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Synthesis, crystal structure and antiproliferative mechanisms of gallium(iii) complexes with benzoylpyridine thiosemicarbazones

We have prepared six thiosemicarbazone ligands and synthesized the corresponding Ga(iii) complexes. The antitumor activity of the ligand increases with its lipophilicity, and the antitumor activity of the Ga(iii) complexes is affected by the ligands. Since C6 has the highest anticancer proliferative activity (0.14 ± 0.01 μM) against HepG-2 (Human hepatocarcinoma cell line), we characterized its structure by X-ray single crystal diffraction and explored its antiproliferation mechanism. Anti-tumor mechanism results show that Ga(iii) complex (C6) promoted HepG-2 cell cycle arrest in the G1 phase by regulating the expression of cell cycle-associated proteins (Cdk 2, cyclin A and cyclin E). Ga(iii) complex (C6) promotes apoptosis by consuming intracellular iron, enhancing intracellular reactive oxygen species (ROS), activating caspase-3/9, releasing cytochromes and apoptotic protease activating factor-1 (apaf-1).


Introduction
Gallium nitrate was introduced into clinical trials in the 1980s due to its good anticancer potential. 1 Ga(III) complexes are important anti-tumor drugs that are superior to platinum due to certain aspects of their biological effects. [2][3][4][5][6] Reports on the detection of poly-phosphates using Ga(III) complexes indicate that gallium has a strong binding affinity for ATP. 7,8 Therefore, Ga(III) complexes may have the potential to inhibit ATP. Similar to iron, Ga(III) enters cells by binding to transferrin, so the solution to increase activity is to increase the bioavailability of Ga. [9][10][11] The ligand-modied Ga(III) complexes exhibit advantageous pharmaceutical properties in various aspects, such as pharmacokinetics, affinity for malignant cells, and anti-tumor proliferative effect. [12][13][14] Ga(III) complexes induce apoptosis in cancer cells by activating caspase activity and modulating cyclin to inhibit the cell cycle. [15][16][17][18] The complexation of Ga(III) with thiosemicarbazone enhances the biological activity of Ga(III) and is considered to be an important strategy for the development of cytotoxic drugs. 10,[18][19][20] Lipophilicity plays a leading role in the rate at which molecules enter cells, and complexation can increase the lipophilicity of Ga(III). 21 At the same time, the biological activity of thiosemicarbazone is closely related to the ability of forming complex with the metal and the lipophilicity of the molecule. [21][22][23][24] Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone) is a potent ribonucleotide reductase inhibitor and is the most prominent representative of this family. [25][26][27] It is currently used in phase I and phase II clinical trials for a variety of malignancies. 25 Another star molecule (di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone, DPC) has entered clinical trials for drug-resistant cancer treatment in 2016. 28 The synthesis and application of thiosemicarbazone and its gallium complex have been paid attention to and become one of the most promising research elds.
In order to further improve the anti-tumor mechanism of thiosemicarbazone-Ga(III) complexes, the following studies were carried out: (1) six thiosemicarbazone ligands and their corresponding Ga(III) complexes are synthesized (Scheme 1); (2) the structure and activity relationships of these compounds have been studied; (3) intracellular ROS, mitochondrial membrane potential, caspase-3/9 activation, and expression of cyt-c and apaf-1 were analyzed and used to investigate the mechanism behind gallium complexes promoted apoptosis; (4) expression of cell cycle-associated proteins (cdk2, cyclin A, cyclin E) which Scheme 1 Synthetic route of Ga(III) complexes.

Results and discussion
Synthesis and crystal structure description The thiosemicarbazone ligands (L1-L6) were prepared as described in the literature, and the prepared ligands were subjected to NMR, mass spectrometry and elemental analysis. Coordination of an equimolar amount of ligand with Ga(III) chloride to synthesize a series of 1 : 1 ligand/Ga(III) complexes, and products were crystallized from methanol in high purity without purication (Scheme 1). The crystal data of C6 was displayed in Table S1, † and the selected angles and bond lengths are listed in Table S1. † C6 crystallized in the monoclinic system, and space group P2 1 /n. The Ga(III) metal center of C6 is coordinated by one S atoms, two Cl atoms (Cl1 and Cl2) and two N atoms (N1 and N2), which formed a deformed trigonal bipyramid (Fig. 1 (7)Å] bonds. The Ga-Cl and Ga-N bonds were shorter than the length of Ga1-S1 [2.3291(7)Å]. In Ga(III) complex, the anion Cl and tridentate thiosemicarbazone coordinate with the central copper ion(II) to form an approximately planar structure. The bond angles and bond lengths in C6 are identical to other known compounds in published articles. 10,11,29 Anticancer properties of Ga(III) complexes A liver hepatocellular carcinoma cell line (HepG-2) was used to evaluate the anticancer activity of the thiosemicarbazide ligand and the Ga(III) complexes. All of the thiosemicarbazide ligands (L1-L6) and Ga(III) complexes (C1-C6) showed potent antiproliferative activity and results were listed in Table 1. The anticancer activity of GaCl 3 (50 mM) was not observed. More importantly, these chemical agents showed very low toxicity to normal cells (LO-2) compared to tumor cells.
As shown in Table 1, the anticancer activity of L1-L6 increased sequentially aer HepG-2 cells were treated for 48 hours. Among them, the antiproliferative activity of L1 (IC 50 , 11.35 AE 0.21 mM) is effectively lower than that of L6 (IC 50 , 0.32 AE 0.02 mM). Similarly, the cytotoxicity of the Ga(III) complex increased synchronously with ligand numbers aer 48 hours of treatment on HepG-2. These data indicate that the antiproliferative activity of Ga(III) complexes is determined by the corresponding ligand, and the anticancer activity of the ligand is enhanced by coordination with gallium. L6 (IC 50 , 0.32 AE 0.02 mM) and C6 (IC 50 , 0.14 AE 0.01 mM) has the most excellent antiproliferative activity against HepG-2 in ligands and complexes, respectively. Aer 48 h of treatment, the cytotoxicity of L1-L6 increases in turn, and the toxicity of the complex is higher than that of the corresponding ligand. However, L6 has the best selectivity indices (SI) in ligands, and the presence of Ga(III) has a better impact on this parameter. The antitumor activity of all Ga(III) complexes was higher than that of cisplatin.

Cell apoptosis assay
Apoptosis is physiological and programmed death process, a basic biological phenomenon of cells. [30][31][32] Lack of apoptosis in cell proliferation is a crucial factor in tumor development. [33][34][35] Therefore, an effective way to treat tumors is to stimulate and restore apoptosis of tumor cells. To assess the effect of concentration on the ability of gallium complexes (C6) to promote apoptosis in HepG-2 cells, Annexin v-tc/PI was used to stain the cells and ow cytometry was used to   detect apoptosis (Fig. 2). Aer incubation for 24 hours at 5 mM of L6 and C6, the percentage of early apoptosis and late apoptosis increased to 24.87% and 40.7% relative to the control.

Cell cycle analysis
We used ow cytometry to evaluate the effect of thiosemicarbazide ligands and Ga(III) complexes on cell cycle, and propidium iodide (PI) was used to stain DNA of HepG-2 cells as well as the cell cycle distribution was performed by ow cytometry (Fig. 3). Aer 24 h of co-cultivation of L6 and C6 with the cells, quantication of the results showed that the percentage of G2 phase cells were reduced compared to the vehicle-treated control. The increase in G1 phase cell proportion was consistent with other data.

Mitochondrial membrane potential assay
Mitochondria are the main site for cellular aerobic respiration, known as "power house", and the dissipation of mitochondrial membrane potential may be one of the causes of cell apoptosis. 36 To examine the effect of the ligands and Ga(III) complexes on mitochondrial membrane potential (Dj m ), changes in mitochondrial membrane potential were monitored by ow cytometry using JC-1 uorescent probe (5,5,6,6 0 -tetrachloro-1,1 0 ,3,3 0 -tetraethyl-imidacarbocyanineiodide). Red uorescence (JC-1 aggregates) occurs when the mitochondrial membrane potential is high, whereas green uorescence (JC-1 monomer) is observed when the mitochondrial membrane potential is low (Fig. 4A). HepG-2 was cultured with 5 mM of L6 and C6, the Dj m was signicantly (p < 0.001) decrease to 22.5% and 73.5% relative to the control, respectively.

Intracellular ROS measurements
The loss of mitochondrial function may cause disorder of antifree radical metabolism and free radical oxidative metabolism in cells. 37 Drug invasion leads to excessive intracellular reactive oxygen species (ROS) production or decreased clearance capacity, resulting in oxidative stress and apoptosis. 38 Aer incubation for 24 hours at 5 mM of L6 and C6, the percentage of intracellular ROS level increased to 17.12% and 27.12% relative to the control (Fig. 6). At the same concentration, C6 promoted the ability of HepG-2 cells to produce ROS signicantly stronger than (Fig. 6) L6.   Detection of changes in protein levels of apaf-1 and cyt-c The apoptotic active substances (cyt-c) will release from the mitochondria through the permeability transition (PT) pore under the action of the mitochondrial membrane potential (Dj m ). 39,40 To conrm this phenomenon, the inuence of L6 and C6 on the expression of apaf-1 and cyt-c in HepG-2 cells were performed by western blot (Fig. 7). The results showed that 5 mM of L6 had signicant effect on the expression of apaf-1 and cyt-c. Aer treated with 5 mM C6, induced a signicant (p < 0.05) increase in apaf-1 and cyt-c protein levels than L6 incubated cells. The increase in pro-apoptotic protein levels implied the occurrence of apoptosis.

Caspase-3/9 activation assay
Upon initiation of apoptosis, cyt-c is released into the cytoplasm and subsequently activates caspase-3/9 protein to form apoptotic bodies. [41][42][43] Thus, the percentage of activated caspase-3/9 was analyzed when HepG-2 cells were treated with L6 and C6, respectively (Fig. 8). Aer incubation with 5 mM of L6 and C6 for 24 hours, the percentage of activated caspase-3 protein level increased to 10.6% and 24.4% compared to the control, and the activated caspase-9 protein level increased to 8.2% and 17%. The results showed that L6 and C6 are potent activators of caspase-3/9, and up-regulation of these pro-apoptotic protein is an important pathway for apoptosis of HepG-2 cells induced by thiosemicarbazide ligands and their gallium complexes.

Detection of changes in protein levels of cell cycle related proteins
Regulation of the cell cycle is inuenced by cyclin, cyclindependent kinase (Cdk), Cdk inhibitor, DNA, and the core mechanism is the activation and regulation of Cdk inhibitors. 44 Therefore, L6 and C6 treated cells were analyzed to reveal the cyclin-dependent kinases (Cdk 2) and cyclins (cyclin A and cyclin E) expression (Fig. 9). HepG-2 cells treated with 5 mM L6 and C6 induced signicant (p < 0.05) decreased in these proteins' expression level.

Material
Chemicals and solvents were purchased from Innochem Co., Ltd. (Shanghai, China), all of which are of analytical grade and used without further purication. Deionized water was used throughout the experiment. The HepG-2 and LO2 cell lines were obtained from Chinese Academy of Sciences. At 298 K, NMR spectra were recorded on a Bruker AVANCE III HD 600 MHz spectrometer. Proton chemical shis were referenced to the central peak of DMSO-d 6. Electrospray ionization mass (ESI-MS) spectrometry were performed on an Agilent 1260-6224 ion trap spectrometer, working in aqueous solution.

Synthesis and characterization of ligands (L1-L6)
The synthesis of ligands (L1-L6) was performed by previous methods. 11 One mole equivalent of 2-benzoyl pyridine and 5 drops of CH 3 COOH were added to 10 mL ethanol (1 mM) solution of thiosemicarbazide and reuxed for 4 h. The formed precipitate was collected by ltration and washed with ethanol, and dried in vacuo.

Synthesis and characterization of Ga(III) complexes
The synthesis of Ga(III) complexes (C1-C6) was performed by previous methods. 11 A solution of thiosemicarbazone in methanol (10 mL, 1 mM) was added GaCl 3 (one mole equivalent) with stirring and set still, waiting for the solvent to slowly emanate. Aer a few days, the Ga(III) complex crystals were obtained and washed with ethanol, followed by vacuum drying. Dichlorido

Determination of structure of Ga(III) complexes
The single crystal of C6 was placed on a glass ber. The cell constants and crystal data are accurately measured by RAPID IP diffractometer or Rigaku AFC5S (Mo Ka radiation, k ¼ 0.71069 A). The crystal structures were solved directly and differential Fourier calculations were performed, followed by full matrix least squares renement using the SHELXL97 and TEXSAN package. All non-hydrogen atoms were rened anisotropically. Crystal data, structural renement and data collection are shown in Table 1.

Cytotoxicity assay (MTT)
The cell lines were cultured in an incubator (ThermoFisher) with 5% CO 2 , 95% air at 37 C. We used a MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) assay to investigate the cell cytotoxicity. Dissolving the thiosemicarbazone ligand and the Ga(III) complex in dimethyl sulfoxide (DMSO) to form a 10 mM stock solution. The DMSO solution in which the complexes were dissolved was diluted with different volumes of cell culture medium to ensure a DMSO concentration of <0.1% so as not to affect cell proliferation. 100 mL of medium containing cells was seeded in 96well plates and adhered for 12 h at 37 C, in 5% CO 2 /95% air. The seeding densities used were 5 Â 10 4 cells per well. Different concentrations of ligand or Ga(III) complex (20, 10, 5 1, 0.5 mM) were added to the wells and incubated for 48 h, then 20 mL 5 mg mL À1 of MTT was added to the wells for another 4 hours. Further added with 100 mL of DMSO, and the optical density (OD) of living cells was measured at a wavelength of 570 nm. The IC 50 (half maximum inhibitory concentration) values were calculated using the nonlinear multi-purpose curve tting program (GraphPad Prism).

Cell cycle distribution analysis
HepG-2 cells (5 mL, 1 Â 10 5 cells per mL) were incubated in a 70 mm culture dish, and then the cells were cultured at 37 C, 5% CO 2 /95% air for 12 hours. Aer treating the cells with L6 (5 mM) or C6 (5 mM) for 24 hours, respectively, the collected cells were washed three times with cold phosphate buffer (PBS, pH ¼ 7.4). Cells were immobilized with 75% EtOH for 12 h at À20 C and washed with iced PBS. The cells were incubated with Ribonuclease (RNase, 2.5 mg mL À1 ) and stained with 50 mg mL À1 propidium iodide for 0.5 hours and analyzed by ow cytometry (BD, Accuri™ C6). Data was analyzed for 10 000 events per sample using MFL 32 soware.

Cell apoptosis assay
Apoptosis events in HepG-2 cells induced by L6 (5 mM) or C6 (5 mM) were assessed by Annexin V staining and PI according to the procedure of the Detection Kit (Bection Dickinson). We used 2 mL 1 Â 10 6 cells per mL, which were incubated with L6 (5 mM) or C6 (5 mM) for 24 h at 5% CO 2 and 37 C. HepG-2 cells were dispersed in 300 mL of mixed buffer (140 mM NaCl, pH 7.4, 2.5 mM CaCl 2 , 10 mM Hepes/NaOH), followed by the addition of 5 mL of PI and Annexin V. Finally, the samples were incubated for 20 minutes at room temperature and assayed by ow cytometry (BD, Accuri™ C6) and analyzed by FlowJo soware.

Intracellular ROS measurements
Evaluation of intracellular ROS was performed by the procedure of active oxygen assay kit (Beyotime, Suzhou) and ow cytometry. Incubate HepG-2 cells with L6 (5 mM) or C6 (5 mM) in serum-free cell culture medium for 24 h at 37 C. 2 mM 2 0 ,7 0dichlorodihydro-uorescein diacetate (H 2 DCF-DA) was added to the cells, and the samples were washed with serum-free medium. Data was obtained by ow cytometry (BD, Accuri™ C6) and FlowJo soware was used to analyze the results.

Caspase-3 and caspase-9 activity analysis
The activities of caspase-3 and caspase-9 were detected by CaspGLOW™ Fluorescein Active Caspase-3 and Caspase-9 Staining Kits. L6 (5 mM) or C6 (5 mM) was added to HepG-2 cells for 24 hours. The controlled cells were harvested from RPMI 1640 medium containing 10% fetal calf serum (FBS), then centrifuged and washed three times with PBS. The cells were uniformly mixed with 300 mL of the culture broth, followed by the addition of 1 mL of FITC-DEVD-FMK, and the sample was allowed to stand at 37 C for an hour. Samples were tested on a BD Accuri™ C6 ow cytometer. The experimental results are expressed as a percentage change in activity between the sample and the untreated control.
Western blot analysis 5 mL of HepG-2 cells (1 Â 10 5 cells per mL) were seeded on a 100 mm diameter dish and incubated for 24 h at 37 C, in 5% CO 2 /95% air atmosphere. Cells were harvested and washed 3 times with ice-cold PBS aer treatment of L6 (5 mM) or C6 (5 mM) for 24 hours. The cells were disrupted with buffer and centrifuged at 10 000g for 15 minutes under 4 C to obtain a supernatant. The protein concentration was determined using a BSA Assay Kit (Beyotime, China), and the samples were detected by SDS-polyacrylamide gel electrophoresis. Under ice bath conditions, the protein was loaded on the polyvinylidene uoride membrane for 3 hours, followed by preparation of skim milk (5%) for the presence of protein-blocked 1 h in TBST buffer at 25 C. Membranes and primary antibodies were incubated overnight at 4 C and washed three times with TBST buffer (20 mM Tris-HCl, 0.05% Tween 20, 150 mM NaCl, pH 8.0). Aer the membranes were incubated with the secondary antibody for 1 h at 25 C, they were washed three times in the same manner. Immunoreactivity was imaged by Amersham ECL Plus (Amersham) and the amount of protein in each lane was determined by b-actin.

Statistical analysis
The signicance of the differences was assessed by the Student's t test. The experimental results are shown as mean AE SD, and when p < 0.05, it is indicated that the data is signicant.