The Effects of Angelica Sinensis Polysaccharide on Tumor Growth and Iron Metabolism by Regulating Hepcidin in Tumor-Bearing Mice Open Access

Iron is involved in a variety of crucial biological processes including energy generation, oxygen transport, DNA synthesis, and enzymatic processes, and acts as an essential element for the majority of the organisms [1]. Iron overload is a severe condition, and increasing evidence indicated that iron overload may contribute to tumor initiation, behavior and recurrence [2-5]. Hepcidin, a peptide hormone is secreted mainly by the liver and function as a key regulator of iron homeostasis [6]. Previous studies have revealed that increased serum hepcidin was related to multiple cancers, such as breast cancer, myeloma, renal cell carcinoma and prostate cancer [7-10]. Hepcidin is homeostatically regulated by iron and erythropoietic activity as well as inflammatory cytokines [9, 11]. Hepcidin inhibits iron absorption from the duodenum and iron egress from macrophages and hepatocytes through binding and inducing degradation of iron exporter ferroportin [12, 13]. Accumulated evidences suggest that therapeutic regimen targeting the hepcidin-ferroportin axis could have important clinical implications for cancer patients with iron overload.

Angelica sinensis is broadly used as Chinese herbal medicine or food in Asian countries such as China, Japan and Korea [14]. ASP is the major bioactive component extracted from roots of angelica. Recently, studies have demonstrated that ASP has various biological functions, including immunomodulation, anti-tumor activity, and hematopoiesis [15, 16]. It was reported that ASP could regulate hepcidin expression and inhibit tumor growth in H22 tumor-bearing mice [17]. However, whether ASP has the ability to decrease iron burden in tumor-bearing mice and the mechanism of ASP regulation hepcidin expression are still unknown. In this study, we confirmed that ASP could potently regulate hepcidin expression and inhibited tumor growth in mice xenografted with 4T1 and H22 cancer cells. Furthermore, we firstly investigated the role of ASP on decrease iron burden in liver, spleen and grafted tumors in mice and explored the potential mechanism of ASP regulating hepcidin via the Janus-kinase/signal transducer and activators of transcription (JAK/STAT) and bone morphogenetic protein-small mothers against decapentaplegic (BMP-SMAD) signaling pathways.

Human normal liver cell line (L-02), hepatocellular carcinoma cell line (HepG2) and mouse breast cancer cell line (4T1) were purchased from the cell bank of Chinese Academy of Sciences. Mouse hepatocellular carcinoma cell line (H22) was obtained from China Center for Type Culture Collection. All cells were authenticated by short tandem repeat (STR) profiling before receipt and were propagated for less than 6 months after resuscitation. These cells were maintained in DMEM (Hyclone, Logan city, USA) supplemented with 10% fetal bovine serum (FBS) and incubated in a 5% CO₂ humidified atmosphere at 37°C. L-02 and HepG2 cells were treated with ASP in different time or concentration. ASP with > 98% purity was purchased from Ci Yuan Biotechnology Co., Ltd. Shanxi (Xian, China).

Balb/c mice (4-5 weeks old) purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Mice didn’t take any treatment as the blank control group. For in vivo tumor growth assay, xenograft tumors were generated by subcutaneous injection of 2×10⁶ 4T1 cells or H22 cells. Tumor-bearing mice were randomly divided into 3 groups: the control group; the CTX group and the ASP group. After 6 days of tumor inoculation, the CTX group was intraperitoneally injected with cytoxan (CTX, Jiangsu Hengrui Medicine Co., Ltd., Lianyungang, china) at 25 mg/kg once daily for 14 days and the ASP group were intraperitoneally injected with ASP at 100 mg/kg once daily for 14 days. The control group was intraperitoneally injected with the same volume of 0.9% normal saline (NS). Dosages and time were determined according to preliminary experiments [18]. Mice body weight and tumor size were monitored every other days.

At the end of the experiment, all mice were humanely euthanized, and the blood samples were collected into Eppendorf tubes without anticoagulant from the angular vein. After 4h standing at room temperature, serum was obtained after centrifugation at 12000 rpm min^–1 for 10 min, and then the supernatant was collected and stored at -20°C. Liver, spleen and tumor were removed, weighed, rinsed and subjected to following analysis.

All animal experiments were approved by the Use Committee for Animal Care and performed in accordance with institutional guidelines.

Iron concentration in mice serum was assayed by automatic biochemistry analyzer (SIMENS Advia 2400). Iron depositions in liver, spleen, and tumor were determined by atomic absorption spectrophotometer (AA240FS series by Varian Australia Pty Ltd).

Liver, spleen, and tumor of mice in each group were harvested and paraffin embedded. 5μm-thick sections were processed with H&E staining for observation of iron depositions. For IHC, liver specimens were incubated with rabbit polyclonal hepcidin-25 antibody (Abcam, Cambridge, UK), tumor specimens were incubated with rabbit Ki67 primary antibody (BD Biosciences, San Jose, CA, USA) at 4°C overnight. Then these were incubated with HRP-conjugated secondary antibodies (Cell Signaling Technology, Beverly, USA) at room temperature for 2h. Immunohistochemically stained sections were reviewed and evaluated by two independent pathologists. All of the slides were observed under a Nikon light microscope (Nikon Corporation, Tokyo, Japan) and representative photographs were nacaptured.

The concentration of Hepcidin (USCN Life Co., Houston, TX, USA), ferritin, Tf, TfR1, TfR2 and IL-6 (Elabscience Biotechnology Co., Ltd, Wuhan, china) in mice serum was determined by ELISA according to the manufacturer’s instructions.

The assay of RT-PCR was carried out as described previously [19]. Primer sequences were as follows: 5'-CCTGACCAGTGGCTCTGTTT-3', 5'-CACATCCCACACTTTGATCG-3' for hepcidin; 5'-GTGGGGCGCCCCAGGCACCA-3', 5'-CTCCTTAATGTCACGCACGATTTC-3' for GAPDH.

Protein lysates were prepared, subjected to SDS–PAGE, transferred onto PVDF membranes and blotted according to standard methods, using anti-hepcidin (Alpha Diagnostic International, San Antonio, USA), anti-p-STAT3 (Invitrogen, Carlsbad, USA), anti-IL-6, anti-p-SMAD1/5/8 (Santa Cruz Biotechnology, Santa Cruz, USA), and anti-JAK2 (Cell Signaling Technology, Beverly, USA), GAPDH and anti-β-actin rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, USA).

Data were presented as mean ± standard deviation (SD) for normal distribution. Groups were compared by one-way Analysis of variance (ANOVA) and multiple comparisons by LSD-t test by SPSS 21.0 (IBM SPSS for Windows, Version 21.0; IBM Corporation, Armonk, NY, USA). Outliers were excluded with larger or smaller than 2 SD. P < 0.05 was considered significant.

In order to investigate the effects of ASP on tumor growth in tumor-bearing mice, volume and weight of tumors were monitored every other day after 4T1 and H22 cells inoculated subcutaneously. As shown in Fig. 1A-C, the tumors in the CTX and ASP groups were smaller in size than those in the saline treated tumor-bearing control group for both 4T1 and H22 cells xenografted mice. Furthermore, immuno- histochemistry (IHC) confirmed that tumors of the control group displayed significantly higher Ki-67 expression than those in the ASP group (Fig. 1D, E). These results indicated that ASP displayed anti-tumor effects and could inhibited tumor growth in vivo.

One of the consequences of iron overload is the deposition of hemosiderin in the liver, spleen, and tumor tissues. Hemosiderin is the compound formed of broken hemoglobin, ferric oxide (unused iron) and ferritin. Iron depositions in liver, spleen, and tumor tissues were determined by hematoxylin-eosin (H&E) staining. The liver and spleen sections from the saline treated tumor-bearing control group mice showed increased hemosiderin deposition compared to the tumor free blank group. However, the liver, spleen, and tumor sections derived from the ASP-treated mice showed fewer hemosiderosis than those of the control group (Fig. 2A, C). Iron concentrations in these organs were detected by atomic absorption spectrophotometer. Results showed that in 4T1 tumor-bearing mice the iron concentration of liver and spleen in control group mice increased 2.71 and 4.55-fold compared to the blank group (P < 0.001). After treatment with ASP, the iron concentration in liver, spleen and tumor tissues were reduced by 38.53%, 32.88% and 55.89% in comparison to the saline treated control group, respectively (P < 0.001) (Fig. 2B). Comparable results were observed in H22 tumor-bearing mice. Iron concentration of liver and spleen in control group increased 3.36 and 7.92-fold when compared to the blank group, respectively (P < 0.001). After treatment of ASP the iron concentration in liver, spleen and tumor reduced by 36.99%, 41.71% and 35.08% compared to the control group, respectively (P < 0.001) (Fig. 2D). We next analyzed the influence of ASP on serum iron concentration of 4T1 and H22 cells xenografted mice. The serum samples were separated from angular vein of these mice. Then the iron concentration in serum was assayed by automatic biochemistry analyzer. The serum iron concentration of the control group was reduced by 36.12% and 28.13% than blank group, respectively (P < 0.001). It was notable that when compared to the saline treated control group, the serum iron level of ASP group had 1.28 and 1.14-fold increase, respectively (P < 0.01) (Fig. 2B, D).

As hepcidin secreted mainly by the liver is the master regulator of systemic iron homeostasis, we analyzed whether ASP regulated hepcidin expression. The HepG2 and L-02 cells were selected and treated with 0, 0.10, 0.20 and 0.40g/L ASP and the expression of hepcidin mRNA was analyzed by RT-PCR after 48h. Results revealed that 0.20 and 0.40g/L of ASP decreased the expression of hepcidin in a concentration dependent manner (Fig. 3E). Next, we investigated the temporal dependence of hepcidin induction in response to 0.40 g/L ASP. The HepG2 and L-02 cells were treated for up to 96h. By means of analyzing hepcidin mRNA expression at different time points using RT-PCR, we discovered that after 48 hours administration of 0.4g/L ASP displayed decrease of hepcidin expression in a temporal dependent manner (Fig. 3E, F). The analogical results were obtained at the protein level. Western blot analysis showed that hepcidin protein level was dramatically down-regulated in HepG2 and L-02 cells treated with ASP (Fig. 3C).

In addition, we determined the impact of ASP on hepcidin expression in vivo. The hepcidin protein level in liver tissues of 4T1 and H22 tumor-bearing mice was investigated by western blot. Expression of hepcidin protein in tumor bearing control group was up-regulated compared to the tumor free blank group, while the hepcidin protein level in ASP group was decreased significantly compared to the control group (Fig. 3D). We next used the IHC method to detect the liver expression of hepcidin in situ. Consistent with our western blot data, IHC results revealed that hepcidin was dramatically over-expressed in the control group compared to the blank group, whereas ASP therapies significantly decreased its abundance in both of the tumor-bearing mice (Fig. 3A).

Finally, we determined the impact of ASP on hepcidin in serum of each tumor- bearing mice by enzyme-linked immunosorbent assay (ELISA). Our results showed that hepcidin level in the control group of 4T1 and H22 tumor-bearing mice increased 1.78-fold and 1.82-fold compared with the blank group, respectively (P < 0.01), and compared to the control group, the levels of hepcidin in the ASP group of 4T1 and H22 tumor-bearing mice were markedly reduced by 54.55% and 47.29%, respectively (P < 0.01) (Fig. 3B). Taken together, our findings revealed that ASP could effectively repress the expression of hepcidin both in vitro and in vivo.

It is well studied that inflammation regulates hepcidin production via the IL-6/JAK2 transducer and activator of transcription JAK/STAT pathway [20]. We first determined the impact of ASP on IL-6 in serum and liver tissues of 4T1 and H22 tumor-bearing mice by ELISA and western blot. Our results showed that serum IL-6 level in the two tumor bearing control group increased 2.21-fold and 2.10-fold in comparison to that of the blank group by ELISA, respectively (P < 0.001), and when compared to the saline treated control group, the levels of IL-6 in the ASP group were markedly reduced by 51.09% and 33.83%, respectively (P < 0.001) (Fig. 4B). Consistent with ELISA data, Western blot analyses showed that the protein level of IL-6 was dramatically increased in the control group compared with the blank group, while ASP therapies significantly decreased its amount both in 4T1 and H22 tumor-bearing mice (Fig. 4A).

Previous reports indicated that JAK2, phospho-STAT3 (p-STAT3), and phospho-SMAD1/5/8 (p-SMAD1/5/8) are the key protein involved in JAK-STAT pathway and BMP-SMAD pathway regulating hepcidin production [21]. These signaling proteins are essential for hepcidin production in response to several stimulants. Therefore, the expression of these signaling molecules was examined at the protein level in liver tissues of 4T1 and H22 tumor-bearing mice. Our results showed that the expression of JAK2, p-STAT3, and p-SMAD1/5/8 was significantly increased in the control group compared with the blank group (Fig. 4A). Interestingly, ASP administration significantly decreased the levels of hepatic JAK2, p-STAT3, and p-SMAD1/5/8.

To determine the effects of ASP on the cytokines related to iron metabolism in serum, we examined ferritin, Tf (Transferrin), TfR1 (Transferrin receptor 1), and TfR2 (Transferrin receptor 2) levels in serum of 4T1 and H22 tumor-bearing mice by ELISA. As shown in Fig. 5, serum ferritin, Tf, TfR1, and TfR2 levels in the 4T1 tumor-bearing mice control group increased 4.27-fold (P < 0.001), 3.45-fold (P < 0.001), 3.83-fold (P < 0.001) and 1.43-fold (P < 0.05) compared to tumor free blank group. And serum ferritin, Tf, TfR1, TfR2 and IL-6 levels in H22 tumor-bearing mice control group increased 3.30-fold (P < 0.001), 3.37-fold (P < 0.001), 3.09-fold (P < 0.001) and 1.50-fold (P < 0.001). However after ASP therapies, the level of these cytokines in serum were significantly reduced compared to the control group. In 4T1 tumor-bearing mice compared to the control group, the levels of serum ferritin, Tf, TfR1 and TfR2 in the ASP group were reduced by 50.73% (P < 0.001), 12.20% (P > 0.05), 69.36% (P < 0.001) and 30.90% (P < 0.01), respectively. In H22 tumor-bearing mice compared to the control group, the levels of serum ferritin, Tf, TfR1, and TfR2 in the ASP group were reduced by 34.31% (P < 0.01), 18.00% (P < 0.05), 39.74% (P < 0.01) and 28.16% (P < 0.001), respectively.

Taken together, our results demonstrated ferritin, Tf, TfR1 and TfR2 levels in serum of 4T1 and H22 bearing mice were increased, and ASP can significantly decrease these cytokines.

As an essential nutrient element, iron is required for the growth of all cells and involved in cell metabolism, division and proliferation [22]. Cancers are often associated with disordered systemic iron homeostasis, which in turn promotes tumor development through various signaling pathways, such as iron-induced cell proliferation and oxidative stress [23, 24]. It has been shown that a low iron diet inhibits the growth of mammary adenocarcinoma cell xenografts in rats [25, 26]. Exposure of cancer cells to iron chelators can lead to cell cycle arrest and repression of cell proliferation both in vitro and in vivo models [27, 28]. In this study we verified that iron was overloaded in tumor-bearing mice, and following treatment with ASP the iron concentration in liver, spleen, and tumor of tumor-bearing mice was significantly reduced in comparison to the control group. Meanwhile, we found that xenografted tumor growth was significantly inhibited following ASP treatment. Hepcidin secreted by liver plays a pivotal role in orchestrating iron metabolism [6]. Ferroportin is well known as export of intracellular iron [7]. Hepcidin controls systemic iron homeostasis through rapid degradation of ferroportin which increase iron retention in macrophages, hepatocytes and tumor cells, and limit the recycling of iron [7, 29]. Recent studies suggest that disordered iron homeostasis in cancers is due to abnormal regulation of hepcidin and ferroportin, both of which have prognostic significance in cancer patients [30]. In previous studies, it was found that high ferroportin and low hepcidin expression favored an extremely higher 10-year survival rate of breast cancer patients [7]_ENREF_29. Ferritin is the major storage form of iron, and it was documented that many solid tumor cells can synthesize or secrete ferritin, such as hepatic cancer, lung cancer, and breast cancer [31]. Moreover, disregulation of ferritin is correlated with breast cancer stage and has a pejorative prognostic value in localized breast cancer [32]. In this study, our results revealed that serum hepcidin and ferritin were increased in tumor-bearing mice, and the reduction of hepcidin and ferritin synthesis was found following ASP treatment, which could robustly diminish tumor iron retention and inhibit tumor growth. Therefore, inhibition of hepcidin by ASP may prove to be a prospective approach to restrain tumor.

Almost all extracellular iron circulating in the plasma is bound to Tf, an abundant iron transport protein with high affinity for iron [33]. Cell surface TfR involving two documented types namely TfR1 and TfR2, and TfR concentration reflects iron demands of the cell. The initial event in the cellular uptake of iron is that Tf carrying iron binds to TfRs on cell surfaces, the Tf–iron–TfR complex is internalized within the cell, and then iron is released into the cell [34]. It was reported that some tumor tissues synthesize Tf for their proliferation and differentiation, such as the breast cancer cell line of MCF-7 [35, 36]. Studies also have proved that cancer cells have higher TfR1 and TfR2 expression compared to normal cells [37-39]. In present study, serum Tf, TfR1 and TfR2 levels of the control group were significantly increased compared to those of blank group. Higher expression of these iron regulatory proteins revealed a faster rate of iron absorption, which was related to an increased iron requirement by tumor cell proliferation [40]. In addition, our study also demonstrated that ASP suppressed the synthesis of these iron regulatory proteins Tf, TfR1 and TfR2, which serve as a good indicator of the intracellular status of the iron pool.

The hepcidin gene transcription in the liver is regulated by three main pathways, which include the inflammatory pathway that increases hepcidin transcription through JAK/STAT signal transduction in response to inflammatory mediators. The BMP/SMAD pathway, which mediates hepcidin upregulation by iron and hypoxia was also documented to be critical for hepcidin production, besides the erythropoietic pathway which decreases hepcidin level following an increase in the rate of erythropoiesis [11, 20, 21]. Cancer stimulates the synthesis of many inflammatory cytokines, including IL-1, IL-6 and TNF-α [41, 42]. A well characterized molecular mechanism by which inflammation regulates hepcidin production is the IL-6/JAK2 transducer and activator of transcription JAK/STAT pathway [20]. Our result suggested that IL-6, JAK2, and p-STAT3 were markedly enhanced in control group compared to blank control, and ASP could significantly down-regulate these signal proteins. Our findings indicate that ASP may mediate hepcidin by affect Janus-kinases that phosphorylate STATs. In hepcidin regulation, BMP signaling stimulate hepcidin expression by enhance the phosphorylation of SMAD1/5/8 [43]. The results presented in this study suggest that p-SMAD1/5/8 was significantly higher in tumor-bearing mice and ASP suppressed phosphorylation of SMAD1/5/8. It is speculated that the inhibition of p-STAT3 and p-SMAD1/5/8 is involved in the ASP-induced suppression of hepcidin expression. The in vivo model using Xenografted 4T1 and H22 cells in mice may not completely correspond to natural growth of tumors in patients. Therefore, further experiments to elucidate the underlying mechanisms how ASP modulating hepcidin pathways in iron metabolism are required.

The authors are grateful to Prof. Yinming Liang from core facility of Immunophenotyping, Xinxiang Medical University for invaluable discussion. This work was supported by Scientific and Technological Projects of Xinxiang City [cxgg16014]; Technological Projects of Henan province [182102310259]; Medical Scientific and Technological Projects of Henan Province [201403132]; Scientific Research Fund of Xinxiang Medical University [2013QN109]; and Key scientific research projects of universities in Henan province [18A310004].

No conflict of interests exists.

Feng Ren and Jian Li contributed equally to this work.