|Year : 2019 | Volume
| Issue : 1 | Page : 185-191
A pilot study of the impact of Vitamin C supplementation with neoadjuvant chemoradiation on regulators of inflammation and carcinogenesis in esophageal cancer patients
Mohamed M.M Abdel-Latif1, Mawash Babar2, Dermot Kelleher3, John V Reynolds2
1 Department of Clinical Pharmacy, Faculty of Pharmacy, Assiut University, Assiut, Egypt; Department of Clinical Medicine, Trinity Centre for Health Sciences, St. James's Hospital and Trinity College Dublin, Dublin, Ireland
2 Department of Clinical Surgery, Trinity Centre for Health Sciences, St. James's Hospital and Trinity College Dublin, Dublin, Ireland
3 Department of Clinical Medicine, Trinity Centre for Health Sciences, St. James's Hospital and Trinity College Dublin, Dublin, Ireland
|Date of Web Publication||13-Mar-2019|
Dr. John V Reynolds
Department of Clinical Surgery, Trinity Centre for Health Sciences, St. James's Hospital and Trinity College Dublin, Dublin 8
Source of Support: None, Conflict of Interest: None
Aims: Vitamin C plays a role in chemoprevention in cancer treatment, and Vitamin C modulates many regulators of inflammation in in vitro studies. The aim of this study is to assess the effect of Vitamin C supplementation with neoadjuvant chemoradiation in esophageal adenocarcinoma on the nuclear factor-kappa B (NF-κB) and associated cytokines.
Materials and Methods: A total of 20 patients undergoing multimodal treatment for esophageal adenocarcinoma were randomized to receive Vitamin C (1000 mg/day) orally for 4 weeks or no supplementation. Pre- and post-Vitamin C endoscopic biopsies were used for the study of NF-κB activity and cytokine analysis.
Results: NF-κB activity along with cytokines was activated in the cancer tissue pretreatment. Down-regulation in NF-κB activity was observed in 25% of cases, two from the Vitamin C arm posttreatment. There was a significant reduction in cytokines levels in the cancer group, and this effect was more pronounced in the Vitamin C group (P < 0.05).
Conclusions: Vitamin C supplementation had a mild protective effect in modulating of regulators of inflammation and carcinogenesis. Further studies with larger numbers of endpoints are needed to evaluate its effect on modulation of chemoradiation responses.
Keywords: Cytokines, esophageal cancer, neoadjuvant chemoradiation, nuclear factor-kappa B, Vitamin C
|How to cite this article:|
Abdel-Latif MM, Babar M, Kelleher D, Reynolds JV. A pilot study of the impact of Vitamin C supplementation with neoadjuvant chemoradiation on regulators of inflammation and carcinogenesis in esophageal cancer patients. J Can Res Ther 2019;15:185-91
|How to cite this URL:|
Abdel-Latif MM, Babar M, Kelleher D, Reynolds JV. A pilot study of the impact of Vitamin C supplementation with neoadjuvant chemoradiation on regulators of inflammation and carcinogenesis in esophageal cancer patients. J Can Res Ther [serial online] 2019 [cited 2020 Aug 6];15:185-91. Available from: http://www.cancerjournal.net/text.asp?2019/15/1/185/230446
| > Introduction|| |
Adenocarcinoma of the esophagus is increasing in incidence considerably in the Western world in recent years., Esophageal cancer is associated with 5-year overall survival rates of approximately 5%. Preoperative treatment in the form of neoadjuvant chemoradiotherapy is increasingly being used for esophageal adenocarcinoma. The current state of cancer chemotherapy is unsatisfactory. In the treatment of esophageal adenocarcinoma, the target for the future is to develop a molecular understanding of the factors that regulate the sensitivity or resistance of a tumor to chemotherapy or radiation therapy. New cancer drugs and combinations continue to be developed to improve the effectiveness of cancer chemotherapies.
In recent years, there is considerable interest in studying the redox mechanisms in carcinogenesis, and the protective role of dietary antioxidants against cancer development, as well as optimizing the effects of cancer therapy., In vitro and animal studies continue to support the hypothesis that antioxidants reduce the risk of gastrointestinal cancers., Vitamin C scavenges oxygen free radicals and reacts with nitrite and converts it to nitrous oxide, thus preventing the formation of such carcinogens., Plasma and tissue levels of Vitamin C were lower in areas of specialized intestinal metaplasia than in squamous mucosa suggesting that oxidative stress may be implicated in the neoplastic progression of Barrett's esophagus. The FINBAR study, a large population-based case–control study from the Northern Ireland Cancer Registry, has confirmed these findings, revealing lower serum levels of the antioxidants including Vitamin C.
Preclinical and clinical studies regenerated interest in ascorbate's role in both enhancing the anticancer effect of chemotherapy and reducing chemotherapy-induced side effects. Epidemiologic evidence suggested that ingestion of ascorbate-rich foods might have an association with reduced cancer incidence.,,, Dietary antioxidants such as Vitamin C, Vitamin E, selenium, and carotenoids are believed to have the potential to reduce tissue and/or DNA damage by scavenging reactive oxygen species and enhancing apoptosis. Thus, a lack of these nutrients may increase cancer risk through oxidative stress, DNA damage, and cell proliferation. Vitamin C was associated with a reduced risk of esophageal adenocarcinoma in most studies.,,, Several studies showed an inverse association between dietary intake of Vitamin C and risk of gastroesophageal reflux disease, Barrett's esophagus and adenocarcinoma of the esophagus.,,,,
The nuclear factor-kappa B (NF-κB) transcription factor has an essential role in inflammation and carcinogenesis., NF-κB is constitutively active in many human malignancies and regulates apoptosis, tumor cell invasion, tumor angiogenesis and proliferation and metastasis, which makes it an attractive therapeutic target in the treatment of human cancers. The inhibition of NF-κB by antioxidants represents an attractive strategy for the treatment of inflammatory diseases. We have reported that NF-κB activation occurs in esophageal cancer cell lines and in patients with Barrett's esophagus and adenocarcinoma. In vitro studies revealed that Vitamin C enhanced the chemosensitivity of esophageal cancer cells by NF-κB inhibition in response to cisplatin and 5-fluorouracil. A study by Kurbacher et al. revealed that Vitamin C potentiates the antineoplastic activity of doxorubicin, cisplatin, and paclitaxel in human breast carcinoma cells in vitro. Vitamin C was also found to augment the chemotherapeutic response of cervical carcinoma HeLa cells by stabilizing p53.
We have recently reported that dietary supplementation of Vitamin C in Barrett's esophagus patients demonstrated a down-regulation of activated NF-κB and cytokines in 8/25 (35%) patients. However, human studies are not supportive as in vitro results, and more clinical work is needed to help clinicians decide on the best treatment strategies. The aim of the present pilot study was to examine the effect of Vitamin C supplementation on NF-κB activation and its associated cytokine profile in patients with esophageal adenocarcinoma undergoing neoadjuvant chemoradiotherapy.
| > Materials and Methods|| |
Patients and tissue collection
The study had the approval of the SJH/AMNCH (St. James's Hospital/Adelaide and Meath incorporating the children's hospital, Tallaght) Institutional Review Board and informed consent was obtained from all patients. Patients diagnosed with esophageal adenocarcinoma and undergoing multimodal treatment were randomized to receive either treatment along with Vitamin C supplementation or multimodal treatment alone. All patients received chemoradiotherapy as previously described. The regimen comprised external beam radiotherapy (40 Gy in 15 fractions over 3 weeks) and chemotherapy given in weeks 1 and 6, consisting of 5-fluorouracil 50 mg/kg for 5 days followed by cisplatin 75 mg/m2 on day 7. Surgery was performed during week 8. The staging was according to the recommendations of the American Joint Committee on Cancer. Samples were snap frozen in liquid nitrogen and stored at −70°C until further analysis. Data concerning the clinical and pathological parameters for all patients were obtained from a detailed database maintained in Microsoft Excel and the Statistical Package for the Social Sciences (SPSS) version 14 (SPSS Inc., Chicago, IL, USA).
Vitamin C supplementation
Esophageal patients undergoing multimodal treatment were randomized to receive either multimodal treatment along with Vitamin C supplementation (n = 9) or multimodal treatment alone (n = 11). This number of patients recruited in both groups was eligible to enroll in the study after strict inclusion and exclusion criteria. Patients received Vitamin C (1000 mg) orally for 4 weeks, and tissue samples were collected before and after supplementation.
Cell isolation and extract preparation
The single cell suspension was prepared from tissue samples of both the diseases tissue and adjacent normal tissue using a modified method of Madrigal et al. In brief, the biopsies were incubated with 2 ml of 120 U/ml collagenase type I (Sigma Aldrich, UK) solution at 37°C tumbling for 1 h. Undigested tissues were removed by centrifugation at a low speed of 200 rpm for 1 min. The supernatants containing the single cells were collected and spun at 2000 rpm. The pellets from this stage were then collected and used for the preparation of total cell and nuclear extracts. Supernatants were obtained and used to measure cytokine expression. Nuclear extracts were prepared from the cells using Active Motif Nuclear Extract Kit (Active Motif, BELGIUM). The protein concentration was determined on nuclear extracts by the method of Bradford.
Colorimetric NFκB assay
The NFκB assay was performed using TransAM Active Motif kits (NF-κB p50 and p65) according to the manufacturer's instructions (Active Motif, BELGIUM). Nuclear extracts from both the esophageal disease tissue (pretreatment and posttreatment) and the adjacent normal tissue as a control were used. In brief, 20 μl of nuclear extract (2 μg/well) diluted in lysis buffer was incubated with 30 μl of binding buffer (4 mM HEPES pH 7.5, 100 mMKCl, 8% glycerol, 5 mM DTT, 0.2% BSA, 0.016% poly d [IC]) in microwells coated with the probes containing the NFκB binding consensus. Thereafter, wells were incubated with 100 μl of primary anti-NFκB for 1 h without agitation. Finally, HRP secondary antibody was added for 1 h, following which the assay was developed, and the absorbance was read on a spectrophotometer at 450 nm with a reference wavelength of 655 nm. We arbitrarily defined ≥10% change in NF-kB levels posttreatment as a primary endpoint.
Total cell extracts and Western blot analysis
Total cell extracts were prepared from both the diseased tissue (pretreatment and posttreatment) and the adjacent normal tissue as a control was used. The cell pellets were collected by centrifugation, and total cell extracts were prepared by lysis the pellets in lysis buffer. Cell extracts (50 μg protein) were resolved by electrophoresis through polyacrylamide gels using 10% separating gels according to the method of Laemmli. Proteins were electrotransferred onto polyvinylidene difluoride membrane, and blots were blocked and incubated overnight at 4°C with IκB-α primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Blots were then incubated with specific anti-horseradish peroxidase-conjugated secondary antibody. Immunodetection was performed using enhanced chemiluminescence. β-actin (Sigma, Poole, Dorset, UK) was used as a loading control.
Quantitative detection of multiple cytokines was performed using Randox Evidence Investigator biochip array according to the manufacturer's instructions. The supernatants prepared from both the diseased tissue (pretreatment and posttreatment) and the adjacent normal tissue as a control were used for cytokine analysis. The Randox Biochip employed a chemiluminescent immunoassay with an array of immobilized antibodies specific to different cytokines. All assays were performed along with Tri-level Cytokine Controls to monitor accuracy and precision and analyzed by comparison with the standard calibration curve. Values were expressed in pg/ml.
Statistical analysis was performed using the Student's t-test, and statistical significance was ascribed to a value of P < 0.05. Mann–Whitney U-test was used for nonparametric analysis.
| > Results|| |
A total of 20 patients were recruited and potentially eligible for the study. The characteristics of the patients with esophageal adenocarcinoma undergoing multimodal treatment are shown in [Table 1]. The median age of the patients was 64.5 years. The ratio of male-to-female was 16:4. The percent of patients with a familial history of cancer was 60%. The association of smoking, alcohol with esophageal adenocarcinoma was 30% and 85%, respectively.
|Table 1: Features of esophageal adenocarcinoma patients undergoing multimodal treatment|
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Vitamin C supplementation and nuclear factor-kappa B expression
NF-κB was activated in the cancer tissue of all the patients' pretreatment. We chose a 10% change in the NF-κB profile as an arbitrary endpoint for analysis. Down-regulation in NF-κBp50 (≥10%) was observed in five patients (5/20; 25%), 2 of them from the Vitamin C arm posttreatment [Figure 1]a. Four patients (4/20; 20%) showed ≤10% down-regulation in NF-κBp50 posttreatment, two from the Vitamin C arm. Similarly, a decrease in NF-κBp65 levels was observed in the same subset of patients (5/20; 25%) in both groups [Figure 1]b. Therefore, down-regulation of activated NF-κB (p50 and p65) was seen in 25% of esophageal cancer patients after neoadjuvant chemoradiation regardless of Vitamin C supplementation.
|Figure 1: Nuclear factor-kappa B status pretreatment and posttreatment with Vitamin C. (a) NF-κBp50 and (b) NF-κBp65 status pretreatment and posttreatment with Vitamin C (9 patients) or without Vitamin C supplementation (11 patients) in esophageal adenocarcinoma patients. Nuclear extracts were prepared pretreatment and posttreatment of esophageal cancer patients and analyzed for nuclear factor-kappa B activity by TransAM Active Motif ELISA-based NF-κBp50 and NF-κBp65 assay. PC: Nuclear extract from HuT78 cells, which contain high levels of constitutive nuclear factor-kappa B, was used as a positive control. (*) denotes to ≥10% down-regulation of NF-κBp50 and NF-κBp65 levels following neoadjuvant chemoradiotherapy treatment with or without Vitamin C supplementation|
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The NF-κB proteins bind to inhibitory IκB-α protein. On activation, IκB-α is phosphorylated and degraded, leading to the translocation of NF-κB to the nucleus. We next examined IκB-α levels in esophageal tumor tissues in patients who showed ≥10% changes in NF-κB levels posttreatment in both groups (five patients; two from the Vitamin C arm). Esophageal tumor specimens had low levels of IκB-α compared to normal esophageal tissues [Figure 2]. On Vitamin C supplementation, IκB-αlevels were raised in the tissues of a subset of patients who showed ≥10% changes NF-κB levels. There was no difference in the mean activated NF-κBp50 and NF-κBp65 expression pretreatment and posttreatment in patients receiving Vitamin C supplementation [Figure 3]. Notably, Vitamin C treatment group (9 patients) collectively revealed a greater trend of NF-κB reduction compared to non-Vitamin C treatment group (11 patients).
|Figure 2: Western blot analysis of IκB-α protein were performed on total cell extracts of patients with ≥10% down-regulation in nuclear factor-kappa B expression (5 patients) of Vitamin C (2 patients) and nonVitamin C (3 patients) groups. Total cell extracts were prepared from tumor specimens and analyzed for IκB-α protein expression. N: normal adjacent esophageal tissue; T: tumor tissue. β-actin was used as a loading control. A representative gel is shown|
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|Figure 3: Mean NF-κBp50 (a) and NF-κBp65 (b) expression in esophageal adenocarcinoma patients pretreatment and post-treatment in Vitamin C (9 patients) and non-Vitamin C (11 patients) groups|
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Vitamin C supplementation and cytokine analysis
Activated cytokines (vascular endothelial growth factor [VEGF], interleukin-8 [IL-8], IL-1α, and IL-1β) were observed in the esophageal cancer tissues relative to NF-κB activation. [Figure 4] shows the mean levels of these cytokines in both groups pre- and post-Vitamin C supplementation. Both treatment arm groups showed a significant reduction in the cytokine profiles, and this effect was more pronounced in the Vitamin C treatment arm (P < 0.05).
|Figure 4: Cytokine (interleukin-8, vascular endothelial growth factor, interleukin-1β and interleukin-1α) levels pretreatment and posttreatment in Vitamin C group (9 patients) and no Vitamin C group (11 patients) (P < 0.05)|
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Correlation of nuclear factor-kappa B status and cytokines expression pretreatment and posttreatment of Vitamin C supplementation in esophageal cancer patients
There was no difference in the clinical cancer staging pretreatment and posttreatment during the period of the pilot study (1-month supplementation of oral Vitamin C). The studies of NF-κB analysis revealed that NF-κBp50 and NF-κBp65 levels were elevated in the cancer tissue of all patients studied (n = 20) pretreatment compared to controls of the adjacent normal tissues. About 45% (9/20) of patients had lower levels of NF-κB posttreatment compared to pretreatment in both treatment groups. No remarkable changes in mean NF-κBp50 and NF-κBp65 levels were observed pretreatment and post-neoadjuvant treatment regardless of Vitamin C in esophageal cancer patients. With regard to the cytokines studied, VEGF, IL-8, IL-1α, and IL-1β were significantly elevated in the cancer tissues compared to controls of the adjacent normal tissues. The cytokines VEGF, IL-8, IL-1α, and IL-1β were significantly (P < 0.05) decreased in both treatment groups. The reduction in the cytokine profile was more pronounced in the Vitamin C treatment arm compared to nonVitamin C treatment group (P < 0.05).
| > Discussion|| |
The use of neoadjuvant chemoradiotherapy before surgery in the treatment of esophageal adenocarcinoma has increased in recent years. Many patients will not respond, however, and the knowledge of molecular factors predicting response or resistance to chemoradiotherapy is required to enhance treatment results. The use of antioxidants in anti-tumor regimens is well-founded. We had previously shown in an in vitro model that Vitamin C down-regulates NF-κB in esophageal cancer cells treated with the anti-cancer drugs cisplatin and 5-flurouracil and the focus of this intervention study was to establish the effects of Vitamin C on NF-κB and related signaling pathways in esophageal adenocarcinoma patients receiving multimodal therapy.
In the present study, we used 1000 mg of Vitamin C orally, and this dose has been reported to produce a sustained plasma level. Constitutive activation of NF-κB and target cytokines was observed in 20 patients undergoing multimodal treatment of esophageal adenocarcinoma patients. NF-κB (p50 and p65) along with pro-inflammatory cytokines (IL-8, VEGF, IL-1α, and IL-1β) were activated pretreatment. Following neoadjuvant treatment, only a subset of the patients showed a modulation of NF-κB and downstream cytokines in tumor tissue, and Vitamin C supplementation had little effect in modulating chemoradiation responses in patients with esophageal adenocarcinoma. It has been reported that similar doses of oral Vitamin C and route of administration have failed to show any benefit. On the other hand, intravenously administrated Vitamin C has shown some benefit. Although a different hypothesis and model, this is discordant with case studies that reveal dietary antioxidant intake to be associated with reduced risk of esophageal adenocarcinoma, or that multivitamins including Vitamin C were associated with a 25% decreased the risk of esophageal cancer.
In general, cancer cells have a defective antioxidant defense system. In addition, low levels of antioxidants, especially Vitamin C, were detected in esophageal adenocarcinoma. The results of in vitro studies revealed that Vitamin C could chemosensitive cancer cells to the actions of the anticancer drugs.,, Second, the amount of Vitamin C supplementation; 1000 mg in our study, is clinically appropriate to reach cells and tissues inconsiderable amount to counteract the increase in reactive oxygen species. Third, the time for the supplementation of the antioxidant; 5 weeks in our study, is suitable to influence the pathology and biology of the tissues of the persons. Fourth, the method of the administration of the antioxidant; oral 1000 mg daily, is an optimum method or the parenteral route will be more efficient in inducing rapid changes and clinical response. Finally, it seems that the interplay between all these factors of antioxidants, neoadjuvant chemotherapy, free radicals, and health status is of great importance in the interpretation of clinical data of dietary supplementation of antioxidants in esophageal adenocarcinoma. Clinically, antioxidant supplements appear to have no beneficial effect on performance and pathologic response, and this could be for many reasons: first, the reduction of the levels of antioxidants in cancer patients to a greater extent which could compromise their actions to demonstrate a notable effect. Therefore, in the case of chemoresistance, high doses of antioxidant is needed to restore the antioxidant defense mechanisms, Taken together, consumption of dietary Vitamin C could be meaningful and play a role as a chemosensitizer in cancer cells.
Activated NF-κB is expressed in the Barrett's and tumor tissue. The activation of NF-κB was found to protect from cell killing, and apoptosis and its inhibition enhanced apoptotic killing by tumor necrosis factor, ionizing radiation and cancer therapy. Consistent with an anti-apoptotic role for NF-κB, the expression of NF-κB was inversely correlated with major or complete pathological responses to neoadjuvant chemotherapy and radiation therapy. The study, however, is negative with respect to hypothesis that oral Vitamin C could impact on tumor biology and response to chemoradiation, notwithstanding, there is some clinical evidence suggests that high-dose intravenous Vitamin C could increase the effectiveness of cancer chemotherapy in vitro and in vivo., Co-administration of Vitamin C and E restored antioxidant status and reduced DNA damage in the VCE supplemented a group of breast cancer patients relative to those of patients receiving chemotherapy alone, suggesting that Vitamin C and E might be useful in protecting against chemotherapy-related side-effects.
| > Conclusion|| |
This pilot translational study based on our existing in vitro data on the effects of Vitamin C on NF-κB and reporting the impact of antioxidants on molecular profile in a prospective and controlled clinical trial did not show significant modulation of this pathway, or clinicopathologic response. We acknowledge that the sample size in our study was small, and this is due to our selection criteria to choose only eligible patients who did not have any supplements and excluding any patient who had multivitamin supplementation or other supplements during carrying out the trial. This small number of patients in our study may explain the small changes of the effects of Vitamin C on inflammatory markers following to neoadjuvant chemoradiation, which requires replication, to prove the beneficial effects of Vitamin C supplement use with chemotherapy as an effective means of augmentation of chemoradiation responses in patients with esophageal cancer. Future studies are required to evaluate the molecular profiles in a larger cohort study, and in particular with parenteral therapy.
Financial support and sponsorship
This work was supported by a grant from Irish Society of Cancer.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]