Lactoferrin Exerts Antitumor Effects by Inhibiting Angiogenesis in a

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Lactoferrin exerts anti-tumor effects by inhibiting angiogenesis in a HT29 human colon tumor model Huiying Li, Ming Li, Chaochao Luo, Jiaqi Wang, and Nan Zheng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03390 • Publication Date (Web): 07 Nov 2017 Downloaded from http://pubs.acs.org on November 8, 2017

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Journal of Agricultural and Food Chemistry

Lactoferrin exerts anti-tumor effects by inhibiting angiogenesis in a HT29 human colon tumor model Hui-Ying Li,‡# Ming Li,‡# Chao-chao Luo, # Jia-Qi Wang,*# Nan Zheng*# # Institute of Animal Sciences of Chinese Academy of Agricultural Sciences, Beijing 100193, People’s Republic of China ‡

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

* Nan Zheng and Jia-Qi Wang should be regarded as the corresponding author

Corresponding author: Nan Zheng, Jia-Qi Wang, Chinese Academy of Agricultural Sciences, 2# Yuanmingyuan west road, Haidian district, Beijing, 100193, China. Tel: +86-10-62816069; Fax: +86-10-62897587 E-mail: [email protected], [email protected].

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ABSTRACT: To investigate the effect and potential mechanisms of lactoferrin on

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colon cancer cells and tumors, HT29 and HCT8 cells were exposed to varying

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concentrations of lactoferrin, and the impact on cell proliferation, migration and

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invasion were observed. Cell proliferation test showed that high dosage of lactoferrin

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(5-100 mg/mL) inhibited cell viability in a dose-dependent manner, with the 50%

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concentration of inhibition at 81.3±16.7 mg/mL and 101±23.8 mg/mL for HT29 and

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HCT8 cells, respectively. Interestingly, migration and invasion of the cells were

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inhibited dramatically by 20 mg/mL lactoferrin, consistent with the significant down

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regulation of VEGFR2, VEGFA, pPI3K, pAkt and pErk1/2 proteins. HT29 was

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chosen as the sensitive cell line to construct a tumor-bearing nude mice model.

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Notably, HT29 tumor weight was greatly reduced in both lactoferrin group (26.5±6.7

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mg) and the lactoferrin/5-Fu group (14.5±5.1 mg), compared with the control one

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(39.3±6.5 mg), indicating that lactoferrin functioned as a tumor growth inhibitor.

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Considering lactoferrin also reduced the growth of blood vessels and degree of

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malignancy, we concluded that HT29 tumors were effectively suppressed by

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lactoferrin, which might be achieved by regulation of phosphorylation from various

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kinases and activation of VEGFR2-PI3K/Akt-Erk1/2 pathway.

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KEYWORDS: Lactoferrin; HT29 cell; HCT8 cell; tumor-bearing model;

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angiogenesis

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1. INTRODUCTION.

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Colorectal cancer is one of the best-understood neoplasms from a genetic perspective,

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yet it remains among leading causes of cancer-related deaths in developed countries.

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Metastastic disease is the primary cause of death among patients with colon cancer.

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The cause of colon cancer has been reported to be associated with dietary habits,

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family history, alcohol, sedentary habits and ulcerative colitis, and several types ? of

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its cancer cells can not be totally eradicated by current therapies.1-5 Colon cancer is

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always accompanied by inflammation, which regulates essential biological pathways

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for cell proliferation, cell differentiation and survival of benign and malignant colon

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tissue, thus, anti-inflammatory therapies are remarkable in prevention and treatment

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of early-stage colon tumors, such as aspirin and celecoxib. 6-7 In the clinical treatment

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of colon malignancies, fluorouracil (5-FU), among the classical chemotherapeutic

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regimens, is reported to have severe toxicity and side-effects to animals and

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humans.8-10

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Lactoferrin (LF, 80 kDa, figure 1) is an iron-binding protein containing 703

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amino-acid residues, which can be found in external secretions such as tears, saliva,

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and milk and the secondary granules of granulocytes. Based on the saturation degree

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of iron (Fe), lactoferrin can be divided into three types: apo-type (without iron atom),

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single iron type (with 1 iron atom) and holo-type (with 2 iron atoms). LF level in

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human milk is much higher than the one of other mammals, and its concentration in

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human milk decreases over the months of lactation.11

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Lactoferrin has multiple biological functions, including anti-inflammatory,

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anti-oxidant, anti-viral, anti-tumor, antibiosis, and anti-parasitic effects. Lactoferrin

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seems to play a role in regulation of the immune system, reduce gastrointestinal

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stimulation, affects the metabolism of Fe and helps to balance the concentration of Fe



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in the body.12-19

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In previous studies, the growth and development of colorectal tumors were proven

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to be highly related to abnormal regulation of cell proliferation, apoptosis, cell cycle

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arrest and angiogenesis.20-23 Lactoferrin suppresses cell proliferation, induces cell

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apoptosis, and down-regulates the expression of the pro-inflammatory cytokines.24-28

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Cytotoxic thymus-dependent lymphocytes (T cells) were also found to be the

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anti-tumor effector of lactoferrin, which was proven to enhance cell immune activity

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and Type I helper T cells (Th1) response, to activate natural killer (NK) cells and

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increase the sensitivity of tumor cells to NK cells.29-32 Lactoferrin can inhibit

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proliferation of several kinds of tumor cells, and it was reported that the increased

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oncogenicity of human cervical endometrium was related to down-regulation of

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lactoferrin, accompanied with increased tumor cell proliferation.33 Rado found there

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was no mRNA expression of lactoferrin in the cells of promyelocytic leukemia and

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myeloblastic leukemia, indicating that the lack of lactoferrin in neutrophile

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granulocytes might cause abnormal proliferation of white blood cells in patients with

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leukemia.34 Additionally, lactoferrin may inhibit growth and metastases of tumors

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through the promotion of

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cell cycle and inhibit cell proliferation by affecting expression of the cyclins, by

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blocking the transfer from G1 to S phase in tumor cells,36 or from G0 to G1 phase by

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P19/cyclinD1 induction.37 In the azoxymethane (AOM)-induced colorectal tumor

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model, lactoferrin increased Fas expression in both mRNA and protein level in

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colonic mucosa, which further activated caspase-8 and caspase-3 and up-regulated

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expressions of Bid and Bax, which then induced apoptosis.38

tumor cells’ differentiation.35 Lactoferrin can also arrest

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Until now, lactoferrin seems to be nontoxic in various animal experiments and

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clinical trials, and it even appears to alleviate toxicity and increase sensitivity when


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combined with chemotherapy drugs. It also reduced drug-resistance and protected the

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blood circulatory system from injury due to chemotherapy drugs.39-41 What’s more,

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when immune- suppressed mice were treated with cyclophosphamide and methopterin

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administered with lactoferrin orally, lymphocytes and myelocytes grew, and

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re-construction of humoral immunity was found.42

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Research on the anti-tumor activity of lactoferrin and its relevant mechanisms is

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well known, while in-vivo research about lactoferrin’s effect in angiogenesis and

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related mechanisms is scarce, and studies on the role of lactoferrin in alleviating

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side-effects in combination with clinical chemotherapeutic agents is even more rarely

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seen. Therefore, the purpose of the present work is to better understand the

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mechanism of colon cancer’s growth and development, to identify the novel target

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molecules which regulate proliferation, invasion and drug-resistance of the colon

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cancer cells, as well as to develop safe and novel therapeutic plans. This work is

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essential in both scientific research and clinical practice. Here, a HT29 tumor bearing

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nude mice model was constructed and lactoferrin’s anti-tumor activity was evaluated,

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as was the role of lactoferrin in tumor angiogenesis and reduction in toxicity.

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Referring to the relationship between iron saturation and biological activity, several

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articles have reported that different iron-saturated forms of bLf (Fe-bLf) showed

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different activities in cancer chemotherapy.43 For example, Jessica A. Gibbons found

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that Fe-bLf (100% iron saturated) induced significantly greater cytotoxicity and

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reduction in cell proliferation in MDA-MB-231 and MCF-7 human breast cancer

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cells.44 While Norrby K. reported that Apo-bLf (4% iron saturated) significantly

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enhanced VEGF-A-mediated angiogenesis.45 In colon cancer cells, only lactoferrin

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(low iron saturated) and low doze lactoferrin were tested for activities of anti-cancer.46

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In our research, lactoferrin (100% iron saturated) were used. Novelty in our research



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may be that our results are supplementary for the previous researches.

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In this study, two kinds of colon cancer cells were utilized in vitro, and proliferation,

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migration, and invasion assays were performed to screen the sensitive cell line to

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lactoferrin and determine its proper dosage. The HT29 tumor-bearing nude mice

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model was constructed to verify lactoferrin’s efficacy in vivo, then pathological

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staining and immunohistochemistry staining were carried out to observe the role of

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lactoferrin in alleviating toxicity and suppressing angiogenesis. To further elucidate

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lactoferrin’s mechanism of action on colon cancer cells, VEGFR2/VEGFA, PI3K/Akt

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and Erk signal pathways which are tightly related to angiogenesis and metastases

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were followed.

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2. MATERIALS AND METHODS.

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2.1. Chemicals. The human colon cancer cell lines HT29 and HCT8 were

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purchased from the Chinese Academy of Science (Shanghai, China), RPMI-1640

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medium was purchased from Gibco (USA), fetal bovine serum (FBS) were purchased

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from Invitrogen(Shanghai, China). Lactoferrin with the purity of above 90%,

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3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium

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Dimethylsulfoxide (DMSO), methanol, crystal violet and formaldehyde solution were

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purchased from Sigma (USA). β-actin, VEGFR2, VEGF-A, PI3K, pPI3K, Akt, pAkt,

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Erk, pErk, CD34 antibodies and secondary antibodies were purchased from Santa

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Cruz (USA). Reagents related with western blotting were purchased from Solarbio

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(Beijing, China). Enhanced chemiluminescence (ECL) reagent was purchased from

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Tanon (Shanghai, China).

bromide

(MTT),

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2.2. Cell culture. HT29 and HCT8 were grown in RPMI-1640 medium

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supplemented with 10 % fetal bovine serum under standard cell culture conditions in

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an incubator (5 % CO2, 37 °C).


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2.3. Cell proliferation assay. HT29 and HCT8 cells (1×104 cells in 100 μL

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medium containing 10 % FBS per well) were seeded into 96-well plates and incubated

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for 24 h at 37 ℃, and then the medium was replaced with 100 μL medium containing

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the indicated concentrations of lactoferrin (1 μg/mL, 10 μg/mL, 100 μg/mL, 1 mg/mL,

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5 mg/mL, 10 mg/mL, 50 mg/mL), followed by 48 h culture. MTT solution (5 mg/mL

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as the final concentration) was added into wells and incubated for 4 h. Then the

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medium was replaced by 200 μL DMSO and the plates were gently shaken for 15 min,

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the optical densities (A value) at 490 nm were measured using a Microplate Reader

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(Thermo, USA). The inhibition rate=[1−(A test-A blank)/(A control-A blank)]× 100%.

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2.4. Transwell migration assay. The migration capacities of HT29 and HCT8 cells

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were detected utilizing transwell chambers (Corning, USA). 5×104 cells were seeded

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into the chamber with 150 μL serum-free medium per well. The outer chambers were

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filled with 450 μL medium containing 10 % FBS. Lactoferrin (20mg/mL as the final

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concentration) was added into the chamber and co-cultured for 12 h. Then the top

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surface of the filter was scrubbed gently with cotton swabs, cells migrating to the

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undersurface were then fixed with icy methanol for 20 min and stained with 0.1 %

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crystal violet prior to PBS buffer washing for three times. The cells on the

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undersurface of each filter were photographed and counted, the mean number of

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migrated cells was calculated by three random fields of each well, and number of

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migrated cells in lactoferrin treatment group and the one in control group were

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compared and analyzed.

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2.5. Wound healing assay. The cells were seeded in a 24-well plate and incubated

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for 24 h, promising the cell density per chamber was above 70 %. A single lesion of

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1-2 mm in width was scratched across the cell monolayer by mechanical scraping.

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Cells were incubated with lactoferrin (20 mg/mL as the final concentration) for 24 h



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and photograghed. For the primary scratch width is the same one at the timepoint of 0

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h, the scratch width in treatment group reflected lactoferrin’s inhibition in cell

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invasion. The recovery rate = scratch width of denuded area in lactoferrin treatment

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group/scratch width of denuded area in control group (0 h) ×100 %.

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2.6. Western blot analyses. Total proteins of the cells (1×106 cells per well) were

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extracted by lysis buffer containing phosphatase and protease inhibitors, then

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centrifuged at 4 °C at 12000 g for 5 min. After catalysis and heat treatment, the

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protein sample was added into the 10 % SDS-polyacrylamide gels, the proteins were

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transferred onto nitrocellulose filters by Trans-Blot machines (Bio-Rad) after

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electrophoresis, and the membrane was blocked with 2 % BSA in TBST buffer for 1 h

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at 25 ℃. Then the proteins were probed with specific antibodies at 4 ℃ overnight,

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including β-actin, VEGFR2, VEGF-A, PI3K, pPI3K, Akt, pAkt, Erk and pErk, the

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β-actin was used as the internal reference to assure equal loading. After three

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washings with PBST buffer (15 min×3), the membrane was incubated with secondary

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antibodies at 25 ℃ for 2 h and then washed (20 min×3). Finally, the membrane was

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detected utilizing an ECL reagent and analysed by Image J software.

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2.7. Animal model. To confirm the role of lactoferrin in inhibiting HT29 tumor,

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nude mice xenograft models were constructed. Thirty 5-6 weeks old BALB/c nude

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mice (male, Beijing Vital River Laboratory Animal Technology Co., Ltd.) were

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selected, and HT29 cells were cultured in a large scale. Then 2×107 cells in 200μL

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matrigel medium (BD, USA) were injected subcutaneously into the right flank of each

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mouse. Once the tumors grew to the volume of 90-100 mm3, mice were randomly

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divided into 5 groups (6 mice per group): control (without any treatment), saline

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group, lactoferrin group (200 mg/Kg, orally administration, once/2d), 5-Fu group (5

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mg/Kg, intraperitoneally administration, once/2 d), combination injection group (200


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mg/Kg lactoferrin together with 5 mg/Kg 5-Fu, once/2 d). Mice in the saline group

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were injected with the same volume of saline as the treated mice every day. All the

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mice were sacrificed on the 25th day. Tumor diameters were recorded with an

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electronic caliper every 4 days, and tumor volume was calculated with the following

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formula: tumor volume (mm3)=0.5×length (mm)×width (mm)2. Relative tumor

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volume (RTV, %)=detected volume/volume before dosing×100%. Relative tumor

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proliferation rate (%)=RTV of treatment group/RTV of control group ×100%. Tumor

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suppression rate (%)=(the average tumor weight of control group-the average tumor

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weight of treatment group)/ the average tumor weight of control group ×100%.

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In order to detect the pathological changes of the excised tumors, hematoxylin and

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eosin (HE) staining and immunohistochemical (IHC) staining for CD34 were

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performed to evaluate the role of lactoferrin on angiogenesis in HT29 tumor model.

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Pathological sections stained with HE were photographed by optical microscopy, and

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those stained with CD34 were photographed using a confocal laser scanning

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microscope. The heart, liver, kidney, spleen and thymus of each mouse were dissected

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and weighed, to evaluate LF’s effect on organ injury index.

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2.8. Statistical analysis. All the data were expressed as mean±standard deviation

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(SD) from several independent experiments (n≥3). Statistical analyses were

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performed

using the software SPSS 13.0 (SPSS Inc, USA). An analysis of variance

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(ANOVA) and independent samples t test were used to determine the differences

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among the treatments. P value less than 0.05 was considered statistically significant

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(*P

Lactoferrin Exerts Antitumor Effects by Inhibiting Angiogenesis in a

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