NO₃] (2) of 8-quinolinecarbaldehyde o-vanilloylhydrazone (H-L1) and 8-quinolinecarbaldehyde salicylhydrazone (H-L2) were hydrothermal synthesized and X-ray structurally characterized. They possessed similar growth inhibitory potency that is in the micromolar range against the different types of tumor cells and slightly lower than that exhibited by cisplatin. Fortunately, low cytotoxicity on the normal liver cells (HL-7702) was observed. Especially, complexes 1 and 2 showed high selectivity against MGC80-3 cells and displayed synergistic effect in cytotoxicity comparing with the free metal ions, H-L1 and H-L2. Further studies demonstrated that complexes 1 and 2 can induce G1 phase arrest in MGC80-3 cells, especially complex 2, as testified by down-regulation of cyclin E and cyclin D1, up-regulation of p27, and finally leading to cell apoptosis through mitochondria-mediated pathway.

Acknowledgements

This work is supported by National Natural Science Foundation of China (No. 21271050 and 51572050), Guangxi Natural Science Foundation (No. 2015GXNSFDA139007), Talent's Small Highland Project of Guangxi Medicine Industry (No. 1509), State Key Laboratory Cultivation Base for the Chemistry and Molecular Engineering of Medicinal Resources, Ministry of Science and Technology of China (CMEM2014-A06), and Scientific Research and Technological Development Project of Guilin (20130122-3).

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Footnote

† Electronic supplementary information (ESI) available: Tables S1 and S2. Table S4: selected bond lengths (Å) and bond angles (°) for 1 and 2. Fig. S1–S6: FT-IR, HRMS (ESI) and NMR spectra of H-L1, H-L2. Fig. S7–S9: FT-IR and HRMS (ESI) of complex 1 and 2. Fig. S11: LC-MS spectra for complex 1 and 2. CCDC 1441396 and 1441392 for complexes 1 and 2, respectively. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c6ra03478k

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