The molecular mechanism of anticancer action of novel octahydropyrazino[2,1-a:5,4-a′]diisoquinoline derivatives in human gastric cancer cells

Methanol and ethidium bromide,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), were purchased from Sigma Chemical Co. (USA). Stock cultures of AGS- CRL-1739 human stomach cancer cells were purchased from the American Type Culture Collection (USA). Ham’s F-12 K (Kaighn’s) Medium and fetal bovine serum (FBS) used in a cell culture were products of Gibco (USA). Glutamine, penicillin and streptomycin were obtained from Quality Biologicals Inc. (USA). [³H]thymidine (6.7 Ci mmol⁻¹) was purchased from NEN (USA), and Scintillation Coctail “Ultima Gold XR” from Packard (USA). Sodium dodecyl sulfate was received from Bio-Rad Laboratories (USA). Acridine orange and ethidium bromide were provided by Sigma Chemical Co (USA). FITC Annexin V Apoptosis Detection Kit II was a product of BD Pharmigen. Topoisomerase II Drug Screening Kit was a product of TopoGEN (Florida, USA).

The octahydropyrazin[2,1-a:5,4-a′]diisoquinoline derivatives (1, 2) were synthesized using previously standardized methods [7‐9].

AGS human gastric adenocarcinoma cells were maintained in a base growth medium – F-12 K, supplemented with fetal bovine serum (FBS) to a final concentration of 10% and 1% antibiotics (penicillin/streptomycin). Cells were cultured in Costar flasks and grown in 5% CO₂ at 37 °C in high humid atmosphere to subconfluence (90–95%). Subconfluent cells were treated with 0.05% trypsin and 0.02% EDTA in calcium free phosphate buffered saline, counted in hemocytometer and seeded at 5 × 10⁵ cells/well in 6-well plates (Nunc) in 2 mL of the growth medium (F-12 K). Cells which reached about 80% of confluency were used in the present study.

Cell growth was assessed in AGS cells following treatment with the compounds tested using MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide), according to the method of Carmichael et al. [10]. The cells were incubated with different concentrations of compounds 1, 2 and etoposide for 24 and 48 h to determine the IC₅₀ value for each drug. Confluent cells, treated for 24 and 48 h with the compounds in 6-well plates, were washed three times with PBS and then incubated for 4 h in 1 mL of MTT solution (5 mg/mL of stock in PBS) at 37 °C in the atmosphere of 5% CO₂ in an incubator. Then, supernatant was discarded and 1 mL of 0.1 mol/L HCl in absolute isopropanol was added to each well and mixed gently. The absorbance of converted dye in living cells was read at a wavelength of 570 nm using spectrophotometer (Helios Gamma UV/VIS Scanning Spectrophotometer, Unicam/ThermoFisher Scientific Inc., Waltham, MA, USA). Untreated cells were also run under identical conditions and served as control. The viability of stomach cancer cells cultured in the presence of the compounds tested was calculated as a percent of control cells.

The incorporation of [³H]thymidine into DNA was used as a measure of cell proliferation. AGS cells were seeded in 6-well plates at a density of 5 × 10⁵ well⁻¹ in a complete growth medium and grown as described above. The cells were incubated for 24 and 48 h with various concentrations of compounds 1, 2 and etoposide in 5% CO₂ at 37 °C before 0.5 μCi of [³H]thymidine was added to each well for 4 h to measure the incorporation of radioactive component into DNA. Then, the cells were harvested by trypsinisation and washed several times with cold phosphate buffered saline (10 min/1500 g) until the dpm in the washes were similar to the control reagent. Radioactivity was determined in a scintillation counter (Packard Tri-Carb Liquid Scintillation Counter 1900 TR, Perkin Elmer, Inc.,Waltham, MA, USA). [³H]thymidine uptake was expressed as dpm well⁻¹.

The distribution of the cell cycle phases was analyzed by flow cytometry. Briefly, AGS-CRL-1739 human gastric cancer cells were seeded into 6-well plates at a density of 2.5 × 10⁵ cells per well and treated with 20 μM of compounds 1, 2 and etoposide for 24 and 48 h. After incubation, the cells were harvested and then fixed with 1 mL of 70% ethanol and kept overnight at −20 °C. Before analysis, the cells were resuspended in PBS, treated with 50 μg/mL of DNase-free RNase A Solution (Promega), and stained with 100 μg/mL of PI. The FACSCanto II flow cytometer (BD Bioscences Systems, San Jose, CA, USA) was used to read the fluorescence.

Supercoiled pHOT DNA (0.5 mg) was incubated with 4 units of human topoisomerase II in the cleavage buffer (30 mM Tris–HCl (pH 7.8), 50 mM KCl, 10 mM MgCl₂, 3 mM ATP, 15 mM mercaptoethanol), in the presence of the compounds tested (100 μM). Reactions were carried out at 37 °C for 1 h and then terminated by the addition of 2 mL of 10% SDS and 2 mL of 50 μg/mL proteinase K. The reaction mixture was subjected to electrophoresis through a 0.8% agarose gel containing 0.5 mg/mL ethidium bromide in TBE buffer (90 mM Tris–borate and 2 mM EDTA). The gels were stained with ethidium bromide and photographed under UV light.

The effect on the induction of apoptosis after 24 and 48 h of incubation with the compounds tested was determined by Becton Dickinson FACSCanto II flow cytometer FACSCanto II (Becton Dickinson Bioscences Systems, San Jose, CA, USA), assessing the loss of asymmetry of the phospholipids on the cell membrane. Cells were trypsinised, resuspended in F12-K and then in binding buffer. Next, they were stained with FITC Annexin V and propidium iodide (PI) for 15 min at room temperature in the dark according to the manufacturer’s instruction (FITC Annexin V Apoptosis Detection Kit II). Cells cultured in a drug-free medium were used as controls. Optimal parameter settings were found using a positive control (cells incubated with 3% formaldehyde in buffer during 30 min on ice). Forward scatter (FS) and side scatter (SC) signals were detected on a logarithmic scale histogram. FITC was detected in the FL1 channel (FL1 539; Threshold–value 52). The results were analyzed with FACSDiva software (Becton Dickinson Biosciences Systems, San Jose, CA, USA).

To confirm the rates of apoptosis, obtained using the FITC Annexin V Apoptosis Detection Kit II, we carried out an assessment of the dual acridine orange/ethidium bromide fluorescent staining, visualized under a fluorescent microscope Nikon Eclipse Ti with an inverted camera (Nikon Instruments Inc., Melville, NY, USA). AGS-CRL-1739 human gastric cancer cells were treated with the tested compounds 1, 2 and etoposide (20 μM) for 24 and 48 h. The cell suspension (250 μl) was stained with 10 μl of the dye mixture (10 μM acridine orange and 10 μM ethidium bromide), which was prepared in PBS. Cells cultured in a drug-free medium were used as controls. The morphology of two hundred cells per sample was examined by fluorescent microscopy within 20 min. The results were analyzed with NIS-Elements software (Nikon Instruments Inc., Melville, NY, USA).

Cells were seeded in BD Falcon™ 96-well black, clear-bottom tissue culture plates at 10.000 cells per well. These plates are optimized for imaging applications. Analysis were performed in three independent experiments. After incubation, the cells were rinsed with PBS and fixed with a 3.7% formaldehyde solution at room temperature for 10 min. Then, they were washed three times with PBS and permeabilized with 0.1% Triton X-100 at room temperature for 5 min. Next, they were washed twice with PBS, and non-specific binding was blocked by incubation in 3% FBS at room temperature for 30 min. The cells were rinsed and incubated with anti-caspase-9 mouse monoclonal antibody (BD Biosciences, San Jose, CA, USA; 1:200), or anti-caspase-3 (Ab-2) mouse monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA; 1:200), or anti- p53 (DO-1) mouse monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA, 1:200), or anti-p-AKT (anti-phospho-PKB (pThr³⁰⁸) rabbit polyclonal antibody (Sigma-Aldrich, St. Louis, MO, USA; 1:200) or anti-ERK1/ERK2 (pT202/pY204) mouse monoclonal antibodies (BD Biosciences, San Jose, CA, USA; 1:200) for 1 h at room temperature. Then, they were washed three times with PBS and incubated with FITC-conjugated anti-rabbit or anti-mouse secondary antibodies (BD Biosciences, San Jose, CA, USA, 1:500) for 60 min in the dark. After washing, nuclei were stained with Hoechst 33,342 (2 μg/ml, blue) and analyzed using a BD Pathway 855 confocal microscope with a 40 × (0.75 NA) objective. The cytoplasmic and nuclear fluorescence intensities of stained cells were analyzed, and images of FITC-labeled cells were acquired using a 488/10 excitation laser and a 515LP emission laser.

Experimental data were presented as mean ± standard deviation (SD) since each experiment was repeated at least three times.

At the beginning, the growth inhibitory effect of the compounds 1, 2 and etoposide on human gastric cancer cells was examined. The results revealed that all compounds exerted a time-dependent growth inhibitory effect. IC₅₀ values for compound 1 were 44 μM and 40 μM at 24 and 48 h. IC₅₀ values for compound 2 were 21 μM and 6 μM after 24 and 48 h of incubation, respectively. The reference compound – etoposide represented the weakest cytotoxic potential with IC₅₀ values of 80 μM and 45 μM at 24 and 48 h, respectively (Fig. 1).

The effect of the compounds on DNA biosynthesis was also investigated. We proved that compound 2 had the strongest time-dependent antiproliferative effect with IC₅₀ values of 16 μM and 6 μM at 24 and 48 h, respectively. Compound 1 at a weaker rate inhibited DNA biosynthesis in the analyzed cells with IC₅₀ values of 54 μM and 44 μM at 24 and 48 h. Etoposide represented the weakest antiproliferative potential with IC₅₀ values of 68 μM and 44 μM at 24 and 48 h (Fig. 2).

The literature data show that etoposide induces G2/M cell cycle arrest and its mechanism of action is associated with inhibition of topoisomerase II [11]. The effect of our novel diisoquinoline derivatives on cell cycle and topoisomerase II inhibition was examined (Figs. 3 and 4). The novel compounds led to the accumulation of cells in the G2/M phase of cell cycle. The percentage of cells in the G2/M phase increased from 10.3% in the untreated control to 13.8% after treatment with compound 1 (20 μM), 25.1% after treatment with etoposide (20 μM) and 26% after treatment with compound 2 (20 μM). Upon prolongation of the exposure time to 48 h, the highest percentage of cells were arrested in the G2/M phase after treatment with compound 2 (56.7%).

The ability of the compounds 1, 2 to inhibit topoisomerase II activity was quantified by detecting Topo II cleavage products, including linear DNA. The obtained results demonstrated that the compounds 1 and 2, the same as a known Topo II poison etoposide (VP-16), stimulated the stabilization of the cleavable complex at the concentration of 100 μM (Fig. 5).

Annexin-V binding assay was used to detect early and late apoptotic cells (annexin-V⁺/PI⁻ and annexin-V⁺/PI⁺), necrotic (annexin-V⁻/PI⁺⁾ as well as viable cells (annexin-V⁻/PI⁻). The test is based on the externalization of phosphatidylserine (PS) at the outer leaflet of the plasma membrane. Annexin V-FITC possess high affinity for the PS, which is exposed to the extracellular environment during programmed cell death. Propidium iodide was used to determine necrotic cells [12]. After 24 h of incubation with compounds 1, 2 and etoposide we observed that compound 2 strongly induced apoptosis in comparison with untreated cells. We detected 19.6% of early and 18.1% of late apoptotic cells, compared to 3.8% of early and 3.9% of late apoptotic cells in the control sample. Etoposide as well as compound 1 insignificantly increased the percentage of early and late apoptotic cells in comparison with untreated cells. The ratio of early and late apoptotic cells was increased from 7.7% to 10.2% after etoposide treatment and from 7.7% to 8.9% after compound 1 treatment (Fig. 6a). After 48 h of incubation a significant proapoptotic effect was observed in gastric cancer cells in all cases. The proportion of viable AGS cells after treatment with compound 2 decreased from 83% to 9.8%. The percentage of viable cells decreased from 83% to 59.9% after etoposide treatment and from 83% to 76.1% after compound 1 treatment (Fig. 6b). These results suggest that cytotoxicity of the compounds against AGS cancer cells is due to the induction of apoptotic cell death.

To reveal the morphology of apoptosis in gastric cancer cells after treatment with the compounds tested, the acridine orange (AO) and propidium iodide (PI) double staining was performed. The untreated cells with normal nuclear structure were displayed as green fluorescence. The early stage of apoptosis was identified as bright green fluorescence, whereas the late apoptosis was identified by reddish-orange color. Our study proved that compound 2 possessed the strongest proapoptotic properties in comparison with untreated cells as well as with cells incubated with compound 1 and etoposide (Fig. 7a). The effect was enhanced after 48 h of incubation (Fig. 7b). In all cases, the changes in cell morphology characteristic of apoptosis, such as chromatin condensation and membrane blebbing, were observed.

The caspases are pivotal players in the best documented mechanism of cancer cell death. Initiator caspases activate executioner caspases that lead to demolishing of key structural proteins and activate other enzymes. DNA fragmentation and membrane blebbing are the morphological hallmarks of apoptosis [13]. The influence of the tested compounds on the expression of initiator and executioner caspases was demonstrated in Figs. 8 and 9. Cells were incubated for 24 h with etoposide and compound 1 at 5 μM, 10 μM and 25 μM concentrations. Compound 2 was very active, so doses were limited to 2.5 μM, 5 μM and 10 μM. All the compounds tested increased the expression of initiator caspase-9 in a dose dependent manner in gastric cancer cells in comparison with control (Fig. 8). The novel diisoquinoline derivatives led to higher expression of caspase-9 in gastric cancer cells. The stronger intensity of red fluorescence was detected in comparison with control and etoposide. Our study confirmed that mitochondrial apoptotic pathway plays a crucial role in the mechanism of cell death.

Caspase-3 is one of the executioner caspases and its expression was also increased in analyzed cancer cells after 24 h of incubation with the compounds tested (Fig. 9). The study confirmed that compound 2 was the most cytotoxic and the strongest activator of caspase-3. The significant effect was detected after treatment with compound 2 at 5 μM concentration compared to cells treated with compound 1 and etoposide in the same doses. The increase of concentration of compound 2 to 10 μM enhanced its cytotoxic properties, but the expression of caspase-3 was also very high.

P53 plays a key role as a regulator of the programmed cell death. It can modulate pivotal control points in death receptors signaling pathway and mitochondrial apoptotic pathwy. It can directly activate the transcription of genes resposible for promotion of apoptosis [14].

The expression of p53 protein was increased in a dose dependent manner after treatment with etoposide and the novel diisoquinoline derivatives 1 and 2, compared to control. The strongest expression of p53 was observed after incubation with compound 2 (Fig. 10).

Finally, the effect of different concentrations of the tested compounds (1, 2) and etoposide on the expression of AKT and ERK1/2 was analyzed in human gastric cancer cells. A large number of studies have suggested that one of the major functions of AKT/PKB is to promote growth factor-mediated cell survival and to block apoptosis. In our study, high intensity of red fluorescence, was observed in control cells without any agents (Fig. 11), thus confirming that AKT expression was higher in untreated cells. Cells exposed to various concentrations of compounds 1, 2 and etoposide decreased the expression of AKT in all cases. The effect was enhanced with an increase in the doses from the lowest to the highest.

ERK activity can promote either intrinsic or extrinsic apoptotic pathways by the induction of mitochondrial cytochrome c release or caspase-8 activation [15]. All the compounds tested decreased the expression of ERK1/2 in comparison with untreated control (Fig. 12). Our research proved that the increase in the doses of etoposide and compound 1 from 5 μM to 25 μM decreased the intensity of red fluorescence. Compound 2 was the most effective inhibitor of kinase expression in the lower spectrum of doses (2.5 μM, 5 μM and 10 μM).