TAM has been a frontline treatment for both early and advanced ER-positive breast cancer in pre- and post-menopausal women [
34‐
36]. A new therapeutic strategy is focus on the combination with other agents that increase efficacy and decrease toxicity of TAM. Here, we evaluate the therapeutic potential of co-treatment of TAM with BD, a natural dietary supplement, in ER-positive human breast cancer. Our results indicate that the combination of 4-OHT and BD or the combination of TAM and BD resulted in the suppression of cell and tumor growth and induction of apoptosis in vitro and in vivo, respectively. Microarray, western blot and immunohistochemistry analyses further demonstrate that the combination treatment regulates expression of proteins involved in the cancer growth and cell death. Importantly, TAM and BD co-treatment significantly suppresses tumor growth in vivo.
The emergence of TAM resistance is almost inevitable, which pose a major clinical problem. Mechanisms may include changes in the expression or function of ER, variation in ER-associated transcription factor recruitment, altered expression of specific microRNAs, and genetic polymorphisms involved in TAM metabolic activity [
37,
38]. Among of them, ER plays the major role in driving resistance [
39]. It has been shown that the enhanced cell proliferation and reduced susceptibility to cell death mediated by ER signaling are in part through the regulation of p21, a key cell cycle break, and Bcl-2, the major anti-apoptotic and pro-survival protein [
40,
41]. Recently, Raha et al. established
de novo and acquired TAM-resistant breast cancer models, which exhibit reduced p21 and elevated Bcl-2 expression [
37]. In clinical studies, loss of p21 is associated with a TAM growth-inducing phenotype and increased Bcl-2 expression is an important phenomenon in metastatic TAM-resistant breast tumors [
42,
43]. Our data demonstrated that 4-OHT/TAM alone had no effect on the expression of p21, but BD and/or combination with 4-OHT/TAM resulted in significant upregulation of p21. Moreover, addition of BD to 4-OHT/TAM leads to enhanced inhibition of Bcl-2. Altered expression of these key proteins may attribute to quercetin, a bioflavonoid presented in BD, which inhibits proliferation and induces apoptosis in ER-positive breast cancer cells via upregulation of p21 and downregulation of Bcl-2 protein expression [
44,
45]. In addition, Oh et al. demonstrate that quercetin suppresses angiogenesis in TAM-resistant breast cancer through inhibition of Pin1 [
46]. Therefore, BD may reverse TAM resistance by enhanced inhibition of Bcl-2 and significant induction of p21, which driving cells into apoptosis. Although we found positive effects in the inhibition of proliferation and induction of apoptosis which was associated with the altered gene expression in MCF-7 cells treated with BD and 4-OH/TAM, these effects were determined in only one ER-positive human breast cancer cells and xenograft model. Therefore, it is possible to expect that other ER-positive human breast cancer cells would also respond to this treatment. Nevertheless, since each cancer cell type has specific and unique genetic make-up, it is plausible that other set of genes would be associated with the anticancer activity of BD and 4-OH/TAM. We have previously demonstrated that therapeutic activity of BD itself was associated with the expression of genes associated with proliferation and metastasis in highly invasive human breast cancer cells MDA-MB-231 and in an animal model of breast-to-lung cancer metastasis [
24,
26]. Another crucial aspect in gene targeting is a temporal gene expression. In our current study, we analyzed gene expression at 6 days because at this time point we detected significant response of BD and 4-OH/TAM in the inhibition of proliferation and induction of apoptosis in MCF-7 cells. Although it is important to evaluate also other time points, in vivo data confirmed the original cell culture data, increased expression of BRAF and p21 and decreased levels of Bcl-2 in tumors after the combined treatment in mouse after 29 days. Indeed, a temporal gene analysis and the use of other human breast cancer cells is necessary for the evaluation of specific molecular targets of BD and their combinations with typical breast cancer drugs. However, these analyses are behind the scope of the present manuscript and will be performed in future studies.
Hormonal therapy using TAM results in menopausal symptoms and serious symptoms not only greatly decrease the quality of life, but also may lead to discontinuation of the treatment [
47,
48]. Hence, non-prescription dietary supplements are often used to relieve TAM-induced side effects. 3,3′-diindolylmethane (DIM), another purified components in BD, is the major product of indole-3-carbinol (I3C) in vivo and has promising activities against ER-positive breast cancer [
49]. Katchamart et al. demonstrated a significant reduction in the
N-oxygenation of TAM catalyzed by liver microsomes in rats fed with DIM, which may actually decrease the toxicity of TAM. Based on the marked shift in the metabolic profiles of TAM, they hypothesize that patients taking TAM in concert with administration of DIM dietary supplements could modulate the risk of developing toxic side effects if there is a similar alteration in humans [
50].
Ganoderma lucidum, a medicinal mushroom in BD, has been used in Asian countries to improve health and promote longevity for centuries [
15]. A pilot clinical trial suggests that spore powder of
G. lucidum may have beneficial effects on cancer-related fatigue and quality of life in ER-positive breast cancer patients undergoing endocrine therapy [
51]. In our present study, mice in the combination group exhibit less fatigue and more energy comparing mice in the TAM group. Definitely, more rigorous experiments are needed to confirm the findings and clarify the specific mechanisms behind them.