Main text
Introduction
Nasopharyngeal carcinoma (NPC) is endemic in Southern China (Chang & Adami, 2006). Patients with NPC are uniformly treated with high-dose radiation therapy, with additional (concurrent chemoradiation) or adjunct chemotherapy for locally advanced diseases (Blanchard et al., 2015; Shield et al., 2017). Unfortunately, 30-50% of patients still relapse within 5 years despite this radical intensive treatment (Sun et al., 2014). The main characteristic of this cancer is the high association between Epstein-Barr virus (EBV) infection and NPC risk, especially type III. Moreover, a hallmark of undifferentiated NPC (the commonest histological type in endemic regions) is substantial infiltration of immune cells into the tumor microenvironment (TME).
Consumption of soy is associated with many health benefits, including a modest reduction in the risk of developing certain cancers (Trock, Hilakivi-Clarke & Clarke, 2006; Yan & Spitznagel, 2005). Soy products provide the main sources of soy phytochemicals, including soy isoflavones which comprise predominately genistein, daidzein and glycitein. Idronoxil (IDX, also known as phenoxodiol) is a synthetic derivative of genistein (Joannou, Kelly, Reeder, Waring & Nelson, 1995). Preclinical studies have shown that IDX causes apoptosis in a wide range of cancer cell lines and is 5-20 times more effective than genistein. IDX is also active in murine cancer models which can sensitize certain types of cancer to chemotherapy (Alvero et al., 2008). The activity of IDX is likely related to its inhibition of enzymes including sphingosine kinase and PI3 kinase which are over-expressed in cancer cells and responsible for regulating cell pro-survival mechanisms (Porter, Fairlie, Laczka, Delebecque & Wilkinson, 2020; Yao et al., 2012). Another target of IDX is a protein found on the outer membrane of all cancer cells known as Ecto-Nicotinamide adenine dinucleotide oxidase disulfide-thiol exchanger type 2 (ENOX2) (Morre, Chueh, Yagiz, Balicki, Kim & Morre, 2007). The binding of IDX to respective receptors can induce cell cycle arrest and promote DNA damage in cancer cells (Aguero, Facchinetti, Sheleg & Senderowicz, 2005).
Whilst clinically tested in the past, IDX’s journey was discontinued as a result of its low bioavailability in humans when administered either intravenously or orally, though strategies to overcome this issue are currently being explored. Strikingly, IDX has previously been reported to inhibit the proliferation and migration of human endothelial cells, thereby inhibiting angiogenesis (Gamble et al., 2006). Moreover, IDX can induce a range of interferon (IFN)-stimulated genes in immortalised mouse embryonic fibroblasts (Yagiz, Wu, Kuntz, James Morre & Morre, 2007). These findings illustrate that IDX may be targeting the apoptotic machinery itself. Indeed, one report described activated T cells (Herst, Davis, Neeson, Berridge & Ritchie, 2009) as a target for IDX and Georgaki et al. have also classified IDX as an immunomodulator through its capacity to stimulate the innate arm of the immune system, specifically the CD56+ natural killer (NK) cell population (Georgaki et al., 2009). Despite the augmentation of NK cell activity and T cells proliferation being critical for the treatment of tumors, the exact mechanisms by which IDX controls the TME and cancer progression remain unclear.
Here we determined the IC50 of IDX on NPC cell lines and showed that it could further sensitize the cells to apoptosis and inhibit the proliferation and migration in conjunction with cisplatin (cis) treatment. Our results also demonstrated a purported ability of IDX to re-educate the TME and elicit anti-tumorigenic effects. In this capacity, IDX itself could induce T-cell migration with augmented cytotoxic functions and proliferative capacity. While IDX treatment did not show significant impact alone, combination of both IDX and cis targeting was synergistic in regulating CD8+ T cell expansion. We next developed spheroid cocultures of NPC cells and immune cells to study the infiltration, activation and functions of these cells towards human tumors. Increased immune cell infiltrates in IDX-treated spheroids were enriched for double-positive (DP) CD4+CD8+ cells, mediated by the up-regulated expression of IFNα/CXCL10. Particularly, the infiltrated DP cells demonstrated downregulated expression of both lymphoid node homing and dysfunctional markers, CD62L and PD-1. Thus, IDX kills cancer cells both directly and also indirectly, by evoking antitumor immunity by modulating T-cell infiltration into and migration within tumors. Additionally, IDX may be a tool to gather further insights concerning TME and the poor response of NPC cells to conventional chemotherapy.