Clin Transl Oncol DOI 10.1007/s12094-015-1281-3
RESEARCH ARTICLE
Does vitamin D mediate inhibition of epithelial ovarian cancer by modulating cytokines? S. Mohapatra • A. Saxena • G. Gandhi B. C. Koner • T. Singh • P. C. Ray
•
Received: 6 June 2014 / Accepted: 4 February 2015 Ó Federacio´n de Sociedades Espan˜olas de Oncologı´a (FESEO) 2015
Abstract Objective Vitamin D deficiency is reported to be involved in pathogenesis of ovarian cancer. But the mechanism is yet to be explored. An imbalance between Th1 and Th2 activity play a crucial role in pathogenesis of many cancers. The purpose of the study is to find out the Th1/Th2 status by estimating TNF-a (Th1 marker) and IL4 (Th2 marker) in ovarian cancer cases and controls and to correlate these with serum vitamin D levels. Materials and methods A case–control study with 50 ovarian cancer cases and 50 healthy controls was conducted. The cytokines TNF-a and IL-4 were estimated by ELISA. Serum vitamin D was measured by electrochemiluminescence immunoassay method. Results Median TNF-a levels (12.2 vs 6.2 pg/ml; p value \0.001) were significantly higher in ovarian cancer patients and mean IL-4 levels (2.22 ± 0.51 vs 2.99 ± 0.68 pg/ml; p value \0.05) were significantly lower as compared to those of controls. Levels of TNF-a and IL-4 did not vary significantly with clinical staging, and histological grading. Vitamin D levels were negatively correlated with TNF-a and positively correlated with IL-4. Conclusions Low vitamin D levels promotes Th1 activity increasing TNF-a levels and inhibits Th2 activity decreasing S. Mohapatra (&) A. Saxena B. C. Koner P. C. Ray Department of Biochemistry, Maulana Azad Medical College, Bahadur Shah Zafar Marg, New Delhi 110002, India e-mail: [email protected] G. Gandhi Department of Obstetrics and Gynecology, Maulana Azad Medical College, New Delhi, India T. Singh Department of Pathology, Maulana Azad Medical College, Bahadur Shah Zafar Marg, New Delhi 110002, India
IL-4 levels in ovarian cancer. These low levels of vitamin D may induce pro-inflammatory micro ambience which might contribute to pathogenesis of ovarian cancer. Keywords Epithelial ovarian cancer Vitamin D IL-4 TNF-alpha
Introduction Ovarian cancer is considered the deadliest of all gynecologic cancers. It is the fifth leading cause of cancer-related death among women. Lifetime risk of dying from invasive ovarian cancer is 1 in 72 with estimated 5 year survival rate being 23 % in patients with distant metastasis [1]. Incessant ovulation has been attributed as one of the important etiological factor in the causation of ovarian cancer. The continuous damage and repair process is characterized by release of chemokines, matrix remodeling enzymes, prostaglandins and recruitment of immune cells. This leads to the generation of a pro-inflammatory microenvironment [2]. Chronic inflammation may lead to dysregulated epithelial cell growth and recruitment of further immune cells like T cells. These cells release various cytokines to further augment inflammation creating a vicious cycle of inflammation, chronic injury, metaplasia and dysplasia causing ovarian cancer. Studies have shown that cancers of the colorectal and respiratory system malignancy have been associated with chronic inflammatory states [3, 4]. The Th1 response creates a pro-inflammatory state. An imbalance between T helper cytokines subset 1 and 2 (Th1 and Th2) response has been observed in various cancers. Studies on lung cancer, breast cancer, renal and bladder cancers point towards a gradual shift of Th1 cytokine profile to Th2 cytokine profile during progressive
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tumour growth [5]. A study in ovarian cancer reported higher Th1/Th2 ratio although no clinical correlation could be found [6]. Tumour necrosis factor-alpha (TNF-a) is produced during Th1 response and is considered as a marker of Th1 response [7]. On the other hand, role of Interleukin-4 (IL-4), an archetypical cytokine for Th2 response, has not been explained in ovarian cancer [8]. Vitamin D is found to modulate immune function by suppressing pro-inflammatory Th1 cytokines and by promoting Th2 subtype differentiation. We have already reported in one of our study that serum vitamin D levels were significantly low in ovarian cancer cases as compared to controls [9] which suggest that low vitamin D3 is a risk factor in epithelial ovarian cancer. There is ecological evidence which attributes high incidence of ovarian cancer to lack of sunlight exposure [10]. Whether the vitamin D deficiency state as observed in ovarian cancer is associated with imbalance in Th1/Th2 response hasn’t yet been explored. In this study, we have endeavored to find out the status of Th1 and Th2 response from serum TNF-a and IL4, respectively, and to correlate Th1 and Th2 response with serum vitamin D levels in ovarian cancer patients.
Materials and methods The study was conducted in Department of Biochemistry, Maulana Azad Medical College and Department of Obstetrics and Gynecology, Lok Nayak Hospital, New Delhi between October 2010 and April 2012. Samples were drawn from patients preoperatively with high suspicion of have ovarian cancer based on clinical diagnosis and radiological investigations. Only those cases were selected for final analysis in whom it was histopathologically confirmed as primary cancer defined by code C56 in the tenth revision of the International Statistical Classification of Diseases and Related Health Problems (ICD-10). Those who had undergone preoperative chemotherapy or radiotherapy, or were suffering from parathyroid diseases, chronic kidney disease and autoimmune disease were excluded. Patients taking calcium, vitamin D supplements were also excluded. Controls were healthy females matched with respect to age, menopausal status and month of blood draw. The study group was subjected to structured questionnaire (regarding demographic, medical, lifestyle and reproductive information) and the study was approved by the institutional ethics committee of Maulana Azad Medical College, New Delhi. Cytokine assay The cytokines analyzed in this study were TNF-a as an indicator for Th1 response and IL-4 as a marker for Th2 response by ELISA technique using Biorad microplate
123
reader (Model 680, Bio-Rad Laboratories Headquarters, Hercules, CA, USA). The kit used for measuring cytokines was the Human IL-4 ELISA kit manufactured by GenProbe Diaclone SAS (Besanc¸on Cedex, France) and human TNF-a ELISA kit from the same manufacturer. Samples from matched case–control sets were assayed together in the same batch to reduce technical variability. All tests were carried out in accordance with the manufacturer’s instructions in duplicates. Inter- and intra-batch coefficient of variation remained less than 10 %. Serum vitamin D estimation The serum vitamin D was measured by electrochemiluminescence immunoassay method using ELECSYS Total vitamin D (25-OHD) kit (Roche diagnostics, Mannheim, Germany) adapted to ELECSYS 2010 (Roche diagnostics, Mannheim, Germany) which measures both 25-OHD2 and 25-OHD3. Statistical analysis Independent T test (for parametric data) and Mann–Whitney U test (for nonparametric data) were used to analyse the data. Linear regression was used to establish and quantify relationship between vitamin D levels and cytokine levels. Statistical analysis was done with SPSS version 17.0 (SPSS Inc., Chicago, IL). Level of significance was considered for p values \0.05.
Results The patient and control characteristics with respect to age, parity, menopausal status, family history of cancer, methods of contraception, tumour marker CA-125 levels, serum calcium, phosphorus and vitamin D levels have been summarized in Table 1. The ovarian cancer cases were between the age group of 20–80 years. The group included 19 women of premenopausal age group and 31 post-menopausal women. The patients were recruited irrespective of the stage of disease. There were 3 patients in stage I, 4 patients in stage II, 40 in stage III and 3 patients in stage IV. The median vitamin D levels in cases and in controls were 20.1 ng/ml and in controls were 24.6 ng/ml, respectively. Cytokine marker results Serum TNF-a and IL-4 levels in ovarian cancer cases and controls are presented in Fig. 1. The TNF-a levels in ovarian cancer cases (median 12.2 pg/ml; range 5.1–21.0 pg/ml) was significantly higher than in controls (median 6.2 pg/ml; range 2.0–12.0 pg/ml). On the other
Clin Transl Oncol Table 1 Characteristics of epithelial ovarian cancer cases and controls
Ovarian cases (n = 50)
Controls (n = 50)
47.9 ± 13.35
47.2 ± 12.4
Pre-menopausal
19 (38 %)
20 (40 %)
Post-menopausal
31 (62 %)
30 (60 %)
3
1
0.50a
Nulliparous
4
1
0.18a
1
4
4
0.13a
2–3
29
36
0.14a
C4
13
9
0.23a
History of use of oral contraceptives Tubal ligation
4 11
6 5
0.37a 0.08a
Vitamin D (ng/ml) median (range)
20.1 (6.93, 61.8)
24.6 (7.3, 47)
0.038b
Calcium (mg/dl)
10.74 ± 1.2
10.71 ± 1.1
0.897c
Phosphorus (mg/dl)
4.03 ± 0.7
4.01 ± 0.7
0.874c
CA-125 (IU/ml) median (range)
674 (5,234, 85.63)
11.82 (72.31, 2.77)
0.001b
LDH (IU/L) median (range)
540 (2,034, 118)
227.5 (472, 112)
0.001b
Age (years)
p value
Menopausal status
Subjects with positive family history of breast or ovarian cancer Parity
Results in bold are significant a
Compared by Chi square test
b
Compared by Mann–Whitney test
c
Compared by Student t test
Fig. 1 Box plot showing distribution of TNF-alpha levels in ovarian cancer cases and controls (method of estimation—Sandwich ELISA Gen-Probe Diaclone SAS, all tests done in duplicates)
hand IL-4 levels in ovarian cancer cases (2.22 ± 0.51 pg/ ml) was significantly lower than in controls (2.99 ± 0.68 pg/ml) (Fig. 2). Association of TNF-a and IL-4 with clinical stage of disease and grade of tumor All the patients with ovarian cancer were assessed according to the FIGO staging. Histological grading was classified as well differentiated, moderately differentiated and un-differentiated ovarian cancer. There was no
significant difference between the levels of TNF-a and IL4 with clinical staging (Table 2) or with histological grade of tumor in our patients (Table 3) but there was some weak association (p value = 0.06) between IL-4 and FIGO staging of ovarian cancer. Relationship between vitamin D and serum TNF-a and IL-4 levels Both serum vitamin D and TNF-a did not follow Gaussian distribution. So after logarithmic transformation it was
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Clin Transl Oncol Fig. 2 Bar graph with standard deviation showing distribution of IL-4 levels in ovarian cases and controls. (Method of estimation—Sandwich ELISA Gen-Probe Diaclone SAS, all tests done in duplicates)
Table 2 TNF-a and IL-4 levels with clinical stage of disease Stage I (n = 3)
Stage II (n = 4)
Stage III (n = 40)
Stage IV (n = 3)
p value
TNF-a (pg/ml) median (range)
12.8 (6.4, 21)
10.2 (5.1, 20)
12.4 (6, 21)
8.4 (8, 12)
0.40a
IL-4 (pg/ml) mean ± SD
2.81 ± 0.26
1.78 ± 0.3
2.2 ± 0.5
2.4 ± 0.2
0.061b
a
Compared by Kruskal–Wallis test
b
Compared by Anova comparison of means
Table 3 TNF-a and IL-4 with histological grade of tumour Grade I (n = 6)
Grade II (n = 29)
Grade III (n = 15)
p value
TNF-a (pg/ml) median (range)
15.45 (8, 21)
12.2 (5.1, 21)
12.3 (6, 20)
0.489a
IL-4 (pg/ml) mean ± SD
2.24 ± 0.5
2.24 ± 0.55
2.17 ± 0.5
0.920b
a
Compared by Kruskal–Wallis test
b
Compared by Anova comparison of means
found that serum vitamin D concentrations were negatively correlated with TNF-a (r2 = -0.0923, p = 0.034). Thus, serum 25(OH) D status explained 9.23 % of the variation in TNF-a concentrations (Fig. 3). Serum vitamin D levels were positively correlated with IL-4 (r2 = 0.2277, p \ 0.001). Thus serum 25(OH) D status explained 22.7 % of the variation in IL-4 levels (Fig. 4).
Discussion High vitamin D levels have been found to play a role in decreasing the incidence of colorectal, pancreatic, esophageal and oral/pharyngeal cancers [11, 12]. However, the role of vitamin D deficiency in pathogenesis of cancer has remained controversial [13, 14]. Recently in a case– control study, we have seen that vitamin D levels were lower in ovarian cancer cases as compared to controls [9] suggesting that vitamin D deficiency is a risk factor for ovarian cancer. Vitamin D receptors are present on human
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immune cells (CD4?, CD8?T lymphocytes, APCs) [15]. Macrophages have 1a hydroxylase which produces active calcitriol [16] and vitamin D is observed to modulate T cell function by either causing proliferation or inhibition [17]. These evidences substantiate that vitamin D modulates the immune function. Immune surveillance mechanism protects us from development of cancer. Hence in the present study, we explored if immune disturbance was associated with low vitamin D levels in ovarian cancer. In this study the median TNF-a levels in ovarian cancer cases was found to be higher than controls which is in concordance with other studies where TNF-a has been reported to be statistically higher in ovarian cancer [18]. TNF-a causes induction of chemokine Monocyte Chemoattractant Protein-1 (MCP-1) and Matrix Metallopeptidase-9 (MMP-9) which dysregulated the macrophage and lymphocyte infiltrate in ovarian tumour environment. Etanercept, a TNF- a antagonist was reported to provide biochemical and radiological evidence for stabilizing ovarian cancer progression [19]. On the
Clin Transl Oncol Fig. 3 Effect of vitamin D on TNF-alpha levels in ovarian cancer cases: linear regression analysis
Fig. 4 Effect of vitamin D on IL-4 levels in ovarian cancer cases: linear regression analysis
other hand, recombinant IL-4 levels showed anti-proliferative activity in human colon cancer, head and neck and glioblastoma cell lines [20]. In a study comparing IL-4 levels in benign prostatic hypertrophy and prostate cancer Goldstein et al. [21] postulated that supraphysiological production of IL-4 within the tumour microenvironment promotes its anti- tumour effect but lower levels of IL-4 (probably produced by infiltrating leucocytes and other cells) promote disease progression. IL-4 has also been to shown to inhibit growth in human colon cancer cell lines by activation of signal transducer and activator of transcription 1 (STAT-1) pathway [22]. In our study IL-4 levels were found to be low. In ovarian cancer TNF-a levels has been consistently shown to have increased expression in different studies but reports on IL-4 are scarce [17, 19, 23]. To our knowledge, only one study estimated serum IL-4 levels in ovarian cancer and was found it to be elevated [24]. The findings of the present study indicate an imbalance between Th1 and Th2 response in ovarian cancer and Th1 activity is more predominant than Th2 response. The negative correlation with vitamin D is in agreement with experimental work in animals where vitamin D has been shown to directly
inhibit TNF-a by down regulating transcription of TNF-a and associated proteins [25]. On linear regression analysis vitamin D levels were positively correlated with IL-4 concentrations. Probable mechanism involves increased transcription factor GATA binding protein-3 (GATA-3) and Th2 cell differentiation by vitamin D and thus it modulates IL-4 levels [26]. Hence this indicates that low vitamin D levels, by modulating Th1 and Th2 response might alter TNF-a and IL-4 levels creating a pro-inflammatory state in ovarian cancer. Although the present study design does not establish a cause and effect relationship, if vitamin D deficient state prevails it could affect the delicate balance between Th1 and Th2 cytokines. Comparison of IL-4 in our study with histological grade and types of ovarian cancer yielded no significant differences although IL-4 has shown strong inverse correlation with tumour grade in Non-Hodgkin lymphoma [27]. The weak association between IL-4 and stages of ovarian cancer needs to be further explored in a larger sample size. We observe that decreased vitamin D levels modulates Th1 and Th2 activity and may thereby induce carcinogenic cytokine ambience to precipitate ovarian cancer. This
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relation is worth investigating in a prospective study with a bigger sample size. Acknowledgments This work was partially supported by the Indian Council of Medical Research as part of its financial assistance for MD/MS/DM/MCH thesis grant. Conflict of interest of interest.
The authors declare that they have no conflict
References 1. American Cancer Society. Cancer Facts and Figures, Atlanta: American Cancer Society; http://www.cancer.org/acs/groups/content/@epidemiologysurveilance/ documents/document/acspc-036845.pdf and http://www.cancer.org/acs/groups/ content/@epidemiologysurveilance/documents/document/acspc-037116.pdf. Accessed 8 Feb 2015. 2. Shan W, Liu J. Inflammation -A hidden path to breaking the spell of ovarian cancer. Cell Cycle. 2009;8(19):3107–11. 3. Itzkowitz SH, Yio X. Inflammation and cancer IV. Colorectal cancer in inflammatory bowel disease: the role of inflammation. Am J Physiol Gastrointest Liver Physiol. 2004;287(1):G7–17. 4. Borm PJ, Driscoll K. Particles, inflammation and respiratory tract carcinogenesis. Toxicol Lett. 1996;88:109–13. 5. Shurin MR, Lu L, Kalinski P, Stewart-Akers AM, Lotze MT. Th1/Th2 balance in cancer, transplantation and pregnancy. Springer Semin Immunopathol. 1999;21(3):339–59. 6. Aoki Y, Tsuneki I, Sasaki M, Watanabe M, Sato T, Aida H, et al. Analysis of TH1 and TH2 cells by intracellular cytokine detection with flow cytometry in patients with ovarian cancer. Gynecol Obstet Invest. 2000;50:207–11. 7. Skinner ML, Schlosser RJ, Lathers D, Neal JG, Woodworth BA, Hall J, et al. Innate and adaptive mediators in cystic fibrosis and allergic fungal rhinosinusitis. Am J Rhinol. 2007;21(5):538–41. 8. Seder RA, Paul WE, Davis MM. De St Groth BF. The presence of interleukin-4 during in vitro priming determines the lymphokine producing potential of CD4?T cells from T cell receptor transgenic mice. J Exp Med. 1992;179:1091–8. 9. Mohapatra S, Saxena A, Gandhi G, Koner BC, Ray PC. Vitamin D and VDR gene polymorphism (FokI) in epithelial ovarian cancer in Indian population. J Ovarian Res. 2013;6(1):37. 10. Mohr SB, Gorham ED, Grant WB, Garland FC. Role of Ultraviolet B Irradiance and vitamin D in Prevention of Ovarian Cancer. Am J Prev Med. 2006;31(6):512–4.
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11. Jenab M, Bueno-De-Mesquita HB, Ferrari P, Van Duijnhoven FJ, Norat T, Pischon T, et al. Diagnostic Circulating vitamin D concentration and risk of colorectal cancer in European populations: a nested case–control study. BMJ. 2010;340:B5500. 12. Giovannucci E, Liu Y, Rimm EB, Hollis BW, Fuchs CS, Stampfer MJ, et al. Prospective study of predictors of vitamin D status and cancer incidence and mortality in men. J Natl Cancer Inst. 2006;98:451–9. 13. Helzlsouer KJ. Over view of the cohort consortium vitamin D pooling project of rarer cancers. Am J Epidemiol. 2010;172(1):4–9. 14. Sperati F, Vici P, Maugeri-Sacca` M, Stranges S, Santesso N, Mariani L, et al. Vitamin D supplementation and breast cancer prevention: a systematic review and meta-analysis of randomized clinical trials. PLoS One. 2013;8(7):e69269. 15. Veldman CM, Cantorna MT, DeLuca HF. Expression of 1,25-dihydroxyvitamin D (3) receptor in the immune system. Arch Biochem Biophys. 2000;374:334–8. 16. Reichel H, Koeffler HP, Bishop JE, Norman AW. 25-Hydroxyvitamin d3 metabolism by lipopolysaccharide-stimulated normal human macrophages. J Clin Endocrinol Metab. 1987;64(1):1–9. 17. Boonstra A, Barrat FJ, Crain C, Heath VL, Savelkoul HFJ, O’Garra A. 1a,25Dihydroxyvitamin d3 has a direct effect on naive CD4?T cells to enhance the development of Th2 cells. J Immunol. 2001;167:974–80. 18. Szlosarek PW, GrimshawMJ Kulbe H, Wilson JL, Wilbanks GD, Burke F, et al. Expression and regulation of tumor necrosis factor A in normal and malignant ovarian epithelium. Mol Cancer Ther. 2006;5(2):382–90. 19. Madhusudan S, Muthuramalingam SR, Braybrooke JP. Study of etanercept, a tumor necrosis factor-a inhibitor, in recurrent ovarian cancer. J Clin Oncol. 2005;23:5950–9. 20. Topp MS, Papadimitriou CA, Eitelbach F, Koenigsmann M, Oelmann E, Koehler B, et al. Recombinant human interleukin 4 has antiproliferative activity on human tumor cell lines derived from epithelial and nonepithelial histologies. Cancer Res. 1995;55(10):2173–6. 21. Goldstein R, Hanley C, Morris J, Cahill D, Chandra A, Harper P, et al. Clinical investigation of the role of interleukin-4 and interleukin-13 in the evolution of prostate cancer. Cancers. 2011;3:4281–93. 22. Chang TL, Peng X, Fu XY. Interleukin-4 mediates cell growth inhibition through activation of Stat1. J Biol Chem. 2000;275(14):10212–7. 23. Balkwill F. Tumor necrosis factor or tumor promoting factor? Cytokine Growth Factor Rev. 2002;13:135–41. 24. Clendenen TV, Lundin E, Zeleniuch-Jacquotte A, Koenig KL, Berrino F, Lukanova A, et al. Circulating inflammation markers and risk of epithelial ovarian cancer. Cancer Epidemiol Biomarkers Prev. 2011;20(5):799–810. 25. Zhu Y, Mahon BD, Froicu M, Cantorna MT. Calcium and 1 alpha, 25-dihydroxyvitamin D3 target the TNF-alpha pathway to suppress experimental inflammatory bowel disease. Eur J Immunol. 2005;35(1):217–24. 26. Cantorna MT, Zhu Y, Froicu M, Wittke A. Vitamin D status, 1,25-dihydroxyvitamin D3, and the immune system. Am J Clin Nutr. 2004;80:1717S–20S. 27. Jones EA, Pringle JH, Angel CA, Rees RC. Th1/Th2 cytokine expression and its relationship with tumour growth in B cel non-Hodgkin’s lymphoma (NHL). Leuk Lymphoma. 2002;43:1313–21.
RESEARCH ARTICLE
Does vitamin D mediate inhibition of epithelial ovarian cancer by modulating cytokines? S. Mohapatra • A. Saxena • G. Gandhi B. C. Koner • T. Singh • P. C. Ray
•
Received: 6 June 2014 / Accepted: 4 February 2015 Ó Federacio´n de Sociedades Espan˜olas de Oncologı´a (FESEO) 2015
Abstract Objective Vitamin D deficiency is reported to be involved in pathogenesis of ovarian cancer. But the mechanism is yet to be explored. An imbalance between Th1 and Th2 activity play a crucial role in pathogenesis of many cancers. The purpose of the study is to find out the Th1/Th2 status by estimating TNF-a (Th1 marker) and IL4 (Th2 marker) in ovarian cancer cases and controls and to correlate these with serum vitamin D levels. Materials and methods A case–control study with 50 ovarian cancer cases and 50 healthy controls was conducted. The cytokines TNF-a and IL-4 were estimated by ELISA. Serum vitamin D was measured by electrochemiluminescence immunoassay method. Results Median TNF-a levels (12.2 vs 6.2 pg/ml; p value \0.001) were significantly higher in ovarian cancer patients and mean IL-4 levels (2.22 ± 0.51 vs 2.99 ± 0.68 pg/ml; p value \0.05) were significantly lower as compared to those of controls. Levels of TNF-a and IL-4 did not vary significantly with clinical staging, and histological grading. Vitamin D levels were negatively correlated with TNF-a and positively correlated with IL-4. Conclusions Low vitamin D levels promotes Th1 activity increasing TNF-a levels and inhibits Th2 activity decreasing S. Mohapatra (&) A. Saxena B. C. Koner P. C. Ray Department of Biochemistry, Maulana Azad Medical College, Bahadur Shah Zafar Marg, New Delhi 110002, India e-mail: [email protected] G. Gandhi Department of Obstetrics and Gynecology, Maulana Azad Medical College, New Delhi, India T. Singh Department of Pathology, Maulana Azad Medical College, Bahadur Shah Zafar Marg, New Delhi 110002, India
IL-4 levels in ovarian cancer. These low levels of vitamin D may induce pro-inflammatory micro ambience which might contribute to pathogenesis of ovarian cancer. Keywords Epithelial ovarian cancer Vitamin D IL-4 TNF-alpha
Introduction Ovarian cancer is considered the deadliest of all gynecologic cancers. It is the fifth leading cause of cancer-related death among women. Lifetime risk of dying from invasive ovarian cancer is 1 in 72 with estimated 5 year survival rate being 23 % in patients with distant metastasis [1]. Incessant ovulation has been attributed as one of the important etiological factor in the causation of ovarian cancer. The continuous damage and repair process is characterized by release of chemokines, matrix remodeling enzymes, prostaglandins and recruitment of immune cells. This leads to the generation of a pro-inflammatory microenvironment [2]. Chronic inflammation may lead to dysregulated epithelial cell growth and recruitment of further immune cells like T cells. These cells release various cytokines to further augment inflammation creating a vicious cycle of inflammation, chronic injury, metaplasia and dysplasia causing ovarian cancer. Studies have shown that cancers of the colorectal and respiratory system malignancy have been associated with chronic inflammatory states [3, 4]. The Th1 response creates a pro-inflammatory state. An imbalance between T helper cytokines subset 1 and 2 (Th1 and Th2) response has been observed in various cancers. Studies on lung cancer, breast cancer, renal and bladder cancers point towards a gradual shift of Th1 cytokine profile to Th2 cytokine profile during progressive
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Clin Transl Oncol
tumour growth [5]. A study in ovarian cancer reported higher Th1/Th2 ratio although no clinical correlation could be found [6]. Tumour necrosis factor-alpha (TNF-a) is produced during Th1 response and is considered as a marker of Th1 response [7]. On the other hand, role of Interleukin-4 (IL-4), an archetypical cytokine for Th2 response, has not been explained in ovarian cancer [8]. Vitamin D is found to modulate immune function by suppressing pro-inflammatory Th1 cytokines and by promoting Th2 subtype differentiation. We have already reported in one of our study that serum vitamin D levels were significantly low in ovarian cancer cases as compared to controls [9] which suggest that low vitamin D3 is a risk factor in epithelial ovarian cancer. There is ecological evidence which attributes high incidence of ovarian cancer to lack of sunlight exposure [10]. Whether the vitamin D deficiency state as observed in ovarian cancer is associated with imbalance in Th1/Th2 response hasn’t yet been explored. In this study, we have endeavored to find out the status of Th1 and Th2 response from serum TNF-a and IL4, respectively, and to correlate Th1 and Th2 response with serum vitamin D levels in ovarian cancer patients.
Materials and methods The study was conducted in Department of Biochemistry, Maulana Azad Medical College and Department of Obstetrics and Gynecology, Lok Nayak Hospital, New Delhi between October 2010 and April 2012. Samples were drawn from patients preoperatively with high suspicion of have ovarian cancer based on clinical diagnosis and radiological investigations. Only those cases were selected for final analysis in whom it was histopathologically confirmed as primary cancer defined by code C56 in the tenth revision of the International Statistical Classification of Diseases and Related Health Problems (ICD-10). Those who had undergone preoperative chemotherapy or radiotherapy, or were suffering from parathyroid diseases, chronic kidney disease and autoimmune disease were excluded. Patients taking calcium, vitamin D supplements were also excluded. Controls were healthy females matched with respect to age, menopausal status and month of blood draw. The study group was subjected to structured questionnaire (regarding demographic, medical, lifestyle and reproductive information) and the study was approved by the institutional ethics committee of Maulana Azad Medical College, New Delhi. Cytokine assay The cytokines analyzed in this study were TNF-a as an indicator for Th1 response and IL-4 as a marker for Th2 response by ELISA technique using Biorad microplate
123
reader (Model 680, Bio-Rad Laboratories Headquarters, Hercules, CA, USA). The kit used for measuring cytokines was the Human IL-4 ELISA kit manufactured by GenProbe Diaclone SAS (Besanc¸on Cedex, France) and human TNF-a ELISA kit from the same manufacturer. Samples from matched case–control sets were assayed together in the same batch to reduce technical variability. All tests were carried out in accordance with the manufacturer’s instructions in duplicates. Inter- and intra-batch coefficient of variation remained less than 10 %. Serum vitamin D estimation The serum vitamin D was measured by electrochemiluminescence immunoassay method using ELECSYS Total vitamin D (25-OHD) kit (Roche diagnostics, Mannheim, Germany) adapted to ELECSYS 2010 (Roche diagnostics, Mannheim, Germany) which measures both 25-OHD2 and 25-OHD3. Statistical analysis Independent T test (for parametric data) and Mann–Whitney U test (for nonparametric data) were used to analyse the data. Linear regression was used to establish and quantify relationship between vitamin D levels and cytokine levels. Statistical analysis was done with SPSS version 17.0 (SPSS Inc., Chicago, IL). Level of significance was considered for p values \0.05.
Results The patient and control characteristics with respect to age, parity, menopausal status, family history of cancer, methods of contraception, tumour marker CA-125 levels, serum calcium, phosphorus and vitamin D levels have been summarized in Table 1. The ovarian cancer cases were between the age group of 20–80 years. The group included 19 women of premenopausal age group and 31 post-menopausal women. The patients were recruited irrespective of the stage of disease. There were 3 patients in stage I, 4 patients in stage II, 40 in stage III and 3 patients in stage IV. The median vitamin D levels in cases and in controls were 20.1 ng/ml and in controls were 24.6 ng/ml, respectively. Cytokine marker results Serum TNF-a and IL-4 levels in ovarian cancer cases and controls are presented in Fig. 1. The TNF-a levels in ovarian cancer cases (median 12.2 pg/ml; range 5.1–21.0 pg/ml) was significantly higher than in controls (median 6.2 pg/ml; range 2.0–12.0 pg/ml). On the other
Clin Transl Oncol Table 1 Characteristics of epithelial ovarian cancer cases and controls
Ovarian cases (n = 50)
Controls (n = 50)
47.9 ± 13.35
47.2 ± 12.4
Pre-menopausal
19 (38 %)
20 (40 %)
Post-menopausal
31 (62 %)
30 (60 %)
3
1
0.50a
Nulliparous
4
1
0.18a
1
4
4
0.13a
2–3
29
36
0.14a
C4
13
9
0.23a
History of use of oral contraceptives Tubal ligation
4 11
6 5
0.37a 0.08a
Vitamin D (ng/ml) median (range)
20.1 (6.93, 61.8)
24.6 (7.3, 47)
0.038b
Calcium (mg/dl)
10.74 ± 1.2
10.71 ± 1.1
0.897c
Phosphorus (mg/dl)
4.03 ± 0.7
4.01 ± 0.7
0.874c
CA-125 (IU/ml) median (range)
674 (5,234, 85.63)
11.82 (72.31, 2.77)
0.001b
LDH (IU/L) median (range)
540 (2,034, 118)
227.5 (472, 112)
0.001b
Age (years)
p value
Menopausal status
Subjects with positive family history of breast or ovarian cancer Parity
Results in bold are significant a
Compared by Chi square test
b
Compared by Mann–Whitney test
c
Compared by Student t test
Fig. 1 Box plot showing distribution of TNF-alpha levels in ovarian cancer cases and controls (method of estimation—Sandwich ELISA Gen-Probe Diaclone SAS, all tests done in duplicates)
hand IL-4 levels in ovarian cancer cases (2.22 ± 0.51 pg/ ml) was significantly lower than in controls (2.99 ± 0.68 pg/ml) (Fig. 2). Association of TNF-a and IL-4 with clinical stage of disease and grade of tumor All the patients with ovarian cancer were assessed according to the FIGO staging. Histological grading was classified as well differentiated, moderately differentiated and un-differentiated ovarian cancer. There was no
significant difference between the levels of TNF-a and IL4 with clinical staging (Table 2) or with histological grade of tumor in our patients (Table 3) but there was some weak association (p value = 0.06) between IL-4 and FIGO staging of ovarian cancer. Relationship between vitamin D and serum TNF-a and IL-4 levels Both serum vitamin D and TNF-a did not follow Gaussian distribution. So after logarithmic transformation it was
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Clin Transl Oncol Fig. 2 Bar graph with standard deviation showing distribution of IL-4 levels in ovarian cases and controls. (Method of estimation—Sandwich ELISA Gen-Probe Diaclone SAS, all tests done in duplicates)
Table 2 TNF-a and IL-4 levels with clinical stage of disease Stage I (n = 3)
Stage II (n = 4)
Stage III (n = 40)
Stage IV (n = 3)
p value
TNF-a (pg/ml) median (range)
12.8 (6.4, 21)
10.2 (5.1, 20)
12.4 (6, 21)
8.4 (8, 12)
0.40a
IL-4 (pg/ml) mean ± SD
2.81 ± 0.26
1.78 ± 0.3
2.2 ± 0.5
2.4 ± 0.2
0.061b
a
Compared by Kruskal–Wallis test
b
Compared by Anova comparison of means
Table 3 TNF-a and IL-4 with histological grade of tumour Grade I (n = 6)
Grade II (n = 29)
Grade III (n = 15)
p value
TNF-a (pg/ml) median (range)
15.45 (8, 21)
12.2 (5.1, 21)
12.3 (6, 20)
0.489a
IL-4 (pg/ml) mean ± SD
2.24 ± 0.5
2.24 ± 0.55
2.17 ± 0.5
0.920b
a
Compared by Kruskal–Wallis test
b
Compared by Anova comparison of means
found that serum vitamin D concentrations were negatively correlated with TNF-a (r2 = -0.0923, p = 0.034). Thus, serum 25(OH) D status explained 9.23 % of the variation in TNF-a concentrations (Fig. 3). Serum vitamin D levels were positively correlated with IL-4 (r2 = 0.2277, p \ 0.001). Thus serum 25(OH) D status explained 22.7 % of the variation in IL-4 levels (Fig. 4).
Discussion High vitamin D levels have been found to play a role in decreasing the incidence of colorectal, pancreatic, esophageal and oral/pharyngeal cancers [11, 12]. However, the role of vitamin D deficiency in pathogenesis of cancer has remained controversial [13, 14]. Recently in a case– control study, we have seen that vitamin D levels were lower in ovarian cancer cases as compared to controls [9] suggesting that vitamin D deficiency is a risk factor for ovarian cancer. Vitamin D receptors are present on human
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immune cells (CD4?, CD8?T lymphocytes, APCs) [15]. Macrophages have 1a hydroxylase which produces active calcitriol [16] and vitamin D is observed to modulate T cell function by either causing proliferation or inhibition [17]. These evidences substantiate that vitamin D modulates the immune function. Immune surveillance mechanism protects us from development of cancer. Hence in the present study, we explored if immune disturbance was associated with low vitamin D levels in ovarian cancer. In this study the median TNF-a levels in ovarian cancer cases was found to be higher than controls which is in concordance with other studies where TNF-a has been reported to be statistically higher in ovarian cancer [18]. TNF-a causes induction of chemokine Monocyte Chemoattractant Protein-1 (MCP-1) and Matrix Metallopeptidase-9 (MMP-9) which dysregulated the macrophage and lymphocyte infiltrate in ovarian tumour environment. Etanercept, a TNF- a antagonist was reported to provide biochemical and radiological evidence for stabilizing ovarian cancer progression [19]. On the
Clin Transl Oncol Fig. 3 Effect of vitamin D on TNF-alpha levels in ovarian cancer cases: linear regression analysis
Fig. 4 Effect of vitamin D on IL-4 levels in ovarian cancer cases: linear regression analysis
other hand, recombinant IL-4 levels showed anti-proliferative activity in human colon cancer, head and neck and glioblastoma cell lines [20]. In a study comparing IL-4 levels in benign prostatic hypertrophy and prostate cancer Goldstein et al. [21] postulated that supraphysiological production of IL-4 within the tumour microenvironment promotes its anti- tumour effect but lower levels of IL-4 (probably produced by infiltrating leucocytes and other cells) promote disease progression. IL-4 has also been to shown to inhibit growth in human colon cancer cell lines by activation of signal transducer and activator of transcription 1 (STAT-1) pathway [22]. In our study IL-4 levels were found to be low. In ovarian cancer TNF-a levels has been consistently shown to have increased expression in different studies but reports on IL-4 are scarce [17, 19, 23]. To our knowledge, only one study estimated serum IL-4 levels in ovarian cancer and was found it to be elevated [24]. The findings of the present study indicate an imbalance between Th1 and Th2 response in ovarian cancer and Th1 activity is more predominant than Th2 response. The negative correlation with vitamin D is in agreement with experimental work in animals where vitamin D has been shown to directly
inhibit TNF-a by down regulating transcription of TNF-a and associated proteins [25]. On linear regression analysis vitamin D levels were positively correlated with IL-4 concentrations. Probable mechanism involves increased transcription factor GATA binding protein-3 (GATA-3) and Th2 cell differentiation by vitamin D and thus it modulates IL-4 levels [26]. Hence this indicates that low vitamin D levels, by modulating Th1 and Th2 response might alter TNF-a and IL-4 levels creating a pro-inflammatory state in ovarian cancer. Although the present study design does not establish a cause and effect relationship, if vitamin D deficient state prevails it could affect the delicate balance between Th1 and Th2 cytokines. Comparison of IL-4 in our study with histological grade and types of ovarian cancer yielded no significant differences although IL-4 has shown strong inverse correlation with tumour grade in Non-Hodgkin lymphoma [27]. The weak association between IL-4 and stages of ovarian cancer needs to be further explored in a larger sample size. We observe that decreased vitamin D levels modulates Th1 and Th2 activity and may thereby induce carcinogenic cytokine ambience to precipitate ovarian cancer. This
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Clin Transl Oncol
relation is worth investigating in a prospective study with a bigger sample size. Acknowledgments This work was partially supported by the Indian Council of Medical Research as part of its financial assistance for MD/MS/DM/MCH thesis grant. Conflict of interest of interest.
The authors declare that they have no conflict
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