Lactoferrin Exerts Antitumor Effects by Inhibiting Angiogenesis in a

Article pubs.acs.org/JAFC

Lactoferrin Exerts Antitumor Effects by Inhibiting Angiogenesis in a HT29 Human Colon Tumor Model Hui-Ying Li,† Ming Li,† Chao-chao Luo, Jia-Qi Wang,* and Nan Zheng*

J. Agric. Food Chem. 2017.65:10464-10472. Downloaded from pubs.acs.org by STOCKHOLM UNIV on 04/24/19. For personal use only.

Institute of Animal Sciences of Chinese Academy of Agricultural Sciences, Beijing 100193, People’s Republic of China ABSTRACT: To investigate the effect and potential mechanisms of lactoferrin on colon cancer cells and tumors, HT29 and HCT8 cells were exposed to varying concentrations of lactoferrin, and the impacts on cell proliferation, migration, and invasion were observed. Cell proliferation test showed that high dosage of lactoferrin (5−100 mg/mL) inhibited cell viability in a dosedependent manner, with the 50% concentration of inhibition at 81.3 ± 16.7 mg/mL and 101 ± 23.8 mg/mL for HT29 and HCT8 cells, respectively. Interestingly, migration and invasion of the cells were inhibited dramatically by 20 mg/mL lactoferrin, consistent with the significant down regulation of VEGFR2, VEGFA, pPI3K, pAkt, and pErk1/2 proteins. HT29 was chosen as the sensitive cell line to construct a tumor-bearing nude mice model. Notably, HT29 tumor weight was greatly reduced in both the lactoferrin group (26.5 ± 6.7 mg) and the lactoferrin/5-Fu group (14.5 ± 5.1 mg), compared with the control one (39.3 ± 6.5 mg), indicating that lactoferrin functioned as a tumor growth inhibitor. Considering lactoferrin also reduced the growth of blood vessels and the degree of malignancy, we concluded that HT29 tumors were effectively suppressed by lactoferrin, which might be achieved by regulation of phosphorylation from various kinases and activation of the VEGFR2-PI3K/Akt-Erk1/2 pathway. KEYWORDS: lactoferrin, HT29 cell, HCT8 cell, tumor-bearing model, angiogenesis

1. INTRODUCTION Colorectal cancer is one of the best-understood neoplasms from a genetic perspective, yet it remains among the leading causes of cancer-related deaths in developed countries. Metastastic disease is the primary cause of death among patients with colon cancer. The cause of colon cancer has been reported to be associated with dietary habits, family history, alcohol, sedentary habits, and ulcerative colitis, and several types of its cancer cells cannot be totally eradicated by current therapies.1−5 Colon cancer is always accompanied by inflammation, which regulates essential biological pathways for cell proliferation, cell differentiation, and survival of benign and malignant colon tissue; thus, anti-inflammatory therapies are remarkable in prevention and treatment of early stage colon tumors, such as aspirin and celecoxib.6,7 In the clinical treatment of colon malignancies, fluorouracil (5-FU), among the classical chemotherapeutic regimens, is reported to have severe toxicity and side-effects to animals and humans.8−10 Lactoferrin (LF, 80 kDa, Figure 1) is an iron-binding protein containing 703 amino-acid residues, which can be found in external secretions such as tears, saliva, and milk and the secondary granules of granulocytes. Based on the saturation degree of iron (Fe), lactoferrin can be divided into three types: apo-type (without iron atom), single iron type (with 1 iron atom), and holo-type (with 2 iron atoms). The LF level in human milk is much higher than the one of other mammals, and its concentration in human milk decreases over the months of lactation.11 Lactoferrin has multiple biological functions, including antiinflammatory, antioxidant, antiviral, antitumor, antibiosis, and antiparasitic effects. Lactoferrin seems to play a role in regulation of the immune system, reduces gastrointestinal © 2017 American Chemical Society

Figure 1. Structure of lactoferrin.

stimulation, affects the metabolism of Fe, and helps to balance the concentration of Fe in the body.12−19 In previous studies, the growth and development of colorectal tumors were proven to be highly related to abnormal regulation of cell proliferation, apoptosis, cell cycle arrest, and angiogenesis.20−23 Lactoferrin suppresses cell proliferation, induces cell apoptosis, and down-regulates the expression of Received: Revised: Accepted: Published: 10464

July 22, 2017 October 11, 2017 October 15, 2017 November 7, 2017 DOI: 10.1021/acs.jafc.7b03390 J. Agric. Food Chem. 2017, 65, 10464−10472

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Journal of Agricultural and Food Chemistry the pro-inflammatory cytokines.24−28 Cytotoxic thymus-dependent lymphocytes (T cells) were also found to be the antitumor effector of lactoferrin, which was proven to enhance cell immune activity and Type I helper T cells (Th1) response, to activate natural killer (NK) cells and increase the sensitivity of tumor cells to NK cells.29−32 Lactoferrin can inhibit proliferation of several kinds of tumor cells, and it was reported that the increased oncogenicity of human cervical endometrium was related to down-regulation of lactoferrin, accompanied by increased tumor cell proliferation.33 Rado found there was no mRNA expression of lactoferrin in the cells of promyelocytic leukemia and myeloblastic leukemia, indicating that the lack of lactoferrin in neutrophile granulocytes might cause abnormal proliferation of white blood cells in patients with leukemia.34 Additionally, lactoferrin may inhibit growth and metastases of tumors through the promotion of tumor cells’ differentiation.35 Lactoferrin can also arrest cell cycle and inhibit cell proliferation by affecting expression of the cyclins and by blocking the transfer from G1 to S phase in tumor cells36 or from G0 to G1 phase by P19/cyclinD1 induction.37 In the azoxymethane (AOM)-induced colorectal tumor model, lactoferrin increased Fas expression in both mRNA and protein level in colonic mucosa, which further activated caspase-8 and caspase-3 and up-regulated expressions of Bid and Bax, which then induced apoptosis.38 Until now, lactoferrin seems to be nontoxic in various animal experiments and clinical trials, and it even appears to alleviate toxicity and increase sensitivity when combined with chemotherapy drugs. It also reduced drug-resistance and protected the blood circulatory system from injury due to chemotherapy drugs.39−41 What’s more, when immune-suppressed mice were treated with cyclophosphamide and methopterin administered with lactoferrin orally, lymphocytes and myelocytes grew, and reconstruction of humoral immunity was found.42 Research on the antitumor activity of lactoferrin and its relevant mechanisms is well-known, while in vivo research about lactoferrin’s effect in angiogenesis and related mechanisms is scarce, and studies on the role of lactoferrin in alleviating side-effects in combination with clinical chemotherapeutic agents are even more rarely seen. Therefore, the purpose of the present work is to better understand the mechanism of colon cancer’s growth and development, to identify the novel target molecules which regulate proliferation, invasion, and drug-resistance of the colon cancer cells, as well as to develop safe and novel therapeutic plans. This work is essential in both scientific research and clinical practice. Here, a HT29 tumor bearing nude mice model was constructed and lactoferrin’s antitumor activity was evaluated, as was the role of lactoferrin in tumor angiogenesis and reduction in toxicity. Referring to the relationship between iron saturation and biological activity, several articles have reported that different iron-saturated forms of bLf (Fe-bLf) showed different activities in cancer chemotherapy.43 For example, Gibbons found that Fe-bLf (100% iron saturated) induced significantly greater cytotoxicity and reduction in cell proliferation in MDA-MB-231 and MCF-7 human breast cancer cells.44 While Norrby reported that Apo-bLf (4% iron saturated) significantly enhanced VEGF-A-mediated angiogenesis.45 In colon cancer cells, only lactoferrin (low iron saturated) and low dose lactoferrin were tested for anticancer activities.46 In our research, lactoferrin (100% iron saturated) was used. The novelty in our research may be that our results are supplementary to the previous research.

In this study, two kinds of colon cancer cells were utilized in vitro, and proliferation, migration, and invasion assays were performed to screen the sensitive cell line to lactoferrin and determine its proper dosage. The HT29 tumor-bearing nude mice model was constructed to verify lactoferrin’s efficacy in vivo, and then pathological staining and immunohistochemistry staining were carried out to observe the role of lactoferrin in alleviating toxicity and suppressing angiogenesis. To further elucidate lactoferrin’s mechanism of action on colon cancer cells, VEGFR2/VEGFA, PI3K/Akt, and Erk signal pathways which are tightly related to angiogenesis and metastases were followed.

2. MATERIALS AND METHODS 2.1. Chemicals. The human colon cancer cell lines HT29 and HCT8 were purchased from the Chinese Academy of Science (Shanghai, China), RPMI-1640 medium was purchased from Gibco (USA), and fetal bovine serum (FBS) was purchased from Invitrogen (Shanghai, China). Lactoferrin with a purity of above 90%, 3-(4,5dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), methanol, crystal violet, and formaldehyde solution were purchased from Sigma (USA). β-actin, VEGFR2, VEGF-A, PI3K, pPI3K, Akt, pAkt, Erk, pErk, CD34 antibodies, and secondary antibodies were purchased from Santa Cruz (USA). Reagents related with Western blotting were purchased from Solarbio (Beijing, China). Enhanced chemiluminescence (ECL) reagent was purchased from Tanon (Shanghai, China). 2.2. Cell culture. HT29 and HCT8 were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum under standard cell culture conditions in an incubator (5% CO2, 37 °C). 2.3. Cell proliferation assay. HT29 and HCT8 cells (1 × 104 cells in 100 μL of medium containing 10% FBS per well) were seeded into 96-well plates and incubated for 24 h at 37 °C, and then the medium was replaced with 100 μL of medium containing the indicated concentrations of lactoferrin (1 μg/mL, 10 μg/mL, 100 μg/mL, 1 mg/ mL, 5 mg/mL, 10 mg/mL, 50 mg/mL), followed by 48 h culture. MTT solution (5 mg/mL as the final concentration) was added into wells and incubated for 4 h. Then the medium was replaced by 200 μL of DMSO and the plates were gently shaken for 15 min, and the optical densities (A value) at 490 nm were measured using a Microplate Reader (Thermo, USA). The inhibition rate = [1 − (A test − A blank)/(A control − A blank) ]× 100%. 2.4. Transwell migration assay. The migration capacities of HT29 and HCT8 cells were detected utilizing transwell chambers (Corning, USA). Five ×104 cells were seeded into the chamber with 150 μL serum-free medium per well. The outer chambers were filled with 450 μL medium containing 10% FBS. Lactoferrin (20 mg/mL as the final concentration) was added into the chamber and cocultured for 12 h. Then the top surface of the filter was scrubbed gently with cotton swabs, cells migrating to the undersurface were then fixed with icy methanol for 20 min and stained with 0.1% crystal violet prior to PBS buffer washing for three times. The cells on the undersurface of each filter were photographed and counted, the mean number of migrated cells was calculated by three random fields of each well, and number of migrated cells in lactoferrin treatment group and the one in control group were compared and analyzed. 2.5. Wound healing assay. The cells were seeded in a 24-well plate and incubated for 24 h, promising the cell density per chamber was above 70%. A single lesion of 1−2 mm in width was scratched across the cell monolayer by mechanical scraping. Cells were incubated with lactoferrin (20 mg/mL as the final concentration) for 24 h and photograghed. For the primary scratch width is the same one at the time point of 0 h, the scratch width in treatment group reflected lactoferrin’s inhibition in cell invasion. The recovery rate = scratch width of denuded area in lactoferrin treatment group/scratch width of denuded area in control group (0 h) × 100%. 2.6. Western blot analyses. Total proteins of the cells (1 × 106 cells per well) were extracted by lysis buffer containing phosphatase 10465

DOI: 10.1021/acs.jafc.7b03390 J. Agric. Food Chem. 2017, 65, 10464−10472

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Journal of Agricultural and Food Chemistry and protease inhibitors, then centrifuged at 4 °C at 12000 g for 5 min. After catalysis and heat treatment, the protein sample was added into the 10% SDS-polyacrylamide gels, the proteins were transferred onto nitrocellulose filters by Trans-Blot machines (Bio-Rad) after electrophoresis, and the membrane was blocked with 2% BSA in TBST buffer for 1 h at 25 °C. Then the proteins were probed with specific antibodies at 4 °C overnight, including β-actin, VEGFR2, VEGF-A, PI3K, pPI3K, Akt, pAkt, Erk and pErk, the β-actin was used as the internal reference to ensure equal loading. After three washings with PBST buffer (15 min ×3), the membrane was incubated with secondary antibodies at 25 °C for 2 h and then washed (20 min ×3). Finally, the membrane was detected utilizing an ECL reagent and analyzed by ImageJ software. 2.7. Animal model. To confirm the role of lactoferrin in inhibiting HT29 tumor, nude mice xenograft models were constructed. Thirty 5−6 weeks old BALB/c nude mice (male, Beijing Vital River Laboratory Animal Technology Co., Ltd.) were selected, and HT29 cells were cultured in a large scale. Then 2 × 107 cells in 200 μL matrigel medium (BD, USA) were injected subcutaneously into the right flank of each mouse. Once the tumors grew to the volume of 90− 100 mm3, mice were randomly divided into 5 groups (6 mice per group): control (without any treatment), saline group, lactoferrin group (200 mg/kg, orally administration, once/2 d), 5-Fu group (5 mg/kg, intraperitoneally administration, once/2 d), combination injection group (200 mg/kg lactoferrin together with 5 mg/kg 5-Fu, once/2 d). Mice in the saline group were injected with the same volume of saline as the treated mice every day. All the mice were sacrificed on the 25th day. Tumor diameters were recorded with an electronic caliper every 4 days, and tumor volume was calculated with the following formula: tumor volume (mm3)=0.5 × length (mm) × width (mm)2. Relative tumor volume (RTV, %)=detected volume/ volume before dosing ×100%. Relative tumor proliferation rate (%) =RTV of treatment group/RTV of control group ×100%. Tumor suppression rate (%)=(the average tumor weight of control group-the average tumor weight of treatment group)/ the average tumor weight of control group ×100%. In order to detect the pathological changes of the excised tumors, hematoxylin and eosin (HE) staining and immunohistochemical (IHC) staining for CD34 were performed to evaluate the role of lactoferrin on angiogenesis in HT29 tumor model. Pathological sections stained with HE were photographed by optical microscopy, and those stained with CD34 were photographed using a confocal laser scanning microscope. The heart, liver, kidney, spleen and thymus of each mouse were dissected and weighed, to evaluate LF’s effect on organ injury index. 2.8. Statistical analysis. All the data were expressed as mean ± standard deviation (SD) from several independent experiments (n ≥ 3). Statistical analyses were performed using the software SPSS 13.0 (SPSS Inc., USA). An analysis of variance (ANOVA) and independent samples t test were used to determine the differences among the treatments. P value less than 0.05 was considered statistically significant (*P < 0.05, **P < 0.01). 2.9. Molecular docking. In order to identify potential targets of lactoferrin in inhibiting angiogenesis, lactoferrin was docked onto molecules in the classical pathway, including VEGFR2, VEGFA, pPI3K, pAkt, and pErk1/2, which are involved in the angiogenesisrelated pathways. All the ligands and water in the protein structures were removed, and each monomer of all the proteins was docked using HEX 8.0. The program searched an initial steric search scan at steps N = 16, followed by a final search at steps N = 25 by default. The best model was selected based on the appropriate electrostatic contribution from the total orientations generated.47−49

showed that low-dosage lactoferrin (1−5 mg/mL) activated proliferation of HT29 cells and HCT8 cells, while high-dosage lactoferrin (5−50 mg/mL) inhibited proliferation of colon cells, and lactoferrin inhibited tumor cells viability in a dosedependent manner (Figure 2). The 50% concentration values of inhibition of lactoferrin were 81.3 ± 16.7 mg/mL and 101 ± 23.8 mg/mL for HT29 and HCT8 cells, respectively (Figure 2).

Figure 2. Effect of lactoferrin on HT29 and HCT8 cells proliferation. The proliferation rate was represented as mean ± SD, *P < 0.05, **P < 0.01, compared with control groups (n = 8).

3.2. Lactoferrin inhibited migration and invasion of human colon cancer cells. Tumor migration and invasion are essential steps in tumorigenesis.50,51 The effects of lactoferrin on the chemotactic motility of HT29 and HCT8 cells were determined utilizing wound-healing and transwell assays. As shown in Figure 3, lactoferrin significantly inhibited cell migration and invasion at 20 mg/mL, compared to the control level (P < 0.01). 3.3. Lactoferrin suppressed tumor growth in nude mice. Data in vivo shows that lactoferrin could inhibit growth and development of HT29 tumors. Nude mice bearing the HT29 tumor were treated with saline, lactoferrin (200 mg/kg) alone, 5-Fu (5 mg/kg) alone, or lactoferrin (200 mg/kg) combined with 5-Fu (5 mg/kg). As shown in Figure 4, on the 25th day, the relative tumor volume and the relative tumor proliferation rate of treated groups reduced significantly compared to the control groups (P < 0.01). Mice in the lactoferrin/5-Fu combination group had the lowest levels of relative tumor volume and proliferation rate. On the 25th day, tumor weight was significantly reduced in the 5-Fu group (18.2 ± 5.6 mg), the lactoferrin group (26.5 ± 6.7 mg), and the lactoferrin/5-Fu group (14.5 ± 5.1 mg), compared with the control level (39.3 ± 6.5 mg). There were also significant differences between the 5-Fu group and the lactoferrin/5-Fu group (P < 0.01) (Figure 4). 3.4. Lactoferrin inhibited degree of tumor malignancy and angiogenesis. Referring to pathological results by HE staining, tumor tissue in each of the treatment groups showed less clustering of neutrophile granulocytes, fewer blood vessels, and less hemorrhage and edematous tissue, compared with the control one (Figure 5A). Blood vessels were counted using computer software, and the total number of blood vessels in the lactoferrin/5-Fu combination treatment was the lowest but was not statistically different from the lactoferrin only treatment, indicating lactoferrin alone could inhibit angiogenesis effectively (Figure 5B and C).

3. RESULTS 3.1. Lactoferrin inhibited proliferation of human colon cancer cells. The effects of lactoferrin on the proliferation of human colon cancer cell lines HT29 and HCT8 were studied in vitro to understand the impact and mechanism underlying the antitumor role of lactoferrin. Results 10466


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Figure 3. Lactoferrin inhibited migration and invasion of HT29 and HCT8 cells. (A) Lactoferrin inhibited wound healing in each cell line (200× magnifications). (B) Quantification of recovery of each scratch width after lactoferrin treatment. Results were expressed as the ratio of the widths of invading cells to the initial widths, and the data was represented as mean ± SD (**P < 0.01, compared with control groups, n = 3). (C) Lactoferrin inhibited migration in each cell line (200× magnifications). (D) Quantification of migrated cells, and the data was represented as mean ± SD (**P < 0.01, compared with control groups, n = 3).

Figure 4. In vivo effect of lactoferrin on HT29 tumor-bearing nude mice. (A) Treatment of lactoferrin decreased the size of HT29 tumors. (B) Relative tumor volume, calculated by average tumor volume (n = 6). Tumor volume (mm3) = 0.5 × length (mm) × width (mm)2. Relative tumor volume (RTV, %) = detected volume/volume before dosing ×100%. (C) Tumor suppression rate, calculated by average tumor weight (n = 6). Tumor suppression rate (%) = (the average tumor weight of control group − the average tumor weight of treatment group)/the average tumor weight of control group × 100%. (D) Relative tumor proliferation rate, calculated by relative tumor volumes of different groups. Relative tumor proliferation rate (%) = RTV of treatment group/RTV of control group × 100%.

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Figure 5. HE staining and IHC staining for CD34 of HT29 tumor tissue. (A) Lactoferrin decreased angiogenesis and malignancy degree of tumor sections shown by HE staining (200× magnifications). (B) Lactoferrin decreased angiogenesis of tumor sections shown by CD34 staining (200× magnifications). (C) Quantification of the microvessel density in tumor sections. The microvessel density is presented as mean number of microvessels per microscopic field ± SEM (**P < 0.01 compared with the control group; ##P < 0.01 compared with the 5-Fu group; n = 3).

Figure 6. HE staining of mice organ tissue (200 × magnifications). (A) Heart tissue staining. (B) Kidney tissue staining. (C) Liver tissue staining.

3.5. Lactoferrin alleviated organ toxicity from 5-Fu and enhanced immunizing power. As Figure 6 showed, there was no obvious injury to the heart, liver, and kidney tissue of LF-treated mice compared to the control group and saline

group. Liver and kidney tissue showed hemorrhage, edema, and cytomorphosis in the 5-Fu group and in the lactoferrin/5-Fu combination group, and this damage in the combination group seemed to be, in part, alleviated by lactoferrin. 10468


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Journal of Agricultural and Food Chemistry Table 1. Mice Organ Injury Index (mg/g)a

a

Group(mg/kg)

heart index

liver index

kidney index

spleen index

thymus index

Control Saline 5-Fu(5) LF(200) LF+5-Fu

0.580 ± 0.08 0.590 ± 0.05 0.680 ± 0.10** 0.580 ± 0.06# 0.640 ± 0.09*

6.14 ± 0.32 6.12 ± 0.29 6.99 ± 0.64** 6.20 ± 0.40# 6.65 ± 0.49*#

1.55 ± 0.30 1.52 ± 0.25 1.97 ± 0.36** 1.51 ± 0.31# 1.77 ± 0.40*#

0.540 ± 0.05 0.530 ± 0.07 0.400 ± 0.09** 0.550 ± 0.09# 0.480 ± 0.08*#

0.0600 ± 0.008 0.0610 ± 0.007 0.0490 ± 0.007** 0.0620 ± 0.011# 0.0550 ± 0.011*#

All the data are represented as mean ± SD (n = 6), compared with control. *p < 0.05. **p < 0.01; compared with 5-Fu. #p < 0.05.

As shown in Table 1, in comparison with the control treatment, the organ index in the LF group showed no significant difference, while the heart/liver/kidney indices of the 5-Fu group increased (P < 0.01), and the spleen/thymus indices decreased significantly (P < 0.01). In the lactoferrin/5Fu combination treatment, the heart/liver/kidney/spleen/ thymus indices were higher than the ones in the control group (P < 0.05), and the liver/kidney/spleen/thymus indices were lower than the ones in the 5-Fu group (P < 0.05). This suggests that the 5-Fu treatment may injure the heart/liver/ kidney and thereby adversely affect the function of the immune system. However, lactoferrin appeared to play some role in protecting the organs from 5-Fu’s toxicity. 3.6. Lactoferrin regulated VEGFR2/VEGFA angiogenesis pathway in HT29 cells and HCT8 cells. Since VEGFRs are key to the function of vascular endothelial cells, a few studies have focused on interrupting the VEGFR2/VEGFA pathway to prevent angiogenesis in tumors, aiming to provide efficient antitumor therapy for clinical practice. In addition, the PI3K/AKT and related pathways are closely related to cell proliferation, differentiation, apoptosis ,and so on, all of which play crucial roles in tumor cell migration, adhesion, and angiogenesis; thus, antitumor therapies specifically targeting PI3K/Akt pathways are also under study. In this study, the effect of lactoferrin on VEGFR2/VEGFA/PI3K/Akt/Erk and related molecules was assessed by Western blot analyses to elucidate the mechanism of the role of lactoferrin on tumor angiogenesis and malignancy. Lactoferrin was found to effectively down-regulate the expression of VEGFR2, VEGFA, pPI3K, pAkt, and pErk1/2 proteins in a dose-dependent manner in HT29 cells and HCT8 cells (Figure 7), while the level of Akt and Erk1/2 seemed to have no obvious change, indicating that lactoferrin might regulate expressions of these signals by kinase phosphorylation. Comparing the two kinds of cells, molecules in HT29 cells seemed to be more sensitive to treatment of lactoferrin (Figure 6). 3.7. Lactoferrin bound the targets in the angiogenesis pathway with high affinity. To confirm potential targets of lactoferrin for inhibiting angiogenesis, lactoferrin was docked into the monomers of VEGFR2, VEGFA, pPI3K, pAkt, and pErk1/2, respectively, which were involved in the angiogenesis-related pathway (VEGFA/VEGFR2-PI3K/AktErk1/2). The results showed that lactoferrin could bind the W subunit of VEGFA (binding free energy −722 kcal/mol), the beta subunit of VEGFR2 (binding free energy −737 kcal/mol), and the alpha subunit of P-Erk1/2 (binding free energy −735 kcal/mol) with high affinity, respectively, which might inhibit the functional multimers formation of these targets. This might be just the reason that lactoferrin could effectively downregulate expression of VEGFR2, VEGFA, pPI3K, pAkt, and pErk1/2 proteins.

##

p < 0.01.

Figure 7. Expression of proteins in the angiogenesis pathway in HT29 and HCT8 cells. (A) Expression of VEGFR2, VEGFA, pPI3K, PI3K, pAkt, Akt, pErk1/2, and Erk1/2. (B) Densitometric quantitation for normalized proteins relative to β-actin (%) in HT29 cells. (C) Densitometric quantitation for normalized proteins relative to β-actin (%) in HCT8 cells. The gray bands in Western blotting were scanned and quantified by ImageJ software, and the data was represented as mean ± SD; all values were calculated from three independent experiments (Compared with the control level, *P < 0.05; **P < 0.01).

4. DISCUSSION Naturally occurring components and nutrients in food have always been important sources for discovering new therapeutic medicines in tumor treatment.52−57 In the present study, as a 10469


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validated to be related with the suppression in angiogenesisrelated pathways, including VEGFR2, VEGFA, PI3K, Akt, and Erk1/2. However, the verification of the efficacious dosages of lactoferrin in clinical trials was still unclear and needed lots of requirements (especially clinical resources, cancer patient volunteers, financing, and so on), which could not be satisifed in our biological lab. With further toxicology and pharmacokinetics research, lactoferrin administration or its use in combination with another chemotherapeutic agent might be an alternative strategy for the clinical treatment of colon cancer.

part of our efforts to develop lactoferrin as a chemotherapeutic agent, its activities in inhibiting proliferation, migration, and invasion of colon cancer cells were verified. There was agreement in in vitro findings of lactoferrin displaying the ability to suppress HT29 tumor growth while in vivo, demonstrating decreased malignancy and angiogenesis of tumor tissue. In cell proliferation rate detection in vitro, we found lowdosage lactoferrin could activate proliferation of HT29 cells and HCT8 cells, which was in coincidence with the results from Roiron-Lagroux (1994) and Amouric (1984).46,58 However, we also found high-dosage lactoferrin inhibited cell proliferation with a dose−effect relationship. HT29 cells seemed to be more sensitive to lactoferrin compared with HCT8 cells; this is shown by the stronger inhibitory effect on cell migration and invasion. Thus, we chose the HT29 cell line to construct a tumor-bearing nude mice model. Results demonstrated that conditions of tumor-bearing mice were normal before administration, including weight, behaviors, and mental state. Within 14 days after injection, tumor volume reached 90 mm3− 100 mm3, and then mice were divided into five groups for administration of treatments. Tumor weight and growth curve were determined to compare the antitumor effect of 5-Fu and lactoferrin, each alone and in combination, on tumor tissue. Heart, liver, and kidney pathological samples were assessed to evaluate lactoferrin’s protection against toxicity of 5-Fu, and new blood vessels were stained by CD34 to elucidate the role of lactoferrin in controlling angiogenesis. Angiogenesis is the growth of new blood vessels from preexisting ones, which is essential for tumor growth and metastasis. Anigiogenesis occurs in physiological conditions (menstrual cycle and wound healing) during inflammatory disorders (allergic disorders and autoimmune diseases) and tumor growth in mammals. The angiogenic process requires tightly regulated interactions among different cell types (e.g., endothelial cells and pericytes), the extracellular matrix, several specific growth factors (e.g., vascular endothelial growth factor (VEGFs), angiopoietins), cytokines, and chemokines.59−62 The VEGF family has been reported to affect endothelial cells involved in angiogenesis and play its role through three receptors: VEGF receptor 1 (VEGFR1), VEGF receptor 2 (VEGFR2), and VEGF receptor 3 (VEGFR3). VEGFR2 has been closely implicated with angiogenesis of solid tumors.63,64 Based on the review of pathology samples which showed decreased blood vessels in tumor sections treated with lactoferrin, we focused the effect of lactoferrin on these pathways to explore its antitumor mechanism. Results revealed that lactoferrin down-regulated expressions of the molecules including VEGFR2, VEGFA, pPI3K, pAkt, and pErk1/2 in a dose-dependent manner, especially in HT29 cells, indicating that lactoferrin might exert its antitumor effect through inhibiting angiogenesis by regulating phosphorylation of these kinases. Information about the direct binding sites of lactoferrin requires further exploration and validation, and the overexpression of the candidate targets and small interfering RNA (SiRNA) treatment of the candidate targets experiments of several targets are in progress in our lab. In summary, we found that lactoferrin exerted a prominent antitumor activity in vitro and in vivo, and lactoferrin was proven to alleviate toxicity and adverse effects when combined with the classical 5-Fu chemotherapeutic agent. What was more, lactoferrin might have a specific role in certain colon tumors cell types, such as HT29, and the mechanism was

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AUTHOR INFORMATION

Corresponding Authors

*Nan Zheng. Tel: +86-10-62816069; Fax: +86-10-62897587; E-mail: [email protected]. *Jia-Qi Wang. Tel: +86-10-62816069; Fax: +86-10-62897587; E-mail: [email protected]. ORCID

Nan Zheng: 0000-0002-5365-9680 Author Contributions †

Hui-Ying Li and Ming Li should be regarded as co-first author.

Funding

We acknowledge financial support from two Agriculture-Key Laboratories, Ministry of Agriculture-Key Laboratory of Quality & Safety Control for Milk and Dairy Products, Ministry of Agriculture-Laboratory of Quality and Safety Risk Assessment for Dairy Products, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R. China. Notes

The authors declare no competing financial interest.

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REFERENCES

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Lactoferrin Exerts Antitumor Effects by Inhibiting Angiogenesis in a

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