RID-B was synthesized as described previously (1,2). Z-VAD-FMK, the caspase-1 and −3 inhibitor, was purchased from Promega Corporation (Madison, WI, USA), and all other general reagents not specified in the following text were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany), Kanto Chemical Co., Inc. (Tokyo, Japan), Nacalai Tesque, Inc. (Kyoto, Japan) and Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

Female Sprague-Dawley rats aged 8 weeks (n=3; weight range, 150–190 g) were obtained from Sankyo Labo Service Corporation, Inc. (Tokyo, Japan). The animals were housed in individual cages supplied with HEPA-filtered air. The ambient temperature in the animal room was maintained at 23±1°C, and the relative humidity at 50–60%. A 12-h light/dark cycle was maintained in the animal room, and food and water were provided ad libitum.

Rat hepatocytes were isolated as described previously (14–16) with some modifications. The liver of the anesthetized rats was perfused in situ via a cannulation of the portal vein. The liver was perfused for calcium removal with 50 ml of 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer containing 3.6 mM NaHCO₃, 5.6 mM glucose and 6.0 mM EGTA, following collagenase perfusion with 50 ml phosphate-buffered saline (PBS) containing 0.5 mg/ml collagenase, 4 mM NaHCO₃, 0.9 mM MgSO₄ and 4.5 mM CaCl₂. The hepatocytes were dispersed and washed three times with Dulbecco's modified Eagle's medium (DMEM) supplemented with antibiotic-antimycotic (100 U/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml amphotericin B; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and gentamicin (10 µg/ml, Thermo Fisher Scientific, Inc.). Cells were then resuspended with DMEM supplemented with antibiotic-antimycotic (100 U/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml amphotericin B), gentamicin (10 µg/ml) and 10% heat-inactivated fetal bovine serum (FBS), and seeded into 96-well plates (1,000 cells/well). After 24 h of incubation at 37°C with 5% CO₂, the culture medium was replenished, and cells were used for a proliferation assay. All animal procedures were conducted according to a protocol approved by the Tokyo University of Science Institutional Animal Care and Use Committee (Tokyo, Japan).

Human hepatoma Huh-7 cells (Japanese Collection of Research Bioresources Cell Bank, Osaka, Japan) were maintained at 37°C with 5% CO₂ in DMEM supplemented with antibiotic-antimycotic (100 U/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml amphotericin B), gentamicin (10 µg/ml) and 10% heat-inactivated FBS. Cells of passage number 70–80 were used throughout the study. Cells were grew to confluence for 2 days following passage.

The Huh-7 cells and normal primary rat hepatocytes were seeded in 96-well plates (1,000 cells/well) and incubated for 24 h at 37°C. Cells were then incubated with RID-B (0.5, 1 and 2 µM) for 48 h, and cell number was estimated by using WST-1 cell proliferation reagent (Cell Counting Kit; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) according to the manufacturer's protocol. The GI₅₀ value of RID-B was calculated using JMP 9 software (SAS Institute, Inc., Cary, NC, USA) using the 4-parameter logistic model (17).

Plasmid DNA and salmon sperm DNA were used subsequently to competitively inhibit the effect of RID-B on Huh-7 cells. Huh-7 cells were seeded in 24-well plates (4×10⁴ cells/well) and incubated for 24 h. Cells were then incubated with RID-B (1 µM) in the presence of pSecTag2B plasmid DNA (0.125, 0.25 and 0.5 mg/ml, respectively; Thermo Fisher Scientific, Inc.) or salmon sperm DNA (0.125, 0.25 and 0.5 mg/ml, respectively; Wako Pure Chemical Industries, Ltd.) overnight at 37°C. Following incubation, cells were stained with trypan blue and live cells were counted immediately under a Nikon TMS Inverted Microscope (Nikon Corporation, Tokyo, Japan) using a Neubauer hemocytometer. The number of cells was determined as the mean of all live cells counted in eight fields (0.1 mm³/field) of view.

Huh-7 cells were seeded in 24-well plates (8×10⁴ cells/well) and incubated for 24 h at 37°C. Cells were then incubated with RID-B (0.5 and 5 µM) for 24 h at 37°C. Following incubation, cells were washed with PBS and lysed with 10 mM phosphate containing 0.15 M NaCl, 5 mM EDTA, 0.1% Nonidet P-40, 0.1% SDS and 0.1% sodium deoxycholate. Cell lysate was incubated with 0.1 mM caspase-3 substrate (acetyl-Asp-Glu-Val-Asp-4-methyl-coumaryl-7-amide; Peptide Institute, Inc., Osaka, Japan) for 3 min at room temperature, and the increase of fluorescence intensity was monitored by using an FP-6200 spectrofluorometer (λ_excitation =380 nm, λ_emission =460 nm; JASCO Corporation, Tokyo, Japan).

Huh-7 cells were seeded on 8-well culture slides (4×10⁴ cells/well, BD Biosciences, San Jose, CA, USA) and cultured for 24 h at 37°C. Following cultivation, cells were pre-incubated with or without 40 µM Z-VAD-FMK for 2 h at 37°C, and were then incubated with RID-B (1 µM) in the presence or absence of 0.5 mg/ml pSecTag2B for 24 h at 37°C. Following a thorough wash with PBS, cells were fixed with 3.7% formaldehyde/PBS for 30 min at room temperature and permeabilized with 0.1% sodium citrate containing 0.1% Triton X-100 for 2 min on ice. Cells were stained using a MEBSTAIN Apoptosis TUNEL Kit Direct (Medical and Biological Laboratories Co., Ltd., Nagoya, Japan), and TUNEL-positive cells were visualized by confocal laser scanning microscopy (LSM710; Carl Zeiss AG, Oberkochen, Germany).

For the preparation of biotinylated double-stranded DNA, complementary oligonucleotides (biotin-TTTTTTATATAT and ATATATAAAAAA; 1 µg/ml each) in distilled water were annealed for 30 min at 25°C. The double-stranded DNA was incubated with RID-B (10 µM) in distilled water for 1.5 h at room temperature, and the reaction product was then incubated with streptoavidin-conjugated magnetic beads (APRO Science, Tokushima, Japan) for 1 h at room temperature. Magnetic beads were washed three times with distilled water to remove RID-B unbound to DNA, and the beads were then boiled for 5 min to separate RID-B from DNA. The supernatant containing RID-B was collected in a 4-tube magnetic rack (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Betaine (5 µg/ml) was added to the supernatant for internal standard, and RID-B, which had bound to DNA, was detected by electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS; micrOTOF-NR-focus; Bruker Daltonik GmbH, Bremen, Germany). The mass spectrometer settings were as follows: Ionization mode, positive; nitrogen gas temperature, 180°C; nebulizer pressure, 5.8 psi; flow rate: 4.0 l/min. The calculated mass (M+H⁺) for C₃₄H₄₃N₂O₂ (RID-B) is 511.3319; the mass for RID-B identified in the experiments was 511.3628 (positive control) and 511.3584 (DNA (+)), respectively. The calculated mass (M+H⁺) for C₅H₁₁NO₂ (betaine) is 118.0868; the mass for betaine identified in the experiments was 118.0991 (positive control), 118.0988 (DNA (+)) and 118.0976 (DNA (−)), respectively.

Data are expressed as the mean ± standard deviation. All experiments were performed at least three times. Two-way analysis of variance with Fisher's protected least significant difference was employed to compare mean values between groups. P<0.05 was considered to indicate a statistically significant difference. Data analysis was performed using JMP 9 software.

To evaluate the anti-proliferative effect of RID-B in hepatoma cells, human hepatoma Huh-7 cells and normal primary rat hepatocytes were incubated with RID-B, and cell numbers were estimated using WST-1 reagent (Fig. 2). Previous study indicated that the mean GI₅₀, for RID-B in 39 human cancer cell lines was 1.17 µM (4). Therefore, the RID-B concentration range of 0.5–2 µM was selected in the current study. RID-B inhibited the growth of Huh-7 cells between the concentrations of 0.5–2 µM (P<0.05 vs. 0 µM RID-B), in an apparent dose-dependent manner; however, RID-B did not inhibit the growth of normal hepatocytes within the same range. The GI₅₀ of RID-B in Huh-7 cells was 1.00±0.16 µM. The GI₅₀ of RID-B in rat hepatocytes could not be calculated as cell number did not fall below 90% (Fig. 2) and was therefore estimated as >2 µM. These results suggested that RID-B was more effective at inhibiting the growth of hepatoma cells than normal hepatocytes.

To clarify the mechanism of cancer cell growth inhibition by RID-B, the effect of caspase inhibitor, Z-VAD-FMK, on RID-B-mediated cell growth inhibition was examined. As depicted in Fig. 3A, the caspase-3 activity of Huh-7 cells was apparently increased dose-dependently between the RID-B concentrations 0.5 and 5 µM. Furthermore, by the pretreatment with Z-VAD-FMK, RID-B-mediated Huh-7 cell growth was significantly attenuated at the RID-B concentration of 1 µM (P<0.05; Fig. 3B). It appears evident from these data that RID-B induced the apoptosis of Huh-7 cells via the activation of caspase-3.

To clarify the mechanism of RID-B-induced apoptosis in cancer cells, the effect of DNA on RID-B-mediated Huh-7 cell growth inhibition was examined. Huh-7 cells were incubated with RID-B in the presence of plasmid DNA (pSecTag2B), and live cells were then counted (Fig. 4A). The addition of plasmid DNA suppressed RID-B-mediated Huh-7 cell growth inhibition; notably, this competitive inhibition by plasmid DNA appeared dose-dependent, as no significant difference was identified between the number of Huh-7 cells incubated with and without RID-B at the DNA concentration of 0.5 mg/ml. Similar results were observed when using salmon sperm DNA instead of plasmid DNA in the same DNA concentration range (data not shown). From these data, it may be suggested that RID-B binds to DNA to induce cancer cell apoptosis.

As in Fig. 3, RID-B induced apoptosis in Huh-7 cells seemingly via the activation of caspase-3. To confirm whether DNA inhibited RID-B-induced apoptosis in cancer cells, Huh-7 cells were pre-incubated with Z-VAD-FMK, and the cells were then incubated with RID-B in the presence of plasmid DNA. By TUNEL assay, it was observed that RID-B caused DNA fragmentation of Huh-7 cells, suggesting that RID-B induced cancer cell apoptosis by causing DNA fragmentation. Z-VAD-FMK had no obvious effect on DNA fragmentation in RID-B-treated Huh-7 cells; whereas the addition of plasmid DNA appeared to completely suppress DNA fragmentation (Fig. 4B).

From the data in Fig. 4A and B, it was apparent that RID-B directly bound to DNA. To verify this hypothesis, RID-B was incubated with biotinylated double-stranded DNA, and DNA-bound RID-B was then collected by streptoavidin-conjugated magnetic beads. DNA-bound RID-B was detected by ESI-TOF-MS (Fig. 4C). The peak derived from RID-B was detected only from the sample incubated with double-stranded DNA, and not from that incubated without DNA, indicating that RID-B directly bound to double-stranded DNA. The ratio of RID-B bound to DNA was 23.0%.