2-Pyridinecarboxylic acid hydrazide was prepared based on a previously reported procedure (12). PPAH was made by refluxing an equal molar ratio of 2-pyridinecarboxylic acid hydrazide and 2-pyridinecarboxaldehyde in ethanol, and the reaction process was monitored by TLC. Pure PPAH was isolated by removing the solvent and re-crystallization in acetonitrile. The ¹HNMR (DMSO-d6, Bruker 400 MHz) of PPAH: ¹HNMR: 12.54(s, H(N-H)), 8.73(d), 8.70(s, (CHO)), 8.62(d), 8.15(d), 8.06(t), 8.0(d), 7.90(t), 7.70(t), 7.43(t). ¹³CNMR(ppm): 161.26, 158.36, 149.97, 149.86, 149.78, 149.03, 138.55, 137.35, 127.65, 124.92, 123.37, 120.45. M/Z: 227.2734 (M+H)⁺. The structures of PPAH and its copper complex are shown in Fig. 1.

The molar ratio of PPAH/copper(II) was determined by spectral methods as previously described (13). Briefly, the stock solutions of 1 mM PPAH in 15% dimethylsulfoxide (DMSO) and 1 mM CuCl₂ in water were prepared. Next, 0.2 ml of the CuCl₂ solution was added to a series of 5-ml volumetric flasks, and then 40, 80, 120, 160, 200, 240, 280, 320, 360 or 400 µl of PPAH was transferred to each flask, respectively. Finally, water was added (5 ml). After mixing and equilibrium, the spectra were recorded on a Shimadzu UV-2450 spectrophotometer.

The stock solution of PPAH (10 mM) was prepared in 25% DMSO, and was diluted to the required concentration with water when used. The copper complex was made by mixing PPAH with a high concentration of CuCl₂ based on 1:1 molar ratio and diluted to the required concentration with water. The human colorectal carcinoma cell line (HCT-116) and liver carcinoma cells (HepG2) were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS) and antibiotics. The cells collected during the exponential phase of growth (1×10⁴/ml) were seeded equivalently into 96-well plates, and 1 µl of PPAH or its copper complex at varied concentrations was added after the cells adhered. Following a 48-h incubation at 37°C in a humidified atmosphere of 5% CO₂, 10 µl MTT solution (1 mg/ml) was added to each well, followed by further incubation for 4 h. The cell culture was removed by aspiration, and 100 µl/well of DMSO was added to dissolve the formazan crystals. The measurement of the absorbance of the solution that was related to the number of living cells was performed on a microplate reader (MK3; Thermo Scientific) at 570 nm. Percent growth inhibition was defined as the percentage of the decrease in absorbance to the appropriate absorbance for each cell line. The same assay was performed in triplet.

The comet assay was adapted as previously described (14). HepG2 cells were treated with or without the investigated compounds (40 and 80 µM for PPAH or 2.5 and 5 µM for the PPAH-Cu complex) followed by a 48-h incubation in a humidified atmosphere of 5% CO₂. The cells were harvested by centrifugation after trypsinization and then embedded in 0.5% low melting point agarose at a final concentration of 1×10⁴ cells/ml. A 20-µl aliquot of this cellular suspension was then spread onto duplicate frosted slides that had previously been covered with 1% normal melting point agarose as a basal layer. The slides were allowed to solidify for 10 min at 4°C before being placed in lysis buffer for 1 h [2.5 M NaCl, 0.1 M ethylenediaminetetraacetic acid (EDTA), 0.01 M Tris, 1% Triton X-100, 10% DMSO, pH 10.0). After lysis, the slides were transferred into alkaline buffer for 40 min (0.001 M EDTA, 0.3 M NaOH, pH >13.0) to allow the DNA to unwind before migration at 0.66 V/cm and 300 mA for 30 min. All these steps were performed in the dark. After neutralization in 0.4 M Tris-HCl pH 7.4, the slides were stored at 4°C until analysis following 24 h. Before analysis, the slides were stained with EB (20 µg/ml) and covered with a coverslip. Images was captured using fluorescence microscopy.

Total RNA was extracted from the cells following treatment with the investigated agents for 24 (or 48 h) using TRIzol reagent (Sangon, Shanghai, China) according to the manufacturer's protocol. Two micrograms of total RNA were used for reverse transcription in a total volume of 20 µl with the M-MLV reverse transcriptase system (LifeFeng Biological Technology Corporation, Shanghai, China). Two microliters of cDNA were subsequently amplified in a total volume of 20 µl using the 2X Taq PCR kit (LifeFeng Biological Technology Corporation) following the conditions recommended by the manufacturer. The sense and antisense primers (synthesized by Shanghai Generay Biological Engineering Corporation, Co., Shanghai, China) for β-actin were: 5′-ACACTGTGCCC ATCTACGAGG-3′ and 5′-CGGACTCGTCATACTCCTG CT-3′ (615 bp) used as an internal control; the sense and antisense primers for caspase 3 were: 5′-GAAGCGAATC AATGGACTCTGG-3′ and 5′-ACATCACGCATCAATTCCA CAA-3′ (241 bp); the sense and antisense primers for caspase 8 were: 5′-AAGTTCCTGAGCCTGGACTACAT-3′ and 5′-ATTT GAGCCCTGCCTGGTGTCT-3′ (227 bp); the sense and antisense primers for p53 were: 5′-GTCTACCTCCCGCCAT AA-3′ and 5′-CATCTCCCAAACATCCCT-3′ (316 bp); the sense and antisense primers for bcl were: 5′-TTACCAAGCAG CCGAAGA-3′ and 5′-TCCCTCCTTTACATTCACAA-3′ (309 bp); the sense and antisense primers for bax were: 5′-TTT TGCTTCAGGGTTTCATC-3′ and 5′-GGCCTTGAGCACCA GTTT-3′ (299 bp); the sense and antisense primers for cyclin A were: 5′-TTAGGGAAATGGAGGTTA-3′ and 5′-CAGAAAG TATTGGGTAAGAA-3′, respectively. RT-PCR was performed on a Nexus Gradient Mastercycler (Eppendorf). The cycling conditions were: 94°C for 2 min, followed by 30 cycles of 94°C for 30 sec, 53–56°C for 30 sec, and 72°C for 1 min, and a final extension of 72°C for 10 min. The PCR products were separated on 1.5% agarose gel and visualized by EB staining. The data were acquired with a Tocan 360 gel imager (version 3.2.1 software) (Shanghai Tiancheng Technology Co., Ltd., China).

HepG2 cells (1×10⁵) were seeded in a 6-well plate and incubated for 24 h at 37°C (5% CO₂), the medium was replaced with fresh medium, supplemented or not (control) with the agents (40 and 80 µM for PPAH, or 2.5 and 5 µM for the PPAH-Cu complex). After 24 h of incubation, the cells were harvested with trypsin, followed by washing with phosphate-buffered saline (PBS), fixed in 70% ethanol and stored at −20°C. The cellular nuclear DNA was stained with propidium iodide (PI). Briefly, after removing the 70% ethanol, the cells were washed with PBS and then suspended in 0.5 ml PBS containing 50 µg/ml PI and 100 µg/ml RNase. The cell suspension was incubated at 37°C for 30 min. DNA flow cytometry was performed in duplicate with a FACSCalibur flow cytometer (Becton-Dickinson, USA). For each sample 10,000 events were collected, and fluorescent signal intensity was recorded and analyzed by CellQuest and ModiFit (Becton-Dickinson).

The nuclear extract from HepG2 cells was prepared as previously described (15). The nuclear extract (0.4 µg) was added to the Top reaction mixture containing 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 150 mM NaCl, 0.1% bovine serum albumin (BSA), 5% glycerol and 0.4 µg pUC18 and 1 µl (or 2 or 3 µl) test agent (0.5 mM for PPAH or 0.3 mM for PPAH-Cu complex in water) at a final volume of 20 µl. Following incubation at 37°C for 30 min, the reaction was terminated by adding 5 µl of stopping buffer (10% SDS, 0.025% bromophenol blue and 5% glycerol). The reaction products were analyzed by electrophoresis on 1% agarose gel using a TBE buffer with 0.1% SDS (89 mM Tris-HCl, 89 mM boric acid and 62 mM EDTA) at 45 V for 3 h, stained by EB (0.5 µg/ml) and photographed using a short wavelength UV lamp on a Tocan 360 gel scanner. The assay was conducted in duplicates.

PPAH has potential coordination atoms. Thus, in the presence of copper ions, a new species may be formed as a copper complex. To determine the molar ratio of PPAH to copper ions, the absorption spectra of varied concentrations of PPAH in water at a fixed copper concentration were recorded. As shown in Fig. 2, the copper(II) solution had no absorption in the investigated range of wavelength (240–400 nm). After addition of PPAH to the copper solution a new absorption at ~350 nm was noted, which was different from the PPAH absorption (~310 nm), indicating that a copper complex had formed. To further determine its composition, a series of solutions was prepared in which the concentration of CuCl₂ was held constant while that of the PPAH was varied. The absorbance of each solution was measured (Fig. 2a) and plotted vs. the molar ratio of Cu(II)/PPAH (Fig. 2b). From Fig. 2, a 1:1 molar ratio of Cu(II)/PPAH was determined. It was reported that the coordination geometry of 2-pyridinecarboxaldehyde benzoyl hydrazone with copper complexes has been described as a tridentate chelator (16). In view of the similarity of PPAH to the above ligand in chemistry, the structure of the PPAH copper complex was tentatively proposed (Fig. 1b).

To determine the potential antitumor activity of the agents, the inhibitory effects on the proliferation of the HepG2 and HCT-116 cell lines was investigated by MTT assay (Fig. 3). For both cell lines, the PPAH exhibited moderate inhibition of proliferation, i.e. 50% growth inhibition at ≥60 µM against HCT-116; ~30% growth inhibition at ≥100 µM against HepG2 (Fig. 3a and b). The copper complex exhibited excellent proliferation inhibition against the cell lines. The IC₅₀ value of the copper complex was 2.75±0.30 µM for the HepG2 cells, an ~45-fold increase in proliferation inhibition compared to that of PPAH. A similar trend against the HCT-116 cell line was also observed. The IC₅₀ value of the copper complex was 1.90±0.2 µM, an ~30-fold increase, which demonstrated that the copper complex exhibited excellent antitumor activity.

ROS play a crucial role in cell growth and apoptosis. To reveal the mechanism of action of PPAH and its copper complex, RT-PCR was conducted to determine changes in expression of apoptotic genes, such as bcl-2, bax, p53, caspase-3 and -8 at the mRNA level after HepG2 cells were treated with PPAH (Fig. 4a) or its copper complex (Fig. 4b) for 48 h. The investigated genes were not evidently regulated by the agents. bcl-2 downregulation and bax upregulation were not observed, which normally occurs in ROS-related apoptosis. Similar analysis was conducted using the HCT-116 cell line. Except for PPAH due to its lesser toxicity, slight changes in the levels of the apoptosis-related genes were observed in the PPAH-Cu-treated cells. To ascertain whether ROS are involved in the proliferation inhibition, ROS production was investigated in vitro. The results indicated that the PPAH-Cu complex was non-redox active and did not lead to DNA fragmentation (data not shown). This was also consistent with the results from the comet assay (Fig. 5). The DNA fragmentation was only observed at a higher concentration and 48 h exposure to PPAH and its copper complex.

As shown above, ROS production was not correlated to the effect on inhibition of proliferation by PPAH and its copper complex. Thus, we hypothesized that they may exert their effect via disturbing the cell cycle. We therefore evaluated the effect of PPAH and its copper complex on the cell cycle using PI staining and flow cytometry. As shown in Fig. 6, PPAH caused an accumulation of cells in the S-phase. The percentage of cells in the S-phase was significantly increased from 11.22 to 28.44 and 22.25% after treatment with 40 and 80 µM PPAH, respectively. The PPAH-Cu complex had a similar effect when compared to PPAH; the S-phase population increased from 11.22 to 26.11 (2.5 µM) and 23.87% (2.5 µM), indicating that the agents have a similar mechanism of action that involves effects on the cell cycle except with differences at various concentrations.

Various transition metal complexes have Top inhibition. To determine whether PPAH and its copper complex recapitulate such activity, pUC18 plasmid DNA was incubated with nucleic extract in the presence of varied concentrations of the investigated agents, and reaction products were analyzed by gel electrophoresis. As shown in Fig. 7, PPAH exhibited very weaker inhibition even at a higher concentration (Top I, >100 µM; Top II >75 µM). The PPAH-Cu complex exhibited dual Top inhibition. For type I, a IC₅₀ value of ~50 µM was noted; while type II inhibition was initialized at 12.5 µM (Fig. 7b and c). To further reveal the action mode of the copper complex in Top inhibition, the reaction mixtures were subjected to electrophoresis on EtB-containing agarose gel. As shown from the gel (Fig. 7d), the topology was dominated by the intercalative dye, all closed circular forms of DNA were positively supercoiled and migrated with a similar rate. The Top II cleavage complex was identified by comparison with the positive control of etoposide. However, in our experiment, the Top II cleavage complex was not observed. This situation may have occurred due to a lower Top II concentration used in the assay. The mode of Top inhibition of the PPAH-Cu complex was not clearly identified, but the cleaved DNA (Fig. 7d) increased with increasing concentrations of the copper complex implying that there was the possibility of catalytic or 'poisoner' inhibition.