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Carboxymethyl chitosan represses tumor angiogenesis in vitro and in vivo
Accepted Manuscript Title: Carboxymethyl chitosan represses tumor angiogenesis in vitro and in vivo Author: Zhiwen Jiang Baoqin Han Hui Li Yan Yang Liu Wanshun PII: DOI: Reference:
S0144-8617(15)00356-2 http://dx.doi.org/doi:10.1016/j.carbpol.2015.04.040 CARP 9871
To appear in: Received date: Revised date: Accepted date:
31-10-2014 13-2-2015 20-4-2015
Please cite this article as: Jiang, Z., Han, B., Li, H., Yang, Y., and Wanshun, L.,Carboxymethyl chitosan represses tumor angiogenesis in vitro and in vivo, Carbohydrate Polymers (2015), http://dx.doi.org/10.1016/j.carbpol.2015.04.040 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1 Carboxymethyl chitosan (CMCS) was non-cytotoxic on HUVECs.
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2 Treatment with CMCS significantly inhibited the migration of HUVECs.
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3 CMCS significantly inhibited tumor growth and CD34 expression of H22 in vivo.
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4 CMCS could regulate the levels of VEGF and TIMP-1 in serum of mice.
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5 The anti-angiogenesis function may contribute to the anti-tumor effects of CMCS.
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Carboxymethyl chitosan represses tumor
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angiogenesis in vitro and in vivo
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Zhiwen Jiang, Baoqin Han*, Hui Li, Yan Yang, Liu Wanshun
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Biochemistry Laboratory, College of Marine Life Sciences, Ocean University of China, Qingdao 266003, PR China
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Abstract chitosan
(CMCS),
with
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Carboxymethyl
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potent
water
solubility,
biocompatibility, and non-toxicity, has emerged as a promising candidate for
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biomedical applications. In this study, the anti-tumor angiogenesis effects of
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CMCS were evaluated in vitro and in vivo. Our results showed that CMCS
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could inhibit the 2-dimensional and 3-dimensional migration of human
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umbilical vein endothelial cells (HUVECs) in vitro. CMCS significantly inhibited
19
the growth of mouse hepatocarcinoma 22 tissues and could promote tumor
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cell necrosis as suggested by pathological observations. The CD34 expression
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in H22 tumor tissue, the levels of vascular endothelial growth factor and tissue
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inhibitor of metalloproteinase 1 in serum was regulated by CMCS treatment.
23
CMCS could significantly improve thymus index, spleen index, tumor necrosis
24
factor α and interferon γ level. In a conclusion, CMCS possessed potent
25
anti-tumor effects by inhibiting tumor angiogenesis, stimulating immune
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functions. Our date provide more foundation for application of CMCS in biomedicine or biomaterials for targeted anticancer drugs delivery. Key words: Carboxymethyl chitosan; anti-tumor; angiogenesis; in vitro; in vivo
1. Introduction
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Angiogenesis, the formation of new capillaries from pre-existing blood
32
vessels (Miura, et al., 2010), is critical for normal embryonic development and
33
also contributes many pathological states including tumor growth and the
34
development of metastases (Park, et al., 2007). Solid tumors, which grow
35
beyond a size of a few millimeters, need new blood vessels to provide oxygen *
Corresponding author. Tel.: +86 532 82032105; fax: +86 532 82032105. E-mail addresses: [email protected] (B. Han). 2
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and nutrients and take metabolic waste away (Folkman, 1972). The inhibition
37
of angiogenesis represents an avenue for blocking tumor growth (Trachsel &
38
Neri, 2006). After Folkman proposed angiogenesis as a target for cancer
39
therapy in the last century, enormous research emerged in the past decades.
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Bevacizumab was approved for the treatment of metastatic colorectal cancer
41
by FDA in 2004, making anti-angiogenesis in tumor therapy a reality. The
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formation of new blood vessels is the result of the mutual regulation of
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pro-angiogenic compounds like vascular endothelial growth factor (VEGF),
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basic fibroblast growth factor (bFGF), transforming growth factor-β (TGF-β),
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platelet-derived growth factor (PDGF), matrix metalloproteinases (MMPs) and
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anti-angiogenesis factors such as angiostatin, endostatin, and tissue inhibitor
47
of metalloproteinase (TIMPs). The balance will be broken under pathological
48
conditions.
Chitosan (CTS) is the N-deacetylated derivative and has been widely
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used as biomedical materials due to its nontoxicity, biocompatibility,
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hemocompatibility and biodegradability (Muzzarelli, 1993; Kumar, Muzzarelli,
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Muzzarelli, Sashiwa, & Domb, 2004). Chitosan is also reported to have
53
anti-tumor effects by inhibiting tumor cell proliferation (Maeda & Kimura, 2004),
54
inducing apoptosis (Pae, et al., 2001), and enhancing immune functions (Yu,
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Zhao, & Ke, 2004). However, the solubility of chitosan greatly restricts its wide
56
application (Faizuloev, et al., 2012). Chitosan has different groups that can be
57
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curcumin, doxorubicin and so on (Anitha, Chennazhi, Nair, & Jayakumar, 2012;
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Anitha, et al., 2011; Jin, et al., 2012; Wang, et al., 2011). Our previous studies
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showed that CMCS could inhibit tumor cells proliferation and tumor tissue
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growth. In this study, we further investigated the anti-tumor angiogenesis
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effects of CMCS. Our data will provide more bases for the clinical application of
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CMCS as biomedical materials in tumor surgery.
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chemically modified. Carboxymethyl chitosan (CMCS) is one of the most important water-soluble chitosan derivates (Muzzarelli, 1988). CMCS has been proven to have favorable properties, such as lower toxicity, better biocompatibility, and biodegradability (Ji, Wu, Liu, Chen, & Xu, 2012). CMCS has been widely used in biomedical fields such as drug carriers, antimicrobial material, gene delivery system and tissue engineering. CMCS has been developed as anti-cancer drug delivery systems such as 5-Flourouracil,
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2. Experimental
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2.1. Reagents and antibodies Chitosan with a degree of deacetylation of 93% and molecular weight of
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350 kDa was purchased from Qingdao Biotemed Biomaterial Co., Ltd
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(Qingdao, China). CMCS were synthesized, purified and identified in our
75
laboratory. Rabbit polyclonal anti-mouse CD34 antibody, Horse Radish
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Peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody,
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3,3-diaminobenzidine (DAB), Mouse VEGF ELISA Kit, Mouse TIMP-1 ELISA
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Kit, Mouse tumor necrosis factor α (TNF-α) ELISA Kit and Mouse Interferon γ
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(IFN-γ) ELISA Kit were purchased from Boster Biological Engineering Co., Ltd
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(Wuhan China). 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide
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(MTT) was obtained from Sigma- Aldrich (St. Louis, MO, USA). RPMI 1640
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medium and fetal bovine serum (FBS) was obtained from Gibco®, Life
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Technologies (Carlsbad, CA, USA). Transwell, 96 well plates and 24 well
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plates were purchased from Corning INC. (Corning, NY, USA). All other
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chemicals and reagents were of the highest commercial grade available.
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2.2. Animals and cell lines
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Forty female Kunming mice (weight range, 18-22 g) were obtained from
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Medicine Inspecting Institute of Qingdao, China, and maintained in the animal
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care facility of the Biochemistry Laboratory of Ocean University of China. All
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animals were kept under a 12h light-dark cycle at consistent temperature
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(25±3) and relative humidity (60-70%). Experiments were performed in accordance with the ethical guidelines of the Shandong Province Experimental Animal Management Committee and were in complete compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Mouse Hepatocarcinoma 22 (H22) cells and Human Umbilical Vein
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Endothelial Cells (HUVECs) were offered by the Institute of Pharmacology,
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Ocean University of China. Endothelial Cells between passage 3 and 10 were
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used in the experiments. The cells were grown on 0.3% gelatin coated dishes
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using RPMI 1640 medium supplemented with 10% fetal bovine serum, 100
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units/ml penicillin and 100 μg/ml streptomycin at 37 °C in a humidified
102
incubator with 5% CO2 in 95% air.
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2.3. Preparation and characterization of CMCS 4
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CMCS was prepared from chitosan according to the previously published
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literature by our laboratory (Zheng, Han, Yang, & Liu, 2011). The sodium salt
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of CMCS was synthesized by chemical reaction with mono-chloroacetic acid
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under alkaline condition, and then treated with HCl in methanol that protonates
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the carboxyl group. (Vaghani, Patel, Satish, Patel, & Jivani, 2012). Carboxyl
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structures and molecular weight was determined by fourier-transformed
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infrared spectroscopy (FTIR) and high performance liquid chromatography
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(HPLC). The IR spectrum of CMCS (~0.5 mg) was acquired on the NEXUE
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470 instrument (Nicolet Co., USA) as KBr pellets at room temperature.
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Standard curves were obtained according to the Tmax and molecular weight of
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Pullulan standards with Agilent 1200 (Agilent Co., USA). The molecular weight
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of CMCS was determined by standard curves and the Tmax. The degree of
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deacetylation and the degree of substitution of carboxymethyl chitosan was
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determined by pH-titration (industry standards of P.R. China, 2005; Mohamed
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& Abd El-Ghany, 2012).
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2.4. In vitro cell proliferation studies
HUVECs were seeded in 96-well culture plates at a density of 4×103
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cells/well in 200 μl of culture medium (plus 10% FBS) and allowed to attach
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and grow for 24 h. The cells were then exposed to 200 μl complete medium
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without or with CMCS at different concentrations. The morphological changes
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of cells were observed and photographed with the T1-SM 100 inverted
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calculated using the percentage of proliferation rate (PR/%). Each assay was
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repeated three times and all experiments were performed in six wells. The PR
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was calculated according to the formula below:
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microscope (Nikon Co. Japan) at 100 magnification. MTT assay was used to measure the effects of CMCS (0.5 mg/ml, 1.0 mg/ml, and 2.0 mg/ml) on HUVECs proliferation 24 h and 48 h after treatment. MTT (5 mg/ml, 20 μl) was added to each well. After 4 h of incubation with MTT, medium was removed and 150 μl of DMSO was added. The plate was then read at 492 nm on Multiskan Go 151 Microplate Scanning Spectrophotometer (Thermo Fisher Scientific, INC, USA). The effects of CMCS on HUVECs proliferation were
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2.5. Wounding migration assay HUVECs suspended in complete medium (plus 10% FBS) were plated in
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24 well plates at 3×104 cells/well and incubated for 12 h to achieve 90%
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confluence. After 12 h incubation in starvation medium (plus 1% FBS), cell
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monolayers were scored vertically down the center of each well with a sterile
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200 μl tip. After wounding, the cultures were washed with phosphate buffer
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solution (PBS) twice to remove detached cells. Fresh growth medium (plus
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10% FBS) without or with CMCS (0.5 mg/ml, 1.0 mg/ml, and 2.0 mg/ml) were
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added. Images of the wound in each well were taken at 0 h, 24 h, and 48 h
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under the inverted microscope at the magnification of 10 ×. Relative migration
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distances on each time point is calculated by subtracting scratch length from
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the initial length of the scratch. The effects of CMCS on the wounding
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migration of HUVECs were calculated using the percentage of inhibition rate
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(IR/%). Each assay was repeated three times and all experiments were
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performed in six wells. The IR was calculated as follows:
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2.6. Transwell migration assay
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HUVECs (5×104 cells /100 μl) re-suspended in RPMI 1640 medium
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containing 1% FBS were added to the cell culture inserts (8 μm pore size) and
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FBS (10% V/V) was used as chemoattractant in the lower wells of the transwell.
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The cells in upper inserts was incubated with CMCS (0.5 mg/ml, 1.0 mg/ml, and 2.0 mg/ml) and allowed to migrate for 14 h at 37 °C. Then the cells were fixed in methanol and stained with crystal violet. The photographs of HUVECs that had migrated to the bottom surface of the insert in different groups were taken at 200 × magnification under a microscope. Cell migration was determined by measuring the absorbance of crystal violet-stained cells at 570 nm. The effects of CMCS on the transwell migrations of HUVECs were
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calculated using the percentage of inhibition rate (IR/%). Each assay was
164
repeated three times and all experiments were performed in six wells. The IR
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was calculated according to the formula below:
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2.7. Anti-tumor experiment in vivo 6
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H22 cells were kept in Kunming moue abdominal cavity for three
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generations. Single-cell suspensions were prepared, washed, and diluted in
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normal saline. Cell suspensions (200 μl, approximately 4×106 H22 cells) were
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injected subcutaneously in the right axilla to each mouse. Then the animals
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were randomizedly assigned into the control group and three experimental
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groups. Different doses of CMCS (75 mg/kg, 150 mg/kg and 300 mg/kg body
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weight) in normal saline were administer to experimental groups peritoneal
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injectionat 1, 3, 5, 7, 9, 11 and 13 days after tumor implantation. Animals in the
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control group were administered with saline. After the last administration, mice
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were fasting for 12 hours. The whole venous blood was collected from the orbit.
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Mice were then killed and tumor tissue, thymus and spleen of each animal
179
were stripped intactly and weighed. Thymus and spleen index is the ratio of
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thymus or spleen weight to the body weight. The inhibition of CMCS on the
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growth of H22 tumor was calculated using the percentage of inhibition rate (IR).
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Each assay was repeated three times and all experiments were performed in
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ten mice. The IR was calculated according to the formula below:
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2.8. Histology of Tumor samples and Immunohistochemical studies
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Tumor tissues were fixed in 4% paraformaldehyde solution for 24 h, and
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then embedded in paraffin for sectioning. For each sample of the four groups,
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some consecutive 4 μm-thick sections were stained with hematoxylin and eosin (HE) for the pathologic examinations, while parallel sections were stained using the endothelium-specific CD34 antibody as described below. The presence of blood vessels was determined by immunohistochemistry using hematopoietic progenitor cell antigen CD34. After three 3-min washes in PBS, sections were incubated with 3% hydrogen peroxide in methanol for 10 min to quench endogenous peroxidase. After a further three washes,
195
nonspecific protein-binding sites were blocked by 20-min incubation with 5%
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bovine serum albumin (BSA) in PBS. The blocking solution was drained and
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the slices were incubated at 4 overnight with a 1:150 dilution of the
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endothelial cell marker CD34 antibody. Negative controls were performed by
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replacing the primary antibody with PBS. After a further three rinses and a
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blocking step, sections were incubated with HRP-conjugated goat anti-rabbit 7
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IgG for 30 min at 37 . The sections were washed and then stained with the
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chromogen diaminobenzidine for 5-10 min, then counterstained with
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hematoxylin. Light microscopy slides were observed and photographed using
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the E200 microscope (Nikon Co. Japan). All photomicrographs were taken with
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DT300 digital camera software.
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2.9. Assay of TIMP-1, VEGF, IFN-γ and TNF-α
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Serum was isolated by centrifugation (2000 × g) with 3K30 Laboratory
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Centrifuge (Sigma, USA). Supernatant was collected and stored at -20 °C for
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further analysis of TIMP-1, VEGF, IFN-γ and TNF-α level by enzyme-linked
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immunosorbent assay (ELISA) kit following the manufacturer’s protocol.
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2.10. Statistical analysis
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All data were expressed as mean ± SD. Data were analyzed statistically
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by the SPSS 11.5 programs software package. *P<0.05 was regarded as
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significant and **P<0.01 was considered as very significant.
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3. Results and discussion
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3.1. Preparation and characteristics of CMCS
As shown in Fig. 1 of the IR spectra of the sodium salt of CMCS, the
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presence of asymmetric and symmetrical stretching vibration of COO– at 1594
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cm-1 and 1410 cm-1 indicated the presence of carboxyl groups, which
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absorption peak at 1737 cm-1 was the stretching vibration of C=O, which
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confirmed the protonation of the carboxyl group. All the results, which are in
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accordance with the work of Xie et al. (Xie, Xu, Wang, & Liu, 2002), indicated
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that substitution occurred mainly at the C-6 position.
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confirmed the carboxymethy-lation of chitosan. Furthermore, the absorption peak at 1064 cm-1 was the stretching vibration of C-O which was reacted by primary alcohol, indicating that the carboxymethy-lation reaction occurs mainly in the C-6 position. Finally, the absorption peak at 2921 cm-1 and 1324 cm-1 was the stretching vibration and bending vibration of C-H respectively, and the strong broad absorption peak at 3376 cm-1 was the stretching vibration of O-H and N-H. As shown in Fig. 1 of the IR spectra of the protonated CMCS, the
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The prepared CMCS was determined to have a molecular weight of 194.6
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kDa with 94.66% degree of deacetylation. The degree of substitution on CMCS
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was found to be 108.41%. Heavy metal content and intracellular toxin content
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were lower than 10 μg/g and 0.5 EU respectively, which was in line with the
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national standards. Specific data on the properties of CMCS were listed in
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Table 1.
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3.2. Effect of CMCS on the growth of HUVECs in vitro
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Tumor angiogenesis, the formation of new capillaries from preexisting
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blood vessels, is considered essential for the spread and metastasis of tumor.
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Proliferation and migration of endothelial cells are essential to angiogenesis
241
(Wang, et al., 2014). According to our previous studies, CMCS exerted growth
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inhibitory effects on BEL-7402, SGC-7901 and Hela cells (P<0.05) (Zheng, et
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al., 2011). The anti-angiogenic effects of CMCS were carried out using
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HUVECs (Mitzner, Lee, Georgakopoulos, & Wagner, 2000) in vitro in this study.
245
To determine whether the growth of HUVECs was influenced by CMCS
246
treatment, morphologic differences and cell proliferation between control group
247
and CMCS treated groups was compared. After HUVECs were incubated with
248
various concentrations of CMCS for 48h, no obvious changes were observed.
249
Cell in each group spread good, rich cytoplasm (Fig. 2A). Cytotoxicity was
250
assessed by MTT-assay in HUVECs after CMCS treatment (Fig. 2B). The
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viability of non-treated cells served as control and was defined to be 100%. No
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significant decreases of cell viability could be observed after 24 h and 48 h
253
incubation with CMCS (P>0.05). Therefore, CMCS was non-toxic at the
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concentrations range from 0.5 mg/ml to 2.0 mg/ml. 3.3. Inhibition of cell migration by CMCS treatment Tumor angiogenesis is the essential process of tumor growth and
metabolism (Zong, et al., 2013), and newborn vessels take tumor metabolic waste away and help tumor metastasis. Studies have showed that drugs can effectively inhibit tumor growth, metabolism and malignant invasion by
260
inhibiting the ability of tumor angiogenesis. The theoretical foundation of
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chemotherapy with the target of endothelial cells is that the tumor cannot grow
262
or infest without the new vessels. Human umbilical vein endothelial cells
263
(HUVECs) was used as model in vitro, and scarification and transwell chamber
264
assay (Wu, Yao, Bai, Du, & Ma, 2010; Yao, Wu, Zhang, & Du, 2014) was
265
carried out to evaluate the impact of CMCS on the migration of HUVECs. 9
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The two-dimensional migration of CMCS had been measured by scratch
267
test, and then three-dimensional migration was performed by transwell
268
migration test. As shown in Fig. 3 (A & B), the scarification in HUVECs of
269
control group was covered by migrating cells after 48 h, while the migration of
270
HUVECs incubated with CMCS had declined in concentration-dependent and
271
time-dependent manner. The number of cells traveled through the 8 μm-
272
membrane was on behalf of the migration capacity of HUVECs. As shown in
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Fig. 3 (C & D), migration of HUVECs was significantly inhibited by treatment
274
with CMCS in a concentration-dependent manner (P<0.05). Results above
275
implied that CMCS could inhibit angiogenesis in vitro.
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3.4. Effects of CMCS on the growth of H22 tumor tissue in vivo
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Since CMCS showed significant anti-tumor and anti-angiogenesis effects
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in vitro, the anti-tumor and anti-angiogenesis effects of CMCS were
279
investigated in vivo using H22 tumor-bearing mice model. The inhibitory effects
280
of CMCS on the growth of H22 tumor tissue in each group was showed in
281
Table 2. The inhibitory rates of CMCS at doses of 75 mg/kg, 150 mg/kg and
282
300 mg/kg were 32.63%, 51.43% and 29.89%, respectively. CMCS at different
283
doses all significantly inhibited tumor growth (P<0.05), compared with the
284
control group.
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3.5. Effects of CMCS on histopathology and CD34 expression in H22 tumor
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treatment groups had an irregular shape and the nuclei of which was twisted
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and dissolved then the tumor necrotic cells die grew into slices. According to
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the data of inhibition on tumor growth and pathological sections of H22 tissues,
295
it was clear that CMCS was effective in repressing H22 growth in vivo.
286 287 288 289 290
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tissue
The effect of the CMCS on histopathology of H22 tumor tissue was
examined by HE staining of paraffin sections. As shown in Fig. 4A, the tumor cells of the control group grew normally in accordance with close-packed rule in the form of antenna or scalene triangle, with prominent nucleoli and abundant cytoplasm. Whereas the majority of the tumor cells in CMCS
Due to the complex molecular mechanism of tumor angiogenesis, the
297
method
for
investigating
angiogenesis
was
imperfect.
Generally,
298
immunochemistry methods have been adopted to label endothelial cells of 10
Page 10 of 25
blood vessels within the tumor to reflect new blood vessels indirectly. Among
300
large amount of vascular endothelial cell markers, CD34 antigen is suitable in
301
studying the newborn blood vessels of malignant hepatic tumor (Folkman,
302
1996). The effects of CMCS treatment on CD34 expression in H22 tumor
303
tissue was observed by immunehistochemistry. As shown in Fig. 4B, positive
304
staining of CD34 was brownish yellow granular, located in vascular endothelial
305
cell membrane and cytoplasm. The expression of CD34 in the control group
306
was strong positive, while CD34 expression was significantly reduced in
307
CMCS treated groups (150 mg/kg and 300mg/kg). Expression of CD34 in
308
CMCS treated groups was significantly decreased (P<0.05, Fig. 4C), indicating
309
that CMCS could inhibit tumor angiogenesis in vivo in a dose-dependent
310
manner.
311
3.6. Effects of CMCS on VEGF and TIMP-1 levels in serum
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Angiogenesis of tumor is the base for tumor growth and invasion. More
313
than 40 tumor angiogenesis-related factors have been reported, including
314
vascular endothelial growth factor VEGF, angiostatin, and angiogenesis
315
inhibitor TIMP-1 and so on (Hanahan & Folkman, 1996).
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VEGF, a highly specific mitogen for vascular endothelial cells, regulates vasculogenesis,
318
embryogenesis and in adults. VEGF is the most potent angiogenesis factor
319
and has been detected in many human tumors. VEGF and its receptor play a
321 322 323 324 325
and
vascular
maintenance
during
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angiogenesis,
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critical role in tumor-associated angiogenesis and have become the new targets of anti-tumor therapy. TIMP-1, the natural inhibitor of matrix metalloproteinase (MMP), could inhibit the degradation of the extracellular matrix in order to reduce tumor angiogenesis and malignant transformation. The effects of CMCS treatment on VEGF and TIMP-1 levels in mouse
serum were evaluated. As shown in Table 3, the level of VEGF was decreased
326
in a dose-dependent manner after 14 d treatment with CMCS. And the level of
327
TIMP-1 was increased at higher doses after 14 d treatment with CMCS. The
328
expressions of VEGF and TIMP-1 can be considered as important factors in
329
regulate tumor angiogenesis, the stimulate effect of the associated key
330
cytokines may be one of the mechanisms of the anti-angiogenesis for CMCS.
331
3.7. Effects of CMCS on mice thymus index, spleen index and serum TNF-α, 11
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IFN-γ levels The body’s immune system including the spleen, the thymus, lymph
334
nodes and lymph ducts build up our body to fight infection and cancer (Effros,
335
2003). In this experiment, thymus and spleen tissues were isolated completely
336
and weighed to calculate thymus index and spleen index (Fig. 5A). It was
337
found that CMCS treatment could increase the thymus index and spleen index
338
of the mice. CMCS significantly increased the thymus index in mice (P<0.05)
339
but no significant influence on spleen index was observed (P>0.05).
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Immune-related cytokines such as TNF-α and IFN-γ have been used in
341
clinical cancers treatment for many years (Aulitzky et al., 1989; Eggermont et
342
al., 1996). TNF-α is a cytokine mediating inflammatory reaction and immune
343
responses, and has various biological activities. TNF-α can enhance host
344
immune function (Ebert, Meuter, & Moser, 2006) and induce the apoptosis of
345
tumor cells (Chang, et al., 2006; Hur, et al., 2003). IFN-γ, produced by
346
activated T cells and NK cells, is a pleiotropic cytokine with immunomodulatory
347
effects. IFN-γ is produced by the immune system to fight tumors by promote
348
tumor cells apoptosis and kill them (Trubiani, Bosco, & Di Primio, 1994; Ahn,
349
Pan, Vickers, & McDonald, 2002). In this experiment, the concentration of
350
IFN-γ and TNF-α in serum was detected by ELISA assay. It was found that
351
CMCS treatment could enhance serum IFN-γ and TNF-α levels compared with
352
the control gourp (Fig. 5B). Our results showed that CMCS treatment could
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improve the immune system. The anti-tumor effects of CMCS maybe partially related with the immune-stimulating functions of CMCS. However, further investigations
are
required
to
understand
the
underlying
molecular
mechanisms by which CMCS regulates the relevant cytokines. 4. Conclusion
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The anti-tumor and anti-angiogenesis effects of CMCS were evaluated in
359
vitro and in vivo to make CMCS a potential biomaterial for many biomedical
360
applications. Our results indicated that CMCS was non-cytotoxic on HUVECs.
361
Treatment with CMCS significantly inhibited the migration of HUVECs
362
compared with the control group (P<0.05) in vitro. CMCS significantly inhibited
363
tumor growth of H22 in vivo compared with control group (P<0.05) as reflected
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by. The immune-stimulating and the anti-angiogenesis functions may
365
contribute to the anti-tumor effects of CMCS.
366
Acknowledgements This work was supported by Ocean University of China and by a grant
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from the National High-tech R & D Program of China (2014AA093605).
369
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as doxorubicin carriers for effective anti-tumor activity: preparation and in vitro evaluation. Colloids and Surfaces B: Biointerfaces, 94, 184-191. Kumar, M. N. V. R., Muzzarelli, R. A. A., Muzzarelli, C., Sashiwa, H., & Domb, A. J. (2004). Chitosan Chemistry and Pharmaceutical Perspectives. Chemical Reviews, 104(12), 6017-6084. Maeda, Y., Kimura, Y. (2004). Antitumor effects of various low-molecular-w eight chitosans are due to increased natural killer activity of intestinal intraepithelial lymphocytes in sarcoma 180-bearing mice. The Journal of Nutrition, 134(4), 945-950. Ministry of Agriculture of the People’s Republic of China, 2005. Chitin and chitosan. Aquatic industry standards of the People’s Republic of China, SC/T 3403-2004, 1-6 (in Chinese). Mitzner, W., Lee, W., Georgakopoulos, D., & Wagner, E. (2000). Angiogenesis in the mouse lung. American Journal Of Pathology, 157(1), 93-101. Miura, S., Mitsui, K., Heishi, T., Shukunami, C., Sekiguchi, K., Kondo, J., et al. (2010). Impairment of VEGF-A-stimulated lamellipodial extensions and motility of vascular endothelial cells by chondromodulin-I, a cartilage-derived angiogenesis inhibitor. Experimental Cell Research, 316(5), 775-788. Mohamed, N. A., & Abd El-Ghany, N. A. (2012). Preparation and antimicrobial activity of some carboxymethyl chitosan acyl thiourea derivatives. International Journal Of Biological Macromolecules, 50 (5), 1280-1285. Muzzarelli. R.A.A. (1988). Carboxymethylated chitins and chitosans. Carbohydrate Polymers, 8(1), 1-21. Muzzarelli. R.A.A. (1993). Biochemical significance of exogenous chitins and chitosans in animals and patients. Carbohydrate Polymers, 20(1), 7-16. Pae, H. O., Seo, W. G., Kim, N. Y., Oh, G. S., Kim, G. E., Kim, Y. H., et al. (2001). Induction of granulocytic differentiation in acute promyelocytic leukemia cells (HL-60) by water-soluble chitosan oligomer. Leuk Res, 25(4), 339-346. Park, H. J., Kim, M. N., Kim, J. G., Bae, Y. H., Bae, M. K., Wee, H. J., et al. (2007). Up-regulation of VEGF expression by NGF that enhances reparative angiogenesis during thymic regeneration in adult rat. Biochim Biophys Acta, 1773(9), 1462-1472. Trachsel, E., & Neri, D. (2006). Antibodies for angiogenesis inhibition, vascular targeting and endothelial cell transcytosis. Adv Drug Deliv Rev, 58(5-6), 735-754. Trubiani, O., Bosco, D., & Di Primio, R. (1994). Interferon-gamma (IFN-gamma) induces programmed cell death in differentiated human leukemic B cell lines. Experimental Cell Research, 215(1), 23-27. Vaghani, S. S., Patel, M. M., Satish, C. S., Patel, K. M., & Jivani, N. P. (2012). Synthesis and characterization of carboxymethyl chitosan hydrogel: application as pH-sensitive delivery for nateglinide. Curr Drug Deliv, 9, 628-636. Wang, Y., Shen, X., Liao, W., Fang, J., Chen, X., Dong, Q., et al. (2014). A heteropolysaccharide, l-fuco-d-manno-1,6-α-d-galactan extracted from Grifola frondosa and antiangiogenic activity of its sulfated derivative. Carbohydrate Polymers, 101, 631-641. Wang, Y., Yang, X., Yang, J., Wang, Y., Chen, R., Wu, J., et al. (2011). Self-assembled nanoparticles of methotrexate conjugated O-carboxymethyl chitosan: Preparation, characterization and drug release behavior in vitro. Carbohydrate Polymers, 86(4), 1665-1670. Wu, H., Yao, Z., Bai, X., Du, Y., & Ma, X. (2010). Chitooligosaccharides inhibit nitric oxide mediated migration of endothelial cells in vitro and tumor angiogenesis in vivo. Carbohydrate Polymers, 82(3), 927-932. Xie. W., Xu. P., Wang. W., & Liu, Q. (2002). Preparation and antibacterial activity of a water-soluble chitosan derivative. Carbohydrate Polymers, 50(1), 35-40. Yao, Z., Wu, H., Zhang, S., & Du, Y. (2014). Enzymatic preparation of κ-carrageenan oligosaccharides and their anti-angiogenic activity. Carbohydrate Polymers, 101, 359-367. Yu, Z., Zhao, L., & Ke, H. (2004). Potential role of nuclear factor-kappaB in the induction of nitric oxide nd tumor necrosis factor-alpha by oligochitosan in macrophages. International Immunopharmacology, 4(2), 193-200. Zamani, A., Henriksson, D., & Taherzadeh, M. J. (2010). A new foaming technique for production of superabsorbents from carboxymethyl chitosan. Carbohydrate Polymers, 80(4), 1091-1101. Zheng, M., Han, B., Yang, Y., & Liu, W. (2011). Synthesis, characterization and biological safety of O-carboxymethyl chitosan used to treat Sarcoma 180 tumor. Carbohydrate Polymers, 86(1), 231-238. Zong, A., Zhao, T., Zhang, Y., Song, X., Shi, Y., Cao, H., et al. (2013). Anti-metastatic and anti-angiogenic activities of sulfated polysaccharide of Sepiella maindroni ink. Carbohydrate Polymers, 91(1), 403-409.
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Table 1 Physicochemical properties of CMCS
471 472 473
Table 2 Effect of CMCS on the growth of H22 tumor tissue * ** data represents mean ± SD, n = 10, P<0.05, P<0.01 significant difference compared with control group.
474 475 476
Table 3 Effects of CMCS on the expression of VEGF and TIMP-1 * ** data represents mean ± SD, n = 10, P<0.05, P<0.01 significant difference compared with control group.
cr us an M d te Ac ce p
477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512
ip t
469 470
15
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513
Table 1 Molecular Water Weight Content
Degree of Carboxymethylation
12.21%
94.66%
108.41%
Viscosity Coefficient 23 mPa· S
Heavy Metal Content ≤ 10 μg/g
Intracellular Toxin Content < 0.5 EU
cr us an M d te Ac ce p
514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551
18.36%
Degree of Deacetylation
ip t
194.6 kDa
Ash Content
552 16
Page 16 of 25
Table 2 Group
Dosage
weight of tumor (g)
(mg/kg)
weight
–
2.10±0.43
–
CMCS
75
1.42±0.36*
32.63%
CMCS
150
1.02±0.21**
51.43%
CMCS
300
1.47±0.41*
29.89%
te
d
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an
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cr
Control
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554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590
inhibitory effect of tumor
ip t
553
17
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591
Table 3 Group
Dosage (mg/kg)
VEGF content (ng/l)
TIMP-1 content (pg/ml)
Control
–
156.43±7.45
5572.22±381.37
CMCS
75
146.98±21.82
5884.17±942.07
CMCS
150
138.03±13.35**
6370.06±941.12*
CMCS
300
136.30±7.30**
6189.44±823.84*
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592 593
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Fig. 2. Effects of CMCS on cell morphology and proliferation of HUVECs. (A) Morphological changes of HUVECs were examined under a light microscope (original magnification, 100×). (a) cells treated with complete medium; (b) cells treated with CMCS at the concentrations of 0.5 mg/ml; (c) cells treated with CMCS at the concentrations of 1.0 mg/ml; (d) cells treated with CMCS at the concentrations of 2.0 mg/ml. (B). MTT test measured the proliferation rate and showed that there was no significant difference of cell viability between CMCS treated groups whose concentrations were below 2.0 * ** mg/ml and control group. data represents mean ± SD, n = 6, P<0.05, P<0.01 significant difference compared with control group.
an
us
cr
Fig. 3. Inhibition on cell migration of HUVECs after incubation with CMCS solutions (A) Scarification changes of HUVECs were recorded using the inverted microscope (original magnification, 100×). (B) Inhibition rate of CMCS on the two-dimensional migration of HUVCE. (C) Photographs of HUVECs migrated through the 8 μm- membranes were taken by the light microscope (original magnification, 100×). (D) Inhibition rate of CMCS on the three-dimensional migration of HUVCE. (a) cells treated with complete medium; (b) cells treated with CMCS at the concentrations of 0.5 mg/ml; (c) cells treated with CMCS at the concentrations of 1.0 mg/ml; (d) cells treated with CMCS at the * ** concentrations of 2.0 mg/ml. data represents mean ± SD, n = 6, P<0.05, P<0.01 significant difference compared with control group.
d
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Fig. 4. Pathological and immunohistochemical detection on H22 tumor tissue (A) Effect of the CMCS on histopathology of H22 tumor tissue was recorded by the light microscope (original magnification, 400×). (B) Photographs of effect on CD34 expression of H22 tumor tissue were taken by the light microscope (original magnification, 400×). (C) Inhibition rate of CMCS on CD34 expression of H22 tumor tissue. (a) mice treated with normal saline; (b) mice treated with CMCS at the dose of 75 mg/kg; (c) mice treated with CMCS at the dose of 150 mg/ kg; (d) mice treated with CMCS at * ** the dose of 300 mg/kg. data represents mean ± SD, n = 10, P<0.05, P<0.01 significant difference compared with control group.
te
594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642
Fig. 1. IR spectra of CTS (A), the sodium salt of CMCS (B) and the protonated CMCS (C)
Fig. 5. Effect of CMCS on thymus index, spleen index and cytokines associated with immunity * ** data represents mean ± SD, n = 10, P<0.05, P<0.01 significant difference compared with control group.
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644
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645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674
Fig. 1
us
643
20
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675
A a
b
ip t
676
us
677 678
d
cr
c
B
60
30
24h 48h
0.0 0.5 1.0 2.0 Concentration of CMCS (mg/ml)
Fig. 2
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te
d
0
679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700
an
90
M
Proliferation rate( %)
120
21
Page 21 of 25
701 702
A 0h
24h
48h
ip t
a
703
cr
b
us
704
an
c
M
705
B
inhibition rate( %)
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50
te
706 707 B 708
d
d
24h
**
30 20
*
*
48h
**
**
10
0
709 710 711
**
40
0.5 1.0 2.0 Concentration of CMCS (mg/ml)
C
22
Page 22 of 25
a
b
ip t
712
d
718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734
50 40 30
*
*
te
0 0.0
d
20 10
M
**
0.5
1.0
1.5
2.0
2.5
Concentration of CMCS (mg/ml)
Ac ce p
716 717
an
D Inhibition rate of migration (%)
713 714 D 715
us
cr
c
Fig. 3
23
Page 23 of 25
A a
b
c
d
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735
B
an
737 738
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cr
736
b
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a
d
739
d
740 741 742
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C
Inhibition rate of CD34 (%)
80
**
60
40
* *
20
0 75
743 744 745 746
150
300
Dosages of CMCS (mg/kg)
Fig. 4
24
Page 24 of 25
A Spleen index
Thymus index 6
15
**
12
*
4
9
3 6
2
3
1 0
Control
300
0
** *
400
**
M
300 200
d
100 0
**
an
**
us
IFN-γ
TNF-α 500
Control
75
150
dosage of CMCS (mg/kg)
1200 1000 800 600 400 200
300
0
Fig. 5
Ac ce p
te
TNF-α content in mouse serum (ng/l)
B
IFN-γ content in mouse serum (ng/l)
750 751 752
150
dosage of CMCS (mg/kg)
748 749
75
ip t
**
Spleen index
Thymus index
5
cr
747
25
Page 25 of 25
S0144-8617(15)00356-2 http://dx.doi.org/doi:10.1016/j.carbpol.2015.04.040 CARP 9871
To appear in: Received date: Revised date: Accepted date:
31-10-2014 13-2-2015 20-4-2015
Please cite this article as: Jiang, Z., Han, B., Li, H., Yang, Y., and Wanshun, L.,Carboxymethyl chitosan represses tumor angiogenesis in vitro and in vivo, Carbohydrate Polymers (2015), http://dx.doi.org/10.1016/j.carbpol.2015.04.040 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1 Carboxymethyl chitosan (CMCS) was non-cytotoxic on HUVECs.
2
2 Treatment with CMCS significantly inhibited the migration of HUVECs.
3
3 CMCS significantly inhibited tumor growth and CD34 expression of H22 in vivo.
4
4 CMCS could regulate the levels of VEGF and TIMP-1 in serum of mice.
5
5 The anti-angiogenesis function may contribute to the anti-tumor effects of CMCS.
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Page 1 of 25
Carboxymethyl chitosan represses tumor
8
angiogenesis in vitro and in vivo
9
Zhiwen Jiang, Baoqin Han*, Hui Li, Yan Yang, Liu Wanshun
10 11
Biochemistry Laboratory, College of Marine Life Sciences, Ocean University of China, Qingdao 266003, PR China
12
Abstract chitosan
(CMCS),
with
cr
Carboxymethyl
13
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7
potent
water
solubility,
biocompatibility, and non-toxicity, has emerged as a promising candidate for
15
biomedical applications. In this study, the anti-tumor angiogenesis effects of
16
CMCS were evaluated in vitro and in vivo. Our results showed that CMCS
17
could inhibit the 2-dimensional and 3-dimensional migration of human
18
umbilical vein endothelial cells (HUVECs) in vitro. CMCS significantly inhibited
19
the growth of mouse hepatocarcinoma 22 tissues and could promote tumor
20
cell necrosis as suggested by pathological observations. The CD34 expression
21
in H22 tumor tissue, the levels of vascular endothelial growth factor and tissue
22
inhibitor of metalloproteinase 1 in serum was regulated by CMCS treatment.
23
CMCS could significantly improve thymus index, spleen index, tumor necrosis
24
factor α and interferon γ level. In a conclusion, CMCS possessed potent
25
anti-tumor effects by inhibiting tumor angiogenesis, stimulating immune
27 28 29 30
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functions. Our date provide more foundation for application of CMCS in biomedicine or biomaterials for targeted anticancer drugs delivery. Key words: Carboxymethyl chitosan; anti-tumor; angiogenesis; in vitro; in vivo
1. Introduction
31
Angiogenesis, the formation of new capillaries from pre-existing blood
32
vessels (Miura, et al., 2010), is critical for normal embryonic development and
33
also contributes many pathological states including tumor growth and the
34
development of metastases (Park, et al., 2007). Solid tumors, which grow
35
beyond a size of a few millimeters, need new blood vessels to provide oxygen *
Corresponding author. Tel.: +86 532 82032105; fax: +86 532 82032105. E-mail addresses: [email protected] (B. Han). 2
Page 2 of 25
and nutrients and take metabolic waste away (Folkman, 1972). The inhibition
37
of angiogenesis represents an avenue for blocking tumor growth (Trachsel &
38
Neri, 2006). After Folkman proposed angiogenesis as a target for cancer
39
therapy in the last century, enormous research emerged in the past decades.
40
Bevacizumab was approved for the treatment of metastatic colorectal cancer
41
by FDA in 2004, making anti-angiogenesis in tumor therapy a reality. The
42
formation of new blood vessels is the result of the mutual regulation of
43
pro-angiogenic compounds like vascular endothelial growth factor (VEGF),
44
basic fibroblast growth factor (bFGF), transforming growth factor-β (TGF-β),
45
platelet-derived growth factor (PDGF), matrix metalloproteinases (MMPs) and
46
anti-angiogenesis factors such as angiostatin, endostatin, and tissue inhibitor
47
of metalloproteinase (TIMPs). The balance will be broken under pathological
48
conditions.
Chitosan (CTS) is the N-deacetylated derivative and has been widely
50
used as biomedical materials due to its nontoxicity, biocompatibility,
51
hemocompatibility and biodegradability (Muzzarelli, 1993; Kumar, Muzzarelli,
52
Muzzarelli, Sashiwa, & Domb, 2004). Chitosan is also reported to have
53
anti-tumor effects by inhibiting tumor cell proliferation (Maeda & Kimura, 2004),
54
inducing apoptosis (Pae, et al., 2001), and enhancing immune functions (Yu,
55
Zhao, & Ke, 2004). However, the solubility of chitosan greatly restricts its wide
56
application (Faizuloev, et al., 2012). Chitosan has different groups that can be
57
63 64
curcumin, doxorubicin and so on (Anitha, Chennazhi, Nair, & Jayakumar, 2012;
65
Anitha, et al., 2011; Jin, et al., 2012; Wang, et al., 2011). Our previous studies
66
showed that CMCS could inhibit tumor cells proliferation and tumor tissue
67
growth. In this study, we further investigated the anti-tumor angiogenesis
68
effects of CMCS. Our data will provide more bases for the clinical application of
69
CMCS as biomedical materials in tumor surgery.
58 59 60 61 62
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chemically modified. Carboxymethyl chitosan (CMCS) is one of the most important water-soluble chitosan derivates (Muzzarelli, 1988). CMCS has been proven to have favorable properties, such as lower toxicity, better biocompatibility, and biodegradability (Ji, Wu, Liu, Chen, & Xu, 2012). CMCS has been widely used in biomedical fields such as drug carriers, antimicrobial material, gene delivery system and tissue engineering. CMCS has been developed as anti-cancer drug delivery systems such as 5-Flourouracil,
3
Page 3 of 25
70
2. Experimental
71
2.1. Reagents and antibodies Chitosan with a degree of deacetylation of 93% and molecular weight of
73
350 kDa was purchased from Qingdao Biotemed Biomaterial Co., Ltd
74
(Qingdao, China). CMCS were synthesized, purified and identified in our
75
laboratory. Rabbit polyclonal anti-mouse CD34 antibody, Horse Radish
76
Peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody,
77
3,3-diaminobenzidine (DAB), Mouse VEGF ELISA Kit, Mouse TIMP-1 ELISA
78
Kit, Mouse tumor necrosis factor α (TNF-α) ELISA Kit and Mouse Interferon γ
79
(IFN-γ) ELISA Kit were purchased from Boster Biological Engineering Co., Ltd
80
(Wuhan China). 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide
81
(MTT) was obtained from Sigma- Aldrich (St. Louis, MO, USA). RPMI 1640
82
medium and fetal bovine serum (FBS) was obtained from Gibco®, Life
83
Technologies (Carlsbad, CA, USA). Transwell, 96 well plates and 24 well
84
plates were purchased from Corning INC. (Corning, NY, USA). All other
85
chemicals and reagents were of the highest commercial grade available.
86
2.2. Animals and cell lines
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Forty female Kunming mice (weight range, 18-22 g) were obtained from
88
Medicine Inspecting Institute of Qingdao, China, and maintained in the animal
89
care facility of the Biochemistry Laboratory of Ocean University of China. All
90
animals were kept under a 12h light-dark cycle at consistent temperature
92 93 94 95 96
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(25±3) and relative humidity (60-70%). Experiments were performed in accordance with the ethical guidelines of the Shandong Province Experimental Animal Management Committee and were in complete compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Mouse Hepatocarcinoma 22 (H22) cells and Human Umbilical Vein
97
Endothelial Cells (HUVECs) were offered by the Institute of Pharmacology,
98
Ocean University of China. Endothelial Cells between passage 3 and 10 were
99
used in the experiments. The cells were grown on 0.3% gelatin coated dishes
100
using RPMI 1640 medium supplemented with 10% fetal bovine serum, 100
101
units/ml penicillin and 100 μg/ml streptomycin at 37 °C in a humidified
102
incubator with 5% CO2 in 95% air.
103
2.3. Preparation and characterization of CMCS 4
Page 4 of 25
CMCS was prepared from chitosan according to the previously published
105
literature by our laboratory (Zheng, Han, Yang, & Liu, 2011). The sodium salt
106
of CMCS was synthesized by chemical reaction with mono-chloroacetic acid
107
under alkaline condition, and then treated with HCl in methanol that protonates
108
the carboxyl group. (Vaghani, Patel, Satish, Patel, & Jivani, 2012). Carboxyl
109
structures and molecular weight was determined by fourier-transformed
110
infrared spectroscopy (FTIR) and high performance liquid chromatography
111
(HPLC). The IR spectrum of CMCS (~0.5 mg) was acquired on the NEXUE
112
470 instrument (Nicolet Co., USA) as KBr pellets at room temperature.
113
Standard curves were obtained according to the Tmax and molecular weight of
114
Pullulan standards with Agilent 1200 (Agilent Co., USA). The molecular weight
115
of CMCS was determined by standard curves and the Tmax. The degree of
116
deacetylation and the degree of substitution of carboxymethyl chitosan was
117
determined by pH-titration (industry standards of P.R. China, 2005; Mohamed
118
& Abd El-Ghany, 2012).
119
2.4. In vitro cell proliferation studies
HUVECs were seeded in 96-well culture plates at a density of 4×103
121
cells/well in 200 μl of culture medium (plus 10% FBS) and allowed to attach
122
and grow for 24 h. The cells were then exposed to 200 μl complete medium
123
without or with CMCS at different concentrations. The morphological changes
124
of cells were observed and photographed with the T1-SM 100 inverted
125
131 132
calculated using the percentage of proliferation rate (PR/%). Each assay was
133
repeated three times and all experiments were performed in six wells. The PR
134
was calculated according to the formula below:
126 127 128 129 130
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microscope (Nikon Co. Japan) at 100 magnification. MTT assay was used to measure the effects of CMCS (0.5 mg/ml, 1.0 mg/ml, and 2.0 mg/ml) on HUVECs proliferation 24 h and 48 h after treatment. MTT (5 mg/ml, 20 μl) was added to each well. After 4 h of incubation with MTT, medium was removed and 150 μl of DMSO was added. The plate was then read at 492 nm on Multiskan Go 151 Microplate Scanning Spectrophotometer (Thermo Fisher Scientific, INC, USA). The effects of CMCS on HUVECs proliferation were
135
5
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136
2.5. Wounding migration assay HUVECs suspended in complete medium (plus 10% FBS) were plated in
138
24 well plates at 3×104 cells/well and incubated for 12 h to achieve 90%
139
confluence. After 12 h incubation in starvation medium (plus 1% FBS), cell
140
monolayers were scored vertically down the center of each well with a sterile
141
200 μl tip. After wounding, the cultures were washed with phosphate buffer
142
solution (PBS) twice to remove detached cells. Fresh growth medium (plus
143
10% FBS) without or with CMCS (0.5 mg/ml, 1.0 mg/ml, and 2.0 mg/ml) were
144
added. Images of the wound in each well were taken at 0 h, 24 h, and 48 h
145
under the inverted microscope at the magnification of 10 ×. Relative migration
146
distances on each time point is calculated by subtracting scratch length from
147
the initial length of the scratch. The effects of CMCS on the wounding
148
migration of HUVECs were calculated using the percentage of inhibition rate
149
(IR/%). Each assay was repeated three times and all experiments were
150
performed in six wells. The IR was calculated as follows:
M
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137
151
2.6. Transwell migration assay
d
152
HUVECs (5×104 cells /100 μl) re-suspended in RPMI 1640 medium
154
containing 1% FBS were added to the cell culture inserts (8 μm pore size) and
155
FBS (10% V/V) was used as chemoattractant in the lower wells of the transwell.
157 158 159 160 161 162
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The cells in upper inserts was incubated with CMCS (0.5 mg/ml, 1.0 mg/ml, and 2.0 mg/ml) and allowed to migrate for 14 h at 37 °C. Then the cells were fixed in methanol and stained with crystal violet. The photographs of HUVECs that had migrated to the bottom surface of the insert in different groups were taken at 200 × magnification under a microscope. Cell migration was determined by measuring the absorbance of crystal violet-stained cells at 570 nm. The effects of CMCS on the transwell migrations of HUVECs were
163
calculated using the percentage of inhibition rate (IR/%). Each assay was
164
repeated three times and all experiments were performed in six wells. The IR
165
was calculated according to the formula below:
166 167
2.7. Anti-tumor experiment in vivo 6
Page 6 of 25
H22 cells were kept in Kunming moue abdominal cavity for three
169
generations. Single-cell suspensions were prepared, washed, and diluted in
170
normal saline. Cell suspensions (200 μl, approximately 4×106 H22 cells) were
171
injected subcutaneously in the right axilla to each mouse. Then the animals
172
were randomizedly assigned into the control group and three experimental
173
groups. Different doses of CMCS (75 mg/kg, 150 mg/kg and 300 mg/kg body
174
weight) in normal saline were administer to experimental groups peritoneal
175
injectionat 1, 3, 5, 7, 9, 11 and 13 days after tumor implantation. Animals in the
176
control group were administered with saline. After the last administration, mice
177
were fasting for 12 hours. The whole venous blood was collected from the orbit.
178
Mice were then killed and tumor tissue, thymus and spleen of each animal
179
were stripped intactly and weighed. Thymus and spleen index is the ratio of
180
thymus or spleen weight to the body weight. The inhibition of CMCS on the
181
growth of H22 tumor was calculated using the percentage of inhibition rate (IR).
182
Each assay was repeated three times and all experiments were performed in
183
ten mice. The IR was calculated according to the formula below:
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2.8. Histology of Tumor samples and Immunohistochemical studies
te
184
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Tumor tissues were fixed in 4% paraformaldehyde solution for 24 h, and
187
then embedded in paraffin for sectioning. For each sample of the four groups,
188 189 190 191 192 193 194
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186
some consecutive 4 μm-thick sections were stained with hematoxylin and eosin (HE) for the pathologic examinations, while parallel sections were stained using the endothelium-specific CD34 antibody as described below. The presence of blood vessels was determined by immunohistochemistry using hematopoietic progenitor cell antigen CD34. After three 3-min washes in PBS, sections were incubated with 3% hydrogen peroxide in methanol for 10 min to quench endogenous peroxidase. After a further three washes,
195
nonspecific protein-binding sites were blocked by 20-min incubation with 5%
196
bovine serum albumin (BSA) in PBS. The blocking solution was drained and
197
the slices were incubated at 4 overnight with a 1:150 dilution of the
198
endothelial cell marker CD34 antibody. Negative controls were performed by
199
replacing the primary antibody with PBS. After a further three rinses and a
200
blocking step, sections were incubated with HRP-conjugated goat anti-rabbit 7
Page 7 of 25
IgG for 30 min at 37 . The sections were washed and then stained with the
202
chromogen diaminobenzidine for 5-10 min, then counterstained with
203
hematoxylin. Light microscopy slides were observed and photographed using
204
the E200 microscope (Nikon Co. Japan). All photomicrographs were taken with
205
DT300 digital camera software.
206
2.9. Assay of TIMP-1, VEGF, IFN-γ and TNF-α
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Serum was isolated by centrifugation (2000 × g) with 3K30 Laboratory
208
Centrifuge (Sigma, USA). Supernatant was collected and stored at -20 °C for
209
further analysis of TIMP-1, VEGF, IFN-γ and TNF-α level by enzyme-linked
210
immunosorbent assay (ELISA) kit following the manufacturer’s protocol.
211
2.10. Statistical analysis
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All data were expressed as mean ± SD. Data were analyzed statistically
213
by the SPSS 11.5 programs software package. *P<0.05 was regarded as
214
significant and **P<0.01 was considered as very significant.
215
3. Results and discussion
216
3.1. Preparation and characteristics of CMCS
As shown in Fig. 1 of the IR spectra of the sodium salt of CMCS, the
218
presence of asymmetric and symmetrical stretching vibration of COO– at 1594
219
cm-1 and 1410 cm-1 indicated the presence of carboxyl groups, which
220
226 227
absorption peak at 1737 cm-1 was the stretching vibration of C=O, which
228
confirmed the protonation of the carboxyl group. All the results, which are in
229
accordance with the work of Xie et al. (Xie, Xu, Wang, & Liu, 2002), indicated
230
that substitution occurred mainly at the C-6 position.
221 222 223 224 225
te
217
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d
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an
212
confirmed the carboxymethy-lation of chitosan. Furthermore, the absorption peak at 1064 cm-1 was the stretching vibration of C-O which was reacted by primary alcohol, indicating that the carboxymethy-lation reaction occurs mainly in the C-6 position. Finally, the absorption peak at 2921 cm-1 and 1324 cm-1 was the stretching vibration and bending vibration of C-H respectively, and the strong broad absorption peak at 3376 cm-1 was the stretching vibration of O-H and N-H. As shown in Fig. 1 of the IR spectra of the protonated CMCS, the
231
The prepared CMCS was determined to have a molecular weight of 194.6
232
kDa with 94.66% degree of deacetylation. The degree of substitution on CMCS
8
Page 8 of 25
was found to be 108.41%. Heavy metal content and intracellular toxin content
234
were lower than 10 μg/g and 0.5 EU respectively, which was in line with the
235
national standards. Specific data on the properties of CMCS were listed in
236
Table 1.
237
3.2. Effect of CMCS on the growth of HUVECs in vitro
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233
Tumor angiogenesis, the formation of new capillaries from preexisting
239
blood vessels, is considered essential for the spread and metastasis of tumor.
240
Proliferation and migration of endothelial cells are essential to angiogenesis
241
(Wang, et al., 2014). According to our previous studies, CMCS exerted growth
242
inhibitory effects on BEL-7402, SGC-7901 and Hela cells (P<0.05) (Zheng, et
243
al., 2011). The anti-angiogenic effects of CMCS were carried out using
244
HUVECs (Mitzner, Lee, Georgakopoulos, & Wagner, 2000) in vitro in this study.
245
To determine whether the growth of HUVECs was influenced by CMCS
246
treatment, morphologic differences and cell proliferation between control group
247
and CMCS treated groups was compared. After HUVECs were incubated with
248
various concentrations of CMCS for 48h, no obvious changes were observed.
249
Cell in each group spread good, rich cytoplasm (Fig. 2A). Cytotoxicity was
250
assessed by MTT-assay in HUVECs after CMCS treatment (Fig. 2B). The
251
viability of non-treated cells served as control and was defined to be 100%. No
252
significant decreases of cell viability could be observed after 24 h and 48 h
253
incubation with CMCS (P>0.05). Therefore, CMCS was non-toxic at the
255 256 257 258 259
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254
cr
238
concentrations range from 0.5 mg/ml to 2.0 mg/ml. 3.3. Inhibition of cell migration by CMCS treatment Tumor angiogenesis is the essential process of tumor growth and
metabolism (Zong, et al., 2013), and newborn vessels take tumor metabolic waste away and help tumor metastasis. Studies have showed that drugs can effectively inhibit tumor growth, metabolism and malignant invasion by
260
inhibiting the ability of tumor angiogenesis. The theoretical foundation of
261
chemotherapy with the target of endothelial cells is that the tumor cannot grow
262
or infest without the new vessels. Human umbilical vein endothelial cells
263
(HUVECs) was used as model in vitro, and scarification and transwell chamber
264
assay (Wu, Yao, Bai, Du, & Ma, 2010; Yao, Wu, Zhang, & Du, 2014) was
265
carried out to evaluate the impact of CMCS on the migration of HUVECs. 9
Page 9 of 25
The two-dimensional migration of CMCS had been measured by scratch
267
test, and then three-dimensional migration was performed by transwell
268
migration test. As shown in Fig. 3 (A & B), the scarification in HUVECs of
269
control group was covered by migrating cells after 48 h, while the migration of
270
HUVECs incubated with CMCS had declined in concentration-dependent and
271
time-dependent manner. The number of cells traveled through the 8 μm-
272
membrane was on behalf of the migration capacity of HUVECs. As shown in
273
Fig. 3 (C & D), migration of HUVECs was significantly inhibited by treatment
274
with CMCS in a concentration-dependent manner (P<0.05). Results above
275
implied that CMCS could inhibit angiogenesis in vitro.
276
3.4. Effects of CMCS on the growth of H22 tumor tissue in vivo
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266
Since CMCS showed significant anti-tumor and anti-angiogenesis effects
278
in vitro, the anti-tumor and anti-angiogenesis effects of CMCS were
279
investigated in vivo using H22 tumor-bearing mice model. The inhibitory effects
280
of CMCS on the growth of H22 tumor tissue in each group was showed in
281
Table 2. The inhibitory rates of CMCS at doses of 75 mg/kg, 150 mg/kg and
282
300 mg/kg were 32.63%, 51.43% and 29.89%, respectively. CMCS at different
283
doses all significantly inhibited tumor growth (P<0.05), compared with the
284
control group.
285
3.5. Effects of CMCS on histopathology and CD34 expression in H22 tumor
291
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te
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an
277
292
treatment groups had an irregular shape and the nuclei of which was twisted
293
and dissolved then the tumor necrotic cells die grew into slices. According to
294
the data of inhibition on tumor growth and pathological sections of H22 tissues,
295
it was clear that CMCS was effective in repressing H22 growth in vivo.
286 287 288 289 290
296
tissue
The effect of the CMCS on histopathology of H22 tumor tissue was
examined by HE staining of paraffin sections. As shown in Fig. 4A, the tumor cells of the control group grew normally in accordance with close-packed rule in the form of antenna or scalene triangle, with prominent nucleoli and abundant cytoplasm. Whereas the majority of the tumor cells in CMCS
Due to the complex molecular mechanism of tumor angiogenesis, the
297
method
for
investigating
angiogenesis
was
imperfect.
Generally,
298
immunochemistry methods have been adopted to label endothelial cells of 10
Page 10 of 25
blood vessels within the tumor to reflect new blood vessels indirectly. Among
300
large amount of vascular endothelial cell markers, CD34 antigen is suitable in
301
studying the newborn blood vessels of malignant hepatic tumor (Folkman,
302
1996). The effects of CMCS treatment on CD34 expression in H22 tumor
303
tissue was observed by immunehistochemistry. As shown in Fig. 4B, positive
304
staining of CD34 was brownish yellow granular, located in vascular endothelial
305
cell membrane and cytoplasm. The expression of CD34 in the control group
306
was strong positive, while CD34 expression was significantly reduced in
307
CMCS treated groups (150 mg/kg and 300mg/kg). Expression of CD34 in
308
CMCS treated groups was significantly decreased (P<0.05, Fig. 4C), indicating
309
that CMCS could inhibit tumor angiogenesis in vivo in a dose-dependent
310
manner.
311
3.6. Effects of CMCS on VEGF and TIMP-1 levels in serum
an
us
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299
Angiogenesis of tumor is the base for tumor growth and invasion. More
313
than 40 tumor angiogenesis-related factors have been reported, including
314
vascular endothelial growth factor VEGF, angiostatin, and angiogenesis
315
inhibitor TIMP-1 and so on (Hanahan & Folkman, 1996).
d
316
M
312
VEGF, a highly specific mitogen for vascular endothelial cells, regulates vasculogenesis,
318
embryogenesis and in adults. VEGF is the most potent angiogenesis factor
319
and has been detected in many human tumors. VEGF and its receptor play a
321 322 323 324 325
and
vascular
maintenance
during
Ac ce p
320
angiogenesis,
te
317
critical role in tumor-associated angiogenesis and have become the new targets of anti-tumor therapy. TIMP-1, the natural inhibitor of matrix metalloproteinase (MMP), could inhibit the degradation of the extracellular matrix in order to reduce tumor angiogenesis and malignant transformation. The effects of CMCS treatment on VEGF and TIMP-1 levels in mouse
serum were evaluated. As shown in Table 3, the level of VEGF was decreased
326
in a dose-dependent manner after 14 d treatment with CMCS. And the level of
327
TIMP-1 was increased at higher doses after 14 d treatment with CMCS. The
328
expressions of VEGF and TIMP-1 can be considered as important factors in
329
regulate tumor angiogenesis, the stimulate effect of the associated key
330
cytokines may be one of the mechanisms of the anti-angiogenesis for CMCS.
331
3.7. Effects of CMCS on mice thymus index, spleen index and serum TNF-α, 11
Page 11 of 25
332
IFN-γ levels The body’s immune system including the spleen, the thymus, lymph
334
nodes and lymph ducts build up our body to fight infection and cancer (Effros,
335
2003). In this experiment, thymus and spleen tissues were isolated completely
336
and weighed to calculate thymus index and spleen index (Fig. 5A). It was
337
found that CMCS treatment could increase the thymus index and spleen index
338
of the mice. CMCS significantly increased the thymus index in mice (P<0.05)
339
but no significant influence on spleen index was observed (P>0.05).
cr
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333
Immune-related cytokines such as TNF-α and IFN-γ have been used in
341
clinical cancers treatment for many years (Aulitzky et al., 1989; Eggermont et
342
al., 1996). TNF-α is a cytokine mediating inflammatory reaction and immune
343
responses, and has various biological activities. TNF-α can enhance host
344
immune function (Ebert, Meuter, & Moser, 2006) and induce the apoptosis of
345
tumor cells (Chang, et al., 2006; Hur, et al., 2003). IFN-γ, produced by
346
activated T cells and NK cells, is a pleiotropic cytokine with immunomodulatory
347
effects. IFN-γ is produced by the immune system to fight tumors by promote
348
tumor cells apoptosis and kill them (Trubiani, Bosco, & Di Primio, 1994; Ahn,
349
Pan, Vickers, & McDonald, 2002). In this experiment, the concentration of
350
IFN-γ and TNF-α in serum was detected by ELISA assay. It was found that
351
CMCS treatment could enhance serum IFN-γ and TNF-α levels compared with
352
the control gourp (Fig. 5B). Our results showed that CMCS treatment could
354 355 356 357
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M
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353
us
340
improve the immune system. The anti-tumor effects of CMCS maybe partially related with the immune-stimulating functions of CMCS. However, further investigations
are
required
to
understand
the
underlying
molecular
mechanisms by which CMCS regulates the relevant cytokines. 4. Conclusion
358
The anti-tumor and anti-angiogenesis effects of CMCS were evaluated in
359
vitro and in vivo to make CMCS a potential biomaterial for many biomedical
360
applications. Our results indicated that CMCS was non-cytotoxic on HUVECs.
361
Treatment with CMCS significantly inhibited the migration of HUVECs
362
compared with the control group (P<0.05) in vitro. CMCS significantly inhibited
363
tumor growth of H22 in vivo compared with control group (P<0.05) as reflected
12
Page 12 of 25
364
by. The immune-stimulating and the anti-angiogenesis functions may
365
contribute to the anti-tumor effects of CMCS.
366
Acknowledgements This work was supported by Ocean University of China and by a grant
ip t
367
from the National High-tech R & D Program of China (2014AA093605).
369
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370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411
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Ac ce p
412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469
14
Page 14 of 25
Table 1 Physicochemical properties of CMCS
471 472 473
Table 2 Effect of CMCS on the growth of H22 tumor tissue * ** data represents mean ± SD, n = 10, P<0.05, P<0.01 significant difference compared with control group.
474 475 476
Table 3 Effects of CMCS on the expression of VEGF and TIMP-1 * ** data represents mean ± SD, n = 10, P<0.05, P<0.01 significant difference compared with control group.
cr us an M d te Ac ce p
477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512
ip t
469 470
15
Page 15 of 25
513
Table 1 Molecular Water Weight Content
Degree of Carboxymethylation
12.21%
94.66%
108.41%
Viscosity Coefficient 23 mPa· S
Heavy Metal Content ≤ 10 μg/g
Intracellular Toxin Content < 0.5 EU
cr us an M d te Ac ce p
514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551
18.36%
Degree of Deacetylation
ip t
194.6 kDa
Ash Content
552 16
Page 16 of 25
Table 2 Group
Dosage
weight of tumor (g)
(mg/kg)
weight
–
2.10±0.43
–
CMCS
75
1.42±0.36*
32.63%
CMCS
150
1.02±0.21**
51.43%
CMCS
300
1.47±0.41*
29.89%
te
d
M
an
us
cr
Control
Ac ce p
554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590
inhibitory effect of tumor
ip t
553
17
Page 17 of 25
591
Table 3 Group
Dosage (mg/kg)
VEGF content (ng/l)
TIMP-1 content (pg/ml)
Control
–
156.43±7.45
5572.22±381.37
CMCS
75
146.98±21.82
5884.17±942.07
CMCS
150
138.03±13.35**
6370.06±941.12*
CMCS
300
136.30±7.30**
6189.44±823.84*
Ac ce p
te
d
M
an
us
cr
ip t
592 593
18
Page 18 of 25
ip t
Fig. 2. Effects of CMCS on cell morphology and proliferation of HUVECs. (A) Morphological changes of HUVECs were examined under a light microscope (original magnification, 100×). (a) cells treated with complete medium; (b) cells treated with CMCS at the concentrations of 0.5 mg/ml; (c) cells treated with CMCS at the concentrations of 1.0 mg/ml; (d) cells treated with CMCS at the concentrations of 2.0 mg/ml. (B). MTT test measured the proliferation rate and showed that there was no significant difference of cell viability between CMCS treated groups whose concentrations were below 2.0 * ** mg/ml and control group. data represents mean ± SD, n = 6, P<0.05, P<0.01 significant difference compared with control group.
an
us
cr
Fig. 3. Inhibition on cell migration of HUVECs after incubation with CMCS solutions (A) Scarification changes of HUVECs were recorded using the inverted microscope (original magnification, 100×). (B) Inhibition rate of CMCS on the two-dimensional migration of HUVCE. (C) Photographs of HUVECs migrated through the 8 μm- membranes were taken by the light microscope (original magnification, 100×). (D) Inhibition rate of CMCS on the three-dimensional migration of HUVCE. (a) cells treated with complete medium; (b) cells treated with CMCS at the concentrations of 0.5 mg/ml; (c) cells treated with CMCS at the concentrations of 1.0 mg/ml; (d) cells treated with CMCS at the * ** concentrations of 2.0 mg/ml. data represents mean ± SD, n = 6, P<0.05, P<0.01 significant difference compared with control group.
d
M
Fig. 4. Pathological and immunohistochemical detection on H22 tumor tissue (A) Effect of the CMCS on histopathology of H22 tumor tissue was recorded by the light microscope (original magnification, 400×). (B) Photographs of effect on CD34 expression of H22 tumor tissue were taken by the light microscope (original magnification, 400×). (C) Inhibition rate of CMCS on CD34 expression of H22 tumor tissue. (a) mice treated with normal saline; (b) mice treated with CMCS at the dose of 75 mg/kg; (c) mice treated with CMCS at the dose of 150 mg/ kg; (d) mice treated with CMCS at * ** the dose of 300 mg/kg. data represents mean ± SD, n = 10, P<0.05, P<0.01 significant difference compared with control group.
te
594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642
Fig. 1. IR spectra of CTS (A), the sodium salt of CMCS (B) and the protonated CMCS (C)
Fig. 5. Effect of CMCS on thymus index, spleen index and cytokines associated with immunity * ** data represents mean ± SD, n = 10, P<0.05, P<0.01 significant difference compared with control group.
Ac ce p
593
19
Page 19 of 25
ip t cr
644
an M d te Ac ce p
645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674
Fig. 1
us
643
20
Page 20 of 25
675
A a
b
ip t
676
us
677 678
d
cr
c
B
60
30
24h 48h
0.0 0.5 1.0 2.0 Concentration of CMCS (mg/ml)
Fig. 2
Ac ce p
te
d
0
679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700
an
90
M
Proliferation rate( %)
120
21
Page 21 of 25
701 702
A 0h
24h
48h
ip t
a
703
cr
b
us
704
an
c
M
705
B
inhibition rate( %)
Ac ce p
50
te
706 707 B 708
d
d
24h
**
30 20
*
*
48h
**
**
10
0
709 710 711
**
40
0.5 1.0 2.0 Concentration of CMCS (mg/ml)
C
22
Page 22 of 25
a
b
ip t
712
d
718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734
50 40 30
*
*
te
0 0.0
d
20 10
M
**
0.5
1.0
1.5
2.0
2.5
Concentration of CMCS (mg/ml)
Ac ce p
716 717
an
D Inhibition rate of migration (%)
713 714 D 715
us
cr
c
Fig. 3
23
Page 23 of 25
A a
b
c
d
ip t
735
B
an
737 738
us
cr
736
b
M
a
d
739
d
740 741 742
Ac ce p
te
c
C
Inhibition rate of CD34 (%)
80
**
60
40
* *
20
0 75
743 744 745 746
150
300
Dosages of CMCS (mg/kg)
Fig. 4
24
Page 24 of 25
A Spleen index
Thymus index 6
15
**
12
*
4
9
3 6
2
3
1 0
Control
300
0
** *
400
**
M
300 200
d
100 0
**
an
**
us
IFN-γ
TNF-α 500
Control
75
150
dosage of CMCS (mg/kg)
1200 1000 800 600 400 200
300
0
Fig. 5
Ac ce p
te
TNF-α content in mouse serum (ng/l)
B
IFN-γ content in mouse serum (ng/l)
750 751 752
150
dosage of CMCS (mg/kg)
748 749
75
ip t
**
Spleen index
Thymus index
5
cr
747
25
Page 25 of 25