Lignan transformation by gut bacteria lowers tumor burden in a gnotobiotic rat model of breast cancer

High dietary lignan exposure is implicated in a reduced breast cancer risk in women. The bacterial transformation of plant lignans to enterolignans is thought to be essential for this effect. To provide evidence for this assumption, gnotobiotic rats were colonized with the lignan-converting bacteria Clostridium saccharogumia, Eggerthella lenta, Blautia producta and Lactonifactor longoviformis (LCC rats). Germ-free rats were used as the control. All animals were fed a lignan-rich flaxseed diet and breast cancer was induced with 7,12-dimethylbenz(a)anthracene. The lignan secoisolariciresinol diglucoside was converted into the enterolignans enterodiol and enterolactone in the LCC but not in the germ-free rats. This transformation did not influence cancer incidence at the end of the 13 weeks experimental period but significantly decreased tumor numbers per tumor-bearing rat, tumor size, tumor cell proliferation and increased tumor cell apoptosis in LCC rats. No differences between LCC and control rats were observed in the expression of the genes encoding the estrogen receptors (ERs) a, ERb and G-coupled protein 30. The same was true for IGF-1 and EGFR involved in tumor growth. The activity of selected enzymes involved in the degradation of oxidants in plasma and liver was significantly increased in the LCC rats. However, plasma and liver concentrations of reduced glutathione and malondialdehyde, considered as oxidative stress markers, did not differ between the groups. In conclusion, our results show that the bacterial conversion of plant lignans to enterolignans beneficially influences their anticancer effects (Authors abstract).
Since high circulating estrogen levels have been considered as a risk factor of breast cancer, dietary interventions that modulate the tumor-promoting estrogen effects may be effective in breast cancer prevention, including lignan-rich food because lignans belong to the non-nutritive plant compounds with estrogenic and/or anti-estrogenic properties. Flaxseed is one of the richest sources of the lignan secoisolariciresinol diglucoside, SDG. Intestinal bacteria are capable of converting plant lignans into enterolignans. The transformation of SDG includes the deglucosylation to secoisolariciresinol (SECO) followed by the demethylation and dehydroxylation of SECO to enterodiol (ED). ED is converted to enterolactone (EL) by dehydrogenation. Women with high EL serum levels and high urinary EL excretion have a significantly reduced breast cancer risk. The essential role of bacterial transformation in the gut for the lignan anticancer effects has so far not been demonstrated in vivo. In previous studies, a consortium consisting of the commensal gut bacteria Clostridium saccharogumia (O-deglycosylation), Blautia producta (O-demethylation), Eggerthella lenta (dehydroxylation) and Lactonifactor longoviformis (dehydrogenation) is capable of forming ED and EL from SDG under in vitro conditions and in rats colonized exclusively with these four bacterial species. In this study, the role of bacterial lignan transformation in breast cancer formation and selected cancer-associated parameters in a 7,12-dimethylbenz(a)anthracene (DMBA)-induced cancer model was investigated. The effects of lignan feeding on breast cancer formation in germ-free rats and in rats colonized exclusively with lignan transforming bacteria were also studied to clarify whether the bacterial activation of SDG to ED and EL is crucial for the cancer-preventing effects of dietary lignans.  The results showed that bacteria of the consortium successfully colonized the intestine of the LCC rats and enterolignans were produced from SDG in the LCC but not in the germ-free rats. Fecal EL excretion by LCC rats was comparable with concentrations in premenopausal women. The conversion of ED to EL by L. longoviformis was very efficient under these experimental conditions. Bacterial lignan transformation did not influence tumor incidence in the model but significantly lowered the number of tumors per tumor-bearing animal and the tumor size. The authors concluded from the Ki-67 index and the transferase-mediated nick end-labeling assay that the reduced tumor growth in LCC rats resulted from a lower proliferation in conjunction with a higher apoptotic rate of the tumor cells. The mechanisms possibly involved in the protective role of lignans in cancer development include anti-angiogenic, pro-apoptotic, antiestrogenic and antioxidant. Taken together, the study results do not support an estrogen-dependent mechanism after feeding a 5% flaxseed diet. To study potential effects of lignans on enzyme systems involved in the degradation of oxidants, the specific activities of GST, SOD and CAT in liver homogenates and in the plasma of LCC and germ-free rats. GSH and MDA were used as markers for oxidative stress in these compartments. Compared with the germ-free rats were measured.  LCC rats displayed higher GST, SOD and CAT activities in liver and plasma suggesting a higher capacity to diminish the concentration of oxidants. However, we found no indication for a protective effect of the increased activities of these enzymes: both GSH and MDA concentrations were similar in experimental and control rats. Thus, it may be speculated that even though the enterolignans enhanced the activity of antioxidant enzymes, they did not reduce the systemic oxidative burden.  In summary, the results are not in favor of an estrogen-dependent mechanism as an explanation for the protective effects of enterolignans observed under our experimental conditions. The increased activity of oxidant-degrading enzyme systems in response to enterolignans did not result in a decrease of oxidative stress markers in liver and plasma. The authors conclude that the bacterial transformation of flaxseed-derived lignans is the prerequisite for their beneficial effects in a rat model of breast cancer and that the underlying mechanisms require further investigation (Editors comments).