TP4O, Triton X-100, staurosporine and ebselen (EB) were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). TP4O was dissolved in ethanol to prepare stock solutions. The compound was administered at a final concentration of 0.0165% ethanol. High-glucose (4.5 g/l) Dulbecco's modified Eagle's medium (DMEM), phenol red-free DMEM and PBS were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Eagle's minimum essential medium (EMEM) was purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). 5,5-dimethyl-l-pyrroline-N-oxide (DMPO) was purchased from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan). N-acetyl-L-cysteine (NAC) and manganese (III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) were purchased from EMD Millipore (Billerica, MA USA) and Funakoshi Co., Ltd. (Tokyo, Japan), respectively. Phenylmethylsulfonyl fluoride was purchased from Roche Diagnostics GmbH (Mannheim, Germany).

Human CRC cell lines (HCT116 and RKO) and a human normal colon epithelial cell line (CCD 841 CoN) were purchased from the ATCC. The cancer cell lines were cultured in DMEM, while CCD 841 CoN cells were cultured in EMEM. The media were supplemented with 10% heat-inactivated foetal bovine serum (FBS) and 1% antibiotics (100 U/ml penicillin). All cell lines were incubated at 37°C and maintained in an incubator with 5% CO₂. All cells were used while within a passage number that ranged between 8 and 21. Cells were seeded at 5,000 cells/well in 96-well plates for the WST-8 and bromodeoxyuridine (BrdU) assays, at 10,000 cells/well in 96-well plates for the lactate dehydrogenase (LDH) release assay, at 2×10⁵ cells/well in 6-well plates for the caspase-3/7 and Annexin V assays, and at 1×10⁶ cells/dish in 6-cm dishes for western blot analysis. Each cell line was seeded in the above culture media and incubated for 24 h prior to each experiment.

To evaluate the effects of TP4O on cell viability and proliferation, WST-8 and BrdU assays were performed. Briefly, cells were pre-incubated for 24 h and treated with various concentrations of TP4O for 24 h (0, 1, 10, 100, 1,000 or 10,000 µM). Cell viability was quantified using a WST-8 assay via the Cell Counting kit-8 (Dojindo Molecular Technologies, Inc.) by measuring the absorbance at 450 nm. Cell proliferation was quantified using a BrdU assay kit purchased from Roche Diagnostics GmbH by measuring the absorbance at 370 nm. The half maximal inhibitory concentration (IC₅₀) values for each cell line were determined over 24 h following TP4O treatment. Cell viability following treatment with antioxidants was also measured. Following pre-incubation of the cells for 24 h at 37°C, the culture medium was exchanged for medium with antioxidants (20 mM NAC, 20 µM EB and 100 µM MnTBAP) 30 min prior to TP4O administration. The results were obtained from three independent experiments.

To evaluate apoptosis, the following flow cytometry experiment was performed. After 24 h of pre-incubation at 37°C, the cells were treated with various concentrations of TP4O (0, 100 or 1,000 µM). Treatment-induced caspase-3/7 activation was examined in HCT116 and RKO cells using the Muse™ Cell Analyzer (Merck KGaA) and Muse™ Caspase-3/7 Assay kit (Merck KGaA), according to the manufacturer's protocol. Following 6 h of treatment, harvested cells were mixed with the Muse™ Caspase-3/7 reagent, which contains a DNA-binding dye that is linked to a DEVD peptide substrate and a dead cell marker [7-aminoactinomycin D (7-AAD)]. Caspase-3/7 activity was detected with the fluorescence of a DNA-binding dye from the Muse™ Caspase-3/7 Assay kit and cell viability was detected with 7-AAD fluorescence using the Muse™ Cell Analyzer. The results were obtained from three independent experiments.

For the Annexin V assay, cells were treated with TP4O (0, 100 or 1,000 µM) for 12 h. Treatment-induced apoptosis was examined using the Muse™ Annexin V & Dead Cell kit (Merck KGaA) according to the manufacturer's protocol. Phosphatidylserine (PS) was detected using Annexin V, and cell viability was detected using a dead cell marker (7-AAD). The results were obtained from four independent experiments.

An LDH-release assay was performed to assess necrosis resulting from TP4O treatment. The LDH Cytotoxicity Detection kit (Takara Bio, Inc., Otsu, Japan) quantifies the activity of LDH released from damaged cells (18). Following 24 h of pre-incubation, the culture medium was exchanged for FBS-free DMEM containing 1% Triton X-100, various concentrations of TP4O (0, 10, 100 or 1,000 µM) and 200 nM staurosporine for 12 h. Following treatment, the plates were centrifuged at 250 × g for 10 min at 4°C. The supernatant was transferred to a clear 96-well plate, and 100 µl of reaction mixture was then added to each well. The plates were incubated in the dark for 30 min at 25°C, the absorbance was measured at 490 nm and the percentage LDH release was calculated. To compare necrotic cell death, samples with 1% Triton X-100 were used as positive controls. To study apoptotic cell death, LDH release induced by 200 nM staurosporine was examined. The results were obtained from three independent experiments.

ROS generation in cells was measured using ESR, as previously described (19). Briefly, 10⁶ cells were seeded on a glass cover slide (49.0×5.0×0.2 mm) and incubated overnight as previously described. Cells were treated with 0 or 1,000 µM TP4O for 15 min. ESR was measured in a respiration buffer containing 10 mM DMPO as the spin-trapping agent. All ESR spectra were obtained using a JES-RE1X X-band spectrometer (JEOL, Ltd., Tokyo, Japan).

The source of superoxide was detected by fluorescence microscopy. A total of 2.5×10⁴ cells/well were seeded in an 8-well cover glass chamber. After 24 h of pre-incubation, cells were treated with 0 or 1,000 µM TP4O for 24 h. Culture medium was exchanged for phenol red-free DMEM, which contained 5 µM MitoSOX Red™ Mitochondrial Superoxide Indicator (Molecular Probes; Thermo Fisher Scientific, Inc., Waltham, MA, USA), and the cells were incubated for 10 min at 37°C. Following one wash with PBS, the cells were incubated for a further 30 min at 37°C with phenol-free DMEM, which contained 100 nM MitoTracker Green FM™ (Molecular Probes; Thermo Fisher Scientific, Inc.). Next, the cells were washed with PBS and observed using a confocal inverted fluorescence microscope (Olympus Corporation, Tokyo, Japan). Localization of ROS derived from mitochondria was detected by MitoSOX Red™ Mitochondrial Superoxide Indicator fluorescence, and localization of mitochondria was detected by MitoTracker Green FM™ fluorescence. Images of bifocal field, MitoSOX Red™ Mitochondrial Superoxide Indicator and MitoTracker Green FM™ were merged.

The quantitative measurement of cellular populations undergoing oxidative stress was measured using the Muse™ Cell Analyzer and Muse™ Oxidative Stress kit (Merck KGaA). Cells were seeded at 2×10⁵ cells/well in a 6-well plate and incubated overnight. Following exposure to 0 or 1,000 µM TP4O for 24 h, according to the manufacturer's protocol, cells were detached, resuspended at 1×10⁶ cells/ml and incubated at 37°C for 30 min with Muse™ Oxidative Stress working solution, which contained dihydroethidium (DHE). DHE is cell permeable, and has been proposed to react with superoxide anions, thus undergoing oxidation upon binding to DNA (20). The number of oxidized cells were counted according to the intensity of red fluorescence using the Muse™ Cell Analyzer. The results were obtained from four independent experiments.

Cells were seeded at 1×10⁶ cells/dish in 6-cm dishes. After 24 h, cells were treated with various concentrations of TP4O (0, 100 or 1,000 µM) for 24 h. Cultured cells were washed with PBS and lysed with lysis buffer that contained 1% Protease Inhibitor Cocktail (100X; Cell Signaling Technology, Inc., Danvers, MA, USA) and 1 mM phenylmethylsulfonyl fluoride. Samples were kept on ice for 2 min, followed by sonication for 5 min and centrifugation at 13,000 × g for 2 min at 4°C; the supernatants were then collected. The samples were subjected to 15% SDS-PAGE and transferred to a polyvinylidene fluoride membrane (Merck KGaA). After 30 min blocking with Tris-buffered saline containing 0.1% Tween-20 (T-TBS) and 5% bovine serum albumin (Iwai Chemicals Company, Ltd., Tokyo, Japan), primary antibodies against superoxide dismutase 2 (SOD2; cat. no. 13141; dilution, 1:1,000) glutathione peroxidase 1 (GPX1; cat. no. 3206; dilution 1:500) and GAPDH (endogenous control; cat. no. 2118; dilution, 1:1,000; all from Cell Signalling Technology, Inc.) were incubated with membranes in Can Get Signal® Immunoreaction Enhancer Solution 1 (Toyobo Co., Ltd., Osaka, Japan) overnight at 25°C. After washing 3 times for 30 min with T-TBS, the membrane was incubated with an anti-rabbit IgG, horseradish perxodiase-linked antibody (cat. no. 7074; dilution, 1:1,000; Cell Signalling Technology, Inc.) in Can Get Signal® Immunoreaction Enhancer Solution 2 (Toyobo Co., Ltd.) for 2 h at 25°C. After washing 3 times for 15 min with T-TBS at 25°C, the bands were incubated with ECL™ Western Blotting Detection reagents as according to the manufacturer's protocol and detected with ImageQuant LAS 4000 mini (both from GE Healthcare Life Sciences, Chalfont, UK). Results were quantified using ImageQuant TL 7.0 software (GE Healthcare Life Sciences).

A total of 14 male ICR-SCID mice aged 6–7 weeks old and weighing 21–25 g were purchased from Charles River Laboratories Japan, Inc. (Yokohama, Japan). The mice were provided with clean water and food ad libitum, and housed in standardized, pathogen-free conditions with a 14 h light/10 h dark cycle, a temperature of 23.5±2.5°C and humidity of 52.5±12.5%. The mice were used after an acclimation period of ≥7 days. All animal experiments were performed with the approval of the Animal Ethics Committee of the University of Tsukuba (Tsukuba, Japan; approval no. 13-385) and according to the guidelines of this committee.

To evaluate the effect of TP4O in vivo, a subcutaneous tumor was induced. HCT116 cells (2×10⁶ cells per mouse) were injected subcutaneously into the right flanks of 14 mice. Tumor volume was calculated using the following formula: 0.5 × length × width². When tumor volume had reached 80–100 mm³, the 12 qualifying mice were randomly divided into two groups (n=6/group) and subcutaneously injected with one of the following: 250 µl saline (control group) or 200 mg/kg TP4O dissolved in 250 µl saline (TP4O group). Each mouse was injected once every 3 days (total, five times). The tumor volume was recorded every 3 days. Body weight was recorded the first day of the injection, on day 7 and at the end of experimentation (day 14). On day 14, blood samples were collected under anesthesia, the mice were sacrificed and the tumors were excized. To evaluate TP4O toxicity, serum alanine aminotransferase (ALT) and serum creatinine levels were measured using an autoanalyzer (Dri-chem 7000 V; Fujifilm, Tokyo, Japan).

The tumor tissue was fixed with 10% formaldehyde. Each 2-µm paraffin-embedded section was stained with an anti-8-hydroxy-2′-deoxyguanosine (8-OHdG) antibody at 4°C overnight (cat. no. MOG-20P; dilution, 1:100, Japan Institute for the Control of Aging, Shizuoka, Japan) or with an anti-cleaved caspase-3 antibody according to the manufacturer's protocol (cat. no. 9661; dilution, 1:300; Cell Signaling Technology, Inc.). 8-OHdG is a marker for oxidative stress (21). To accurately quantify 8-OHdG- and cleaved caspase-3-positive areas, slides from three randomly selected high-power fields (magnification, ×200) of each tumor were visualized using a BZ-X710 microscope (Keyence Corporation, Osaka, Japan). An automated software analysis program, BZ-X analyser version 1.3.0.3 (Keyence Corporation), was used to determine the percentage of stained areas in the digital photomicrographs.

The data are expressed as the mean ± standard deviation. For comparison of >2 groups, analysis of variance (ANOVA) was performed. If the ANOVA results were significant, a Dunnett's test for comparison with the control was used. For comparison of two groups, an unpaired Student's t-test was performed. All indicated P-values were two-sided, and P<0.05 was considered to indicate a statistically significant difference. All statistical analyses were performed using GraphPad Prism software version 5.01 (GraphPad Software, Inc., La Jolla, CA, USA).

To evaluate cytotoxicity following TP4O treatment, cell viability was measured using a WST-8 assay. TP4O significantly decreased the viability of the human CRC cell lines HCT116 at 100, 1,000 and 10,000 µM, and RKO at 1,000 and 10,000 µM (Fig. 1A). The effect of TP4O was dose dependent. The IC₅₀ values of TP4O in HCT116 and RKO cells were 661 and 381 µM, respectively. By contrast, the IC₅₀ value of TP4O in the normal human colon epithelial cell line CCD 841 CoN was 5,347 µM. In addition, TP4O significantly inhibited cell proliferation in a dose-dependent manner in CRC cell lines and CCD 841 CoN cell line, particularly in the CRC cell lines at 1,000 µM (Fig. 1B).

TP4O-induced apoptotic cell death was determined using a caspase-3/7 activity assay and Annexin V assay. DNA fragmentation and exposure of PS are typical phenomena observed in apoptotic cell death (22). Caspase-3/7 activity was enhanced in TP4O-treated cells in a dose-dependent manner (Fig. 2A). The distribution of apoptotic cells, which were located in the top- and bottom-right quadrants, is presented in a bar graph; the percentage of apoptotic cells was significantly increased in cells treated with 1,000 µM TP4O compared with 0 µM TP4O (P<0.05; Fig. 2B). Although there was no statistical difference, the percentage of Annexin V-positive cells tended to increase in HCT116 and RKO cell cultures treated with 1,000 µM TP4O (Fig. 2C and D). The lower level of LDH release following TP4O treatment indicates that TP4O did not induce necrotic LDH release in HCT116, RKO (Fig. 2E) or CCD 841 CoN (data not shown) cell lines.

In the presence of DMPO, the hydroxyl radical can be detected as as four spikes in ESR (23). ESR measurements revealed that treatment with 1,000 µM TP4O for 15 min induced the generation of the hydroxyl radical in CRC cells (Fig. 3A), whereas the hydroxyl radical was barely detectable in CCD841 CoN cells (data not shown). The increase in oxidative stress was quantified by DHE reaction. TP4O administration led to an increase in the number of cells with high ROS (Fig. 3B). There was a significant increase in oxidative stress in HCT116 cells (P<0.05; Fig. 3C). This result implies that TP4O enhances the accumulation of intracellular ROS in HCT116 cells. There was a non-significant increase observed in RKO cells following the administration of TP4O.

To determine the source of TP4O-induced ROS, CRC cells were observed by confocal inverted fluorescence microscopy. The location of the mitochondria was represented by MitoTracker Green FM™ fluorescence, while accumulated ROS were represented by MitoSOX Red™ Mitochondrial Superoxide Indicator fluorescence. The merged image demonstrated that the ROS induced by TP4O were generated by mitochondria (Fig. 4).

Protein levels of intrinsic oxidant scavengers were analysed by western blotting. Protein levels of SOD2 and GPX1 tended to increase following exposure to TP4O (Fig. 5A). However, there was no significant difference in comparison with that of 0 µM TP4O (Fig. 5B). The results suggested that ROS accumulation was not due to suppression of ROS scavengers, but due to increased ROS production. CRC cells were exposed to specific antioxidants (NAC, EB and MnTBAP), which prevented significant TP4O-derived cell death (Fig. 5C), demonstrating that ROS accumulation was the cause for cell death.

A schematic representation of the experimental design is provided in Fig. 6A. Mice treated with TP4O developed significantly smaller tumors compared with the group treated with vehicle (P<0.05; Fig. 6B). TP4O administration did not cause marked complications or body weight loss, and the serum levels of ALT and creatinine were not different in the TP4O group compared with those in the control group (Fig. 6C). For the histological analysis, tumors were stained using antibodies against the oxidative stress marker 8-OHdG. The areas of anti-8-OH-dG staining were larger in tissues from the TP4O group compared with those from the control group (Fig. 6D). In addition, areas stained by anti-cleaved caspase-3 antibody were larger in the TP4O group than in the control group (Fig. 6D).