MTT, di-2-pyridylketone, 3-methylad-enin (3-MA), Pifithrin-α, RPMI-1640 and other chemicals were purchased from Sigma-Aldrich. LC3 antibody was obtained from Proteintech Group (Wuhan, China); antibodies p53, caspase-8, β-actin, cytochrome c, Bax, Mdm2 and Bcl-2 were purchased from Wuhan Boster Biological Technology, Ltd. (Wuhan, China).

DpdtaA (generated by ACDLabs: 3-[({2-[di(pyridin-2-yl)methylidene] hydrazinyl} carbonothioyl) sulfanyl] acetic acid) was made by three-step reactions as indicated in Fig. 1: the first two steps were as previously described (19). In addition, the final product (DpdtaA) was prepared by reaction of di-2-pyridylhydrazone dithiocarbamate (1 mmol) with 2-bromo acetic acid in absolute ethanol (5 ml), the yellow solid was filtered and washed with ethanol. TLC tracing (ethyl acetate/petroleum ether = 3:1) showed the reaction completed. Following a flash chromatography the purity of the compound was achieved to 98%. Yield: 80%; mp: 148.6°C; composition: C₁₄H₁₂N₄O₂S₂; ¹HNMR (Bruker, D₆-DMSO): 8.86 (d, H, J = 4 Hz), 8.64 (d, H, J = 4 Hz), 8.02 (m, 3H, J = 4, 8 Hz), 7.63 (m, 2H, J = 8 Hz), 7.55 (ddd, H, J = 4, 8 Hz), 4.12 (s, 2H). ¹³CNMR: 199.25, 169.87, 155.09, 151.14, 149.20, 148.94, 145.43, 138.24, 137.92, 128.04, 125.72, 124.93, 123.96, 36.80. IR (cm⁻¹): IR (KBr, cm⁻¹): 3088, 1709, 1584, 1458, 1435, 1417, 1393, 1326, 1292, 1234, 1202, 1188, 1127, 1058, 1018, 959, 905, 802, 791, 750, 695, 654, 640, 596, 472. ESI-MS (microTOF-Q III, Bruker): m/z: 371.0043 (M+K, calcd: 371.00389).

A 10 mM DpdtaA in 70% dimethyl sulfoxide (DMSO) was diluted to the required concentration with culture. The copper complex was prepared by mixing DpdtaA with CuCl₂ (at high concentration) based on 1:1 molar ratio and diluted to required concentration with DMSO. The MTT assay was conducted as previously described (20). Briefly, the HepG2 (5×10³/ml) cells were seeded equivalently into 96-well plate and the various amount of DpdtaA (or its copper complex) was added after the cells adhered. Following 48-h incubation at 37°C in a humidified atmosphere of 5% CO₂, 10 μl MTT solution (5 mg/ml) was added to each well, and incubated further for 4 h. After removing the cell culture, 100 μl DMSO was added in each well to dissolve the formed formazan. The absorption of the solution that was related to the number of live cells was performed on a microplate reader (MK3; Thermo Fisher Scientific, Waltham, MA, USA) at 490 nm. Percent growth inhibition was defined as percent absorbance inhibition within appropriate absorbance in each cell line. The same assay was performed in triplicate. Morphologic study was conducted under inverted microscope (Shanghai Batuo Instrument Co., Ltd., Shanghai, China), the photographs of HepG2 cells exposed to DpdtaA (1.56 or 3.12 μM DpdtaA or DpdtaA-Cu for 24 or 48 h were recorded (objective size: 10×20).

ROS production assessment was conducted as previously reported (21). Briefly, H₂DCF-DA was first converted to dichlorofluorescin (DCF) by NaOH, following neutralization, the hydrolysate was used for the assay. Reaction system contained either single reagent or multicomponent in 50 mM sodium phosphate buffer (pH 7.4) with total 4 ml volume, i.e., 0.4 μM DCF, or with 6.25 μM (NH₄)₂Fe(SO₄)₂ (or CuCl₂ or DpdtaA) and 200 μM H₂O₂ (1 mM) for the Fenton reactions. The fluorescence was detected by a FC-960 spectrofluorimeter (excitation at 488 and emission at 525 nm; Shanghai Lengguang Technology Co., Ltd., Shanghai, China) in a 10-min time course at room temperature.

The intracellular ROS assay was measured as company recommended (Beyotime Institute of Biotechnology, Beijing, China). Briefly, ~10⁶ HepG2 cells were collected and washed by phosphate-buffered saline (PBS), then the cells were stained by H₂DCF-DA in serum-free culture medium for 30 min, followed by washing of serum-free culture to remove excess H₂DCF-DA. Then, 100 μl the cell suspension was transferred to individual PCR tube and the test compound (or positive control) was added, following 1-h incubation, the cell suspension was used directly for ROS detection on a FC-960 spectrofluorimeter (excitation at 488 nm and emission at 525 nm; Shanghai Lengguang Technology).

Briefly, 1×10⁷ HepG2 cells treated with or without the DpdtaA or DpdtaA-Cu were scraped off in lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1.0% NP-40, 10% glycerol and protease inhibitors) and subjected to sonication, following spin down by centrifugation at 14,000 × g. The clear supernatant was stored at −80°C. The protein concentration was determined using a colorimetric Bio-Rad DC protein assay on a microplate reader MK3 at 570 nm. Proteins (50 μg) were separated on a 13% sodium dodecyl sulfate-polyacrylamide gel at 200 V for 1 h. The separated proteins were subsequently transferred onto a PVDF membrane at 60 V for 1 h. The membrane was washed three times with Tris-buffered saline (TBS) and was then blocked for 2 h in TBS containing 0.1% Tween-20 and 5% non-fat skimmed milk. The membrane was incubated at 4°C overnight with the primary monoantibody used at a dilution of 1:300 in TBS plus 0.1% Tween-20 (TBST). The membrane was washed several times with TBST and was subsequently incubated with HRP-conjugated secondary antibody (1:2,000 in TBST) for 1 h at room temperature. After another wash of the membrane with TBST, the protein bands were detected using a super sensitive ECL solution (Wuhan Boster Biological Technology), and visualized on an Amersham Imager 600 (GE Healthcare Life Sciences, Fairfield, CT, USA).

Cells were seeded into a 6-well flask and treated as described above for the cell viability assay. The cells were treated with different concentrations of the agents (0.78 and 1.56 μM DpdtaA or 3.12 and 6.25 μM DpdtaA-Cu) for 24 h. Then the cell culture was removed, following PBS washing, trypsin digestion, finally the Annexin V and propidium iodide (a kit from Dojindo Laboratories, Kumamoto, Japan) were added as recommended by the company. The stained cells were subjected to cytoflow analysis.

The structure of human type MDM2 (3jzk) was obtained from the RCSB Protein Data Bank. The structure of DpdtaA was generated by Chemdraw (Chemdraw Ultra 8.0; CambridgeSoft, Cambridge, MA, USA). The energy minimization was conducted by Chem3D (Ultra 8.0; CambridgeSoft). PyMol and LigPlot displayed the conformation, interaction between bound ligands and residue around the binding cavity (22,23).

Molecular docking studies were performed by AutoDock Vina and AutoDock Tools based on the recommended procedure (24). Grid box was set to the center of Yin model, and the grid box size for DpdtaA was set to 22, 24 and 28 for x-, y- and z-axes, respectively. The DpdtaA was set as a flexible ligand by using the default parameters of the AutoDock Tools. The optimal conformation of the ligand was generated by AutoDock Vina.

Cells were seeded into a 24-well flask and treated as described above in the cell viability assay. The cells were treated with different concentrations of the agents (0.78 and 1.56 μM DpdtaA or 3.12 and 6.25 μM DpdtaA-Cu) for 24 h. For detection of the acidic cellular compartment, acridine orange (or LysoTracker Red; Invitrogen) was used, which emits bright red fluorescence in acidic vesicles but green fluorescence in the cytoplasm and the nucleus. After treatment of the cells with the agent, acridine orange was added at a final concentration of 1 μg/ml (the concentration of LysoTracker Red, as recommended) for a period of 15 min. Following PBS washing, the fluorescent micrographs were captured using an inverted fluorescence microscope.

A three-step reaction as described in Fig. 1 was used to generate the DpdtaA. Firstly, hydrazine reacted with carbon disulfide to form hydrazine dithiocarbamate (HdtC, I), then following addition of dipyridineketone, the dipyridylhydrazone dithiocarbamate (DpdtC, II) was made as previously described (19). The DpdtaA (III) was prepared by DpdtC reaction with 2-bromoacetic acid. The new compound was traced by TLC, purified by flash chromatography. Finally, the chemical structure of DpdtaA was characterized by NMR and HRMS spectra. HPLC and NMR showed that agents have adequate purity (>98%, data not shown), indicating that the new prepared DpdtaA can be used for biological assay.

We are interested in the biological activity of new synthetic DpdtaA, thus, antiproliferative activity of DpdtaA against hepatocellular carcinoma cell lines was screened (Fig. 2F–G), the dose-response curves are depicted in Fig. 2. As shown in Fig. 2F–G, DpdtaA exhibited significant growth inhibition for HepG2 and Bel-7402 (IC₅₀ ≤6 μM), but the cell line dependence in dose-response was also obvious. For HepG2 cells, the maximal inhibition of ~80% can be achieved at 12.5 μM (Fig. 2F), but only 40% inhibition for Bel-7402 at the same concentration of DpdtaA (Fig. 2G). In view of the important role of copper ion in dithiocarbamates activity, the proliferation inhibition of DpdtaA in the presence of copper ion was also investigated. As previously reported, an antagonistic effect in growth inhibitions was observed for both cell lines, leading to an ~3-fold decrease in HepG2 cells based on IC₅₀ value, but this was not evident for Bel-7402 cells except maximal inhibition, which was few for dithiocarba-mate derivatives. To gain details, the morphologic change was further investigated. As shown in Fig. 2A–D, the DpdtaA caused rounded HepG2 cells, but the addition of copper ion significantly attenuated the action, showing an antagonistic effect as we previously reported (19). We speculated that the 'antagonistic effect' might stem from a new species which was confirmed at DpdtaA/Cu =1:1 ratio in composition by spectral titration (data not shown).

It is prevailingly accepted concept that antiproliferation inhibition (or cytotoxicity) of many drugs stem from generation of reactive oxygen species (ROS). DpdtaA may chelate iron or copper, thus, involved in Fenton or Fenton-like reaction. The ROS production in vitro is shown in Fig. 3A, fluorescence intensities of DCF in the presence of DpdtaA and Fe²⁺ was higher than that of Fe²⁺, indicating that iron DpdtaA complex is redox active and could produce more ROS. In contrast, DpdtaA-Cu has weaker ability to generate ROS, however, in the reduced environment [presence of ascorbic acid (Vc)], the DpdtaA-Cu induced highest content of ROS. To further confirm the in vitro results, the drugs inducing intracellular ROS were investigated, as shown in Fig. 3B, both DpdtaA and its copper complex induced ROS generation in a concentration-dependent manner. It was noted that the fluorescent intensities of DCF from copper complex treated cells were significantly greater than that of DpdtaA, which might be related to the redox feature of Cu^2+/1+ complexes, because the copper (I) complex could also react with oxygen molecule to form superoxide radical except with H₂O₂ (25).

As mentioned above, both DpdtaA and its copper complex induced ROS generation, whether the ROS generation was involved in antiproliferative activity was unclear. Thus, the correlation between ROS and growth inhibition, the effect of reducing agent, N-acetylcysteine (NAC) on growth inhibition of DpdtaA was investigated. The morphologic changes induced by DpdtaA in the absence or presence of NAC are shown in Fig. 4. It was clear that the numbers of rounded cells after addition of DpdtaA were decreased with increasing NAC (Fig. 4A–C), indicating that the NAC can protect the HepG2 cells from ROS oxidative damage. However, a contrary phenomenon in the DpdtaA-Cu treated cells was also observed, i.e., rounded cells increased (Fig. 4D–E) with addition of NAC. The situation was speculated to attribute to the redox feature of Cu²⁺ which can be reduced to Cu¹⁺ by NAC, and the resulting DpdtaA-Cu¹⁺ reacted with O₂ to generate more ROS. To confirm the speculation, the growth inhibition in the presence of NAC was further evaluated. As shown in Fig. 4F, the antiproliferative activity of DpdtaA was significantly decreased from ~90 to 60%, but the growth inhibition of DpdtaA-Cu dramatically increased, indicating the antiproliferative action of the agents was ROS mediated.

It has been well documented that the excess intracellular ROS induce apoptosis whether the ROS induced by the agents involved in apoptosis was required to be determined. To measure the apoptosis populations at early and late stages, the Annexin V/propidium iodide (PI) staining was performed, which measures externalization of phosphatidylserine on the cell surface of apoptotic cells specifically. The flow cytometric analyses showed that the DpdtaA induced early apoptosis and later apoptosis in a concentration-dependent manner (Fig. 5A–C, from 5.8 to 11.1%), but the apoptotic ratios were smaller after 24-h treatment of DpdtaA. Similar situation for DpdtaA-Cu, just the percentage was less (5.8–9.8%) even at higher concentration.

To further support that the growth inhibition may involve apoptosis, the expressions of apoptosis related proteins were investigated by western blotting. The alterations of bcl-2 and bax proteins before and after treatment with DpdtaA at different concentrations were recorded (Fig. 6). It was clear that the increased bax was observed in both DpdtaA and DpdtaA-Cu treated HepG2 and Bel-7402 cells compared to that of control (Fig. 6A-a and B-a), on the contrary bcl-2, a pro-survival protein was downregulated in the agent treated cells. For comparative purposes, the ratio of bax/bcl-2 was normalized (Fig. 6A-b and B-b). As shown in Fig. 6A-b the difference in ratio of bax/bcl-2 was obvious, 23–35-fold increase for DpdtaA, 4- to 17-fold for DpdtaA-Cu. Similar results were observed in Bel-7402 cells except smaller folds (Fig. 6B-b). The increased bax/bcl2 determined that apoptosis occurred when the cells were treated with the agents. To correlate the apoptosis with ROS, the ROS scavenger NAC was added to the Bel-7402 cells either individually or combined. As shown in Fig. 6C-a, the NAC did decrease the ratio of bax/bcl2 compared to the DpdtaA (DpdtaA-Cu) treated group only, indicating that the agent-induced apoptosis was ROS-dependent.

The change of permeability of the mitochondria and cytochrome c release from the mitochondria are a key step in the process of apoptosis and is one of markers in intrinsic apoptosis, the increase of cytochrome c in cytoplasm (Fig. 6A-a and B-a), indicating that intrinsic apoptosis was involved in the growth inhibition induced by the agents. To determine whether apoptosis was due to p53 regulation, the expression of p53 was assessed, as shown in Fig. 6, the upregulated p53 may hint that there was a correlation between p53 and apoptosis. On the other hand, the upregulation of caspase-8 may also indicate that an extrinsic pathway protease was involved.

The DpdtaA and its copper complex induced alterations in apoptosis related proteins in both cell lines were similar, and could upregu-late p53. Hence, the role of p53 required to be further investigated. Pifithrin-α (PFT-α) is a specific wild-type p53 inhibitor, and the expression of wild-type p53 in HepG2 cells was monitored in the presence or absence of 10 μM PFT-α. Western blot analysis revealed that the expression of p53 was significantly attenuated by PFT-α treatment or combination with the agents, compared with the treatment of agents alone (Fig. 7A). These results indicate that p53 played a role in agent-induced apoptosis. It has been accepted that cellular p53 levels is primarily controlled through its ubiquitin-mediated proteasomal degradation (26,27) and the mouse double minute protein 2 (Mdm2) as the principal endogenous E3-ligase with high specificity for p53 (28). The interaction of Mdm2 with p53 led to p53 degradation. Thus, the assessment of expression of Mdm2 was necessary. As shown in Fig. 7A, the downregulation of Mdm2 was observed in treatment of the cell with the agents, indicating that DpdtaA and its copper complex upregulated p53 via Mdm2 downregulation pathway. Since p53 exerts its induction of apoptosis mainly through the regulation of bcl-2 family proteins, the bcl-2 and bax protein levels were assessed. The relative ratio of bax/bcl-2 is shown in Fig. 7B, such ratio may determine the cell sensitivity or resistance to the apoptotic stimuli (7). On the other hand, p53 and Mdm2 interacted reciprocally, the interaction is hydrophobic interaction, disrupting the interaction would benefit the upregulation of p53. It has been demonstrated that some of small molecules can disrupt their interaction (29), so the theoretical simulation by molecular docking was conducted. The crystal structure of human type Mdm2 (PDB ID: 3jzk) was from the RCSB Protein Data Bank. In order to evaluate the accuracy of our docking protocol, the Yin (ligand in 3jzk) was re-docked into the Mdm2 complexes based on recommended procedure (Fig. 8A). As shown in Fig. 8, the docked Yin was almost fully superimposed on the native co-crystallized one. Thus, following the same protocol the DpdtaA was individually docked into the Mdm2 (Fig. 8B), the simulating affinity energies were −9.4 for Yin and −6.9 kcal/mol for DpdtaA, respectively. The interaction of DpdtaA with residues of Mdm2 is depicted in Fig. 8C. The data implied that the medium interaction between DpdtaA and Mdm2 might also contribute to the upregulation of p53.

Previous report revealed that bax could be translocated from the cytosol to the lysosomal membrane, which led to alteration in lysosomal membrane integrity (30). To test this hypothesis, LysoTracker Red, which can accumulate within the lysosomes, was employed to assess the LMP (31). As shown in Fig. 9A–C, the treatments of DpdtaA and DpdtaA-Cu significantly increased the red fluorescence density in HepG2 cells compared to without treatment, indicating that LMP was altered. Furthermore, the changes in LMP may be a response to autophagy; thus, the formation of autophagosomes was measured by acridine orange staining. As shown in Fig. 9D, E and G, the accumulated red granular fluorescence in the acidic vacuoles was observed in the agent-treated groups, implying that the formation of autophagosome was increased, and autophagy occurred. To support the above conclusion, an autophagy inhibitor, 3-methyladenin (3-MA) was added to agent-treated cells and clearly the red granular fluorescence in the acidic vacuoles decreased compared to that the agent only (Fig. 9F and H). The LC3 (microtuble-associated protein light chain 3) is an autophagosome molecular marker, its change was further assessed by western blot analysis. As expected, the increase of cleaved LC3-II and decrease of LC3-I were observed compared to that of control, indicating that there was autophagy response after exposure of the agents (Fig. 9I). The changes of LC3-II were parallel to the results from acridine orange staining and demonstrated the DpdtaA and its copper complex had similar action in induction of autophagy.