Role of ZIP8 in regulation of cisplatin sensitivity through Bcl-2

Highlights

• ZIP8 expression is associated with cisplatin toxicity.

• Overexpression of ZIP8 leads to elevated cisplatin sensitivity in MEF cells while ZIP8 knockout induces cisplatin resistance.

• ZIP8 overexpression does not increase cisplatin accumulation in cells and in ZIP8 transgenic mice.

• ZIP8 expression is reversely associated with Bcl-2 expression and cisplatin induced apoptosis.

Abstract

ZIP8 is a membrane transporter that facilitates the uptake of divalent metals (e.g., Zn, Mn, Fe, Cd) and the mineral selenite in anionic form. ZIP8 functionality has been recently reported to regulate cell proliferation, migration and cytoskeleton arrangement, exhibiting an essential role for normal physiology. In this study, we report a ZIP8 role in chemotherapy response. We show ZIP8 regulates cell sensitivity to the anti-cancer drug cisplatin. Overexpression of ZIP8 in mouse embryonic fibroblast (MEF) cells induces cisplatin sensitivity, while knockout of ZIP8 in leukemia HAP1 cells leads to cisplatin resistance. In ZIP8 altered cells and transgenic mice, we show cisplatin is not a direct ZIP8 substrate. Further studies demonstrate that ZIP8 regulates anti-apoptotic protein Bcl-2. ZIP8 overexpression decreases Bcl-2 levels in cultured cells, mice lung and liver tissue while loss of ZIP8 elevates Bcl-2 expression in HAP1 cells and liver tissue. We also observe that ZIP8 overexpression modulates cisplatin-induced cell apoptosis, manifested by the increased protein level of cleaved Caspase-3. Since Bcl-2 elevation was previously discovered to induce cisplatin drug resistance, our results suggest ZIP8 may modulate cisplatin drug responses as well as apoptosis through Bcl-2. We therefore conclude ZIP8 is a new molecule to be involved in cisplatin drug responses and is predicted as a genetic factor to be considered in cisplatin therapy.

Introduction

ZIP8 is a multifunctional membrane transporter that regulates the cellular influx of divalent metals including: Zn²⁺, Fe²⁺, Co²⁺, Mn²⁺ and toxic Cd ²⁺(He et al., 2006; Liu et al., 2008; He et al., 2009; Wang et al., 2011; Wang et al., 2012). ZIP8 transports divalent metal ions through coupling with anionic bicarbonate (HCO₃⁻). ZIP8 is also identified as the major transporter for inorganic selenite, which is a monovalent anion (HSeO₃⁻) at physiological pH. The ZIP8 selenite transport requires both Zn²⁺ and bicarbonate as co-substrates (McDermott et al., 2016).

ZIP8 is an essential gene in mice and is involved in organogenesis (Galvez-Peralta et al., 2012). Aberrant expression of ZIP8 is related to multiple human diseases, such as low HDL-cholesterol, elevated blood pressure, increased body mass index, osteoarthritis, asthma and increased risk of schizophrenia (Speliotes et al., 2010; Waterworth et al., 2010; Kim et al., 2014; Zhang et al., 2016; Costas, 2018; Mak et al., 2018). ZIP8 is also known to be highly responsive to inflammatory stimuli, such as lipid polysaccharide (LPS), tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1β) (Besecker et al., 2008; Liu et al., 2013). Our results show ZIP8 is involved in regulation of cytoskeleton arrangement, proliferation and migration (Geng et al., 2018). ZIP8 deficiency in liver decreases liver selenium and expression of anti-oxidant selenoproteins (Liu et al., 2018). However, as a transporter for multiple functional essential biometals, ZIP8 may regulate many downstream targets and pathways, which remain to be elucidated.

Platinum-based drugs, such as cis-diamminedichloroplatinum(II) (CCCD, cisplatin), have been used to treat various malignancies, including testicular, ovarian, head and neck, colorectal, bladder, and non-small cell lung cancers (Prestayko et al., 1979; Lebwohl and Canetta, 1998; Galanski, 2006). Cisplatin exerts anticancer effects via multiple mechanisms - the best understood mechanism involves the generation of DNA lesions and the induction of DNA damage (Dasari and Tchounwou, 2014). In addition, cisplatin can also elevate oxidative stress and induce cell mitochondrial apoptosis (Dasari and Tchounwou, 2014).

Despite a consistent rate of initial responses, cisplatin resistance forms a therapeutic impediment for its clinical efficacy. Numerous mechanisms are currently reported to be involved in tumor cell resistance to cisplatin. The mechanisms include: 1) decreased intracellular Pt accumulation via drug influx and efflux pumps, e.g., down-regulation of copper transporter receptor 1 (CTR1) (Ishida et al., 2002), organic cation transporters (OCT3) (Guttmann et al., 2018), up-regulation of multidrug resistance-associated protein (Kool et al., 1997), CTR2 (Huang et al., 2014) and Copper-transporting ATPases (ATP7A and ATP7B) (Samimi et al., 2004; Yoshizawa et al., 2007), 2) enhanced DNA repair capability (Furuta et al., 2002), 3) elevated detoxification, e.g., increased glutathione (GSH) level, which conjugates to platinum rendering inactivation of cisplatin (Zhang et al., 2001), Glutathione S-transferase (GST) which catalyzes conjugation of GSH to drug(Townsend et al., 2009), overexpressed superoxide dismutase 1 (SOD1) which protects resistant-cells against apoptosis(Kim et al., 2010) and elevated metallothioneins (MTs) that bind to cisplatin contributing to resistance (Hagrman et al., 2003), 4) altered cell signaling pathway, e.g., activation of MEK/ERK and PI3K/AKT pathway (Fu et al., 2014) and 5) altered apoptotic signaling pathway, e.g., up-regulation of anti-apoptotic proteins and down-regulation of pro-apoptotic proteins (Stewart, 2007). In addition to these mechanisms, proteomic studies have identified additional proteins that are significantly correlated with cisplatin drug resistance from clinical specimen, which involve many previously uncharacterized candidates (Severi et al., 2018).

In the present study, we found that ZIP8 expression is negatively associated with cisplatin resistance; overexpression of ZIP8 increases cisplatin sensitivity while downregulation of ZIP8 leads to cisplatin tolerance. We have excluded the possibility that ZIP8 may serve as a direct cisplatin transporter in ZIP8 overexpressed cells and ZIP8 transgenic mice. Further studies have shown that ZIP8 deficiency induces upregulation of anti-apoptotic Bcl-2, while ZIP8 overexpression downregulates Bcl-2 levels in cells and mouse tissue. Bak, the apoptotic signal, is elevated following ZIP8 overexpression. ZIP8 overexpression triggers cisplatin induced apoptosis, shown by elevation of cleaved Caspase-3. Based on these results, we conclude that ZIP8 overexpression modulates Bcl-2 expression and cisplatin-induced apoptosis, resulting in altered cisplatin drug responses.