We sought to determine whether use of the selenomethionine supplementation or the celecoxib treatment given in the Linxian Chemoregression Study altered cytokine levels; whether baseline cytokine levels were associated with baseline dysplasia (t0), dysplasia at the end of the ten month intervention (t10), or a change in dysplasia during this period (t10-t0); and whether changes in cytokine levels, especially those that were associated with active treatment, were associated with changes in dysplasia. Such associations would support the possibility that the effects of treatment on dysplasia that were observed in the parent trial were mediated by their effects on cytokine levels. We measured 22 cytokines simultaneously in each serum sample, and found that selenomethionine supplementation was associated with lower IL-2 levels and celecoxib treatment was associated with lower IL-7 levels and higher IL-13 levels.
None of the baseline cytokine levels were associated with dysplasia status at baseline or after 10 months of intervention, or with the change in dysplasia from baseline to 10 months after intervention. However, an increase in IL-2 or IL-7 between baseline and the end of intervention was associated with an increase in dysplasia grade. These associations were most evident in participants who had mild dysplasia at baseline and were given selenomethionine treatment. Since selenomethionine treatment was associated with a reduction in dysplasia grade in the parent trial and a reduction in IL-2 levels in this study, and changes in IL-2 level paralleled the changes in dysplasia grade, it is plausible that the beneficial effect of selenomethionine treatment on dysplasia observed in the original trial may have been partially mediated by its effects on lowering IL-2. This hypothesis would be consistent with reports of a direct relationship between IL-2 serum levels and disease progression in other tumors (23
). The same hypothesis cannot be made for IL-7, because selenomethionine treatment did not affect its levels in this trial.
There are several possible mechanisms for anticarcinogenic effects of selenium compounds, including immunologic modulation (24
). The potential therapeutic importance of enhancing immunologic function, as may have occurred with the selenomethionine treatment in this trial, stems from the fact that host-derived cytokines can modulate an anti-tumor response and, consequently, can play a beneficial role in suppressing tumor formation. This response has been associated with a reduced frequency of metastasis and improved patient survival in early-stage cancers of several types (25
Of particular relevance to the current findings are several possible mechanisms by which a selenium-induced reduction in IL-2 might protect against tumor development. Selenium inhibits NF-kappa B expression, which can result in reduced expression of NF-kappa B's target genes, including IL-2 (19
). A reduction in serum IL-2 might be especially protective against IL-2 receptor alpha (IL-2Ra)-expressing tumors (26
). These IL-2 promoted tumors include squamous cell carcinoma of the lung and cervical intraepithelial neoplasias, entities with histologic and etiologic similarities to esophageal squamous cell carcinoma (23
). And in the evaluation of cervical intraepithelial neoplasia, IL-2Ra expression was also associated with the grade of squamous dysplasia (27
), suggesting that serum IL-2 may specifically help regulate pre-invasive squamous disease, which was the focus of the Linxian chemoprevention trial.
Selenium-induced reduction in IL-2 might also protect against tumor development by reducing the activity of regulator T-cells (Tregs), which are T-lymphocytes that can impair cancer immunosurveillance. IL-2 activated Tregs protect against autoimmune reactivity but suppress the activation, proliferation, and effector functions of tumor infiltrating lymphocytes (TILs) (28
), which may be particularly relevant in modulating immune responses during the earliest stages of neoplastic disease. Thus, the reduction in IL-2 seen with selenomethionine treatment could lead to reduced Treg activity and enhanced tumor immune surveillance.
In a separate set of studies, we have previously shown that the people of Linxian are highly exposed to polycyclic aromatic hydrocarbons (PAHs), carcinogenic products of incomplete combustion of organic matter (32
). It is interesting to note that PAHs can also have harmful effects on immunologic function. The intracellular receptor for PAHs is the aryl hydrocarbon receptor (AhR), and increased exposure to PAHs stimulates the production of AhR target genes (35
). AhR in turn can be immunotoxic, because it stimulates Tregs, which impair cancer immunosurveillance. Thus PAH exposure and selenium exposure are probably both impacting cancer immunosurveillance in Linxian (in opposite directions) via their effects on Tregs.
IL-7 decreased with celecoxib supplementation and was positively associated with a worsening of preneoplastic disease. IL-7 is a member of the common gamma-chain family of cytokines which are involved in homeostatic proliferation and survival (36
). It has a potential regulatory role affecting the interaction between mucosal lymphocytes and intestinal epithelium (38
), is constitutively expressed by gastrointestinal epithelial cells (39
), and is involved in the development and persistence of chronic inflammatory bowel disease (40
). Thus, the decrease in IL-7 seen with celecoxib supplementation could serve to lessen a potentially adverse chronic inflammatory response that might potentiate cancer formation (41
IL-13, which was increased with ten months of celecoxib supplementation, also plays an important role in various inflammatory diseases, including cancer, asthma, and allergy (42
). In this study, IL-13 was not associated with a change in preneoplastic disease and, thus, the potential clinical significance of its association with celecoxib could not be further assessed.
A significant strength of the current study was the original intervention trial, with its accurate, paired, endoscopic categorization of existing esophageal disease (44
) before and after an intervention that showed a significant beneficial effect. Thus, this study provided an opportunity to explore potential mechanisms to explain the observed changes in preneoplastic disease in the parent intervention trial.
A limitation of the study was the relatively high within-plate and plate-to-plate variability, the reasons for which remain unclear. This variation was similar to that recently reported by others but still deemed successful at yielding high intraclass correlation coefficients that were useful for epidemiologic studies (45
). Another limitation of all cytokine studies is the fact that serum cytokine levels may change rapidly over time in an individual in response to changes in diet and other factors, so that a single serum measurement may not represent long-term cytokine levels. This issue may be less important in a population with chronic nutrient deficiencies such as our study population. Finally, it is also possible that some of the associations that we observed may have occurred by chance, since we examined 22 cytokines and did not adjust for multiple comparisons. Thus our observations must be regarded as exploratory and need independent confirmation.
In conclusion, we explored 22 cytokines and found that selenomethionine treatment decreased serum IL-2 levels and that celecoxib treatment decreased IL-7 and increased IL-13 levels over a 10-month intervention conducted in Linxian, China. Importantly, we found that increased IL-2 and IL-7 levels during the trial were associated with neoplastic progression to increased grades of esophageal squamous dysplasia at the end of the trial. Thus, the favorable effects of selenomethionine treatment on dysplasia that were observed in this trial may have been mediated in part by the reduction in IL-2 levels associated with this treatment.