OC cell lines PA-1 (PA1; (ATCC^® CRL-1572™) and NIH:OVCAR-3 (OVCAR3; ATCC^® HTB-161™), and a normal lung fibroblast cell line WI-38 (ATCC^® CCL-75™) were purchased from ATCC and cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C in a humidified 5% CO₂ chamber (HF90; Heal Force Bio-Meditech Holdings Ltd.). Cells were observed under an inverted phase contrat microscope (Zeiss Axiovert. A1; magnification, ×20).

Cell viability was assessed by MTT assay. PA1 and OVCAR3 cells were seeded into 96-well plates at a density of ~1×10⁴ cells/well and treated with 0–100 µM Siomycin A for 24, 48 or 72 h. Subsequently, 20 µl of MTT (Sigma-Aldrich; Merck KGaA) solution, from the stock of 5 mg/ml in PBS, was added to each well and incubated for 3 h at 37°C. The purple formazan crystals were dissolved using 100 µl of DMSO and cell viability was analyzed at a wavelength of 570 nm, using a MultiskanEX plate reader (Labsystems Diagnostics Oy) (15). The cytotoxicity of Siomycin A to normal cells was determined by the same method on cultured WI-38 cells. Cell viability of PA1 and OVCAR3 cells were also assessed using the anticancer drug cisplatin (Sigma-Aldrich; Merck KGaA; 0–50 µM) (and following co-treatment with the IC₅₀ dose of cisplatin and Siomycin A for 48 h using MTT assay. All experiments were performed in triplicate.

Induction of apoptosis in Siomycin A-treated OC cells was determined by the Cell Death Detection ELISA^PLUS (Sigma-Aldrich; Merck KGaA) according to the manufacturer's instructions as well as a published protocol (16). Briefly, PA1 and OVCAR3 cells (~1×10⁴ cells/ml) of 60 mm plates were treated with 2.5 and 5 µM Siomycin A for 48 h. Following treatment, untreated and Siomycin A-treated cells were harvested, lysed with RIPA lysis buffer (Thermo Fischer Scientific, USA) and subjected to subcellular fractionization using the components of the kit. The cytoplasmic fraction was collected by centrifugation at 10,000 × g at 4°C for 15 min, and ~20 µl of the lysate containing 15 µg protein as assessed by the Bradford assay, was added to the streptavidin-coated microplate. Subsequently, 80 µl of buffer mixture containing anti-histone-biotin and anti-DNA peroxidase was added, and the reaction mixture was incubated for 2 h at room temperature with continuous shaking. Finally, the chromogenic substrate 2,2′-azino-di-(3-ethylbenzthiazoline-6-sulfonic acid) was added to develop the color, and the absorbance was measured at 405 nm using an ELISA reader (Molecular Devices, LLC).

PA1 and OVCAR3 cells (~1×10⁴ cells/ml) of 60 mm plates were treated with 2.5 and 5 µM Siomycin A for 48 h. Following treatment, the cells were incubated with 3 µg/ml JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide; Abcam) for 20 min at 37°C (17). The cells were washed with chilled PBS, and JC1 fluorescence was assessed at 530 and 590 nm using a Fluoroskan microplate fluorimeter (Thermo Fischer Scientific, Inc.). The ratio of red/green fluorescence intensity (aggregate/monomer) was analyzed using the instrument Scanlt 2.0.7 software. All experiments were performed in triplicate.

Cultured PA1 and OVCAR3 cells (~1×10⁴ cells/ml) of 60 mm plates were treated with Siomycin A in two sets: i) 5 µM for 6, 12, 24 and 48 h; and ii) 2.5, 5 and 10 µM for 24 h. Following treatment, the cells were stained with 10 µM DCFDA (Abcam) for 30 min in the dark at room temperature (18). Subsequently, the cells were washed twice with cold PBS, and fluorescence was assessed at the excitation/emission wavelengths of 485/535 nm using a Fluoroskan microplate fluorimeter. All experiments were performed in triplicate. For ROS generation-associated assays, the cells were pretreated with 200 µM N-acetylcysteine (NAC) for 6 h, followed by the treatment with the putative drug Siomycin A, as aforementioned.

The PA1 and OVCAR3 cells treated with Siomycin A were collected and resuspended in 200 µl RIPA lysis buffer (Thermo Fischer Scientific, Inc.) for lysis and subsequent extraction of cellular proteins. The cells were sonicated on an ice bath, thrice for 15 sec each pulse to extract the cellular proteins from the supernatant, and were used for subsequent antioxidant assays. All experiments were performed in triplicate.

A total of 20 µl cell extract was added to 100 µl sample buffer (50 mM potassium phosphate buffer, pH 7.0) and mixed with 180 µl 30 µM H₂O₂ (cat. no. 107209; MilliporeSigma). For blank setup, 20 µl sample buffer was used instead of the cell extract. The decomposition of H₂O₂ to H₂O and O₂ by CAT present in the cell lysates was monitored at 240 nm using a T8001S Double beam UV/Vis spectrophotometer (Shanghai Yoke Instrument Co., Ltd.). Catalase enzyme activity was calculated as the number of µmol of H₂O₂ consumed per min per 1 mg protein against a standard curve (19).

A total of 2.35 ml buffer A [0.1 mol/l Tris-HCl (cat. no. 648313; MilliporeSigma) buffer solution with 1 mM EDTA (MilliporeSigma), pH 8.2] was added to 2.00 ml deionized water; subsequently, 0.15 ml buffer B (4.5 mmol/l pyrogallol solution in HCl; cat. no. 100612; MilliporeSigma) was added, and the solution was vortexed for 2 min at room temperature. Absorbance was measured at 420 nm in 1-min intervals. The difference in absorbance between two aliquots, ∆A420 (min −1), indicated the rate of pyrogallol autoxidation in the blank sample. For all experimental samples, the same procedure was followed with the addition of sample before buffer B, and absorbance was recorded using a T8001S Double beam UV/Vis spectrophotometer. The SOD enzyme activity was based on the autooxidation rate of pyrogallol and the inhibition of this autooxidation by SOD, where 50% inhibition corresponded to one unit of enzyme activity (20).

A total of 10 µl sample buffer (50 mM potassium phosphate buffer, pH 7.0) was added to potassium phosphate buffer (170 µl) with 1 mM EDTA and 2 mM sodium azide (pH 7.0; cat. no. 106688; MilliporeSigma) and used as the blank sample. For all experimental samples, 10 µl cell extract was added in place of sample buffer. A total of 90 µl master mix with 30 µl (10 mM) glutathione (GSH; cat. no. 3541 MilliporeSigma); mixed with 30 µl (2.4 U/ml) glutathione reductase (cat. no. 359960; MilliporeSigma) and 30 µl nicotinamide adenine dinucleotide phosphate [1.5 mM NADPH (cat. no. 481973; MilliporeSigma) in 0.1% sodium bicarbonate (cat. no. 106329; MilliporeSigma) solution] was added to each sample and incubated at 37°C for 10 min. A total of 30 µl (2 mM) H₂O₂ was added to each sample and the decomposition of H₂O₂ was monitored by observing the rate of NADPH consumption at 340 nm for 10 min in a T8001S Double beam UV/Vis spectrophotometer. The GPx enzyme activity was calculated as NADPH (in nM) consumed per min per 1 mg protein (21).

A total of 230 µl sample buffer (50 mM potassium phosphate buffer, pH 7.0) was added to 40 µl potassium phosphate buffer with 1 mM EDTA and used as the blank sample. A total of 10 µl cell extract and 30 µl (20 mM) glutathione disulfide (cat. no. 3542; MilliporeSigma) was added to the mixture for each experimental sample. All samples were incubated at 37°C for 3 min, and the reaction was started by adding 30 µl 1.5 mM NADPH in 0.1% sodium bicarbonate. The subsequent consumption of NADPH was monitored at 340 nm for 5 min at 37°C in a T8001S Double beam UV/Vis spectrophotometer. The GR enzyme activity was calculated as NADPH (in nM) consumed per min per 1 mg cellular protein (21).

A total of 200 µl of assay mixture [0.84 mM 5,5′ dithiobis-(2-nitrobenzoic acid) (DTNB; cat. no. 322123; MilliporeSigma) and 0.28 mM NADPH dissolved in 100 mM sodium phosphate buffer, pH 7.5, with 5 mM EDTA] was added to 20 µl sample buffer (50 mM potassium phosphate buffer, pH 7.0) in a quartz cuvette and incubated at 37°C for 5 min. For the experimental samples, 20 µl cell extract was added instead of the sample buffer. The reaction was initiated by adding 40 µl glutathione reductase, and the reduction of DTNB and formation of TNB was monitored for 10 min at 412 nm using a T8001S Double beam UV/Vis spectrophotometer (22). The GSH content was determined with reference to a GSH standard curve using known concentrations of GSH (0–50 µM).

PA1 and OVCAR3 cells were seeded at a density of 1×10⁴ cells/ml, grown to confluency in 60 mm plates and treated with 2.5 and 5 µM Siomycin A treatment for 48 h. Following treatment, the adherent cells were detached using trypsin (Sigma-Aldrich; Merck KGaA) and subsequently centrifuged in various buffers to separate the cytosolic phase (supernatant of final step) from the rest of the cell pellet as previously described (23). The amount of total protein from the cytosolic extracts of control and treated PA1 and OVCAR3 cells was estimated using the Bradford reagent (Abcam), and equal amounts (40 µg) of protein were loaded in each well for the detection of cytochrome c by western blotting. All experiments were performed in triplicate.

PA1 and OVCAR3 cells in 60 mm plates at a concentration of ~1×10⁴ cells/ml were treated with 2.5 and 5 µM of Siomycin A for 48 h. Following treatment, the cells were incubated in lysis buffer (Thermo Fischer Scientific, Inc.). The total protein concentrations were determined by Bradford assay using a standard curve, and equal amount of proteins (30–50 µg) were separated by 10–12% SDS-PAGE. Following separation, the cellular proteins were transferred to nitrocellulose membranes (MilliporeSigma) and blocked with 5% skimmed milk for 1 h at room temperature. The membranes were incubated with primary antibodies (all 1:1,000 dilution) against Bax (cat. no. sc-7480), Bcl2 (cat. no. sc-492), cleaved caspase −3 (cat. no. CST- 9661), cytochrome c (cat. no. sc-13156) and β-actin (cat. no. sc-47778) for 16 h at 4°C, followed by incubation with a corresponding horseradish peroxidase-linked secondary antibody (1:3,000 dilution; goat anti-mouse-HRP secondary antibody, cat. no. 32430 and goat anti-rabbit HRP secondary antibody, cat. no. 31460) Invitrogen; Thermo Fisher Scientific, Inc.)] for 2 h at room temperature and washed using TBS and TBST buffers (Tris buffer saline; BioRad Laboratories, Inc.). Bands were developed using the Clarity Western ECL substrate luminol assay kit (cat. no. 1705060; BioRad Laboratories, Inc.), and chemiluminescence was recorded in Chemidoc™ Gel Imaging System (Bio-Rad Laboratories, Inc.). The densitometric analysis was performed using ImageJ 1.52a software (National Institutes of Health). All experiments were performed in triplicate.

Data are presented as the mean ± standard error of the mean of at least three independent experiments. Two-way ANOVA followed by Sidak's or Bonferroni correction was performed to analyze the effects of the dose of Siomycin A, NAC treatment and the interaction between the dose of Siomycin A and NAC. All other data were analyzed by one-way ANOVA followed by Tukey's test. GraphPad Prism 5.0 software (GraphPad Software, Inc.) was used for analysis. P<0.05 was considered to indicate a statistically significant difference.

The present study evaluated the antitumor effects of the thiopeptide antibiotic Siomycin A (Fig. 1A) on OC cells. OC cell lines PA1 and OVCAR3 were treated with a 0–100 µM Siomycin A for 24, 48 and 72 h. The results of the MTT assay demonstrated that Siomycin A decreased the PA1 and OVCAR3 cell viability and the effects appeared to increase at higher doses and longer incubation times (Fig. 1B and C), with the IC₅₀ values at 5 µM for PA1 cells and 2.5 µM for OVCAR3 cells following 72-h treatment (P<0.05). The cytotoxicity of Siomycin A in normal cells was also evaluated. Previous studies have used the human normal lung fibroblast WI-38 cell line to assess the cytotoxicity of anticancer drugs (10,24,25). In the present study, WI-38 cells were treated with 0–10 µM Siomycin A for 72 h, and no significant loss of cell viability was determined by MTT assay (Fig. 1C). These results demonstrated that Siomycin A exerted an inhibitory effect on OC cells, and the low IC₅₀ values indicated that it may be a potential option for treatment due to limited toxicity on the surrounding normal cells.

Treatment of PA1 and OVCAR3 cells with 2.5 and 5 µM Siomycin A resulted in the alteration of the cellular morphology. The cells began to lose their spindle-shape nature and formed shrunken spherical structures upon treatment; the change was moderate for 2.5 µM and more prominent for 5 µM. The cell density also significantly decreased following treatment with 5 µM Siomycin A in both cell lines (Fig. 2A). Siomycin A treatment of PA1 and OVCAR3 cells significantly induced of apoptosis at 48 h, as determined by ELISA. In the presence of 2.5 µM Siomycin A, the absorbance increased by ~2.3-fold for PA1 and ~5.1-fold for OVCAR3 cells, whereas following treatment with 5 µM Siomycin A, the increase in absorbance was ~4.0-fold for PA1 and ~6.4-fold for OVCAR3 cells (Fig. 2B and C; P<0.05.

These results demonstrated that Siomycin A exerted proapoptotic effects on the OC cell lines by initially altering the cell morphology and changing the adherent defined structures to rounded shrunken clots. These features, along with the reduced cell density and decreased viability, were indicative of apoptosis, which was further confirmed by ELISA to reveal that Siomycin A induced apoptosis, with high occurrence of cell death at 5 µM for both PA1 and OVCAR3 cells.

In order to determine the cellular changes that induce apoptosis in OC cells following Siomycin A treatment, the roles of mitochondria were examined. The changes in the mitochondrial membrane potential were analyzed by staining PA1 and OVCAR3 cells with the mitochondria-specific dye JC-1. Fluorimetric analysis of the Siomycin A-treated PA1 and OVCAR3 cells revealed a decrease in the red fluorescence population and a simultaneous increase in the green fluorescence population, resulting in the increase in the green/red ratio of 3.2- and 6.6-fold for PA1 cells following 2.5 and 5 µM Siomycin A treatment, respectively, compared with that in the untreated control group (P<0.05 Fig. 3A), and an increase of 5.3- and 8.9-fold in OVCAR3 cells with the two doses of Siomycin A, respectively, compared with those in the untreated control group (P<0.05 Fig. 4A), which indicated the loss of the mitochondrial membrane potential. The decrease in the mitochondrial membrane potential is associated with the release of cytochrome c in the cytosol (26). Thus, the levels of cytochrome c in the cytosol were determined in the present study by western blotting (Fig. 3B), and the results demonstrated that with increasing concentrations of Siomycin A, the protein levels of cytosolic cytochrome c increased significantly in PA1 cells (P<0.05 Fig. 3C). Siomycin A treatment also resulted in the alterations of the expression levels of the proapoptotic protein Bax and the antiapoptotic protein Bcl-2; the upregulation of Bax (P<0.05) and the downregulation of Bcl-2 (P<0.05) in PA1 cells treated with Siomycin A compared with those in the untreated cells, as demonstrated by western blotting, indicated an altered Bax/Bcl-2 ratio, suggesting that apoptosis was triggered (Fig. 3B and C). The activation of Bax leads to functional translocation of Bax to the mitochondria, increasing the permeability of the mitochondrial membrane, and thus facilitating the translocation of mitochondrial cytochrome c into the cytosol pool (27). This augmented release of cytochrome c serves a crucial role in increasing the levels of caspase-3, which leads to apoptosis by further activating the other caspases (27). In the present study, increased levels of Bax were observed with increases of cytochrome c in the cytosol as well as the protein levels of caspase-3 (P<0.05) following Siomycin A treatment compared with those in the untreated cells, and the effects were observed in a dose-dependent fashion (Fig. 3C and D). Similar results were observed for the pro- and antiapoptotic proteins in OVCAR3 cells where the levels of Bax (P<0.05), cytochrome c (P<0.05) and cleaved caspase-3 (P<0.05) were increased, and the levels of Bcl-2 (P<0.05) were reduced significantly following Siomycin A treatment (Fig. 4B and C). These results suggested that Siomycin A may target the mitochondria of OC cells to induce caspase-mediated cell death.

Among the limited number of studies that have reported the activity of Siomycin A on cancer cells, ROS generation has not been clearly indicated as a mechanism of Siomycin A-induced cell death. Therefore, the present study investigated whether Siomycin A enhanced ROS generation in OC cells. The results demonstrated that DCF fluorescence, which is a standard indicator of cellular ROS generation, was significantly increased following Siomycin A treatment in PA1 and OVCAR3 cells (P<0.05 with peak increases at 24 h (Fig. 5A and C) and with a 5-µM dose (Fig. 5B and D) in the two cell lines compared with those in the untreated cells. The generation of ROS is one of the key mechanisms of anticancer agents against various types of cancer cells (28), and the results of the present study demonstrated that Siomycin A is may be a potent ROS generator in OC cells, disrupting the cellular homeostasis in these cells and eventually leading to cell death.

Since the production of antioxidant enzymes is a key regulator of oxidative stress response (29), the present study evaluated the levels of major antioxidant enzymes that may be effective in detoxification of electrophilic and oxidative species in Siomycin A-treated OC cells. Treatment of PA1 and OVCAR3 cells (Figs. 6 and 7, respectively) with 1, 2.5 and 5 µM Siomycin A for 24 h significantly reduced the levels of the antioxidant enzymes CAT (P<0.05; Fig. 6A; P<0.05; Fig. 7A), SOD (P<0.05; Fig. 6B; P<0.05; Fig. 7B), GPx (P<0.05; Fig. 6C; P<0.05; Fig. 7C) and GR (P<0.05; Fig. 6D and 7D) compared with those in the untreated control cells. Combined with the aforementioned increases of ROS levels, these results indicated that Siomycin A may inactivate the antioxidant machinery in OC cells, leading to an uninhibited increase of ROS that causes the loss of cell viability and cell death.

The antioxidant enzymes GPx and GR form a well-regulated system to maintain the intracellular levels of GSH, which is the major non-protein thiol that acts as an antioxidant and a redox regulator in cells (30). Thus, the present study estimated the intracellular GSH levels and demonstrated that treatment with Siomycin A significantly decreased GSH levels in PA1 (P<0.05; Fig. 6E) and OVCAR3 (P<0.05; Fig. 7E) cells compared with the untreated control sets. Therefore, Siomycin A may be as a redox-active compound that alters the levels of intracellular GSH to regulate redox signaling. High ROS levels cannot be balanced by the decreasing antioxidant enzyme status in the Siomycin A-treated OC cells, creating an imbalance in redox homeostasis and promoting cancer cell death.

To determine whether ROS generation may be a potent cytotoxic mechanism in Siomycin A-treated OC cells, pretreatment with the standard antioxidant 200 µM NAC for 6 h was performed prior to treatment with Siomycin A. The results demonstrated that treatment of PA1 cells with NAC + Siomycin A significantly reduced the ROS levels by 4.24-fold in the NAC + 2.5 µM (P<0.05 and 9.4-fold in the NAC + 5 µM (P<0.05) Siomycin A groups relative to Siomycin A treatment at the corresponding doses alone, as indicated by the DCF fluorescence measurement (Fig. 8A). In addition, the MTT assay revealed that pretreatment with NAC restored the viability of Siomycin A-treated PA1 cells by 2.5 fold in the NAC + 2.5 µM (P<0.05) and 4.2 fold in the NAC + 5 µM (P<0.05) Siomycin A groups compared with Siomycin A treatment alone at both doses (Fig. 8B). Similarly, in OVCAR3 cells, NAC + Siomycin A significantly reduced the ROS levels by ~5-fold in the NAC + 2.5 µM (P<0.05) and ~10-fold in the NAC + 5 µM (P<0.05) Siomycin A groups relative to Siomycin A treatment alone at both concentrations (Fig. 8C). Pretreatment with NAC restored the viability of Siomycin A-treated OVCAR3 cells, as demonstrated by MTT assay, by ~13% in the NAC + 2.5 µM (P<0.05) and ~25% in the NAC + 5 µM (P<0.05) Siomycin A groups compared with that in cells treated with Siomycin A alone at both doses (Fig. 8D). Therefore, ROS may serve a key role in Siomycin A-mediated cytotoxicity of OC cells, and administration of the antioxidant NAC abrogated this process and reversed the loss of cell viability.

Since a number of novel drug candidates act synergistically with existing anticancer drugs to increase their efficacy in targeting cancer cells, the present study further aimed to determine whether Siomycin A may be paired with cisplatin, a drug used in OC treatment that may lead to tumors acquiring platinum resistance and result in a poor prognosis (31). We hypothesized that Siomycin A may sensitize OC cells to cisplatin therapy, therefore decreasing their chances of survival in a tumor. The results of the present study demonstrated that when PA1 cells were co-treated with 5 µM Siomycin A and 15 µM cisplatin, the cell viability was reduced to 15% of that in the control group (P<0.05, which was lower compared with Siomycin A (52%; P<0.05) or cisplatin (70%; P<0.05) treatment alone (Fig. 9A). OVCAR3 cells were highly susceptible to the co-treatment, as their viability was reduced to 10% of that in the control group by co-treatment with 5 µM Siomycin A and 10 µM cisplatin (IC₅₀ for OVCAR3; data not shown) (P<0.05), which was lower compared with Siomycin A (40%; P<0.05) and cisplatin (60%; P<0.05) alone (Fig. 9B).

For the combination study, single doses of Siomycin A and cisplatin were selected against OC cells. Given that it is not possible to determine whether the effect is additive or synergistic from a single dose-combination, more doses of these two drugs need to be included in prospective studies to determine the combination index. Thus, these results demonstrated that co-treatment with Siomycin A and cisplatin significantly increased the viability inhibition in OC cells compared with monotherapy.