Original ArticlesApigenin inhibits the inducible expression of programmed death ligand 1 by human and mouse mammary carcinoma cells
Introduction
Although there have been substantial advances in the screening and treatment of breast cancer (BC), the disease remains the most common cancer in women and has the second highest rate of mortality [1]. The ability to evade detection and elimination by the immune system has been identified as an important factor in cancer progression [2]. In this regard, constitutive or induced expression of programmed death-ligand 1 (PD-L1), also known as B7-H1, is among the immune evasion mechanisms observed in many cancers, including BC, and is typically associated with a poor prognosis [3], [4], [5], [6], [7], [8], [9].
Interferon (IFN)-γ is a potent inducer of PD-L1 on cancer cells [10], acting via MyD88/tumor necrosis factor receptor-associated factor 6/extracellular signal-regulated kinase (ERK)-dependent activation of signal transducer and activator of transcription 1 (STAT1) [11], the phosphoinositide 3-kinase (PI3K)/ERK signaling pathway [12], and the IFN-γ receptor-associated Jak1/Jak2/STAT1 signaling cascade [13]. Since the PD-1 ligand for PD-L1 is expressed by most tumor-infiltrating T cells [14] and IFN-γ is among the cytokines present in the microenvironment of some breast carcinomas and other tumors [15], [16], it is believed that IFN-γ-induced upregulation of PD-L1 on tumor cells suppresses the cell-mediated anti-tumor immune response [17]. For example, tumor cell expression of PD-L1 is correlated with decreased tumor infiltration by CD8+ cytotoxic T cells, reduced T cell activation and interleukin (IL)-2 production, and T cell anergy or apoptosis [18], [19], [20]. In addition, tumor growth in mouse models of cancer is inhibited by antibody (Ab)-mediated blockade of PD-1 or PD-L1, DNA vaccination with the extracellular region of PD-1, genetic elimination of the PD-1 gene, RNA interference, or expression of recombinant soluble PD-1 and PD-L1 proteins [21], [22], [23]. A number of clinical trials have investigated the effects of various drugs that interrupt the PD-1/PD-L1 pathway on cancer progression [24], [25], [26], [27]. Based on strong evidence that the PD-1/PD-L1 pathway is a suitable target for cancer treatment, MPDL3280A (anti-PD-L1 Ab) has been granted breakthrough therapy status for the treatment of bladder cancer [24]. Patients who respond to PD-1/PD-L1 inhibitors have higher numbers of CD8-, PD-1- and PD-L1 expressing cells at the invasive tumor margin and within the tumor itself [27]. Since immune evasion promotes tumor development and progression, agents that have the potential to interfere with the PD-1/PD-L1 pathway may be the basis for new cancer treatments.
Epidemiological studies have established that a diet rich in fruits and vegetables is associated with a lower risk of certain cancers, including BC [28], [29], [30]. Many of the disease-preventing components of fruits and vegetables belong to the flavonoid class of phytochemicals [31]. Apigenin is a bioavailable flavone found in a variety of fruits, vegetables, and beverages, including parsley, onions, grapefruit, oranges, and chamomile tea [32], [33]. Apigenin protects against carcinogen-induced mammary tumor development in rats [34] and inhibits the proliferation of BC cells and other cancer cell types by causing G2/M cell cycle arrest and, at higher concentrations, inducing apoptosis [35], [36], [37], [38], [39]. Importantly, apigenin does not harm normal, healthy cells [35]. However, the immunomodulatory properties of apigenin are less understood. Since apigenin and its metabolite luteolin reduce microglia-mediated inflammation via inhibition of IFN-γ-induced STAT1 phosphorylation [40], we predicted that apigenin would also inhibit STAT1-dependent PD-L1 expression induced in BC cells following exposure to IFN-γ or other cytokines that trigger STAT-1 phosphorylation. The present investigation focuses on the effect of the phytochemical apigenin on inducible and constitutive PD-L1 expression by mammary carcinoma cells. We provide evidence that apigenin-mediated inhibition of IFN-γ-induced PD-L1 expression by BC cells may allow T cells to mount an anti-tumor immune response.
Section snippets
Cell culture
MDA-MB-231 human BC cells were kindly provided by Dr. S. Dover (Memorial University of Newfoundland, St. John's, NL). Dr. P. Lee and Dr. G. Dellaire generously provided MDA-MB-468 and SK-BR-3 human BC cells, respectively (Dalhousie University, Halifax, NS). 4T1 mouse mammary carcinoma cells were provided by Dr. D. Waisman (Dalhousie University). All BC cell lines were authenticated by short tandem repeat profiling performed by ATCC (Manassas, VA). Human mammary epithelial cells (HMEC) were
Apigenin inhibits IFN-γ-induced PD-L1 expression by mammary carcinoma cells
Consistent with findings from earlier studies [8], [10], we observed that IFN-γ-treated MDA-MB-468 BC cells showed a time- and dose-dependent increase in PD-L1 expression (Fig. 1A and Appendix: Supplementary data S1). Optimal induction of PD-L1 was obtained after 24 h treatment with 10 ng/ml IFN-γ, although PD-L1 expression was also increased after 12 h exposure to IFN-γ. As shown in Fig. 1B, pretreatment of MDA-MB-468 cells with 30 µM apigenin, which does not affect the viability of the
Discussion
Apigenin has been extensively studied for its direct inhibitory effects on the growth of cancer cells [35], [36], [37], [38], [39]; however, to our knowledge, the present investigation is the first to provide evidence of a potential role for apigenin in enhancing a T cell-mediated anti-tumor immune response against BC cells by targeting the PD-L1/PD-1 checkpoint, which is considered a promising target for immunotherapy [17]. Many solid tumors use IFN-γ, which is often present within the tumor
Conflict of interest
All authors declare that they have no conflicts of interest.
Acknowledgments
This work was supported by the Dalhousie Medical Research Foundation and the Canadian Breast Cancer Foundation-Atlantic Region. M. Harrison was the recipient of a Frederick Banting and Charles Best Canada Graduate Scholarship, a Canadian Imperial Bank of Commerce Graduate Scholarship in Medical Research and a Trainee Award from The Beatrice Hunter Cancer Research Institute as part of the Terry Fox Strategic Health Research Training Program in Cancer Research at the Canadian Institutes of Health
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These individuals contributed equally to this work.