PB was purchased from TCI (Shanghai) Development Co., Ltd. Chlorambucil was obtained from Tokyo Chemical Industry (TCI) Co., Ltd. Dihydroxy chlorambucil was purchased from SynZeal Research Pvt Ltd. Z-VAD-FMK was obtained from Promega Corporation. Diaminofluorescein-FM diacetate (DAF-FM DA) was purchased from Goryo Chemical, Inc. Necrostatin and N-acetyl-L-cysteine (NAC) were procured from FUJIFILM Wako Pure Chemical Corporation. GSK872 was obtained from Abcam. Necrosulfonamide was purchased from Funakoshi Co., Ltd. Antibodies against caspase-3 (product no. 9662S), caspase-7 (product no. 9492S), poly (ADP-ribose) polymerase (PARP)-1 (product no. 9542S), CHOP (product no. 2895S), β-actin (cat. no. 3700S), and HRP-conjugated anti-rabbit (product no. 7074P2) were purchased from Cell Signaling Technology, Inc.

A mixture of chlorambucil (250 mg, 0.822 mmol) and AgNO₃ (558 mg, 3.29 mmol) in CH₃CN (16 ml) was stirred at 70°C overnight. After being cooled to room temperature, the suspension was filtered and the solvent was evaporated. The residue was purified by flash column chromatography on silica gel (DCM/MeOH, 99:1 to 92:8, v/v) to yield NPB (Fig. 1) (254 mg, 0.711 mmol, 86% yield) as a pale yellow oil. ¹H-NMR (500 MHz, CDCl₃): δ=7.10 (d, J=8.6 Hz, 2H), 6.66 (d, J=8.6 Hz, 2H), 4.60 (t, J=6.0 Hz, 4H), 3.71 (t, J=5.7 Hz, 4H), 2.59 (t, J=7.4 Hz, 2H), 2.37 (t, J=7.4 Hz, 2H), 1.90-1.96 (m, 2H). ¹³C-NMR (126 MHz, CDCl₃): δ=179.8, 144.1, 131.2, 129.8, 112.9, 69.9, 48.9, 33.8, 33.2, 26.3. MS (ESI): m/z calculated for C₁₄H₂0N₃O₈ [M+H]⁺ 358.1250, found 358.1241.

The human pancreatic cancer cell lines, AsPC1 (CRL-1682) and BxPC3 (CRL-1687), were obtained from the American Type Culture Collection. The cells were cultured in the recommended medium, consisting of RPMI-1640 (FUJIFILM Wako Pure Chemical Corporation), supplemented with 10% heat-inactivated fetal calf serum (Capricorn Scientific GmbH), penicillin (100 U/ml), and streptomycin (100 µg/ml) (FUJIFILM Wako Pure Chemical Corporation), and grown at 37°C in 95% humidified air with 5% carbon dioxide.

Live and apoptotic cell numbers were determined using the MUSE Annexin V and Dead Cell kit (Luminex Corporation) according to the manufacturer's instructions. Briefly, AsPC1 and BxPC3 cells were seeded in a 6-well plate with 1×10⁵ cells per well, and then, incubated at 37°C overnight. Following incubation, the cells were exposed with various concentrations of NPB (100, 300, and 500 µM) for 48 h or with 500 µM for 24, 48 and 72 h. Necrostatin, GSK872, Necrosulfonamide and NAC were used at concentrations of 20, 1 and 1 µM and 1 mM respectively. After treatment, the cells were washed twice with phosphate-buffered saline (PBS), trypsinized, and mixed well with the Muse Annexin V and Dead Cell Assay kit reagents. Reactions, which were conducted in triplicate, and analyzed using a MUSE Cell Analyzer (Luminex Corporation).

AsPC1 and BxPC3 were seeded in round bottom 96-well plates with 5×10⁵ cells per well. After confirming the formation of spheroids, 500 M of NPB was added to the each well, and then incubated for 1 week at 37°C. Spheroid structure and the spheroid area were analyzed by fluorescence microscopy.

The nitrite and nitrate (NOx) levels were quantified by the Griess method [NO₂/NO₃ Assay kit-C II (Colorimetric); Dojindo Laboratories, Inc.]. The NOx levels were assessed at 0, 3, 24, 48, 72 and 96 h after 100 µM of NPB was dissolved in PBS containing 10% MeOH. Samples were read at 540 nm in a 96-well plate using a Spectra Microplate Auto reader (Bio-Rad Model 680; Bio-Rad Laboratories, Inc.). For microscopic observation, AsPC1 and BxPC3 cells were seeded in a 6-well plate with 5×10⁵ cells per well. Following overnight incubation, the culture medium was replaced with 10 µM of DAF-FM DA, and then cells were incubated at 37°C for 1 h. After incubation, cells were washed with PBS three times. Following washing, the cells were treated with 500 µM NPB for 5 min and observed using a fluorescence microscope. For assessment of fluorescence intensity of DAF-FM DA, cells were seeded in a black 96-well plate with 5×10⁴ cells per well. Following overnight incubation at 37°C, the culture medium was replaced with 10 µM DAF-FM DA, followed by incubation at 37°C for 1 h. Following incubation, the cells were washed with PBS three times. After washing, the cells were treated with 500 µM of NPB with PBS for 5 min and measured by using fluorescence plate reader (ex. 495 nm and em. 515 nm).

BxPC3 cells were seeded in a black 96-well plate with 1×10⁴ cells per well. Following overnight incubation at 37°C, the cells were treated with 500 µM of NPB for 6 and 24 h. Subsequently, 100 µl of Caspase-Glo 3/7 Reagent (Promega Corporation) was added to each well. After mixing gently, the cells were incubated at room temperature for 1 h. Finally, luminescence of each sample was measured by a multifunctional microplate reader (Infinite 200 Pro; Tecan Group, Ltd.).

BxPC3 cells were seeded in a 6-well plate with 5×10⁵ cells per well. After overnight incubation at 37°C, the cells were treated 500 µM of NPB for 6 and 24 h. Following treatment, the cells were lysed with RIPA buffer (Thermo Fisher Scientific, Inc.), including a Protease/Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific, Inc.). Protein concentration of each lysate was determined by BCA method. Aliquots of protein (30–40 µg) were subjected to SDS-PAGE (12% of acrylamide for PARP and 10% for caspase-3/7 and β-actin), transferred to a polyvinylidene difluoride, (PVDF) membrane. After blocking with 5% skim milk in PBS including 0.1% Tween-20 for 3 h at 37°C, the membrane was processed for incubation with caspase-3, caspase-7, PARP, CHOP or β-actin antibody (all 1:1,000) for 12 h at 4°C, followed by anti-rabbit IgG antibodies for 1 h at 4°C. Membranes were reacted with a chemiluminescence reagent (GE Healthcare UK Ltd.; Cytiva). Band density values were normalized to β-actin.

BxPC3 cells were seeded in a white 96-well plate with 1×10⁴ cells per well. Following overnight incubation at 37°C, cells were treated with 500 µM of NPB for 48 h, and then 100 µl of intracellular ATP Reagent (TOYO-B Net Co., Ltd.) was added to each well. After being gently mixed, luminescence of each sample was measured using a plate-reading luminometer (Infinite 200 Pro; Tecan Group, Ltd.). To correct for variations in cell number, the protein content of each sample was measured using the BCA protein assay kit (Thermo Fisher Scientific, Inc.) and the ATP content was normalized to the protein content.

For the cell cycle analysis, propidium iodide-based nuclear staining was carried out using Muse Cell Cycle Kit (Luminex Corporation) according to the manufacturer's protocol. Briefly, AxPC1 and BxPC3 cells were cultured in a 6-well plate for 24 h, and then treated with 500 µM of NPB for 24 h. After the treatment, cells were fixed in 70% ethanol and stored at −20°C for at least 3 h. Fixed cells were washed with cold PBS and centrifuged at 300 × g for 5 min and stained using a Muse™ Cell Cycle Kit for 30 min at room temperature in dark conditions. After the staining, analysis was performed by Muse Cell Analyzer (Luminex Corporation).

A total of 10 male, six-week-old BALB/c nude mice (20–25 g) were obtained from Japan SLC, Inc., and raised in a laminar mouse house, with 50±5% humidity, 25°C and a 12-h light/dark cycle. The mice were fed standard rodent food and mineral water. BxPC3 cells (5×10⁶ cells/mouse) were s.c. injected into the right flank. When tumor volumes reached 50 mm³, NPB (10 mg/kg) or saline were administered via the tail vein once. Following treatment, tumor formation was monitored by measuring the width and length of the mass, and the tumor volume (TV) was calculated as follows: TV (mm³)=(L × W²)/2, with L as the longest and W as the shortest radius of the tumor. Animals were euthanized by cervical dislocation after seven weeks from the administration of NPB. The death of the animals was confirmed by checking for cardiac arrest, decreased body temperature, and no movement. All experiments were approved by the Animal Ethics Committee of Sojo University (Kumamoto, Japan) and carried out according to the Laboratory Protocol for Animal Handling of Sojo University.

For continuous variables, unpaired Student's t-test or one-way analysis of variance (one-way ANOVA) was performed. The pairwise t-test with Holm's adjustment for post hoc test was employed after one-way ANOVA. In addition, a linear mixed model was used to analyze longitudinal data such as tumor volume and body weight. The mean ± standard deviation was used to present statistical outcome.

These analyses were performed using R version 4.0.3 (The R Foundation for Statistical Computing; http://www.r-project.org/foundation/). P<0.05 was considered to indicate a statistically significant difference.

First, the effects of NPB on the cell death of human pancreatic cancer cells were examined. Human pancreatic cancer cell lines, AsPC1 and BxPC3, were exposed to 500 µM NPB for 24-72 h (Fig. 2A and B) or 100-500 µM for 48 h (Fig. 2C and D) and the number of Annexin-positive cells was determined. In both cell lines, NPB significantly induced cell death in a time- and concentration-dependent manner. Next, the effect of NPB on the growth inhibition of AsPC1 and BxPC3 spheroids was examined. Even 7 days after the addition of 500 µM of NPB, spheroid growth was significantly inhibited in both cell lines compared to their controls. In addition, disruption of spheroid surface structure was observed in AsPC1 cells (Fig. 3).

NO radical detecting agent, DAF-FM DA, was used to examine intracellular NO release from NPB. As revealed, more cells in the NPB-exposed cells had DAF-FM DA-derived fluorescence compared with the control, indicating that NO radical is generated within NPB-exposed cells (Fig. 4A and B). It is known that a part of nitrite ions released from the NO-donor compound are reduced to NO under anaerobic conditions in tumors but are oxidized to nitrate ions under aerobic conditions. Quantitative assessment of NOx released from NPB was performed (Fig. 4C). Notably, increased NOx was observed at least up to 96 h after dissolving NPB in PBS. Moreover, no cell death-inducing effect was observed for OH-PB, in which the NO₂ moiety of NPB was replaced by an OH group, and PB (Fig. 4D), suggesting that NO released from NPB was mainly involved in the cell death-inducing effect of NPB.

Cell death is induced by various pathways, including apoptosis and necrosis. To date, NO donors have been reported to induce apoptosis (9–11,23–25). To investigate the involvement of caspase, which plays a central role in apoptosis, the cell death effects of NPB in the presence of a caspase inhibitor, Z-VAD-FMK (Fig. 5A) were examined. The results revealed no significant effect of Z-VAD-FMK on cell death induction by NPB. Assessment of caspase-3/7 activity after NPB exposure exhibited no activation at 6 h or 24 h after the addition of NPB (Fig. 5B). Results of western blotting also showed no degradation of PARP or caspase-3/7 (Fig. 5C). These results indicated that apoptosis was not involved in the induction of cell death by NPB. Necrosis is characterized by cell swelling and a decrease and depletion of intracellular ATP (26). Thus, the amount of ATP after the addition of NPB was determined. A total of 48 h after addition of NPB, the amount of ATP was significantly decreased compared with the control (Fig. 5D), suggesting cell death was due to necrosis. Necrostatin, GSK872 and Necrosulfonamide, which are necroptosis inhibitors, did not suppress the cell death effect by NPB (Fig. S1).

Since NPB was observed to inhibit cell proliferation (Fig. S2), the effect of NPB on the cell cycle was examined and it was determined that cells exposed to NPB for 24 h exhibited a significant accumulation of S-phase cells compared with the control (Fig. 6). This indicated that NPB also induced cell cycle arrest.

The antitumor effect of NPB was investigated in vivo (Fig. 7). Tumor-bearing mice with BxPC3 tumors (50 mm³) implanted subcutaneously were prepared, and after a single dose of NPB (10 mg/kg) by intravenous tail injection, the tumor volumes and body weights were evaluated. There were no significant differences in body weight and no mice succumbed during the observation period. Notably, significant tumor suppression was observed even up to 7 weeks after NPB administration compared with the control group.