CPF was purchased from Shuangma Fine Chemical Co., Ltd. (Nantong, China) with the purity of >99.99%. The THP-1 human macrophage, HepG2 human hepatic carcinoma and HEK293T human embryonic kidney cell lines were obtained from the Shanghai Cell Bank of Type Culture Collection of Chinese Academy of Sciences. The cells were cultured in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS) and 100 U/ml penicillin/streptomycin (both from HyClone, Logan, UT, USA) in a humidified incubator (Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 5% CO₂ at 37°C. Differentiation of THP-1 cells into macrophages was induced with 1 μg/ml PMA (Promega, Madison, WI, USA) in complete medium for 18 h.

Cell viability was determined through the Alamar blue assay according to the manufacturer’s instructions (Thermo Fisher Scientific, Inc.). Briefly, THP-1 and HepG2 cells were seeded in 96-well plates at a concentration of 5.0×10³ cells/well. The cells were then treated with CPF for 24 h, followed by emission detection at 590 nm with excitation at 530 nm.

Cells were harvested and washed twice with cold phosphate-buffered saline (PBS). Total proteins were then extracted with RIPA lysis buffer (Solarbio, Beijing, China) supplemented with protease inhibitor cocktail (Roche Applied Science, San Francisco, CA, USA). Equal amounts of protein lysates (30–50 μg/sample) were subjected to 10% SDS-PAGE and western blot analysis was performed as described previously (24). Antibodies included anti-ferroprotin (1:500; Sigma-Aldrich, St. Louis, MO, USA) and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:1,000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Intensity of bands was assessed with the ImageJ software (http://rsb.info.nih.gov/ij/). GAPDH was used as an internal control for normalization.

Total RNAs were isolated from cells with TRIzol according to the manufacturer’s instructions (Invitrogen Life Technologies, Carlsbad, CA, USA). The first strand of cDNA was synthesized from 2 μg total RNAs using oligo(dT) primer. Primer sequences used for the PCR reaction were: hepcidin, 5′-CCTGACCAGTGGCTCTGTTT-3′ (forward) and 5′-CACATCCCACACTTTGATCG-3′ (reverse); GAPDH, 5′-GAAGGTGAAGGTCGGAGT-3′ (forward) and 5′-GAAGATGGTGATGGGATTTC-3′ (reverse). GAPDH was used as an internal control.

A DNA fragment with ferroportin promoter region was subcloned into the pGL3-Promoter luciferase reporter vector (Promega) to replace the SV40 promoter. The final construct was validated by DNA sequencing (Invitrogen). Then, 0.8 μg target construct plus 80 ng Renilla luciferase construct were co-transfected into HEK293T cells in 24-well plates using Lipofectamine™ 2000 (Invitrogen). After 24 h, the cells were washed with PBS, and assayed for luciferase activity using a dual-luciferase reporter assay system according to the manufacturer’s instructions (Promega). Relative firefly luciferase activities were subsequently normalized to those of Renilla luciferase.

Intracellular LIP was measured as previously described (25). Briefly, the cells were collected and washed following treatments, and subsequently incubated with 0.5 μM calcein acetoxymethyl ester (CA-AM) (Sigma-Aldrich) for 15 min at 37°C. After washing with PBS, the cells were equally divided into two groups. One group was treated with 100 μM desferoxamine (DFO; Sigma-Aldrich) for 1 h at 37°C, and the second group was left untreated. The cells were subjected to FACS analysis (FACSCalibur; BD Biosciences, San Jose, CA, USA), where calcein was excited at 488 nm and measured at 525 nm. Intracellular LIP was obtained through deduction of the cellular fluorescence of DFO-treated cells from that of the untreated cells.

Significance of data between two groups was determined by the two-tailed independent t-test. One-way analysis of variance (ANOVA) was employed to assess the mean differences among groups relative to the control. Experimental data were shown as mean ± standard error (SE). P<0.05 was considered to indicate a statistically significant result.

To examine the potential effects of CPF exposure on iron metabolism, two cardinal types of cells were employed to determine systemic iron flux, macrophages and hepatocytes (16,26,27). The THP-1 human macrophage and HepG2 human hepatic carcinoma cell lines were used. In order to dissect the non-toxicity biological effects from cytotoxicity, in other words, to focus on the biological effects of CPF without exerting significant injuries on cells, concentrations that did not induce significant toxicity to cells were initially obtained. THP-1 and HepG2 cells were treated with different concentrations of CPF for 24 h, and cell viabilities were measured using the Alamar blue assay. No alterations of cell viability were observed when the concentrations of CPF were <80 μM for THP-1 cells, while cell viability was reduced >10% at 80 μM (P<0.05) and subsequently reduced by >30% at 100 μM (P<0.05) (Fig. 1A). With respect to HepG2 cells, no detectable cytotoxicity occurred when the cells were treated with CPF up to 100 μM CPF (P>0.05) (Fig. 1B). Therefore, in the present study, a non-toxic (i.e., sublethal) concentration, 20 μM, was selected for the subsequent experiments. Notably, the medium of 20 μM CPF contained 0.001% DMSO only. Medium with 0.001% DMSO caused no toxicity to cells compared to the complete blank control, and it served as a control in the present study (designated as control or cont in the text and figures).

Ferroportin is the only known iron exporter in mammal animals, and its dysfunction is involved in various iron disorders, such as thalassemias and hemochromatosis (16,27,28). We therefore investigated the effect of CPF treatment on ferroportin concentration in THP-1 macrophages. Following treatment with CPF at 20 μM for 24 h, the ferroportin concentration was significantly induced by >2-fold in CPF-treated cells compared to the untreated cells (Fig. 2A). Ferric ammonium citrate (FAC) (100 μM), as a positive control, was used to increase ferroportin concentration (29) (Fig. 2A). To investigate the mechanism underlying the increase of ferroportin concentration, a transcription inhibitor, actinomycin D, was used to attenuate global mRNA transcription. Actinomycin D treatment alone greatly reduced ferroportin concentration in THP-1 cells, suggesting marked transcriptional inhibition by actinomycin D (Fig. 2B). The increase of ferroportin concentration by CPF was markedly reduced when cells were simultaneously treated with actinomycin D compared to that in cells treated with CPF only (Fig. 2B). The ferroportin concentration in the cells treated with CPF + actinomycin D was greater than that in the cells treated with actinomycin D only (Fig. 2B). These results suggested that CPF promoted the ferroportin level through a transcriptional regulatory mechanism. FAC was used as a positive control to elevate ferroportin, and DFO (100 μM), a negative control (30), was used to impede the ferroportin increase (Fig. 2B).

To substantiate the regulation of ferroportin by CPF at the transcriptional level, we performed the luciferase reporter assay to examine ferroportin expression driven by its own promoter upon CPF (Fig. 3). Following exposure to CFP at 20 μM for 24 h in HEK293T cells, luciferase activity was measured. Compared to the control, CPF treatment increased luciferase activity by ~2.5-fold (P<0.05) (Fig. 3), supporting the transcriptional regulation of ferroportin by CPF. These data together identified a novel effect of CPF on promoting ferroportin expression in macrophages.

To understand the biological effects of CPF on iron metabolism, hepatic hepcidin expression was investigated. As the master regulator of systemic iron homeostasis, hepcidin is predominantly secreted by hepatocytes (31). Additionally, hepcidin directs iron flow by suppressing ferroportin-conducted iron release (32). Hepcidin reduction is associated with increased iron egress and intracellular iron depletion. HepG2 cells were similarly treated with CPF at 20 μM. After 24 h, we found that the hepcidin mRNA level was greatly reduced by CPF (Fig. 4A). The quantified data showed an ~50% reduction of the hepcidin mRNA level in CPF-treated cells relative to the untreated cells (P<0.05) (Fig. 4B). Consistent with previous studies (33), DFO was used as a control to inhibit hepcidin expression (P<0.05) (Fig. 4). These results demonstrated a considerable inhibitory effect of CPF on hepatic hepcidin expression.

The available section of intercellular iron occurred in loosely-bound divalent iron form (i.e., LIP). It was readily and rapidly involved in the synthesis of iron-sulfur clusters, heme and other iron-containing proteins (34,35). LIP was largely determined by the concentrations of iron exporter ferroportin and iron-storage protein ferritin (35). Thus, the intracellular iron concentration was sensitively reflected by the availability and abundance of cellular LIP. To elucidate the consequential effects on iron storage following CPF exposure in THP-1 and HepG2 cells, we assayed LIP in the two types of cells following CPF treatment at 20 μM for 24 h. As shown in Fig. 5A the level of intracellular LIP was markedly reduced by ~3-fold in THP-1 cells in CPF-treated cells relative to the untreated cells (P<0.001), consistent with the significant induction of ferroportin subsequent to CPF treatment (Fig. 2). The level of intracellular LIP was greatly reduced by >2-fold in HepG2 cells treated with CPF compared to the untreated cells (Fig. 5B) (P<0.001), concomitant to hepcidin reduction in these cells following CPF (Fig. 4). These data signified the biological consequences of CPF on iron flow in macrophages and hepatocytes, by modulating ferroportin and hepcidin expression, respectively.