The Effects of Analgesics and Local Anesthetics on Gene Transcription Mediated by NFATc2 and Sp1 in Pancreatic Carcinoma

Background/Aim: Recent research has identified the transcription factors NFATc2 and Sp1 as key regulators in the carcinogenesis of pancreatic carcinoma. This study aimed to examine the effect of clinically achievable dosages of analgesics including ketamine, s-ketamine, metamizole, and paracetamol as well as that of sufentanil, ropicavaine, and lidocaine on pancreatic carcinoma cells and the expression of NFATc2 and Sp1. Materials and Methods: The effects of analgesics on the expression of NFATc2 and Sp1 were investigated with immunoblotting. Cell proliferation was measured with the ELISA BrdU assay. Results: In PaTu8988t pancreatic carcinoma cells, 48 h stimulation with ketamine and s-ketamine significantly inhibited proliferation and decreased expression of NFATc2 in the nucleus. The addition of metamizole and lidocaine reduced proliferation of PaTu8988t cells after 48 h. Conclusion: New treatment concepts target specific signaling and transcription pathways. The extent to which drugs influence these mechanisms in pancreatic carcinoma cells needs to be investigated in future studies.

Pancreatic adenocarcinoma is the fourth leading cause of cancer-related deaths worldwide (1). This type of cancer is known for its extremely poor long-term survival rates of 5 years which is only about 7% of all affected patients as well as for its almost identical incidence and mortality rates (2). The treatment of choice next to chemotherapy or radiation is surgical removal of the tumor (3). However, research carried out in the past few years has shown that the perioperative period is a particularly vulnerable phase in which tumor progression and metastasis are facilitated (4). Medication, surgery, and perioperative immunosuppression induce the constitutive activation of important signaling pathways and change the expression of transcription factors (5). Thus, it is hardly surprising that approximately 80% of affected patients show tumor recurrence even after surgery (2) and that the mean survival rate is reduced to approximately 9 months only (6).
Key elements in the carcinogenesis of pancreatic carcinoma are NFAT transcription factors (7). NFAT is the abbreviation of 'nuclear factor of activated T cells' that was first described by Shaw et al. in 1988 (8). NFATs control gene expression during activation and differentiation of T lymphocytes (9). In addition, these proteins are also expressed in a wide range of cells and tissue types and regulate genes involved in cell cycle, apoptosis, angiogenesis, and metastasis (10). As transcription factors, NFATs also control the expression of central genes involved in the control of growth and differentiation in pancreatic carcinoma (9). Furthermore, NFAT has been shown to interact with the transcription factor Sp1 (11).
It is still unclear to what extent the oncogenic transcription factors NFATc2 and Sp1 are influenced by agents given during the perioperative period for anesthesia or pain therapy.
The purpose of this study was to conduct an in vitro analysis of the impact of clinically achievable dosages of the analgesics ketamine, s-ketamine, metamizole, and paracetamol as well as of sufentanil, ropicavaine, and lidocaine on the pancreatic carcinoma cell lines PaTu8988t in dependency to NFATc2 and Sp1.
Cell proliferation. Quantification of cell proliferation was based on the measurement of BrdU incorporation during DNA synthesis. The test was performed according to the manufacturer's protocol (Cell proliferation ELISA BrdU, Roche, Basel, Switzerland). In brief, cells were incubated with 100 μl of the test compounds for 0, 24, 48 and 72 h. After 8, 32, or 56 h of incubation, cells were additionally treated with BrdU labeling solution for the last 16 h. The culture medium was removed, cells were fixed, and DNA was denatured. Afterwards, cells were incubated with Anti-BrdU-POD solution for 90 min, antibody conjugate was removed by flicking off and wells were rinsed with three washing cycles (PBS). Immune complexes were detected by means of TMB substrate (3,3,5,5-Tetramethylbenzidine Liquid Substarte) for 15 min and quantified by measuring the absorbance at 405 nm and 490 nm. All tests were performed in duplicates with eight wells per treatment group and repeated three times.

Results
Effects of ketamine and s-ketamine. PaTu8988t pancreatic cancer cells were stimulated with 5 μM ketamine or 5 μM sketamine for 0, 24, 48 and 72 h. Cell proliferation was significantly decreased after 48 h stimulation with ketamine and s-ketamine ( Figure 1a).
The protein expression of NFATc2 and Sp1 in PaTu8988t pancreatic carcinoma cells was assayed by western blotting. Cells treated with FCS showed presence of NFATc2 in the nucleus. Twenty-four and 48 h stimulation with 5μM ketamine or 5 μM s-ketamine reduced the expression levels of NFATc2 in the nucleus of pancreatic carcinoma cells. Concurrently, stimulation with ketamine increased the expression of NFATc2 in the cytoplasm. After 72 h stimulation, expression of NFATc2 in the cell nucleus was significantly increased again. The expression levels of Sp1 remained unchanged, and Lamin B and β-actin served as a loading control (Figure 1b). Effect of ropivacaine, lidocaine and sufentanil. PaTu8988t pancreatic cancer cells were stimulated with 5 μM ropivacaine, 1.5 nM sufentanil, 5 μM ropivacaine, 1.5 nM sufentanil or 10 μM lidocaine for 0, 24, 48 and 72 h each (Figure 3a, 3b, 3c). Stimulation with 10 μM lidocaine for 48 h resulted in a slight but statistically significant decrease in cell proliferation of PaTu8988t cell (Figure 3a). Stimulation for 0, 24 or 72 h or use of the other test substances did not change the proliferation rate in comparison to untreated control (Figure 3a).
Western blot analysis did not yield any effects at the protein levels of NFATc2 and Sp1 after stimulation for 0, 24, 48 or 72 h. Furthermore, subcellular localization of NFATc2 remained unchanged. Columns 3 and 4 depict the loading control (Figure 3b and c).

Discussion
Medications given in the context of anesthesia and postoperative pain therapy of pancreatic carcinoma, such as ketamine, s-ketamine, metamizole, and paracetamol but also sufentanil, ropicavaine, and lidocaine, exert different effects and their administration requires careful consideration. The purpose of this study was to examine the effect of clinically relevant drug concentrations to facilitate the transfer of experimental data to clinical practice.
As an NMDA receptor antagonist, ketamine in narcotic dosages causes profound analgesia; thus, this drug is not only successfully used for the management of cancer pain and opioid-refractory pain but also in preventive pain therapy (12,13). Plasma concentrations achievable in clinical settings range between 0.1 and 0.42 μg/ml (≈2.3-9.5 μM) (14). The main impact of ketamine and s-ketamine is based on the non-competitive blockade of the NMDA receptor complex. In the process, ketamine as well as its racemate sketamine bind to the binding site of phencyclidine (PCP) inside the NMDA channel, thus inhibiting the effect of NMDA antagonists (15). This process results in the reduction of the intracellular calcium concentration and in the inactivation of Ca2+-dependent cytosolic guanylate cyclase (16). Several studies have described the influx of calcium into the cell as a key trigger or regulator of cellular processes relevant to tumor progression including proliferation and apoptosis (17). Interestingly, 24h and 48h stimulation with ketamine and s-ketamine initially reduced the levels of NFAT transcription factors in the nucleus of pancreatic carcinoma cells PaTu8988t; at the same time, stimulation with ketamine increased the levels of NFATc2 in the cytoplasm.
In dormant inactive cells, NFATc2 proteins are present in the cytoplasm in a phosphorylated form. The proteins have only a low affinity for DNA (18) and are activated by stimulation with FCS. The resulting intracellular increase in calcium activates the protein serine/threonine phosphatase calcineurin, which can thus bind to the PxIxIT motifs located at the N-terminal of NFAT proteins. As a result, NFAT proteins are dephosphorylated on 13 serine residues (19). Due to the resulting conformational change, NFAT proteins are translocated into the nucleus and bind to specific DNA- binding sequences (GGAAA) (20). NFAT proteins eventually interact with other transcription factors and exert their carcinogenic effect (21). Both 24 and 48 h stimulation with ketamine or s-ketamine seem to be able to inhibit this protein activation cascade. NFAT localizes into the cytoplasm in a dormant stage, and cell proliferation decreases after stimulation for 48 h.
The pyrazolone derivative metamizole (dipyrone) and the aminophenol derivative paracetamol (acetaminophen) are nonacidic, non-opioid analgesics (22,23). The administration of these drugs is a key element of the WHO's cancer pain ladder (24) and an important part of postoperative analgesia (25). Oral administration of 1g metamizole results in a maximum plasma concentration of 17.3±7.5 mg/l (≈50-75 μM metamizole) and the intravenous injection of 1g paracetamol results in a plasma concentration of 95±36 μM (26,27).
According to the literature, metamizole and paracetamol primarily inhibit cyclooxygenase activity (COX), thus influencing prostaglandin synthesis as the central regulator of inflammation and inhibiting the transformation of arachidonic acid into endoperoxide, the precursor of prostaglandin, thromboxane A2, and prostacyclin (28,29).
In this study, proliferation of PaTu8988t pancreatic carcinoma cells was inhibited by the administration of metamizole. Paracetamol has been shown to have a slight but significant anti-proliferative effect in PaTu8988t and Panc-1 pancreatic carcinoma cells in an earlier study (30). Stimulation with metamizole and paracetamol did not change the levels of expression of NFATc2 or Sp1.
It has recently been shown that COX-2 inhibitors increase Sp1 protein degradation (31). Tolfenaminic acid is a nonsteroidal anti-inflammatory drug (NSAID) that additionally activates the degradation of Sp1, Sp3, and Sp4 and decreases the expression of several Sp regulated growth-promoting, angiogenic, survival, and inflammatory gene products (32). These characteristics are of particular significance given the long existing assumption that Sp1 expression is a key factor in tumor development, growth, and metastasis. In some types of cancers, Sp1 overexpression is associated with poor survival (33). Ropivacaine and lidocaine are amide local anesthetics (34) that block voltage-gated sodium channels of neuronal axons. The local anesthetics bind inside of the inactivated sodium channel, thus impeding the fast sodium influx into the cell that is important for depolarization (35). The conduction of stimuli in the nerve is inhibited, thus stopping the transmission of pain (36). Several studies have shown that plasma concentrations of 0.61-4 μg/ml (≈1.6-10.9 μM) are achievable with ropivacaine and 1-5 μg/ml (≈2.3-11.5 μM) with lidocaine (37,38). In peridural anesthesia, ropivacaine is often combined with the opioid sufentanil (39). As a pure agonist, sufentanil binds to the opioid receptors of the nervous system (40) and has been proven to improve the quality of analgesia. In peridural anesthesia, the addition of opioids to local anesthetics results in a faster onset of effects and reduces the dosage of the individual drugs (41). Plasma concentrations achievable with sufentanil are 0.40±0.14 ng/ml (≈1.5 nM) (42). Similar effects can also be observed following intravenous injections of lidocaine in large abdominal surgical interventions in contrast to singular general anesthesia. The reduction in peri-and postoperative pain significantly decreases the requirement of anesthetics and opioid analgesics (43). In our study, the administration of lidocaine decreased proliferation after 48 h, but ropivacaine, sufentanil, and lidocaine had no effect on the expression of the transcription factors NFATc2 and Sp1 in pancreatic carcinoma cells.

Conclusion
Pancreatic adenocarcinoma is one of the most aggressive cancers. Its oncogenic potential is mainly marked by extremely fast growth trigged by the activation of important signaling cascades during vulnerable phases. Thus, new therapeutic concepts also target the efficient modulation of specific signaling and transcription pathways. A wide variety of inhibitors is being investigated in the context of preclinical studies or is currently being established in clinical practice (34,35). One possible novel therapeutic concept for pancreatic carcinoma cells is the inhibition of the interaction between NFATc2 and Sp1. The extent to which medication influences mechanisms in vulnerable phases of pancreatic carcinoma needs to be investigated in future studies. The basis of novel therapeutic approaches to any disease is detailed knowledge of the carcinogenesis and profound molecular and biological understanding of the mechanisms.

Conflicts of Interest
The Authors declare that they have no competing interests regarding this study.

Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Authors' Contributions
All Authors have made substantial contributions to the conception, design, analysis, and the interpretation of this research article. They have been involved in the critical revision of the manuscript with regard to important intellectual content. All Authors have given their final approval for the version to be published 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.