Journal of Cancer Research and Therapeutics

: 2021  |  Volume : 17  |  Issue : 1  |  Page : 211--217

Effects of Vitamin E on the immune system and tumor growth during radiotherapy

Yeun-Hwa Gu1, Ki-Mun Kang2, Takenori Yamashita3, Jin Ho Song4,  
1 Department of Radiological Science, Faculty of Health Science, Junshin Gakuen University, Fukuoka, Japan
2 Department of Radiation Oncology, Gyeongsang National University School of Medicine and Gyeongsang National University Changwon Hospital, Changwon; Institute of Health Science, Gyeongsang National University, Jinju, Republic of Korea
3 Department of Radiological Science, Faculty of Health Science, Suzuka University of Medical Science, Suzuka, Mie, Japan
4 Department of Radiation Oncology, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea

Correspondence Address:
Jin Ho Song
Department of Radiation Oncology, Seoul St. Mary's Hospital, Catholic University of Korea, 222 Banpo-daero, Seocho-gu, Seoul 06591
Republic of Korea
Yeun-Hwa Gu
Department of Radiological Science, Faculty of Health Science, Junshin Gakuen University, 1-1-1 Chikushigaoka, Minami-ku, Fukuoka 815-8510


Purpose: The purpose of this study is to evaluate the effects of Vitamin E (VE) on the immune system and tumor growth during radiotherapy (RT) in mice model. Methods: C57BL/6NCrSlc mice were randomly distributed in four groups (control, VE alone, RT alone, and VE + RT). In the VE and VE + RT groups, VE was administered in the diet at 500 mg/kg. Radiation was delivered at 2 Gy in a single fraction on the whole body or at 6 Gy in three fractions locally in the RT and VE + RT groups. Changes in leukocytes and T lymphocytes were counted and compared between the four groups. To evaluate the effects on tumor growth, Ehrlich carcinoma cells were injected into the thighs of mice, and tumor volumes and growth inhibition rates were compared. Results: The number of leukocytes was increased in the VE group compared with that in the control group. The magnitude of leukocyte recovery after RT was also increased by VE. This change was affected largely by alterations in lymphocytes and monocytes rather than that in granulocytes. Both CD4+ and CD8+ T lymphocytes were positively affected by VE. The tumor growth was inhibited not only by RT but also by VE alone. If RT was delivered with VE, tumor growth was markedly inhibited. Conclusion: VE could increase the number of leukocytes, primarily lymphocytes, even after RT was delivered. VE also inhibited the tumor growth in addition to RT. Thus, VE may be a useful radioprotective supplement in radiotherapy without inducing tumor growth.

How to cite this article:
Gu YH, Kang KM, Yamashita T, Song JH. Effects of Vitamin E on the immune system and tumor growth during radiotherapy.J Can Res Ther 2021;17:211-217

How to cite this URL:
Gu YH, Kang KM, Yamashita T, Song JH. Effects of Vitamin E on the immune system and tumor growth during radiotherapy. J Can Res Ther [serial online] 2021 [cited 2021 May 19 ];17:211-217
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Full Text


For several decades, various technical improvements have been made in the treatment of cancer. In the field of radiotherapy (RT), intensity-modulated radiation therapy, image-guided radiation therapy, and proton therapy have been developed and are frequently used in clinical settings.[1] These physical techniques were developed to deliver higher doses to the tumor and lower doses to the normal organs. However, some healthy tissue is still expected to be irradiated, which may cause several radiation-induced complications. Therefore, several biologically acting radioprotective compounds have been investigated.[2],[3] Despite these studies, no drugs have been widely accepted or used in the clinic because of toxicity and concerns regarding the influence of the compounds on tumor growth.[2],[3]

Recently, researchers have become interested in cancer immune therapy.[4],[5] The central concept of traditional radiobiology is the cytotoxic effects of radiation on tumor cells due to the induction of DNA double-strand breaks, which leads to apoptosis, necrosis, and autophagy in damaged cells. In addition, a new form of cell death, designated the immunogenic cell death, is also emerging.[5] The representative effect of immunogenic cell death is the “abscopal” effect, a term describing radiotherapy-induced tumor regression in targets away from a targeted lesion. However, this is not observed in lymphocyte-deficient nude mice.[5],[6] Many studies have reported that host immunity and T cells are essential for induction immunogenic cell death.[5]

Vitamin E (VE) is a commonly consumed vitamin because of its well-known antioxidant capacity and many health benefits.[7],[8] VE is a lipid-soluble vitamin class encompassing all tocopherols and tocotrienols,[7] and is abundant in wheat germ oil, sunflower, and safflower oil. Several recent studies have reported that VE exhibits powerful antioxidant properties by scavenging free radicals, which is critical in radiation-induced cell damage.[9] However, our understanding of the effects of VE on the immune system and tumor growth is still limited. Therefore, in this study, we explored the effects of VE on the immune system and tumor growth in conjunction with RT using a mouse model.


Animals and maintenance

All experiments were performed in accordance with the guidelines and regulation, which were approved by the Suzuka University of Medical Science Animal Research Ethics Committee (Ref: 07/625/36). C57BL/6NCrSlc mice (3 weeks of age; body weight: 8–13 g) were purchased from Japan SLC (Shizuoka, Japan) and were housed in a temperature-controlled room maintained at 22°C ± 3°C with a relative humidity of 60% on a 12-h light/dark cycle. Mice were given experimental diets (CE-2) and water ad libitum. All mice were acclimated to laboratory conditions for 1 week before the experiments.

Radiation delivery

Radiation was delivered using an MG226/4.5 X-ray generator (Philips, Inc., Tokyo, Japan), which was developed for animal irradiation. Radiation was delivered with a tube voltage of 200 kV, dose rate of 0.35 Gy/min, and applying an additional filter composed of 0.1 mmCu and 1 mmAl. Radiation was delivered for local thigh irradiation or for whole-body irradiation. The prescribed dose was 6 Gy in 3 fractions with application every-other-day for local thigh irradiation or 2 Gy in a single fraction for whole body irradiation.

For local irradiation, only the right thigh of the mouse was exposed to the round irradiation field, and other sites were completely blocked by a lead shield to reduce the scattered radiation. For whole-body irradiation, the mice were confined to a small plastic dish, and the dish was rotated at a constant speed.

Experiment 1: Changes in leukocyte numbers

For this experiment, four groups of mice (control, VE, RT, and RT + VE groups) were used, with 10 animals in each group. VE was administered through the diet in the VE and RT + VE groups at 500 mg/kg until the end of the experiment. In the control and RT alone groups, purified water was added. On day 14, 2 Gy of whole-body radiation was delivered in a single fraction. Ten μL peripheral blood was obtained from the caudal vein with a capillary tube, and the number of leukocytes were counted using an automated hematology analyzer (Celltac-a MEK-6318, Nihonkouden Co., Ltd. Tokyo). This analyzer differentiates between leukocytes and the WBC subsets by electrical resistance and histogram calculation. The counts of total leukocytes, granulocytes, lymphocytes, and monocytes were recorded for 5 weeks (day 35). We observed the effects of VE during both the deteriorative period and recovery period.

Experiment 2: Changes in T lymphocytes

The experimental groups were same as those in Experiment 1. Same dose of VE was administered in the VE and RT + VE groups, and 2 Gy of whole-body radiation was delivered on day 14. Whole blood was collected from the hearts of anesthetized mice into heparin-containing tubes just before RT delivery (day 14), on day 21, and on day 24. Flow cytometry reagents for lymphocyte subset measurement were added into the lymphocyte suspension in phosphate-buffered saline (PBS), and the mixture was stained for immunofluorescence for about 30 min at 4°C in a dark room. After the reaction, the solution was rinsed three times with PBS, and CD3, CD4, and CD8 subsets were analyzed with a FACS Caliber flow cytometer (Becton Dickinson, San Jose, CA, USA). To analyze T lymphocyte subsets, Multicolor Flowcytometry (FCS) System (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) was employed, and CD3+, CD4+, and CD8+ T lymphocytes in the peripheral blood were counted in three-color flow cytometry using anti-CD3-PE-Cy5.5, anti-CD4-FITC, and anti-CD8-PE.

Experiment 3: Effect on tumor growth

The same four groups were used to evaluate the effects on tumor growth. Ehrlich carcinoma cells (1 × 106 cells) were injected into the right lateral thighs of the mice. Same dose of VE was administered in the diet in the VE and RT + VE groups until the end of the experiment. Beginning of day 14, radiation was delivered locally on the right lateral thigh at 6 Gy in 3 fractions for the RT and RT + VE groups. From day 7, the shortest and longest diameters of the tumors were measured every-other-day using Vernier calipers, and the tumor volume was calculated as follow:

Tumor volume (mm3) = 0.5× (longest diameter) × (shortest diameter)2

On the last day (day 35), the tumor was removed to measure the tumor weight, and the tumor growth inhibition rate was calculated as follow:

Tumor growth inhibition rate (%) = (WC − WT)/WC × 100

where WC is the mean tumor weight of the control group, and WT is the mean tumor weight of the treated group (RT, VE, and VE + RT groups).

Experiment 4: Effects on tumor necrosis factor-alpha levels

To test the effects of VE on tumor necrosis factor-alpha (TNF-α) levels, the same four groups were compared, with five mice in each group. VE was administered in the diet in the VE and RT + VE groups at the same dose level. Radiation was delivered locally as described in Experiment 3 beginning on day 14 in the RT and RT + VE groups. The level of TNF-α was measured 1 week after RT (day 21) by enzyme-linked immunosorbent assays (ELISAs) using a mouse TNF-α ELISA kit (Pierce Biotechnology, Inc.). Fifty μL of the sample and the standard were added to each well and incubated at room temperature (20°C –25°C) for 120 min after covering with a plate cover. Then, 50 ml of biotinylated antibody reagent was added to each well, and samples were incubated at room temperature for 2 h. After washing five times with wash buffer, 100 ml of horseradish peroxidase solution was added, and wells were covered with a plate cover. After incubation at room temperature for 30 min, washing was carried out five times with wash buffer. One hundred microliters of 3,3'-5,5'-tetramethylbenzidine solution was added, and incubation was carried out for >30 min. Incubation time was determined according to the extent of development of blue color. After incubation, 100 ml stop buffer was added. Absorbance at 450 nm was measured using a Labsystems Multiskan MSUV (Dainippon Pharmaceutical Co., Ltd) within 30 min after the addition of stop buffer, and the amount of TNF-α was estimated based on absorbance using a standard calibration curve.


Experiment 1: Changes in leukocyte numbers

Changes in blood leukocytes are shown in [Figure 1] and [Figure 2]. The number of leukocytes was increased significantly in the VE group compared with that in the control group [P < 0.01; [Figure 1]a]. In both irradiated groups (RT and RT + VE group), the number of leukocytes decreased until 1 week after RT and then recovered. However, the magnitude of recovery was greater in the RT + VE group than in the RT alone group [P < 0.05; [Figure 2]a]. As shown in [Figure 1] and [Figure 2], the changes in leukocytes were affected primarily by changes in lymphocytes [Figure 1]b and [Figure 2]b and monocytes [Figure 1]c and [Figure 2]c rather than changes in granulocytes. The number of granulocytes did not show significant differences between the control and VE group [Figure 1]d. Recovery of granulocytes after RT was also not different between the RT and RT + VE groups [Figure 2]d. The number of granulocytes was higher in the RT alone group on days 24 and 36 compared to the RT + VE group.{Figure 1}{Figure 2}

Experiment 2: Changes in T lymphocytes

Since the protective effects of VE were greatest for lymphocytes, we analyzed which type of lymphocytes was increased. The flow cytogram and dot plot of CD4+ and CD8+ cells are shown in [Figure 3], and the numbers of lymphocytes are presented in [Table 1].{Figure 3}{Table 1}

CD4+ cells were increased by 92% in the VE group compared with that in the control group. VE also increased the numbers of CD4+ cells in the irradiated groups by 32% on day 21 (1 week after RT) and by 26% on day 24 (10 days after RT) [Figure 4]a.{Figure 4}

CD8+ cells also showed similar results. The CD8+ cell number in the VE group was higher than that in the control group. In the irradiated groups, the CD8+ cell number in the VE + RT group was twice than that in the RT group on day 21 and on day 24 [Figure 4]b.

Experiment 3: Effect on tumor growth

Tumor volumes in the control and VE groups are shown in [Figure 5]a. Even without any cytotoxic treatment, the tumor volume in the VE group was smaller than the control group after day 23. The difference became larger over time. The tumor volumes in the irradiated groups (RT and RT + VE groups) are shown in [Figure 5]b. The tumor volume in the VE + RT group was also smaller than that in the RT group after day 25, and the difference also became larger over time.{Figure 5}

Tumor weights and tumor growth inhibition rates of each group are shown in [Table 2]. The average tumor weight in the control group was 1.28 g. In the RT group, the average tumor weight was 0.05 g, and in the VE group, it was 0.29 g. When VE and RT were both administered, the average tumor weight was only 0.01 g. The tumor growth inhibition rates were 77.3% in the VE group, 95.8% in the RT group, and 98.8% in the VE + RT group.{Table 2}

Experiment 4: Effects on tumor necrosis factor-alpha levels

TNF-α levels are shown in [Table 3]. The TNF-α level was higher in the VE groups (VE and VE + RT groups) than in the corresponding no − VE groups (control and RT groups). TNF-α levels in the VE group were than twice those in the control group (P < 0.01). TNF-α was also increased as RT was administered and was higher in the VE + RT group than in the RT alone group (P < 0.05).{Table 3}


Hematologic toxicity is the most common complication observed in patients with cancer treated by chemotherapy or RT.[10] Especially, when leukocytopenia develops, the patient can suffer from severe infection or delayed treatment. However, with regard to cancer immune therapy, hematologic toxicity is not just a complication. Several studies have reported that the immune system plays an important role in the prevention and control of cancer. Indeed, recently published studies have highlighted the role of lymphocytes in tumor control.[6],[11] These studies indicate that patients who have intense lymphocytic infiltration around the tumor exhibit improved tumor control and survival. In addition, survival rates were poor in patients with comorbidities that cause immunosuppression or decreased lymphocyte levels.[11],[12] A high neutrophil-lymphocyte ratio is also correlated with poor prognosis in several cancers owing to a higher rate of metastasis and recurrence.[12],[13] Although the mechanism is unclear, it may be associated with increased neutrophil-dependent inflammation and reduced lymphocyte-mediated tumor responses.[13],[14] Therefore, protecting lymphocytes from cytotoxic therapies is important for not only reducing treatment complication but also increasing the probability of cancer cure.

In our study, VE increased the number of leukocytes overall due to an increase in lymphocytes rather than granulocytes. Both CD4+ and CD8+ cells were increased by administration of VE. This effect has also been demonstrated in irradiated mice. Lymphocytes are the most radiosensitive cells in the body, and in vitro studies have suggested that the dose required to kill 50% of the population (D50) of lymphocytes is approximately 1 Gy, whereas the D90 is approximately 2 Gy.[15] In our study, the recovery of overall leukocytes and lymphocytes was faster in the RT + VE group than in the RT alone group. Several studies have reported that for proper activation of host immunity, both CD8+ cytotoxic T cells and CD4+ helper T cells are essential to show immune reactions in tumors.[16],[17]

Although the mechanism through which VE increases the number of lymphocytes is unclear, one explanation is the anti-oxidant effect of VE.[8],[9] Davis et al. reported reduced toxicities in VE administered patients of chemotherapy and RT by reducing free-radicals.[18] Alternatively, some studies have shown that VE can directly stimulate T cell-mediated immunity and increase the levels of several pro-inflammatory cytokines.[8],[18]

One of the limitations of our study is that we did not directly demonstrate, through a dose-response experiment, whether a 500 mg/kg oral dose of VE is optimal. This dose was selected based on the study conducted by Moriguchi et al.[19] They administered different doses of VE in the diet, checked the α-tocopherol levels in the plasma, and evaluated its effects on the immune system. They concluded that at least 500 mg/kg of VE is required to increase the α-tocopherol level in the plasma and to enhance the phagocytic activity of alveolar macrophages.

All materials that can act as radioprotectors must also be assessed for their effects on tumor control because the compounds may also protect the tumor from RT damage, leading to a decrease in local control.[20] Amifostine, the only radioprotective drug approved by the Food and Drug Administration can also have hazardous effects on tumor control, although these findings are still controversial.[3],[21] However, in our study, VE did not negatively affect tumor control; instead, VE showed tumor cell killing when used alone or in combination with RT, with the latter showing additive effects.

The exact mechanism through which VE affects tumor control is unknown. However, based on the finding of increased TNF-α levels and lymphocyte number in the VE group, we assumed that VE could increase the immune reaction against the tumor. TNF-α is a cytokine involved in acute systemic inflammation and is produced mainly by activated macrophages, CD4+ lymphocytes, and natural killer (NK) cells.[22] Other studies, which demonstrate an increased immune response induced by VE, suggest that the antioxidant effect of VE is associated with reduced production of prostaglandin E2 (PGE2). Since PGE2 inhibits lymphocyte proliferation and NK cell activity, it is possible that this is one immunomodulatory mechanism of VE. This could also be the mechanism by which VE induces an increase in TNF-α levels.[8],[23],[24] In most cases, TNF-α induces apoptosis in several types of tumors, although some studies showed that TNF-α can sometimes promote tumor growth by stimulating proliferation and migration.[23],[24] VE also have antiangiogenic effects through down-regulating vascular endothelial growth factor expression.[25] Further studies are, therefore, required to establish the exact role of VE-induced TNF-α, and the exact mechanism of tumor control by VE.


Based on our results, we concluded that VE could increase the number of leukocytes, mainly lymphocytes, and therefore prevent hematologic toxicity in patients treated with RT. VE also suppressed tumor growth in addition to RT, potentially through the increased lymphocyte numbers and TNF-α levels. The study results support the use of VE to reduce tumor growth and protect normal tissues from RT in vivo. More clinical studies are needed to establish VE as a safe and effective compound that may increase cancer treatment.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.


1Bortfeld T, Jeraj R. The physical basis and future of radiation therapy. Br J Radiol 2011;84:485-98.
2Citrin D, Cotrim AP, Hyodo F, Baum BJ, Krishna MC, Mitchell JB. Radioprotectors and mitigators of radiation-induced normal tissue injury. Oncologist 2010;15:360-71.
3Bourhis J, Blanchard P, Maillard E, Brizel DM, Movsas B, Buentzel J, et al. Effect of amifostine on survival among patients treated with radiotherapy: A meta-analysis of individual patient data. J Clin Oncol 2011;29:2590-7.
4Galluzzi L, Kepp O, Kroemer G. Immunogenic cell death in radiation therapy. Oncoimmunology 2013;2:e26536.
5Golden EB, Apetoh L. Radiotherapy and immunogenic cell death. Semin Radiat Oncol 2015;25:11-7.
6Stanton SE, Disis ML. Clinical significance of tumor-infiltrating lymphocytes in breast cancer. J Immunother Cancer 2016;4:59.
7Peh HY, Tan WS, Liao W, Wong WS. Vitamin E therapy beyond cancer: Tocopherol versus tocotrienol. Pharmacol Ther 2016;162:152-69.
8Pekmezci D. Vitamin E and immunity. Vitam Horm 2011;86:179-215.
9Singh PK, Krishnan S. Vitamin E analogs as radiation response modifiers. Evid Based Complement Alternat Med 2015;2015:741301.
10Crawford J, Dale DC, Lyman GH. Chemotherapy-induced neutropenia: Risks, consequences, and new directions for its management. Cancer 2004;100:228-37.
11Grossman SA, Ellsworth S, Campian J, Wild AT, Herman JM, Laheru D, et al. Survival in patients with severe lymphopenia following treatment with radiation and chemotherapy for newly diagnosed solid tumors. J Natl Compr Canc Netw 2015;13:1225-31.
12Sun J, Chen X, Gao P, Song Y, Huang X, Yang Y, et al. Can the neutrophil to lymphocyte ratio be used to determine gastric cancer treatment outcomes? A systematic review and meta-analysis. Dis Markers 2016;2016:7862469.
13Tang L, Li X, Wang B, Luo G, Gu L, Chen L, et al. Prognostic value of neutrophil-to-lymphocyte ratio in localized and advanced prostate cancer: A systematic review and meta-analysis. PLoS One 2016;11:e0153981.
14Yang JJ, Hu ZG, Shi WX, Deng T, He SQ, Yuan SG. Prognostic significance of neutrophil to lymphocyte ratio in pancreatic cancer: A meta-analysis. World J Gastroenterol 2015;21:2807-15.
15Nakamura N, Kusunoki Y, Akiyama M. Radiosensitivity of CD4 or CD8 positive human T-lymphocytes by an in vitro colony formation assay. Radiat Res 1990;123:224-7.
16Farhood B, Najafi M, Mortezaee K. CD8+cytotoxic T lymphocytes in cancer immunotherapy: A review. J Cell Physiol 2019;234:8509-21.
17Liu M, Guo F. Recent updates on cancer immunotherapy. Precis Clin Med 2018;1:65-74.
18Lamson DW, Brignall MS. Antioxidants in cancer therapy; their actions and interactions with oncologic therapies. Altern Med Rev 1999;4:304-29.
19Moriguchi S, Kobayashi N, Kishino Y. High dietary intakes of vitamin E and cellular immune functions in rats. J Nutr 1990;120:1096-102.
20Johnke RM, Sattler JA, Allison RR. Radioprotective agents for radiation therapy: Future trends. Future Oncol 2014;10:2345-57.
21Wasserman TH, Brizel DM, Henke M, Monnier A, Eschwege F, Sauer R, et al. Influence of intravenous amifostine on xerostomia, tumor control, and survival after radiotherapy for head-and- neck cancer: 2-year follow-up of a prospective, randomized, phase III trial. Int J Radiat Oncol Biol Phys 2005;63:985-90.
22Clark IA. How TNF was recognized as a key mechanism of disease. Cytokine Growth Factor Rev 2007;18:335-43.
23Jeng KC, Yang CS, Siu WY, Tsai YS, Liao WJ, Kuo JS. Supplementation with Vitamins C and E enhances cytokine production by peripheral blood mononuclear cells in healthy adults. Am J Clin Nutr 1996;64:960-5.
24Zidi I, Mestiri S, Bartegi A, Amor NB. TNF-alpha and its inhibitors in cancer. Med Oncol 2010;27:185-98.
25Aykin-Burns N, Pathak R, Boerma M, Kim T, Hauer-Jensen M. Utilization of vitamin E analogs to protect normal tissues while enhancing antitumor effects. Semin Radiat Oncol 2019;29:55-61.