Abstract
Background/Aim: Nitric oxide (NO) has antitumor activity against various solid tumor cell types in addition to its vasodilatory effect. In the current study, we investigated the effect and mechanism of the synthetic nitrated form (NO2-NAT) of nateglinide, a hypoglycemic agent, on the induction of cell death in human pancreatic cancer cells. Materials and Methods: NO production was evaluated by measuring nitrite (NO2) and nitrate (NO3) (NOx). Apoptotic cell numbers were determined using annexin V. Results: NO2-NAT released nitrate and nitrite ions immediately upon dissolving in aqueous solution. NO2-NAT caused significant extracellular leakage of lactate dehydrogenase (LDH) in the human pancreatic cancer cell lines AsPC1 and BxPC3, and increased annexin-positive cells in a time- and concentration-dependent manner. NO2-NAT also significantly increased the activity of caspases 3 and 7. Exposure to Z-VAD-FMK, a caspase inhibitor, significantly suppressed NO2-NAT-induced cell death. Conclusion: These results indicated that NO2-NAT induces apoptosis in human pancreatic cancer cells and may serve as a new NO-based chemotherapeutic agent for the treatment of pancreatic cancer.
Pancreatic cancer is the fourth- and third-leading cause of cancer-related deaths in Japan and the United States, respectively (1, 2). Pancreatic cancer is one of the most lethal types of cancer, with a 5-year relative survival rate of less than 8% in Japan (2). Standard chemotherapy regimens consisting of tegafur, gimeracil and oteracil potassium (TS-1); oxaliplatin, irinotecan, fluorouracil, and leucovorin (FOLFIRINOX); gemcitabine plus nab-paclitaxel; and gemcitabine plus erlotinib are widely used as first-line therapies to treat patients with advanced and metastatic pancreatic cancer; however, the anti-tumor effects of these regimens are remarkably lower compared to other solid tumors. One of the causes of this discrepancy is the inefficiency of drug delivery to pancreatic tumors. This inefficiency reflects both the fact that blood flow around pancreatic tumors is low, and that the pancreas is inherently an organ with low blood flow. In addition, tumor-associated stromal tissues create a barrier for delivery of drugs from blood vessels to the tumors (3, 4).
Nitric oxide (NO) has broad effects on initiation, progression and metastasis of cancer. In the past 20 years, many papers have reported the cell death-inducing effect of nitrated compounds, demonstrating that these effects are mediated via intracellular signals such as Ras, extracellular signal-regulated kinase (ERK), and mechanistic target of rapamycin (mTOR) (5-9). Modification of aspirin, a non-steroidal anti-inflammatory drug, with a nitro group has been reported to significantly potentiate the induction of apoptosis in cancer cells (10). Similarly, addition of a nitro group to doxorubicin has been shown to attenuate the activity of mitochondria-associated ABC transporters, thereby decreasing drug resistance to doxorubicin and enhancing cytotoxicity (11). Other nitrated compounds include nitroglycerin and isosorbide dinitrate, which are classical drugs aimed at vasodilatory action. Together, these observations suggest that the vasodilatory effect of NO may dilate new blood vessels surrounding the tumor and increase the efficacy of drug uptake. Recently, Isram et al. reported that nitroglycerin improves the efficacy of anticancer drugs when used in combination with such treatments (12). Chen et al. also found that NO exposure resulted in depletion of stroma, resulting in improvement of the enhanced permeability and retention (EPR) effect (13). These findings indicate that nitrated compounds increase the therapeutic effect not only of other anticancer agents, but also of the nitrated compound itself.
Nateglinide (NAT) is a compound that is used to treat diabetes; this agent stimulates pancreatic islet β-cells with residual insulin secretory capacity to increase insulin secretion (14). Previously, nitrated NAT (NO2-NAT) was reported to have anti-ischemic cardioprotective activities in vitro and in vivo (15, 16). In the current study, we investigated the effects and mechanism of NO2-NAT (Figure 1) on cell death in human pancreatic cancer cells.
Chemical structure of nitrated nateglinide (NO2-NAT). NO2-NAT was synthesized by reacting Nateglinide with 4- (hydroxymethyl) benzyl nitrate in the presence of N, N’-Dicyclohexylcarbodiimide and 4-Dimethylaminopyridine.
Materials and Methods
Cell cultures and reagents. The human pancreatic cancer cell lines AsPC-1 and BxPC3 were obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in the recommended medium, consisting of RPMI1640 (Fujifilm, Wako, Japan), supplemented with 10% heat-inactivated fetal calf serum (Capricorn Scientific, Ebsdorfergrund, Germany), penicillin (100 units/ml), and streptomycin (100 μg/ml) (Fujifilm), and grown at 37°C in 95% humidified air with 5% carbon dioxide. NO2-NAT was synthesized according to the method of Digiacomo et al. (15). All other reagents were of analytical grade.
Lactose dehydrogenase (LDH) assay. Cell toxicity was evaluated using the Cytotoxicity LDH Assay Kit-WST (Dojindo Laboratories, Japan) according to the manufacturer’s protocol. Briefly, cancer cells were seeded at a density of 3,000 cells/well in 96-well white, flat-bottom plates. After overnight incubation, the cells were exposed to NAT or NO2-NAT for 72 h. Subsequently, the cells were incubated for 1 h at room temperature with the LDH reagent, and luminescence was measured. Cytotoxicity was estimated using the ratio between the activities of LDH in the culture medium and the whole cell. All experiments were repeated three times.
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).
Annexin V and dead cell assay. Live and apoptotic cell numbers were determined using the MUSE Annexin V and Dead Cell kit (Luminex, 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, inhibitors were added to each well, and the plates were incubated for 24 h. The cells then 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, were analyzed using a MUSE Cell Analyzer (Luminex).
Caspase-3/7 activity assay. Cellular caspase-3/7 activity was measured using the Caspase-Glo 3/7 Reagent (Promega, USA). Briefly, cells were seeded at a density of 1.0×104 cells/well in 96-well plates. After incubation with NO2-NAT for 6 h, Caspase-Glo 3/7 Reagent was added to each well. After incubation at room temperature for 1 h, luminescence of each sample was measured in a plate-reading luminometer. Assays were conducted in triplicate.
Statistical analysis. For continuous variable, Student’s t-test or one-way analysis of variance (one-way ANOVA) were performed. The pairwise t-test with Holm’s adjustment or Dunnett’s test for post-hoc test was employed after one-way ANOVA. In addition, repeated measures ANOVA was used to analyze longitudinal data.
These analyses were performed using R version 4.0.3 (The R Foundation for Statistical Computing, Vienna, Austria). A value of p<0.05 was considered statistically significant.
Results
Release of NO from NO2-NAT. Upon dissolving in aqueous solution, NO2-NAT is hydrolyzed, resulting in the release of NO2, which has been shown to have NO-mediated vasorelaxing effects in isolated endothelium-denuded rat aortic rings (16). NO2 released from nitro compounds is reduced to NO in anaerobic environments such as within tumors; alternatively, the liberated NO2 can be oxidized to stable NO3 in aerobic environments (17). To confirm the release of NO2 from NO2-NAT, nitrite and nitrate (NOx) were detected using the Griess method (Figure 2). Immediately after dissolving NO2-NAT at 50 μM in PBS, the release of 8 μM NOx (16% of the mass of the parent compound) was observed. By 24 h after NO2-NAT was dissolved in PBS, the release of 12 μM NOx (24% of the mass of the parent compound) was confirmed. These results suggested that NO2-NAT immediately releases much of the NO2 that can be released after dissolution.
Release of nitrite and nitrate (NOx) from nitrated nateglinide (NO2-NAT) in aqueous solution. After dissolving NO2-NAT to 50 μM in phosphate-buffered saline (PBS), NOx was measured at each of the indicated time points. Each value indicates the mean±S.D. from three independent measurements. p<0.05 using repeated measures ANOVA.
Cytotoxicity of NO2-NAT in human pancreatic cancer cells. To investigate the cytotoxicity of NO2-NAT in human pancreatic cancer cells, the relative activity of lactate dehydrogenase (LDH) in the medium to that of whole cells LDH was assessed as cytotoxicity at 72 h after adding NO2-NAT to AsPC1 (Figure 3A) or BxPC3 (Figure 3B). In both cell lines, cytotoxicity was observed in the presence of 25 μM NO2-NAT and increased up to 8% at 50 μM, whereas NAT did not show significant cytotoxicity. Therefore, cytotoxicity appears to be attributable to the NO released from NO2-NAT.
Cytotoxicity of nitrated nateglinide (NO2-NAT) in human pancreatic cancer cells. Cytotoxicity was evaluated by measuring the degree of lactate dehydrogenase (LDH) activity in medium at 72 h after adding nateglinide (NAT) or NO2-NAT in (A) AsPC1 and (B) BxPC3 cells in different concentrations. N.D: Not detected. Data are presented as the mean±S.D. of three independent experiments. *Represents significant differences vs. NAT, p<0.05 using Student’s t-test.
Effect of NO2-NAT on apoptosis induction in pancreatic cancer cells. Next, we investigated whether NO2-NAT induced apoptotic cell death in the AsPC1 and BxPC3 human pancreatic cancer cell lines. In both cell lines, the proportion of annexin-positive cells was nominally increased in a concentration-dependent manner as the concentration of NO2-NAT rose, although these changes did not achieve statistical significance (Figure 4A). Specifically, exposure to 50 μM NO2-NAT resulted in a time-dependent 2.5-fold increase (compared to control) in the percentage of annexin-positive BxPC3 cells (Figure 4B). To investigate whether caspase, an inducer of apoptosis, is involved in NO2-NAT induced-cell death, the effect of a non-specific caspase inhibitor, Z-VAD-FMK, was examined (Figure 4C). Z-VAD-FMK significantly attenuated the increase in annexin-positive cells induced by NO2-NAT. Furthermore, 6-h exposure to NO2-NAT significantly increased caspase 3/7 activity (Figure 4D) compared to that seen in the control cells.
Effect of nitrated nateglinide (NO2-NAT) on apoptosis induction in pancreatic cancer cells. (A) Cell death (%) was measured at 6 h after adding NO2-NAT (0-50 μM) to cultures of AsPC1 or BxPC3. One-way ANOVA with Dunnett’s post-hoc test was performed. (B) Cell death (%) was measured at 3, 6, and 9 h after adding NO2-NAT (50 μM) to AsPC1 or BxPC3. (C) Cell death (%) was measured at 6 h after adding NO2-NAT (50 μM) to BxPC3 with or without the Z-VAD-FMK caspase inhibitor (20 μM). One-way ANOVA and the pairwise t-test with Holm’s adjustment was applied for post-hoc analysis. (D) Caspase 3/7 activity was measured at 6 h after adding NO2-NAT (50 μM) to BxPC3. Data are presented as the mean±S.D. of three independent experiments. Student’s t-test was used. *represents significant differences vs. control or Z-VAD-FMK caspase inhibitor, p<0.05.
Discussion
Pancreatic cancer has extremely low uptake of anticancer drugs due to low blood flow and the presence of a fibrotic interstitium. Therefore, there is an urgent need to develop a new drug delivery system for the treatment of pancreatic cancer. Nitrated compounds are widely known to have antitumor effects, in addition to possessing conventional vasodilatory effects (18). Therefore, we conjectured that NO2-NAT, the compound characterized in the present study, may not only promote the blood flow around the tumor, thereby increasing NAT uptake into the tumor, but also induce cell death in the stroma and cancer cells. In this study, we investigated the effect of NO2-NAT on the cell death of human pancreatic cancer cells.
NO2-NAT released NOx immediately after dissolution, although no significant further increase in release was observed during the subsequent 24 h. The maximum released amount of NOx was 12 μM, representing 24% of the initial 50-μM concentration of NO2-NAT. We propose that NO2 released from the nitrated compound subsequently is oxidized to NO3 under the aerobic experimental conditions. On the other hand, under hypoxic conditions like those that exist in tumors, NO2 can be reduced to NO, which in turn exerts effects on cancer cells. Therefore, future experiments will need to examine the effect of NO2-NAT in a hypoxic environment. In our preliminary experiments, we observed that NO2-NAT generates NO radicals in cell culture. This finding suggests that the effect of NO2-NAT in the current study may be due to the partial reduction to NO in cancer cells.
The present work revealed that NO2-NAT induces apoptotic cell death in culture. The effect was observed after 6 h and is consistent with the observed quick release of NOx. In a study using LDH as a marker for cytotoxicity, the activity of LDH in medium is indicative of release of the enzyme due to structural breakdown of the cell membrane. Such disruption of selective permeability of cell membranes is the most typical feature of necrosis, whereas in apoptotic cells, exposure to phosphatidyl serine is visible on the cell membrane surface, which reacts with annexin V. When cytotoxicity was induced, no significant decrease in intracellular ATP content (a marker of necrosis) was observed at 24 h after adding NO2-NAT (relative ATP content to control: 1.01±0.10). These results suggest that NO2-NAT-induced cell death is mediated primarily via apoptosis and has limited involvement in necrosis. Cell death caused by nitrated compounds has been attributed variously to oxidative stress (19), endoplasmic reticulum stress (20), DNA modification, protein nitration (21), and inhibition of autophagy (22). Further work will be needed to elucidate the detailed mechanism of NO2-NAT-induced cell death.
The results obtained in the present study revealed that the exposure of human pancreatic cancer cells to NO2-NAT induces caspase-mediated apoptosis via NO generation. Given that the vasodilatory effect of NO2-NAT that has been reported previously (15, 16), we propose that NO2-NAT may serve as a novel chemotherapeutic agent that is able to increase drug uptake by tumors by improving blood flow around the lesion.
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 contributed to the synthesis and the structural validation of NO2-NAT. Takuro Beppu, Shotaro Uchibori, Ayana Yano, Yu Ishima and Kenji Tsukigawa contributed to data collection. Tokunori Ikeda contributed to the statistics analysis. 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 December 21, 2021.
- Revision received January 19, 2022.
- Accepted January 31, 2022.
- Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
