A new Schiff base coordinated copper(II) compound induces apoptosis and inhibits tumor growth in gastric cancer

Human gastric cancer cell lines, SGC 7901 (TCHu46) and BGC823 (TCHu11), were purchased from the Cell Bank of Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). The catalogue number of each cell line is indicated in the bracket following its name. Dulbecco’s modified Eagle’s medium and RPML-1640 culture medium were obtained from Thermo Scientific Co. (USA). The gastric cancer cells, SGC 7901 and BGC823, were maintained in their respective medium in a 5% CO₂ incubator at 37 °C. 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) agent was purchased from Promega Corporation (USA). The annexin V-FITC cell apoptosis detection kit and the cell cycle kit were obtained from Sigma-Aldrich (St. Louis, MO, USA). Acridine orange/ethidium bromide (AO/EB) staining solution, Hoechst 33258, N-acetyl cysteine (NAC), and 4,6-diamidino-2-phenylindole, dihydrochloride (DAPI), N_ω-nitro-l-arginine methyl ester (L-NAME), ammonium pyrrolidine dithiocarbamate (PDTC) were purchased from Sigma (St. Louis, MO, USA). Antibodies for detection of nuclear factor (NF)-κBp65, IκB, and p-IκB were obtained from Cell Signaling Technology (Danvers, MA USA). Antibodies for detection of LC3I, LC-3II, and Beclin-1 as well as the secondary antibody were from Shanghai Biological Engineering Company Limited (Shanghai, China).

For synthesis of the SBCCC, 2 mmol of salicylic aldehyde (0.402 g) and 2 mmol of salicylic hydrazide were dissolved in 80 mL of anhydrous ethanol and heated in a water bath under reflux for 7 h. Next, the sediment was filtered at atmospheric pressure and recrystallized twice to afford the Schiff base ligands. The copper coordinated Schiff base complexes were prepared by dissolving the Schiff base ligands (2 mmol) and copper sulfate (1 mmol) in anhydrous ethanol (70 mL). The mixture was heated in a water bath under reflux for 8 h, filtered at atmospheric pressure, and then recrystallized. The synthesis equation for SBCCC was shown in Fig. 1.

MTS assay was performed to determine cell proliferation and the tested samples were triplicated. In brief, cells were seeded at 1 × 10⁴ cells/well in a 96-well plate. When 80% confluence was reached, the cells were washed with phosphate-buffered saline (PBS) and treated with SBCCC at different concentrations (0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, and 12.8 μM) diluted in culture medium. After 24 h treatment, 10 μL MTS was added into each well. Optical density (OD) value was determined at 490 nm in each test well using a microplate reader (BioTek, Winooski, VT, USA). Cell growth inhibition rate was calculated according to the equation: cell growth inhibition rate = (1 − OD of SBCCC treated cell/OD of control cell) × 100%. Half maximal inhibitory concentration (IC50) was calculated by GraphPad Prism 7.

Cell death was determined by flow cytometric analysis (FACS Calibur, BD, USA) using an Annexin V-FITC Apoptosis Detection kit according to the manufacturers’ instructions. Cell growth arrest was detected by flow cytometry using propidium iodide (PI) staining. In brief, cells were seeded in a 6-well culture plate at a density of 1 × 10⁵ cells per well. When 80% confluence was reached, the cells were treated with SBCCC at a final concentration of 1 μM. Only culture medium was added as control cells. After treatment for 12, 24, and 36 h, the cells were collected and washed twice with PBS, and incubated with 100 μL of RNase A at 37 °C in a water bath for 30 min. The cells were then incubated with PI at 4 °C in the dark for another 30 min. After completion of all staining steps, the cells were washed with incubation buffer and resuspended in 500ul incubation buffer for flow cytometric analysis. FlowJo 7.6.1 software was used for the data analysis.

For the AO/EB staining and apoptosis analysis, cells were seeded in 6-well culture plates at a density of 1 × 10⁴ cells per well in a 5% CO₂ incubator at 37 °C. When 80% confluence was reached, the cells were washed with PBS and treated with 200 μL of the SBCCC at 1 μM for 36 h, while only culture medium was added as the control cells. The treated cells were washed with PBS, and then stained with respective solution, 50 μg/mL dual AO/EB solution, 10 μg/mL Hoechst 33258, and 5 μg/mL DAPI were added to each well. The positive staining and cell morphology were examined and photographed using an inverted fluorescence microscope (LX71, Olympus, Japan).

For the MDC staining, the cells were cultured as described above. The cells were treated with SBCCC at a final concentration of 1 μM for 12, 24 and 36 h. After treatment, the cells were washed with PBS stained with MDC at 50 μM. Cells were incubated for 1 h in a 5% CO₂ incubator at 37 °C. The positive staining and cell morphology were examined and photographed using an inverted fluorescence microscope (LX71, Olympus, Japan).

For the colony formation assay, SGC-7901 cells and BGC-823 cells were seeded in 6-well culture plates at a density of 1 × 10⁴ cells per well and were cultured in a 5% CO₂ incubator at 37 °C. When 80% confluence was reached, the cells were washed with PBS and treated with 1 μM SBCCC, 50 μM PDTC, 1 μM SBCCC + 50 μM PDTC respectively for 24 h. After treatment, the cells were collected and reseeded in 6-well plates at a cell density of 200 cells/well. The cells were then cultured for 3 weeks under saturated humidity conditions of 5% CO₂ at 37 °C, and the culture medium was changed every 3 days. Cell colony formation was examined by hematoxylin staining and the colony numbers were counted.

Total protein was extracted from cells using lysis buffer containing 20 mM Tris–HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1% Triton-X 100, 1% dithiothreitol, and 1% protease inhibitor cocktail. The protein sample was loaded at 40 μg and separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane (Millipore, USA). Membranes were blocked with 5% (w/v) nonfat dry milk dissolved in Tris-buffered saline plus Tween-20 (TBS-T; 0.1% Tween-20; pH 8.3) at room temperature for 1 h, then incubated with respective primary antibodies (NF-κB p65, 1:150; I κB, 1:1000; p-I κB, 1:1000; LC3I, 1:1000; LC-3II, 1:1000; Beclin-1, 1:1000; Bcl-2, 1:500; Bax, 1:500; caspase-3, 1:1000; cleaved caspase-3, 1:1000; cleaved poly(ADP-ribose) polymerase (PARP), 1:1000; cytochrome C, 1:1000; iNOS, 1:1000; JNK, 1:1000; p-JNK, 1:1000) at 4 °C overnight. After washing with TBS-T, the membranes were incubated with polyclonal anti-horseradish peroxidase-labeled sheep or rabbit IgG (1:5000) secondary antibodies for 2 h at room temperature. Immunobands were visualized using an enhanced chemiluminescence kit (GE Healthcare, USA), according to the manufacturer’s instructions, and exposed to X-ray film. β-Actin was used as a loading control.

For the measurement of ROS generation, cells were seeded in 6-well culture plates at a density of 1 × 10⁴ cells per well and were cultured in a 5% CO₂ incubator at 37 °C. When 80% confluence was reached, the cells were washed with PBS and treated with 1 μM SBCCC for 12, 24 and 36 h. For untreated controls, only culture medium were added. After treatment, the cells were washed with PBS and then added 1 mL of freshly prepared DCFH-DA was added at a final concentration of 10 μM. The cells were incubated in the dark for 20 min at 37 °C. The positive staining and cell morphology were examined and photographed using an inverted fluorescence microscope (LX71, Olympus, Japan).

Twelve male BALB/c nude mice were purchased from the Guangdong Medical Laboratory Animal Center (Foshan, China). The animals were housed four per cage, given indicated chow and tap water, and maintained at 22 °C and on a 12-h light/dark cycle. To established tumor model, SGC-7901 cells were inoculated subcutaneously at 10⁶ cells/mouse into the right flank. Tumor growth was monitored and tumor sizes were determined by measuring the length and width using a caliber. Tumor sizes were calculated using the formula: tumor volume = length × width × width/2. When the average tumor size reached at 5–6 mm³. The 9 tumor-burden BALB/c nude mice were injected with SBCCC at 40 µM/kg bodyweight, i.p., twice per day. Three control mice were treated with same volume saline. The SBCCC treated mice were sacrificed at week 1 and week 2 and the harvested tumor were fixed in formalin. At week 3, the last 3 SBCCC treated mice along with the saline treated controls were sacrificed and the harvested tumor tissues were fixed in formalin for a week, then the tumor size was determined and tumor gross anatomy was photographed. This animal experimental protocol was approved by the Animal Care and Use Committee (College of Basic Medicine, Jilin University).

With SBCCC treatment, there were significantly increases of inhibitory rates in the gastric cancer cells when SBCCC dosing increased from 0.4 to 12.8 µm (P < 0.05). Both SGC-7901 cells and BGC-823 cells showed a similar growth inhibition trend, in a dose-dependent manner (Fig. 2a, left). IC50 of SGC-7901 cells and IC50 of BGC-823 cells were 1.101 μM and 0.9864 μM, respectively. When the logarithms of SBCCC concentrations were plotted versus the inhibition rates, IC50 showed as 1 μM for both gastric cancer cell lines (Fig. 2a, right). Therefore, we chose 1 μM as an approximate IC50 to continue the further experiments.

To investigate the cellular events, apoptosis and cell growth arrest were determined by Flow cytometry. With SBCCC treatment, the apoptotic rates (%) for SGC-7901 cells were 25.11 ± 1.34 at 12 h, 32.53 ± 2.54 at 24 h, and 53.03 ± 5.67 at 36 h, respectively. Similarly, the apoptotic rates (%) for BGC-823 cells were 28.87 ± 2.34 at 12 h, 39.62 ± 3.61 at 24 h, and 64.33 ± 6.25% at 36 h, respectively. The apoptotic rates for both cell lines significantly increased in a time-dependent manner compared to control cells (P < 0.05) (Fig. 2b left). The growth arrest was further evaluated in both SGC-7901 cells and BGC-823 cells with SBCCC treatment. For both cell lines, the percentage of cells in the G1 phase continually increased during the SBCCC treatments for 12, 24, and 36 h. The increases of G1 phase cells were found to be statistically significant compared to control cells (P < 0.05). In contrast, the cells in the S phase continually decreased during the SBCCC treatments for 12, 24, and 36 h. The decreases of S phase cells were found to be statistically significant compared to control cells (P < 0.05). No changes were observed in the percentage of cells in the G2 phase from both cell lines treated with SBCCC (Fig. 2b right). Based on these results, the growth arrest was happened at G1 phase for both gastric cancer cells, implying that the SBCCC blocked the cell progression to S phase.

The copper(II) complexes facilitate the DNA cleavage, and exhibited substantial cytotoxic activity against cancer cells [19]. Dual AO/EB, Hoechst 33258 and DAPI staining were performed in both SGC-7901 and BGC-823 cells treated with SBCCC. AO, a cell-permeant dye, emitted green fluorescence when bounded to dsDNA but emitted red fluorescence when bounded to ssDNA. This unique characteristic of AO staining allowed us to evaluate the cell-DNA structure affected by SBCCC. EB only entered cells with damaged membranes and emitting orange-red fluorescence when bound to concentrated DNA fragments. Therefore, dual AO/EB staining allowed us to detect early apoptotic cells, late apoptotic cells, and dead cells. The result indicated that gastric cancer cells with 1 μM SBCCC treatment showed gradually orange color for 12 h. The numbers of orange-stained cells were increased at 24 h, and a portion of the SBCCC treated cells turned to red and orange-red at 36 h (Fig. 3a). Both Hoechst 33358 and DAPI were minor groove-bound and UV-excited. In chromosome condensation or DNA cracking changes, increased binding of Hoechst 33258 dye and DNA shows strong blue compared to normal chromosome cells. DAPI dye is not completely permeability. The ability of permeability for DAPI is improved in apoptotic cells, in which high blue fluorescence is stained in condensed chromosome and cracking DNA, shown abnormal margin of nucleus. When SGC-7901 and BGC-823 cells were treated with 1 μM SBCCC for 36 h, strong blue fluorescence of Hoechst 33258 and DAPI was found. In DAPI staining with high magnification, pyknosis and abnormal margin of nuclei was identified (Fig. 3a). Taken together, SBCCC interfering with DNA structure could be an important mechanism for its cell-killing effect.

Monodansylcadaverine (MDC) was reported as a specific in vivo marker for autophagic vacuoles [20]. Therefore, MDC staining was further performed to detect the autophagic vacuoles in both of SGC-7901 and BGC-823 cells challenged by 1 μM SBCCC. The result indicated that the autofluorescent MDC (green fluorescence) gradually increased in the two cell lines, in a time-dependent manner. Computer-image quantification for the MDC green fluorescence indicated that there was significantly increases of the fluorescence intensity of MDC in cancer cells with SBCCC treatment for 12, 24 and 36 h, compared to untreated controls (P < 0.05) (Fig. 3b).

Based on the in vitro data, the anti-tumor effect of SBCCC was further tested in vivo using a moue tumor model established by SGC-7901 cells xenograft. The result indicated that SBCCC treated mice showed a significantly decreased in tumor size compared to the untreated mice. As shown in Fig. 3c, the tumor volume in untreated group was 1463 mm³ ± 87.79 versus that in the treatment groups 783 mm³ ± 66.91 (P < 0.05) at week 134 mm³ ± 18.82 (P < 0.05) at week 2, and 67 mm³ ± 6.81 (P < 0.05) at week 3.

As the SBCCC induced apoptosis had been demonstrated by above experiments, we further investigated the important components of apoptotic signaling in term of apoptotic initiators and effectors to elucidate the potential anti-tumor mechanism(s). As expected, SBCCC treatment significantly reduced the protein level of Bcl-2 but increased the protein levels of Bax, and reduced the release of cytochrome C from mitochondria to cytosolc, in a time-dependent manner in both cell lines. Moreover, leaved caspase-3, and cleaved PARP-1, a hallmark of apoptosis [21], were found significantly increased in the SBCCC treated gastric cancer cells compared to the controls (Fig. 4a). Because activation of NF-κB signaling was well established in gastric cancer [22], the critical components in NF-κB signaling were further evaluated in the two gastric cancer cell lines challenged by SBCCC. Because pyrrolidine dithiocarbamate (PDTC) could prevent degradation of I-κB and translocation of NF-κB from the cytoplasm into the nucleus [23], it was used a selective NF-κB inhibitor to investigate if the anti-cancer effect of SBCCC was mediated through NF-κB inhibition. The results indicate that treatments with either 1 μM SBCCC or 50 µM PDTC significantly inhibited (P < 0.05) I-κB phosphorylation and NF-κB nucleus translocation (Fig. 4b), indicating that the SBCCC caused cell death, at least in part, was mediated by inhibition of NF-κB signaling because of less anti-apoptosis products being produced. However, the cell colony formation number in the cells with PDTC treatment was not as less as that in the cells with SBCCC treatment (Fig. 4c), indicating that other pathways might contribute to the cell death by SBCCC, in addition to its NF-κB inhibitory effect. Previous report showed that the copper(II) complexes induced apoptosis could be dependent on ROS generation [24]. Therefore, we further examined the generation of ROS in the gastric cancer cells with SBCCC treatment. As presented in Fig. 4d, ROS generation in SGC-7901 and BCG-823 cells was significantly increased in a time-dependent manner. SBCCC treatment significantly increased the expressions of iNOS and phosphorylated JNK and the increased expressions were inhibited by two inhibitors, N-acetyl-l-cysteine (NAC, an inhibitor of ROS) and L-NAME (an inhibitor of NOS) but not the NF-κB inhibitor PDTC.

NF-κB was reported as a pleiotropic transcription factor activated by low levels of ROS but inhibited by antioxidants [25]. Further experiment was performed to determine if the SBCCC induced apoptosis could be ROS-dependent in addition to NF-κB signaling. Interestingly, the ROS inhibitor (NAC) alone or in combination with SBCCC did not significantly affect NF-κB signaling (Fig. 5a). Although ROS could act as upstream signaling of the NF-κB pathway, our results indicated that the cell-killing effect of SBCCC were from both ROS-dependent DNA cleavage and inhibition of NF-κB signaling. As that SBCCC induced increases of autophagic vacuoles (MDC staining), the important molecular targets of autophagy were further evaluated. The protein expressions of Beclin-1 and LC-3II were significantly increased in SGC-7901 and BGC-823 cells with SBCCC treatment compared to the control cells (Fig. 5b). To further elucidate the autophagy signal linking to NF-κB or ROS pathways, NAC and PDTC were used in combination with the SBCCC to treat SGC-7901 and BGC-823 cells. PDTC did not affect SBCCC induced increases of Beclin-1 and LC-3II (Fig. 5c), but NAC significantly attenuated the SBCCC induced protein expressions of Beclin-1 and LC-3II (Fig. 5d). These results suggested that SBCCC associated autophagy was ROS-dependent but not via the NF-κB signaling.