Abstract
Background/Aim: Nitric oxide (NO) has various physiological activities. In this study, diclofenac (DF) which has a high affinity for human serum albumin (HSA) was nitrosylated to a novel NO donor (NDF). The cytotoxic effects and the mechanism of NDF were investigated. Materials and Methods: Binding experiments of NDF to HSA were performed by the ultrafiltration method. NO was measured by the Griess method. The number of dead cells were measured using annexin V. Apoptosis and endoplasmic reticulum stress were evaluated by western blotting. Results: NDF competitively inhibits the binding of DF to HSA, suggesting that NDF and DF have equivalent binding characteristics. NDF rapidly released NOx after being dissolved. At 200 μM, NDF induced cell death in human pancreatic cancer cells. Western blotting showed that NDF promoted the cleavage of PARP, caspase-3, and caspase-7. Inhibitors of caspase-1 and caspase-9 significantly suppressed NDF-induced cell death, as did a non-specific caspase inhibitor (Z-VAD). In addition, NDF significantly increased the expression of the endoplasmic reticulum stress marker, CHOP. Conclusion: NDF induces apoptotic cell death by causing endoplasmic reticulum stress. The findings of this study suggest that NDF may become a promising compound for the treatment of pancreatic cancer.
Pancreatic cancer is difficult to detect early, and the five-year relative survival rate in Japan is less than 10% due to very low chemotherapy response rates (1). This is thought to be due to the low blood flow in the pancreas and around the tumor and the rich stroma surrounding the tumor, which decrease the amount of drug that can reach the tumor. Nitric oxide (NO) has not only vasodilator effects, but also cytotoxic effects (2). These effects could increase the degree of drug delivery to pancreatic cancer cells by improving blood flow and shrink tumors and their stroma. Therefore, NO donors are expected to be effective anticancer candidates in the treatment of pancreatic cancer. However, due to their low blood retention and tumor accumulation, they have not yet been clinically applied.
To solve this problem, we focused on human serum albumin (HSA). HSA is one of the representative drug-binding proteins with high blood retention (3-6). In addition, macromolecules such as HSA can migrate from peritumoral blood vessels to tumors due to the enhanced permeability and retention (EPR) effect and reach tumor cells via specific receptors, such as SAPRC and gp60 (7). These characteristics of HSA suggest that HSA is a useful carrier for delivering small-molecule drugs to tumors. We previously reported that the nitrosylated form of phenylbutyrate, which binds to HSA with high affinity, showed antitumor effects in vitro and in vivo (8, 9).
In the current study, a nitrosylated form (NDF) of diclofenac (DF) was newly synthesized (Figure 1), which bound to HSA with a high affinity, as well as phenylbutyrate, and we aimed to investigate the cell death effects of this compound as well as its mechanisms of action.
Scheme of synthesis of NDF from DF. 2-(2,6-Dichloroanilino)phenylacetic Acid (1.093 g, 3.69 mmol) was added to a solution of 4-(hydroxymethyl)benzyl nitrate (901 mg, 5.26 mmol) in dry DCM (25 ml), followed by DCC (761 mg, 3.69 mmol) and 4-(dimethylamino)pyridine (DMAP) (10 mg, 0.0819 mmol). The reaction mixture was stirred at room temperature for 14 h. After filtration, the solvent was evaporated to dryness under reduced pressure. The crude product was purified by flash column chromatography on amino silica gel (Hexane/EtOAc=95:5 to 85:15) to give the target product (846 mg, 1.834 mmol, 49.7% yield) as a yellow oil. 1H-NMR (500 MHz, CHLOROFORM-D) δ 7.38-7.32 (m, 6H), 7.24 (dd, J=7.4, 1.7 Hz, 1H), 7.13 (td, J=7.7, 1.3 Hz, 1H), 6.97 (q, J=7.4 Hz, 2H), 6.79 (s, 1H), 6.55 (d, J=8.0 Hz, 1H), 5.41 (s, 2H), 5.19 (s, 2H), 3.86 (s, 2H). NDF: Nitrosylated form of diclofenac (DF).
Materials and Methods
Cell cultures and reagents. The human pancreatic cancer cell line, BxPC3, was obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in the recommended medium, consisting of RPMI1640 (Fujifilm Wako, Osaka, Japan), supplemented with 10% heat-inactivated fetal calf serum (Capricorn Scientific, Germany), penicillin (100 units/ml), and streptomycin (100 μg/ml) (Fujifilm Wako), and grown at 37°C in 95% humidified air with 5% carbon dioxide. NDF was synthesized according to the method of Digiacomo et al. (10). Z-VAD FMK (non-specific caspase inhibitor), belnacasan (caspase-1 inhibitor), Gly-Phe β-naphthylamide (caspase-8 inhibitor), and Z-LEHD-FMK TFA (caspase-9 inhibitor) were purchased from Selleck Chemicals LLC (Houston, TX, USA). All other reagents were of analytical grade.
Evaluation of NO-releasing properties. NO production was evaluated by measuring nitrite (NO2) and nitrate (NO3), stable end-products of NO, using the NO2/NO3 Assay Kit-C II (Dojindo Laboratories, Kumamoto, Japan). NOx was measured according to the manufacturer’s protocol, at 0, 24, 48 and 72 h after dissolving NDF in phosphate-buffered saline (PBS).
Annexin V and dead cell assay. Live and apoptotic cell numbers were determined using the MUSE Annexin V and Dead Cell kit (Cytek Biosciences, Fremont, CA, USA), according to the manufacturer’s instructions. Briefly, cells were seeded at a density of 2.0×105 cells/well in six-well plates. After 12 h NDF was added to each well in the presence or absence of 50 μM of caspase inhibitors, and the plates were incubated for 24 h. The cells were then washed twice with PBS, trypsinized, and mixed well with the Muse Annexin V and Dead Cell Assay kit reagents. Reactions, which were conducted in triplicate, were analyzed using a MUSE Cell Analyzer (Cytek Biosciences).
Western blotting. After treatment with the appropriate concentration of NDF for 24 h, BxPC3 cells were washed twice with PBS and then lysed with RIPA buffer (ThermoFisher Scientific, San Jose, CA, USA), including a Protease/Phosphatase Inhibitor Cocktail (Cell Signaling Technology, Beverly, MA, USA). Aliquots of protein (30-40 μg) were subjected to SDS-PAGE (12%), transferred to a polyvinylidene difluoride (PVDF) membrane, and processed for incubation with PARP (#9452), caspase-3 (#9662), and caspase-7 (#9492) antibodies (Cell Signaling Technology) for 24 h, followed by anti-rabbit IgG antibodies for 1 h. Membranes were reacted with a chemiluminescence reagent (Nakarai Chemicals, Kyoto, Japan). Band density values were normalized to β-actin antibody (#3700) (Cell Signaling Technology). Assays were conducted in triplicate.
Statistical analysis. The differences between groups were determined by one-way analysis of variance (ANOVA). Probability values of p<0.05 were considered significant.
Results
NOx release. To confirm the release of NO from NDF, the concentrations of nitrate and nitrite (NOx) were measured after dissolving in PBS using the Griess method (Figure 2). It was observed that NDF released NOx immediately after dissolving. It was also found that the amount of released NOx was dependent on the NDF concentration, and the release rate finally reached 10%.
Release of NOx from NDF in aqueous solution. (A) After dissolving NDF to be 200 μM in PBS, NOx was measured at each of the indicated time points. (B) At 72 h after dissolving NDF to 100, 200, 400 and 500 μM in PBS, NOx was measured. Each value indicates the mean±S.D. from three independent measurements. NOx: Nitrite and nitrate; NDF: nitrosylated form of diclofenac (DF).
Binding of NDF to HSA. DF is widely known to bind to the binding site, site II, of HSA (11-13). Therefore, whether NDF also binds to site II of HSA was investigated. The replacement experiment showed that the binding constant (K) of DF was significantly decreased in the presence of NDF, whereas the number of binding sites (n) was not (Table I). This result indicates that NDF replaces DF competitively, and NDF also binds to site II of HSA specifically.
Binding constants (K) and the number of binding sites (n) of DF in the presence or absence of NDF.
Cytotoxicity of NDF against human pancreatic cancer cells. To investigate the effect of NDF on the survival of human pancreatic cancer cells, the annexin-positive cells observed in apoptotic or necrotic cells were measured by flow cytometry (Figure 3), and NDF significantly induced cell death at 200 μM. To investigate the mechanism of cell death induction by NDF, the degradation of apoptosis markers, PARP, caspase-3, and caspase-7 was evaluated by western blotting (Figure 4). Degradation of PARP, caspase-3, and caspase-7 was observed with 200 μM of DF. These results suggest that NDF induces apoptosis in human pancreatic cancer cells. Furthermore, to investigate the detailed apoptosis induction pathway, the effects of various caspase inhibitors on the cell death induction effect of NDF were examined (Figure 5). The nonspecific inhibitor Z-VAD FMK and the selective inhibitor of caspase-9, Z-LEHD-FMK TFA, significantly suppressed cell death. These results suggest that the caspase-9 pathway is involved in NDF-induced apoptosis. Previous reports have shown that endoplasmic reticulum stress is involved in the activation of caspase-9 (14). Therefore, the expression level of CHOP, an endoplasmic reticulum stress marker, was examined in cells treated with NDF by western blotting (Figure 6). It was found that NDF significantly increased the expression level of CHOP. This result indicates that NDF induces endoplasmic reticulum stress, which results in the activation of caspase-9.
Effect of NDF on cell death induction in pancreatic cancer cells. Cell death (%) was measured 24 h after adding NDF (50, 100, and 200 μM) to cultures of BxPC3 cells. Significantly different vs. control. *p<0.05. NDF: Nitrosylated form of diclofenac (DF).
NDF-induced cell death mechanism. BXPC3 cells were treated with the indicated amounts of NDF for 24 h, and cell lysates were then analyzed by western blotting to detect cleaved PARP, caspase-3, and caspase-7 levels, as described in Materials and Methods. β-actin was probed to indicate the relative amounts of loaded proteins. NDF: Nitrosylated form of diclofenac (DF).
Effect of caspase inhibitors on NDF-induced cell death. Cell death (%) was measured 24 h after adding NDF (200 μM) to cultures of BxPC3 cells in the presence or absence of caspase inhibitor (50 μM). Significantly different vs. control, #p<0.05. Significantly different vs. NDF alone, ##p<0.05.
Involvement of endoplasmic reticulum stress in NDF induced-cell death. BxPC3 cells were treated with the indicated amounts of NDF or 5 μM of thapsigargin as a positive control for 24 h, and cell lysates were then analyzed by western blotting to detect CHOP levels, as described in the Materials and Methods section. GAPDH was probed to indicate the relative amounts of loaded proteins. NDF: Nitrosylated form of diclofenac (DF).
Discussion
In human pancreatic cancer, anticancer drugs have difficulty reaching the tumor due to the low blood flow rate of the pancreas and pancreatic tumors and the presence of abundant stroma. To date, many nitric oxide donors have been developed as candidate anticancer drugs (15), but they have not been used in clinical applications due to poor results in terms of blood retention and tumor accumulation. Previously, we focused on HSA, which remains in blood for a long time and accumulates in tumors, and synthesized a nitrosylated form of phenylbutyric acid that has high binding affinity to HSA (16-18), and reported that this NO donor has high antitumor activity in vitro and in vivo (9). In the present study, a nitrosylated form of DF, which has high binding affinity to HSA, was synthesized, and its cell death induction effect and its mechanism were investigated.
Nitrosylated DF (NDF) released approximately 10% NO immediately after dissolution in PBS, and the degree was concentration-dependent. This result indicates that NDF releases NO by hydrolysis, with almost constant efficiency. In the binding experiment of NDF to HSA, it was suggested that NDF competitively inhibited DF binding. In particular, NDF significantly decreased the binding constant of DF, suggesting that NDF has high binding affinity to HSA, equivalent to DF.
In the study of the toxicity of NDF to human pancreatic cancer cells, NDF significantly increased the number of annexin-positive cells and induced the degradation of PARP, caspase-3, and caspase-7. These phenomena are observed during apoptosis, suggesting that NDF induces cell death through apoptosis. The study using selective inhibitors of caspases-1, -8, and -9 showed that an inhibitor of caspase-9 significantly suppressed cell death caused by NDF. Caspase-9 is activated by endoplasmic reticulum stress (14). The increase in intracellular CHOP expression observed after NDF treatment also suggested that endoplasmic reticulum stress is involved in cell death caused by NDF. Previously, it was reported that NO nitrosylates intracellular proteins, triggering the unfolding protein response and inducing endoplasmic reticulum stress (19) or activating intracellular signaling pathway (20, 21). Therefore, NDF may cause endoplasmic reticulum stress by nitrosylating some intracellular proteins. However, an inhibitor of caspase-8 enhanced the cell death-inducing effect of NDF. Inhibition of caspase-8 is known to activate the necroptotic pathway by inhibiting apoptosis (22, 23). Therefore, necroptosis may also be involved in part of the cell death-inducing effect of NDF in the presence of a caspase-8 inhibitor.
Conclusion
In this study, NDF, a nitrosylated form of DF, was synthesized and shown to exhibit albumin binding activity equivalent to that of DF. It was suggested that NDF induces apoptosis in human pancreatic cancer cells by inducing endoplasmic reticulum stress. These results suggest that NDF may be a useful candidate anticancer drug for the treatment of human pancreatic cancer.
Acknowledgements
This work was supported by JSPS KAKENHI Grant Number 20K07193.
Footnotes
Authors’ Contributions
Koji Nishi contributed to the design of this study, and data collection and interpretation, and wrote the initial draft of the manuscript. Shuhei Imoto and Hirotaka Murase contributed to the synthesis and the structural validation of NDF. Ryo Kanda, Kaho Takasaki, Ayano Tamori, Yoshifumi Arimura contributed to data collection. Kenji Tsukigawa, Masaki Otagiri and Keishi Yamasaki contributed to the design of this study, interpretation, and critically reviewed the manuscript. All Authors approved the final version of the manuscript and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.
Conflicts of Interest
The Authors declare no conflicts of interest.
- Received June 17, 2024.
- Revision received July 4, 2024.
- Accepted July 12, 2024.
- Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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