Abstract
Background/Aim: To ascertain whether preoperative neo-adjuvant nutritional therapy (NANT) using eicosapentaenoic acid (EPA) supplementation can provoke a rise in blood levels of EPA capable of restricting NF-
B nuclear translocation in resected specimens. Patients and Methods: Patients were allocated to two groups depending on individual preference: Patients in the treatment group received 2 g of EPA daily for two weeks prior to surgery (NANT group, n=18). Patients in the control group had a normal diet (CONT group, n=26). NF-
B translocation rate, in specimens collected, was investigated by histopathology. Five hundred malignant cells were counted, and tissues with 10% or higher NF-
B nuclear translocation were determined to be positive. Results: The EPA blood concentration rose significantly in the NANT group (p<0.01). The positive rate of NF-
B nuclear translocation in cancer cells was 11.1% in the NANT group compared with 50% in the CONT group. This difference was statistically significant (p<0.01). Conclusion: Increased blood concentrations of EPA after preoperative supplementation was associated with suppression of NF-
B nuclear translocation in malignant cells. These results suggest that intake of EPA-containing supplements before surgery can control NF-
B activation and by extension, cancer aggressiveness.
Excessive surgical stress and operative complications may induce a cytokine storm during the perioperative period. Cytokine storm has been shown to enhance cancer metastasis in an experimental model and may contribute to a poorer prognosis, a phenomenon known as “surgical oncotaxis” (1). The mechanism underlying “surgical oncotaxis” is complicated, but the accompanying rise in corticosterone level, increased coagulability, immune-suppression, and raised reactive oxygen species generation were attributed to activation of NF-
B (1).
Eicosapentaenoic acid (EPA) is well known to attenuate the production of pro-inflammatory cytokines (2). We demonstrated similar actions in the esophageal cancer cell line TE-1 and reported a suppressive effect of EPA on IL-6 production (3).
Whereas stress promotes nuclear translocation of NF-
B and induction of pro-inflammatory states in various cells, the intake of EPA before excessive surgical stress has not been investigated with regards to its impact on NF-
B translocation in malignant cells. Therefore, the control of NF-
B activation is quite important in cancer surgery. Here, we investigated the impact of preoperative EPA intake, named as neo-adjuvant nutritional therapy (NANT), on NF-KB nuclear translocation in cells of resected cancer specimens.
Patients and Methods
Patient recruitment and group allocation. Forty-four patients with esophageal cancer, from 2009 through 2013, agreed to participate in this clinical study. Of these, 18 cases agreed to intake the ω3 fatty acid (eicosapentaenoic acid EPA, docosahexaenoic acid DHA)-containing oral nutrition supplement (ONS; Prosure®), 2 packs daily for two weeks prior to surgery (NANT group). Twenty-six cases declined this ONS and were assigned to the control group (CONT group).
The oral nutrition supplement (ONS) Prosure® consisted of the following per bottle; energy 280 kcal, protein 14.6 g, lipids 5.6 g, carbohydrates 42.2 g, fiber 2.1 g, salt 0.6 g, calcium 326 mg, EPA 1.0 g, DHA 0.4 mg, oligosaccharide 2.4 g, L-carnitine 22 mg.
Operative method. Operative methods were selected by tumor status and location. Operative procedures included transthoracic esophagectomy (TTE), video-assisted transthoracic esophagectomy (V-TTE), and the trans-diaphragmatic esophagectomy (TDE). The steroid (methylpredonisolone, 250 mg) was given preoperatively to suppress possibility of cytokine storm occurrence. Enteral feeding was started on postoperative day one via an enteral feeding tube and oral food intake resumed on day seven after inspection of anastomotic condition.
Monitoring of EPA blood concentrations. Three cases were chosen randomly from each group and after patient consent obtained, EPA blood concentrations were measured. EPA levels were measured before EPA intake, prior to surgery and on postoperative days one, three, and seven. Measurement of EPA was conducted by SRL Co., Ltd. (Tokyo, Japan). It was economically feasible to limit sampling to three cases.
Sample collection. Resected specimens were formalin-fixed immediately postoperatively and paraffin embedded prior to tumor sectioning and immunohistochemical staining of NF-
B.
Immunohistochemistry. Formalin-fixed, paraffin embedded tumors were sectioned and 4 μm thick sections were de-waxed in xylene and rehydrated in graded ethanol concentrations. Sections were subjected to heat-induced antigen activation using 0.01 M citrate buffer (pH 6.0) in a pressure cooker for 10 min. After air cooling for 30 min, they were cooled under running water. Slides were treated with 3% hydrogen peroxide/methanol solution for 5 min to block endogenous peroxidase activity. To block non-specific IgG reactions, they were incubated with antibody diluent with background reducing components (Agilent, Santa Clara, CA, USA) for 60 min at room temperature. Slides were incubated with NF-
B p65 (D14E12) XP®Rabbit mAb (1:100, Cell Signaling Technology, Danvers, MA, USA) primary antibody overnight at 4°C. The following day, incubation with the secondary antibody, Dako REAL™ EnVision™ Detection System, Peroxidase/DAB+, and Rabbit/Mouse (Agilent) was performed for 60 min at room temperature, followed by application of diaminobenzidine chromagen for 1 min. Mayer hematoxylin (Muto Pure Chemicals Co., Ltd., Tokyo, Japan) was used as a nuclear stain for 1 min. Slides were dehydrated in ethanol, treated with xylene, and cover slipped. Sections were examined using an IX83 microscope (Olympus, Tokyo, Japan). A densely stained nucleus was deemed to be positive for nuclear translocation (Figure 1). Five hundred cells were observed, and specimens with 10% or more nuclear translocation were determined to be NF-
B positive.
Positive staining for nuclear factor-kappa B in the nuclei of formalin-fixed paraffin embedded tumors (black arrow).
Statistical analysis. Continuous variables were compared using the Mann–Whitney U-test. Categorical variables were compared using the chi-square test or Fisher’s exact probability test. All reported p-values were two-sided, and p-values of <0.05 were defined as statistically significant. Analyses were performed using the JMP software (version 13.2.1 for Windows; SAS Institute Inc., Cary, NC, USA).
Ethical disclosure statement. The protocol for this research project was approved by Kawasaki Medical School and conformed to the Declaration of Helsinki’s provisions (approval number 5525-00).
This study was supported in part by a Research Project Grant 20-4191 from Kawasaki Medical School. Prosure® was offered from Abbott Japan Co., Ltd (Minato-ku, Tokyo, Japan).
Results
NANT and CONT groups consisted of 18 and 26 cases, respectively. Both groups showed no differences by age, sex, American Society of Anesthesiologists-physical status (ASA-PS), and operative method (Table I). Blood loss and complication rates including postoperative anastomotic leakage did not differ between both groups (Table II).
Patient demographics and tumor staging.
Surgical procedures, postoperative complications according to Clavien–Dindo classification.
Prosure® intake showed a clear elevation in EPA blood concentrations. After discontinuation of Prosure®, EPA blood levels gradually decreased, returning to baseline levels 7 days postoperatively (Figure 2).
Blood concentrations of eicosapentaenoic acid (EPA). ■ NANT n=3, ◆ CONT n=3. CONT: Control; NANT: neo-adjuvant nutritional therapy.
NF-
B nuclear translocation rate was 11.1% in the NANT group compared with 50% in the CONT group, demonstrating a statistically significant difference (p<0.01) (Table III).
Immunohistochemical staining of NF-
B.
Discussion
EPA, a polyunsaturated fatty acid derived from fish oils, has been used therapeutically in the management of occlusive arteriosclerosis and dyslipidemia. Furthermore, its antitumor (4) and anti-inflammatory effects (5) are becoming increasingly recognizable.
NF-
B is activated by nuclear translocation through exogenous stimulation, and contributes to cancer cell invasion, angiogenesis, metastasis, and anti-apoptotic activity (6). Additionally in rats, excessive surgical stress related to thoraco-laparotomy induced marked increases in NF-
B positive nuclei even in normal cells of the lung, liver, and spleen (7). Therefore, in cancer-related surgical procedures, prevention of cytokine storm, namely the control of the NF-
B nuclear translocation, is regarded as critically important. We previously reported that EPA modulates IL-6 production and the proliferative ability of an esophageal cancer cell line, TE-1 (6). Furthermore, EPA suppressed the chemotactic ability of TE-1 cells, revealed using EZ-TAXIScan® (8). On the basis of these results, we hypothesized that pre-surgical EPA intake may have an anti-inflammatory effect, thereby reducing cancer metastasis and proliferation through suppression of NF-
B nuclear translocation resulting in the control of “surgical oncotaxis”.
Combined administration of EPA and cisplatin (CDDP) to TE-1 cells, has been shown to lead to cell cycle arrest at either the S phase or G2/M phase, in association with increased apoptosis (9). These results suggested that neoadjuvant chemotherapy plus EPA might reinforce the effect of adjuvant therapy on surgical intervention.
Our data showed a clear rise in blood concentrations of EPA after daily supplementation with 2 g of EPA. Furthermore, concentrations greater than 300 μM in vitro demonstrated a clear antitumor effect (1). In this study, continued EPA intake was required to maintain blood concentrations, given the rapid decrease after cessation. However, preoperative EPA dosing led to a significant suppression of NF-
B nuclear translocation in excised tumor samples.
Our results suggest that intake of EPA-containing supplements before surgery may control NF-
B activation, although, because we did not assess postoperative inflammatory cytokine levels, the clinical impact of preoperative EPA intake on “surgical oncotaxis” is yet unknown. In this study, a randomized controlled study could not be performed because some patients refused EPA supplementation. However, after having explained the significance of cytokine storm prevention and preliminary data demonstrating a beneficial effect of EPA, it is likely a randomized controlled trial may be possible in future.
Acknowledgements
The Authors would like to thank all participants who kindly agreed to provide the data for this study. The Authors also thank Liwen Bianji (Edanz) (www.liwenbianji.cn) for editing the English text of a draft of this manuscript.
Footnotes
Authors’ Contributions
HK, TH, HM designed this study. Data generation and analysis were undertaken by AO and MH. SE, YF and TU prepared the draft manuscript and all Authors contributed to the final manuscript version.
Conflicts of Interest
The Authors declare no conflicts of interest associated with this manuscript.
- Received February 21, 2023.
- Revision received March 6, 2023.
- Accepted March 7, 2023.
- Copyright © 2023 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
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