The present study has demonstrated that oral administration of Fe-Lf improves tamoxifen therapy in a mouse model of basal-like breast cancer, and overcomes tamoxifen-mediated immunosuppression. Orally fed Fe-Lf augmented tamoxifen therapy to delay the appearance of palpable tumors in the breasts of female Balb/c mice, and inhibited their subsequent growth. It augmented tamoxifen-mediated inhibition of the metastasis of tumors to the liver and lung. Oral Fe-Lf increased serum levels of IL-18 and IFN-γ and the numbers of cells expressing IL-18 and IFN-γ in intestinal tissues, and prevented their reduction by tamoxifen. It attenuated the loss of body weight caused by tamoxifen and cancer cachexia.
The 4T1 tumor cell line employed here was derived from the 410.4 cell line obtained from a spontaneously arising mouse mammary epithelial tumor [41, 44], and hence cannot be regarded as a tumor that has been forced to acquire tamoxifen-resistance. It is resistant to the effects of tamoxifen by virtue of the fact that it intrinsically expresses very low levels of ER , and accordingly does not respond to estrogen . Thus, the study describes the impact of co-treatment with Lf and tamoxifen on the development of established ER-ve disease.
The anti-tumor activity of Lf is largely dependent on its ability to stimulate anti-tumor immunity when taken orally, by promoting both innate and adaptive immune responses [33, 45]. Each of the Fe-Lf-induced cytokines IL-18 and IFN-γ might be expected to play a role in Fe-Lf-mediated antitumor immunity. Thus, orally administered Lf was previously reported to exhibit antitumor activity through production of IL-18 in the intestinal mucosa . Iron-saturated bLf was chosen for the current study, as we have previously shown that it has superior antitumor activity compared to native bLf when combined with chemotherapeutic agents . When fed to C57BL6 mice bearing a variety of different tumor types, it increased antitumor cytotoxicity, tumor apoptosis and the infiltration of tumors by leukocytes. It bound to the intestinal epithelium and was preferentially taken up within Peyer’s patches. It increased the production of Th1 and Th2 cytokines within the intestine and tumor, including TNF, IFN-γ, as well as nitric oxide that have been reported to sensitize tumors to chemotherapy. Importantly, it restored both red and white peripheral blood cell numbers depleted by chemotherapy, potentially fortifying the mice against cancer . The presence of iron may have several beneficial effects, including rendering Fe-Lf more resistant to proteolysis as it passes through the gastrointestinal tract , and enhancing lymphocyte function .
Here we further demonstrated that oral ingestion of bovine Fe-Lf leads to increases in the Lf content of macrophages, NK and T cells in the intestinal lamina propria, and B and T cells in Peyer’s patches. An interesting phenomenon was the finding that many of the T and B cells, and NK cells that infiltrated into tumors in response to feeding of Fe-Lf contained high levels of Lf. The most plausible explanation is that these cells are derived from the populations of cells in the intestine that contain high levels of Lf [36, 49].
Reanalysis of the results of the Royal Marsden Hospital study of primary breast cancer prevention  showed that obese women treated with tamoxifen gained significantly less body weight over a 6-year period than obese women given placebo, indicating that tamoxifen can cause weight loss . Tamoxifen has an effect on energy homeostasis in rodents such that it markedly decreases food intake and body weight [42, 43]. Tamoxifen-induced anorexia in rats was associated with fatty acid synthase inhibition in the ventromedial nucleus of the hypothalamus and accumulation of malonyl-CoA . Tamoxifen induces rapid atrophy and metaplasia in mouse stomach . In the present study, body weight loss was observed, particularly in the first week after tamoxifen administration in mice fed the control diet. The Fe-Lf diet attenuated the body weight loss induced by tamoxifen.
The 4T1 model of metastatic breast cancer represents a model of cancer cachexia, which is a serious problem for cancer patients as it physically weakens patients and reduces their response to treatment. Here, we also showed that oral Fe-Lf attenuated cancer cachexia, as evidenced by reduced loss of body weight, and increased weights of gastrocnemius muscle and ovarian adipose tissue in tumor-bearing animals. The anti-tumor activity of Fe-Lf may partly contribute to the ability to inhibit cachexia, as the sizes of primary and metastatic tumors were significantly smaller in Fe-Lf-treated mice, thus reducing energy wasting by tumor cells. Effects of Fe-Lf in preventing fatty acid synthase inhibition and accumulation of malonyl-CoA in the hypothalamus, and stomach atrophy may also contribute, but additional studies will be required to determine their relevance.
Tamoxifen forms DNA adducts in human colon after administration, and may elevate the risk of gastrointestinal cancers . It inhibits the growth of normal human colon epithelial cells . bLf has the potential to improve the overall physiological condition due to its beneficial effects on the gut epithelium, as it has been shown to inhibit chemically-induced carcinogenesis in the colon , and human and bovine Lf stimulate the proliferation and differentiation of crypt cells and enterocytes [56, 57]. It improves the microbial intestinal environment by inhibiting the growth of pathogens and stimulating the establishment of beneficial microflora .
The dose of Fe-Lf used in the present study equates to a readily consumable dose of 2.5 g/day for humans, based on equivalent surface area. The dose of tamoxifen used equates to a human dose of 25 mg every two days, which is slightly less than the 20 mg/day dose given to breast cancer patients. The results indicated that Fe-Lf was slightly superior to tamoxifen in inhibiting the growth of tumors, albeit the difference was not significant. The added effects of tamoxifen and Fe-Lf in inhibiting the growth of ER-ve 4T1 cells can be explained by the chemotherapeutic properties of tamoxifen, and the ability of Fe-Lf to stimulate anti-tumor immunity and overcome tamoxifen-induced immunosuppression.