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A Huaier polysaccharide restrains hepatocellular carcinoma growth and metastasis by suppression angiogenesis
International Journal of Biological Macromolecules 75 (2015) 115–120
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
A Huaier polysaccharide restrains hepatocellular carcinoma growth and metastasis by suppression angiogenesis Cong Li a , Xia Wu b,∗ , Honghai Zhang a , Gengxia Yang a , Meijun Hao a , Shoupeng Sheng a , Yu Sun a , Jiang Long a , Caixia Hu a , Xicai Sun c , Li Li d , Jiasheng Zheng a,∗ a
Intervention Therapy Center of Liver Diseases, Beijing You An Hospital, Capital Medical University, Beijing 100069, China Department of Infectious Disease, the Second Affiliated Hospital of Harbin Medical University, Huanghe Road, Harbin 150081, China c School of Medicine, Tsinghua Center for Life Sciences, Tsinghua University, Beijing 100084, China d Institute of Liver Diseases, Beijing You An Hospital, Capital Medical University, Beijing 100069, China b
a r t i c l e
i n f o
Article history: Received 28 May 2014 Received in revised form 26 November 2014 Accepted 8 January 2015 Available online 15 January 2015 Keywords: Huaier polysaccharide Metastasis Angiogenesis
a b s t r a c t Hepatocellular carcinoma (HCC) is a highly metastatic cancer. Huaier polysaccharide (TP-1) is a naturally occurring bioactive macromolecule, found in Huaier fungus and has been shown to exert in vitro antitumor and antimetastasis for HCC, but no study has addressed in vivo efficacy and mechanisms of action. Presently, we found that TP-1 at doses of 0.5, 1 and 2 mg/kg significantly inhibited tumor growth and metastasis to the lung in mice bearing HCC SMMC-7721 tumors without toxicity. The analysis of tumors by immunohistochemistry demonstrated that TP-1 inhibited PCNA expression, increased the number of TUNEL-positive cells and reduced microvessel density (MVD) to achieve this effect. Furthermore, TP-1 administration reduced the protein expression of hypoxia-inducible factor (HIF)-1alpha, vascular endothelial growth factor (VEGF), AUF-1 and AEG-1, in tumor tissues. Taken together, our data suggested that the antitumor and anti-metastatic activities of TP-1 might be at least partially through down-regulation of HIF-1alpha/VEGF and AUF-1/AEG-1 signaling pathways. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver and the third leading cause of cancer-related death worldwide [1]. Although chemotherapy, hepatectomy, and liver transplantation are representative treatments for HCC, these treatments have a limited range of applications [2]. Over the last several decades, the common treatment of liver cancer is suboptimal and the prognosis of patients is poor [3]. Therefore, it is necessary to seek for a multi-targeted therapeutic approach for novel HCC therapies, including regulation of tumor angiogenesis, tumor cell proliferation and survival through modulation of different protein targets. It has been well established that angiogenesis is not only involved in tumor growth and distant metastasis, but also plays an important role in carcinogenesis by supplying nutrients and oxygen [4–7]. Once a tumor becomes vascularized, the growth of the tumor is dramatically accelerated. Emerging studies over the last
∗ Corresponding authors. Tel.: +86 45156854562. E-mail addresses: hepato [email protected] (X. Wu), [email protected] (J. Zheng). http://dx.doi.org/10.1016/j.ijbiomac.2015.01.016 0141-8130/© 2015 Elsevier B.V. All rights reserved.
several decades provide substantial evidence that HCC in one of the most hypervascular tumors with a high tendency for vascular invasion [8]. Angiogenesis is a complex multistep process initiated and regulated by a number of angiogenic factors from malignant cells [9–11]. Consequently, anti-angiogenesis may inhibit the development of HCC and prolong the lives of patients. Hypoxia-inducible factor (HIF)-1alpha and vascular endothelial growth factor (VEGF) are important angiogenic factors and their elevated production by cancer cells directly correlates with tumor angiogenesis and tumor development [12–15]. The HIF-1alpha/VEGF signaling pathway has been demonstrated to involve in the progression of hepatocarcinogenesis [16].Therefore, compounds showing inhibitory effects on the HIF-1alpha/VEGF signaling pathway can be used as promising drug candidates for HCC therapy. In our previous clinical study, we have reported that the poor prognosis in HCC patients was closely associated with high RNA-binding factor 1 (AUF-1) expression, suggesting that the AUF-1 may play an important role in HCC progression [17]. An unpublished data in our continuous work had confirmed that a Huaier polysaccharide (TP-1) exhibited a potent growth inhibitory effect on HCC cells in vitro, and achieved this effect via modulating RNA-binding factor 1 (AUF-1)/astrocyte elevated gene-1 (AEG-1)/EMT signaling pathways to induce apoptosis. However, there are no reports on the effect of TP-1 on
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HCC-induced angiogenesis and apoptosis-related protein on HCC xenograft models. In this context, we are trying to evaluate whether TP-1 can inhibit HCC induced angiogenesis and suppress tumor growth and metastasis in nude mice bearing HCC xenografts, and to further explain the possible mechanisms that may be involved by detecting the expression of HIF-1alpha/VEGF, PCNA, AUF-1, AEG-1, E-cadherin and N-cadherin in tumor tissues.
2. Materials and methods 2.1. Materials and chemicals Huaier extract was donated by Qidong Gaitianli Pharmaceutical Co., Ltd. (Jiangsu, China). All reagents used in the ELISA assay and VEGF ELISA kit were purchased from R&D systems (Minneapolis, MN). Anti-CD34, anti-VEGF, anti-HIF-1alpha, anti-PCNA, anti-AUF-1, anti-AEG-1, anti-E-cadherin, anti-N-cadherin and anti-actin antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Fetal calf serum (FCS) and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Gibco Invitrogen Co (Grand Island, NY, USA). DEAE-cellulose-52 and Sepharose CL-6B were from Amersham (Sweden). All other chemicals were of the highest commercial grade available 2.2. Isolation and purification of polysaccharide TP-1 Huaier extract (8%) suspended in distilled water was added with three-fifth volume of Sevage reagent to remove the protein [18] and then exhaustively dialyzed against water for 2 days. The dialysate was concentrated and precipitated by the addition of 95% ethanol to a final concentration of 40% (v/v) at 4 ◦ C overnight. Thereafter, the collected precipitates were washed in turn with absolute ethanol, acetone and ether to yield the crude polysaccharide (TCP-40). The filtrate of TCP-40 through a 0.45 m millipore membrane was loaded onto a DEAE-cellulose-52 column (2.6 cm × 30 cm), which was equilibrated with deionized water. After equilibration, the column was eluted with 0, 0.1 and 0.5 M NaCl aqueous solution in 3 L for each concentration at a flow rate of 2 ml/min. Each fraction was collected and combined by the detection for polysaccharide with the phenol–sulfuric acid method at 490 nm absorbance. The fraction (TCP-40-1) eluted with 0.1 M NaCl was further fractioned on a Sepharose CL-6B column (2.6 cm × 100 cm) eluted with 0.15 M NaCl at a flow rate of 1 ml/min to yield one purified polysaccharide, named as TP-1. The results of chemical analysis indicated that TP1 contained 93.2% of carbohydrate and was free of proteins and uronic acid.
by the Institutional Ethical Committee of Harbin Medical University. 2.4. In vivo antitumor experiments The implanted tumor tissues for establishment of the nude mouse xenograft model was achieved by subcutaneous injection of hepatocellular carcinoma SMMC-7721 cells (1 × 107 cells per mouse, in 0.1 ml PBS) into the left armpit of one nude BALB/c mouse. Two weeks later, the exuberantly proliferating tumor tissue was removed from mice and chopped into a volume of 1.5 mm3 , which was subcutaneously transplanted into right axillary fossa of nude mice. When the tumors grew to 100–300 mm3 , the animals (20 in all) were randomly allocated into four groups with five animals in each group. Mice were administered with TP-1 (0.5, 1, 2 mg kg−1 ) or PBS via intragastric administration daily for consecutive 15 days one day after tumor implantation. The size of each solid tumor was measured every three days until the day of sacrifice. The mice were killed on the twelfth day after treatment, and the local tumors were removed carefully. The tumor volume (TV) and relative tumor volume (RTV) was calculated by the following formula: TV (mm3 ) = length × width2 /2. RTV = Vt /V0 (in which V0 is the TV at the day when the drug were given and Vt is the TV at the day when mice were sacrificed). Tumor inhibitory efficiency in mice was expressed as follows: TIR (%) = (1 − RTV of the TP-1 administration group/RTV of control group) × 100%. Each tumor was split into two halves: one was fixed in 4% buffered, freshly prepared paraformaldehyde, embedded in paraffin, and make into paraffin sections (5 m thick), and the other stored at −80 ◦ C in a freezer. 2.5. In vivo lung metastasis experiments To establish an in vivo pulmonary metastasis model, SMMC7721 cells suspended in 200 l PBS were injected into BALB/c nude mice via the tail vein (5 × 106 cells for each mouse). To ensure all mice bore actively growing lung tumors before the drug treatment, pulmonary metastasis was allowed to develop for 10 days. On day 11, the mice were grouped and treated as described previously. Drug administration began 24 h after transplantation and lasted for 14 days. All the mice were sacrificed at the end of the study and lungs were removed for the examination of lung metastasis. The metastatic nodes in the lungs of mice from all groups were visualized by fixing them in Bouin’s solution (saturated picric acid:formalin:acetic acid = 15:5:1) for 24 h. After being fixed, the occurrence rate and tumor nodules of lung metastasis were numbered. 2.6. Quantitation of PCNA proliferation index
2.3. Animals and tumor cells Human hepatocellular carcinoma SMMC-7721 cell line was obtained from Shanghai Institute of Cell Biology in the Chinese Academy of Sciences (Shanghai China). The cells were routinely grown in DMEM supplemented with 10% heat inactivated FCS, 100 U/ml penicillin and streptomycin, 1% sodium pyruvate and 2 mM glutamine at 37 ◦ C in a humidified atmosphere of 5% CO2 and 95% air. Male athymic BALB/c nude mice, weighting 18–20 g, aged between 4 and 6 weeks, were provided by the Experimental Animal Center of Harbin Medical University (Harbin, China). All the animals were kept at room temperature at 25 ± 2 ◦ C, 12 h dark–light cycle and a relative humidity of 60–70%. All the mice were fed with normal mice chow and water ad libitum under specific pathogen free (SPF) conditions. The use and treatment of mice were in accordance with institutional guideline for Laboratory Animal Care approved
Five m thickness of paraffin sections from formalin-fixed paraffin-embedded tumour xenografts were used for immunohistochemical staining. After deparaffinisation, anti-PCNA mouse monoclonal Ab was used at a dilution of 1:200 to perform the immunohistochemical analysis according to the manufacturer’s instructions. PCNA density was quantified using the Image-Pro Plus image analysis software and the integral optical density (IOD) of every visual field was calculated for each sample. 2.7. Apoptosis detection Apoptosis in HCC tumors were assayed using commercially available TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP-FITC nick end-labeling) assay kits (Wako Pure Chemical Industries, Osaka, Japan) according to the instructions provided by the manufacturer. The TUNEL positive cells were counted within
C. Li et al. / International Journal of Biological Macromolecules 75 (2015) 115–120
3
6
9
12
15
RTV
Days after treatment Fig. 1. The relative tumor volume (TV) of the mice in different groups. Data are presented as mean ± SD (n = 5).
3.2. Effect of TP-1 on tumor metastasis in nude mice To confirm inhibitory effect of TP-1 on HCC metastasis in vivo, we next transferred SMMC-7721 cells into nude mice by tail vein injection. Two weeks after injection, the mice were killed and lungs were removed to undergo histological examinations. The number of metastatic tumor foci on the lung surface of control and TP-1 treated mice was counted under microscopy. The data in Fig. 2 showed that transplantation of SMMC-7721 cells via the tail vein resulted in an increase of metastasis focuses on the surface of the mouse lungs, whilst the number of lung metastases nodules was dose dependently decreased by TP-1 treatment, which was significant at all doses compared to the control (P < 0.05 or P < 0.01). 3.3. Effects of TP-1 on cell proliferation, apoptosis, and MVD in nude mice Considering the tumor growth inhibitory and anti- metastatic effect of TP-1 in nude mice, we subsequently analyzed tumors for cell proliferation, apoptosis, and MVD in nude mice using immunohistochemistry. Specific staining of tumor sections by PCNA (PCNA is regarded as a cell proliferation marker) antibody demonstrated that the number of PCNA-positive cells in tumors was significantly lower in the TP-1-treated groups compared to the control group (P < 0.05 or P < 0.01) (Fig. 3A). TP-1 at a dose of 0.5 mg/kg reduced the proliferation index by 28%, 1 mg/kg by 44%, and 2 mg/kg by 51%, compared to the control (Fig. 3B). Western blot result showed that the protein expression of PCNA in the tumor tissues decreased significantly after treatment with TP-1 (Fig. 3C). Representative
150
*
100 ** **
50
m
g/ kg )
g/ kg )
0
TP -1
(2
m
Using the implanted SMMC-7721 xenograft rat’s model, in vivo anti-tumour efficacy of different doses of TP-1 was evaluated. The tumor sizes were measured by a caliper every three days until the end of experiment to monitor the in vivo therapeutic efficiency. As shown in Fig. 1, the tumor sizes of control mice keep a higher growth rate during all the experiment than those of TP-1 treated mice. A slight time-dependent increase in RTV was observed in PBS or 0.5, 1, and 2 mg/kg of TP-1 treated groups and their average RTV value were 7.5, 5.0, 3.7 and 3.3, respectively, on day 15. The results in Table 1 also confirmed that treatment with TP1 significantly inhibited primary tumor growth compared with control group, especially at 1 and 2 mg/kg doses (P < 0.05). The relative TIR obtained with TP-1 was 33.3% for 0.5 mg/kg, 50.7% for 1 mg/kg, and 56.0% for 2 mg/kg, respectively. In addition, physiological behaviour, appetite and weight of mice were not disturbed during the time.
0
(1
3.1. Effect of TP-1 on tumor growth in nude mice
0
TP -1
3. Results and discussion
2
g/ kg )
The statistical significance was analysed by one-way ANOVA using the SPSS 16.0 software. Differences with a P value less than 0.05 were considered statistically significant.
4
m
2.10. Statistical analysis
TP-1 (2 mg/kg/day)
(0 .5
Transplanted HCC tumor tissues were homogenized on ice in lysis buffer and debris was removed by centrifugation. The protein content was determined according to the Bradford assay with bovine serumalbumin as a standard [20]. Samples containing approximately 30 g of total protein were subjected to electrophoresis on a 10–12% (v/v) SDS-PAGE gel, and then transferred onto nitrocellulose membranes (Millipore, Bedford, MA). After brief incubation with 5% skim milk in PBST for 1 h to block nonspecific binding of the membranes, the blots were incubated with primary antibodies against HIF-1alpha, VEGF, AUF-1, AEG-1 and -actin overnight at 4 ◦ C. After washing three times with PBST, the membrane was incubated with secondary peroxidase-conjugated antibody for 1 h at room temperature. Finally, the protein bands on the membrane were visualized with the enhanced chemiluminescence (ECL) system (Amersham Biosciences). The density of each band was analyzed on the Gel Image Analysis System.
TP-1 (1 mg/kg/day)
6
on tr ol
2.9. Western blotting analysis
TP-1 (0.5 mg/kg/day)
TP -1
Vascularization in tumor tissues was quantified by immunostaining with a monoclonal antibody against endothelial cell antigen CD34. Microvessel density (MVD) was determined according to the method reported by Weidner et al. [19]. Briefly, the slides were scanned at low magnification (×100) magnification to identify the areas with the highest density of CD34-positive vessels and these areas were then counted with a 200 times magnification in 10 blindly chosen slices. The average vessel number in 10 randomly chosen fields was used to indicate the MVD of the tissue.
Control
8
C
2.8. Assessment of microvessel density (MVD)
10
The number of metastatic nodules on lung surface
ten randomly selected fields under a confocal microscope at a magnification of ×200. The Percentage of TUNEL-positive staining in each field was calculated according to the following formula: Apoptosis index (AI) = the number of TUNEL-positive cells/the number of total cells × 100%.
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Fig. 2. In vivo anti-metastasis activity of TP-1 in the HCC pulmonary metastasis model. Data are presented as mean ± SD (n = 5). * P < 0.05, ** P < 0.01 significantly different from control group.
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Table 1 Inhibitory effect of TP-1 on the growth of human liver cancer SMMC7721 cell xenograft in nude mice. Group
Control TP-1
Doses (mg/kg/day)
PBS 0.5 1 2
Body weight (g) Before
After
18.92 19.20 18.52 19.45
23.54 24.85 24.72 26.07
Increase of body weight (g)
4.62 5.65a 6.20a 6.62a
TV (mm3 ) Before
After
235 ± 90 245 ± 97 268 ± 102 254 ± 98
1765 ± 456 1225 ± 335 985 ± 312 848 ± 212
RTV
TIR (%)
7.5 5.0 3.7 3.3
/ 33.3 50.7a 56.0a
Data are presented as mean ± SD (n = 5). a P < 0.05 significantly different from control group.
TUNEL-positive cells from control and TP-1 administrated animals were shown in Fig. 3D. The number of TUNEL-positive cells in tumors was significantly greater in TP-1 treatment group compared to the control group (P < 0.05 or P < 0.01), suggesting that TP-1 could induce apoptosis in tumor tissue. To monitor the effect of TP-1 on tumor angiogenesis, we examined the intratumoral MVD by immunostaining with anti-CD34 antibody. The number and size of blood vessel profiles were demonstrated by CD34 antigen which is considered to be a marker of capillary endothelial cells [21]. A significant decrease in MVD was evident in the TP-1treated groups compared to the control group (P < 0.05 or P < 0.01), particularly in the high-dose group that had very few stained microvessels (Fig. 4A and B), suggesting a significant decrease in new vessel formation.
3.4. Effect of TP-1 on HIF-1alpha, VEGF, AUF-1 and AEG-1 protein expression in nude mice We next examined the expressions of an array of pro-tumor mediators related to angiogenesis and tumorigenesis, including HIF-1alpha, VEGF, AUF-1 and AEG-1, in tumors from control and TP1-treated mice. As shown in Fig. 5, TP-1 administration significantly repressed the expression of HIF-1alpha and VEGF in HCC transplanted tumors. Consistently, substantial decreases in the protein levels of AUF-1 and AEG-1 could be observed in SMMC-7721 tumor xenograft from PBS or TP-1 treated mice. These data suggested the capacities of TP-1 in suppressing tumor growth and distant metastasis in vivo might be at least partially through down-regulating the protein expression of HIF-1alpha, VEGF, AUF-1 and AEG-1.
Fig. 3. (A) Effect of TP-1 on cell proliferation mark PCNA in tumors as determined by immunohistochemical analysis. The photographs are representative images taken at a magnification of 200× (B) Quantification of immunohistochemical staining for PCNA in tumors. (C) Effect of TP-1 on the protein expression of PCNA in tumors. Lane 1: Control; Lane 2: 0.5 mg/kg TP-1; Lane 3: 1 mg/kg TP-1 and Lane 4: 2 mg/kg TP-1. (D) TUNEL-positive cells in tumors upon TP-1 treatment. Data are presented as mean ± SD (n = 5). * P < 0.05, ** P < 0.01 significantly different from control group.
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Fig. 4. (A) Effect of TP-1 on the tumor microvessel density (MVD) in tumors. The photographs are representative images taken at a magnification of 200×. (B) Quantification of immunohistochemical staining for CD34 in tumors.
proliferation, induction of tumor cell apoptosis and angiogenesis), which are at least in part mediated through inhibition of HIF1alpha/VEGF and AUF-1/AEG-1 signaling pathways. In the future, preclinical and clinical developments are needed to explore its possible clinical uses in cancer therapy. Acknowledgements This research is funded by National Natural Science Foundation of China (grant No. 81472328), Liver disease AIDS Foundation of You An Hospital, Capital Medical University (BJYAH2011-034), National Science and Technology Support Project (2012BAI15B08), and Key Project of National Communicable Disease (2012ZX10002015-002). References Fig. 5. Western blot analysis of HIF-1alpha, VEGF, AUF-1 and AEG-1 protein expression in tumors. Lane 1: Control; Lane 2: 0.5 mg/kg TP-1; Lane 3: 1 mg/kg TP-1 and Lane 4: 2 mg/kg TP-1.
4. Conclusions Angiogenesis occurs in both physiological and pathological conditions for new blood vessel formation and is a necessary event for tumor growth and metastasis [22]. VEGF is a tumor-secreted cytokine with grave importance in tumor angiogenesis and invasion [23], which acts as an angiogenic factor in neo-angiogenesis by promoting the proliferation and migration of endothelial cells, as well as the formation of new blood vessels [24,25]. Recent studies have demonstrated that HCC is generally considered to be a hypervascular tumor and that inhibition of angiogenesis via downregulation of VEGF has become a promising approach for the treatment of HCC [26–28]. Furthermore, lager amount of evidence has shown that HIF-1alpha, AFU-1 and AEG-1 play an important role in the growth of highly vascularized tumors, such as HCC [29,30,17,31,32]. Therefore, the combination regimens targeting HIF-1alpha/VEGF and AUF-1/AEG-1 might be an effective therapeutic strategy in the prevention as well as treatment of human HCC. In conclusion, our work provides novel in vivo evidence that TP-1 attenuates tumor growth and pulmonary metastasis by exerting a range of prominent antitumor effects (inhibition of cell
[1] J.S. Lee, I.S. Chu, J. Heo, D.F. Calvisi, Z. Sun, T. Roskams, A. Durnez, A.J. Demetris, S.S. Thorgeirsson, Hepatology 40 (2004) 667–676. [2] B.A. Cahill, D. Braccia, Clin. J. Oncol. Nurs. 8 (2004) 393–399. [3] H.B. El-Serag, K.L. Rudolph, Gastroenterology 132 (2007) 2557–2576. [4] S. Coulon, F. Heindryckx, A. Geerts, C. Van Steenkiste, I. Colle, H. Van Vlierberghe, Liver Int. 31 (2011) 146–162. [5] V. Granci, Y.M. Dupertuis, C. Pichard, Curr. Opin. Clin. Nutr. Metab. Care 13 (2010) 417–422. [6] S.S. Verbridge, N.W. Choi, Y. Zheng, D.J. Brooks, A.D. Stroock, C. Fischbach, Tissue Eng. A 16 (2010) 2133–2141. [7] J.A. Chen, M. Shi, J.Q. Li, C.N. Qian, Hepatol. Int. 4 (2010) 537–547. [8] M. Varela, M. Sala, J.M. Llovet, J. Bruix, Cancer Treat. Rev. 29 (2003) 99–104. [9] D. Aoki, S. Ueno, F. Kubo, T. Oyama, T. Sakuta, K. Matsushita, I. Maruyama, T. Aikou, Anticancer Res. 25 (2005) 133–138. [10] J. Folkman, J. Natl. Cancer Inst. 82 (1990) 4–6. [11] J. Folkman, Y. Shing, J. Biol. Chem. 267 (1992) 10931–10934. [12] M.P. Barr, D.J. Bouchier-Hayes, J.J. Harmey, Int. J. Oncol. 32 (2008) 41–48. [13] C. Tzao, S.C. Lee, H.J. Tung, H.S. Hsu, W.H. Hsu, G.H. Sun, C.P. Yu, J.S. Jin, Y.L. Cheng, Dis. Markers 25 (2008) 141–148. [14] A. Basu, A.G. Contreras, D. Datta, E. Flynn, L. Zeng, H.T. Cohen, D.M. Briscoe, S. Pal, Cancer Res. 68 (2008) 5689–5698. [15] C. Lin, R. McGough, B. Aswad, J.A. Block, R. Terek, J. Orthop. Res. 22 (2004) 1175–1181. ¨ G. Finkenzeller, B. Wiedenmann, S. [16] Z. von Marschall, T. Cramer, M. Hocker, Rosewicz, Gut 48 (2001) 87–96. [17] Y. Yang, P. Kang, J. Gao, C. Xu, S. Wang, H. Jin, Y. Li, W. Liu, X. Wu, Tumor Biol. 35 (2014) 2747–2751. [18] A.M. Staub, Methods Carbohydr. Chem. 5 (1965) 5–6. [19] N. Weidner, J.P. Semple, W.R. Welch, J. Folkman, N. Engl. J. Med. 324 (1991) 1–8. [20] M.M. Bradford, Anal. Biochem. 72 (1976) 248–254. ˜ [21] N.G. Ordónez, Adv. Anat. Pathol. 19 (2012) 281–295. [22] J.L. Masferrer, K.M. Leahy, A.T. Koki, B.S. Zweifel, S.L. Settle, B.M. Woerner, D.A. Edwards, A.G. Flickinger, R.J. Moore, K. Seibert, Cancer Res. 60 (2000) 1306–1311.
120
C. Li et al. / International Journal of Biological Macromolecules 75 (2015) 115–120
[23] R.S. Finn, A.X. Zhu, Expert Rev. Anticancer Ther. 9 (2009) 503–509. [24] A.E. Frankel, P.S. Gill, Leuk. Res. 28 (2004) 675–677. [25] M.H. Oak, J. El Bedoui, V.B. Schini-Kerth, J. Nutr. Biochem. 16 (2005) 1–8. [26] D. Ribatti, B. Nico, D. Mangieri, V. Longo, D. Sansonno, A. Vacca, F. Dammacco, Histol. Histopathol. 22 (2007) 285–289. [27] I.T. Chiang, Y.C. Liu, W.H. Wang, F.T. Hsu, H.W. Chen, W.J. Lin, W.Y. Chang, J.J. Hwang, In Vivo 26 (2012) 671–681. [28] J.A. Chen, M. Shi, J.Q. Li, C.N. Qian, Hepatol. Int. 29 (2010) 537–547.
[29] H. Tanaka, M. Yamamoto, N. Hashimoto, M. Miyakoshi, S. Tamakawa, M. Yoshie, Y. Tokusashi, K. Yokoyama, Y. Yaginuma, K. Ogawa, Cancer Res. 66 (2006) 11263–11270. [30] S. Yasuda, S. Arii, A. Mori, N. Isobe, W. Yang, H. Oe, A. Fujimoto, Y. Yonenaga, H. Sakashita, M. Imamura, J. Hepatol. 40 (2004) 117–123. [31] J. Zheng, C. Li, X. Wu, Y. Yang, M. Hao, S. Sheng, Y. Sun, H. Zhang, J. Long, C. Hu, Tumour Biol. 35 (2014) 2265–2269. [32] J. Zheng, C. Li, X. Wu, M. Liu, X. Sun, Y. Yang, M. Hao, S. Sheng, Y. Sun, H. Zhang, J. Long, Y. Liang, C. Hu, Int. J. Biol. Macromol. 64 (2014) 106–110.
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
A Huaier polysaccharide restrains hepatocellular carcinoma growth and metastasis by suppression angiogenesis Cong Li a , Xia Wu b,∗ , Honghai Zhang a , Gengxia Yang a , Meijun Hao a , Shoupeng Sheng a , Yu Sun a , Jiang Long a , Caixia Hu a , Xicai Sun c , Li Li d , Jiasheng Zheng a,∗ a
Intervention Therapy Center of Liver Diseases, Beijing You An Hospital, Capital Medical University, Beijing 100069, China Department of Infectious Disease, the Second Affiliated Hospital of Harbin Medical University, Huanghe Road, Harbin 150081, China c School of Medicine, Tsinghua Center for Life Sciences, Tsinghua University, Beijing 100084, China d Institute of Liver Diseases, Beijing You An Hospital, Capital Medical University, Beijing 100069, China b
a r t i c l e
i n f o
Article history: Received 28 May 2014 Received in revised form 26 November 2014 Accepted 8 January 2015 Available online 15 January 2015 Keywords: Huaier polysaccharide Metastasis Angiogenesis
a b s t r a c t Hepatocellular carcinoma (HCC) is a highly metastatic cancer. Huaier polysaccharide (TP-1) is a naturally occurring bioactive macromolecule, found in Huaier fungus and has been shown to exert in vitro antitumor and antimetastasis for HCC, but no study has addressed in vivo efficacy and mechanisms of action. Presently, we found that TP-1 at doses of 0.5, 1 and 2 mg/kg significantly inhibited tumor growth and metastasis to the lung in mice bearing HCC SMMC-7721 tumors without toxicity. The analysis of tumors by immunohistochemistry demonstrated that TP-1 inhibited PCNA expression, increased the number of TUNEL-positive cells and reduced microvessel density (MVD) to achieve this effect. Furthermore, TP-1 administration reduced the protein expression of hypoxia-inducible factor (HIF)-1alpha, vascular endothelial growth factor (VEGF), AUF-1 and AEG-1, in tumor tissues. Taken together, our data suggested that the antitumor and anti-metastatic activities of TP-1 might be at least partially through down-regulation of HIF-1alpha/VEGF and AUF-1/AEG-1 signaling pathways. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver and the third leading cause of cancer-related death worldwide [1]. Although chemotherapy, hepatectomy, and liver transplantation are representative treatments for HCC, these treatments have a limited range of applications [2]. Over the last several decades, the common treatment of liver cancer is suboptimal and the prognosis of patients is poor [3]. Therefore, it is necessary to seek for a multi-targeted therapeutic approach for novel HCC therapies, including regulation of tumor angiogenesis, tumor cell proliferation and survival through modulation of different protein targets. It has been well established that angiogenesis is not only involved in tumor growth and distant metastasis, but also plays an important role in carcinogenesis by supplying nutrients and oxygen [4–7]. Once a tumor becomes vascularized, the growth of the tumor is dramatically accelerated. Emerging studies over the last
∗ Corresponding authors. Tel.: +86 45156854562. E-mail addresses: hepato [email protected] (X. Wu), [email protected] (J. Zheng). http://dx.doi.org/10.1016/j.ijbiomac.2015.01.016 0141-8130/© 2015 Elsevier B.V. All rights reserved.
several decades provide substantial evidence that HCC in one of the most hypervascular tumors with a high tendency for vascular invasion [8]. Angiogenesis is a complex multistep process initiated and regulated by a number of angiogenic factors from malignant cells [9–11]. Consequently, anti-angiogenesis may inhibit the development of HCC and prolong the lives of patients. Hypoxia-inducible factor (HIF)-1alpha and vascular endothelial growth factor (VEGF) are important angiogenic factors and their elevated production by cancer cells directly correlates with tumor angiogenesis and tumor development [12–15]. The HIF-1alpha/VEGF signaling pathway has been demonstrated to involve in the progression of hepatocarcinogenesis [16].Therefore, compounds showing inhibitory effects on the HIF-1alpha/VEGF signaling pathway can be used as promising drug candidates for HCC therapy. In our previous clinical study, we have reported that the poor prognosis in HCC patients was closely associated with high RNA-binding factor 1 (AUF-1) expression, suggesting that the AUF-1 may play an important role in HCC progression [17]. An unpublished data in our continuous work had confirmed that a Huaier polysaccharide (TP-1) exhibited a potent growth inhibitory effect on HCC cells in vitro, and achieved this effect via modulating RNA-binding factor 1 (AUF-1)/astrocyte elevated gene-1 (AEG-1)/EMT signaling pathways to induce apoptosis. However, there are no reports on the effect of TP-1 on
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HCC-induced angiogenesis and apoptosis-related protein on HCC xenograft models. In this context, we are trying to evaluate whether TP-1 can inhibit HCC induced angiogenesis and suppress tumor growth and metastasis in nude mice bearing HCC xenografts, and to further explain the possible mechanisms that may be involved by detecting the expression of HIF-1alpha/VEGF, PCNA, AUF-1, AEG-1, E-cadherin and N-cadherin in tumor tissues.
2. Materials and methods 2.1. Materials and chemicals Huaier extract was donated by Qidong Gaitianli Pharmaceutical Co., Ltd. (Jiangsu, China). All reagents used in the ELISA assay and VEGF ELISA kit were purchased from R&D systems (Minneapolis, MN). Anti-CD34, anti-VEGF, anti-HIF-1alpha, anti-PCNA, anti-AUF-1, anti-AEG-1, anti-E-cadherin, anti-N-cadherin and anti-actin antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Fetal calf serum (FCS) and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Gibco Invitrogen Co (Grand Island, NY, USA). DEAE-cellulose-52 and Sepharose CL-6B were from Amersham (Sweden). All other chemicals were of the highest commercial grade available 2.2. Isolation and purification of polysaccharide TP-1 Huaier extract (8%) suspended in distilled water was added with three-fifth volume of Sevage reagent to remove the protein [18] and then exhaustively dialyzed against water for 2 days. The dialysate was concentrated and precipitated by the addition of 95% ethanol to a final concentration of 40% (v/v) at 4 ◦ C overnight. Thereafter, the collected precipitates were washed in turn with absolute ethanol, acetone and ether to yield the crude polysaccharide (TCP-40). The filtrate of TCP-40 through a 0.45 m millipore membrane was loaded onto a DEAE-cellulose-52 column (2.6 cm × 30 cm), which was equilibrated with deionized water. After equilibration, the column was eluted with 0, 0.1 and 0.5 M NaCl aqueous solution in 3 L for each concentration at a flow rate of 2 ml/min. Each fraction was collected and combined by the detection for polysaccharide with the phenol–sulfuric acid method at 490 nm absorbance. The fraction (TCP-40-1) eluted with 0.1 M NaCl was further fractioned on a Sepharose CL-6B column (2.6 cm × 100 cm) eluted with 0.15 M NaCl at a flow rate of 1 ml/min to yield one purified polysaccharide, named as TP-1. The results of chemical analysis indicated that TP1 contained 93.2% of carbohydrate and was free of proteins and uronic acid.
by the Institutional Ethical Committee of Harbin Medical University. 2.4. In vivo antitumor experiments The implanted tumor tissues for establishment of the nude mouse xenograft model was achieved by subcutaneous injection of hepatocellular carcinoma SMMC-7721 cells (1 × 107 cells per mouse, in 0.1 ml PBS) into the left armpit of one nude BALB/c mouse. Two weeks later, the exuberantly proliferating tumor tissue was removed from mice and chopped into a volume of 1.5 mm3 , which was subcutaneously transplanted into right axillary fossa of nude mice. When the tumors grew to 100–300 mm3 , the animals (20 in all) were randomly allocated into four groups with five animals in each group. Mice were administered with TP-1 (0.5, 1, 2 mg kg−1 ) or PBS via intragastric administration daily for consecutive 15 days one day after tumor implantation. The size of each solid tumor was measured every three days until the day of sacrifice. The mice were killed on the twelfth day after treatment, and the local tumors were removed carefully. The tumor volume (TV) and relative tumor volume (RTV) was calculated by the following formula: TV (mm3 ) = length × width2 /2. RTV = Vt /V0 (in which V0 is the TV at the day when the drug were given and Vt is the TV at the day when mice were sacrificed). Tumor inhibitory efficiency in mice was expressed as follows: TIR (%) = (1 − RTV of the TP-1 administration group/RTV of control group) × 100%. Each tumor was split into two halves: one was fixed in 4% buffered, freshly prepared paraformaldehyde, embedded in paraffin, and make into paraffin sections (5 m thick), and the other stored at −80 ◦ C in a freezer. 2.5. In vivo lung metastasis experiments To establish an in vivo pulmonary metastasis model, SMMC7721 cells suspended in 200 l PBS were injected into BALB/c nude mice via the tail vein (5 × 106 cells for each mouse). To ensure all mice bore actively growing lung tumors before the drug treatment, pulmonary metastasis was allowed to develop for 10 days. On day 11, the mice were grouped and treated as described previously. Drug administration began 24 h after transplantation and lasted for 14 days. All the mice were sacrificed at the end of the study and lungs were removed for the examination of lung metastasis. The metastatic nodes in the lungs of mice from all groups were visualized by fixing them in Bouin’s solution (saturated picric acid:formalin:acetic acid = 15:5:1) for 24 h. After being fixed, the occurrence rate and tumor nodules of lung metastasis were numbered. 2.6. Quantitation of PCNA proliferation index
2.3. Animals and tumor cells Human hepatocellular carcinoma SMMC-7721 cell line was obtained from Shanghai Institute of Cell Biology in the Chinese Academy of Sciences (Shanghai China). The cells were routinely grown in DMEM supplemented with 10% heat inactivated FCS, 100 U/ml penicillin and streptomycin, 1% sodium pyruvate and 2 mM glutamine at 37 ◦ C in a humidified atmosphere of 5% CO2 and 95% air. Male athymic BALB/c nude mice, weighting 18–20 g, aged between 4 and 6 weeks, were provided by the Experimental Animal Center of Harbin Medical University (Harbin, China). All the animals were kept at room temperature at 25 ± 2 ◦ C, 12 h dark–light cycle and a relative humidity of 60–70%. All the mice were fed with normal mice chow and water ad libitum under specific pathogen free (SPF) conditions. The use and treatment of mice were in accordance with institutional guideline for Laboratory Animal Care approved
Five m thickness of paraffin sections from formalin-fixed paraffin-embedded tumour xenografts were used for immunohistochemical staining. After deparaffinisation, anti-PCNA mouse monoclonal Ab was used at a dilution of 1:200 to perform the immunohistochemical analysis according to the manufacturer’s instructions. PCNA density was quantified using the Image-Pro Plus image analysis software and the integral optical density (IOD) of every visual field was calculated for each sample. 2.7. Apoptosis detection Apoptosis in HCC tumors were assayed using commercially available TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP-FITC nick end-labeling) assay kits (Wako Pure Chemical Industries, Osaka, Japan) according to the instructions provided by the manufacturer. The TUNEL positive cells were counted within
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3
6
9
12
15
RTV
Days after treatment Fig. 1. The relative tumor volume (TV) of the mice in different groups. Data are presented as mean ± SD (n = 5).
3.2. Effect of TP-1 on tumor metastasis in nude mice To confirm inhibitory effect of TP-1 on HCC metastasis in vivo, we next transferred SMMC-7721 cells into nude mice by tail vein injection. Two weeks after injection, the mice were killed and lungs were removed to undergo histological examinations. The number of metastatic tumor foci on the lung surface of control and TP-1 treated mice was counted under microscopy. The data in Fig. 2 showed that transplantation of SMMC-7721 cells via the tail vein resulted in an increase of metastasis focuses on the surface of the mouse lungs, whilst the number of lung metastases nodules was dose dependently decreased by TP-1 treatment, which was significant at all doses compared to the control (P < 0.05 or P < 0.01). 3.3. Effects of TP-1 on cell proliferation, apoptosis, and MVD in nude mice Considering the tumor growth inhibitory and anti- metastatic effect of TP-1 in nude mice, we subsequently analyzed tumors for cell proliferation, apoptosis, and MVD in nude mice using immunohistochemistry. Specific staining of tumor sections by PCNA (PCNA is regarded as a cell proliferation marker) antibody demonstrated that the number of PCNA-positive cells in tumors was significantly lower in the TP-1-treated groups compared to the control group (P < 0.05 or P < 0.01) (Fig. 3A). TP-1 at a dose of 0.5 mg/kg reduced the proliferation index by 28%, 1 mg/kg by 44%, and 2 mg/kg by 51%, compared to the control (Fig. 3B). Western blot result showed that the protein expression of PCNA in the tumor tissues decreased significantly after treatment with TP-1 (Fig. 3C). Representative
150
*
100 ** **
50
m
g/ kg )
g/ kg )
0
TP -1
(2
m
Using the implanted SMMC-7721 xenograft rat’s model, in vivo anti-tumour efficacy of different doses of TP-1 was evaluated. The tumor sizes were measured by a caliper every three days until the end of experiment to monitor the in vivo therapeutic efficiency. As shown in Fig. 1, the tumor sizes of control mice keep a higher growth rate during all the experiment than those of TP-1 treated mice. A slight time-dependent increase in RTV was observed in PBS or 0.5, 1, and 2 mg/kg of TP-1 treated groups and their average RTV value were 7.5, 5.0, 3.7 and 3.3, respectively, on day 15. The results in Table 1 also confirmed that treatment with TP1 significantly inhibited primary tumor growth compared with control group, especially at 1 and 2 mg/kg doses (P < 0.05). The relative TIR obtained with TP-1 was 33.3% for 0.5 mg/kg, 50.7% for 1 mg/kg, and 56.0% for 2 mg/kg, respectively. In addition, physiological behaviour, appetite and weight of mice were not disturbed during the time.
0
(1
3.1. Effect of TP-1 on tumor growth in nude mice
0
TP -1
3. Results and discussion
2
g/ kg )
The statistical significance was analysed by one-way ANOVA using the SPSS 16.0 software. Differences with a P value less than 0.05 were considered statistically significant.
4
m
2.10. Statistical analysis
TP-1 (2 mg/kg/day)
(0 .5
Transplanted HCC tumor tissues were homogenized on ice in lysis buffer and debris was removed by centrifugation. The protein content was determined according to the Bradford assay with bovine serumalbumin as a standard [20]. Samples containing approximately 30 g of total protein were subjected to electrophoresis on a 10–12% (v/v) SDS-PAGE gel, and then transferred onto nitrocellulose membranes (Millipore, Bedford, MA). After brief incubation with 5% skim milk in PBST for 1 h to block nonspecific binding of the membranes, the blots were incubated with primary antibodies against HIF-1alpha, VEGF, AUF-1, AEG-1 and -actin overnight at 4 ◦ C. After washing three times with PBST, the membrane was incubated with secondary peroxidase-conjugated antibody for 1 h at room temperature. Finally, the protein bands on the membrane were visualized with the enhanced chemiluminescence (ECL) system (Amersham Biosciences). The density of each band was analyzed on the Gel Image Analysis System.
TP-1 (1 mg/kg/day)
6
on tr ol
2.9. Western blotting analysis
TP-1 (0.5 mg/kg/day)
TP -1
Vascularization in tumor tissues was quantified by immunostaining with a monoclonal antibody against endothelial cell antigen CD34. Microvessel density (MVD) was determined according to the method reported by Weidner et al. [19]. Briefly, the slides were scanned at low magnification (×100) magnification to identify the areas with the highest density of CD34-positive vessels and these areas were then counted with a 200 times magnification in 10 blindly chosen slices. The average vessel number in 10 randomly chosen fields was used to indicate the MVD of the tissue.
Control
8
C
2.8. Assessment of microvessel density (MVD)
10
The number of metastatic nodules on lung surface
ten randomly selected fields under a confocal microscope at a magnification of ×200. The Percentage of TUNEL-positive staining in each field was calculated according to the following formula: Apoptosis index (AI) = the number of TUNEL-positive cells/the number of total cells × 100%.
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Fig. 2. In vivo anti-metastasis activity of TP-1 in the HCC pulmonary metastasis model. Data are presented as mean ± SD (n = 5). * P < 0.05, ** P < 0.01 significantly different from control group.
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Table 1 Inhibitory effect of TP-1 on the growth of human liver cancer SMMC7721 cell xenograft in nude mice. Group
Control TP-1
Doses (mg/kg/day)
PBS 0.5 1 2
Body weight (g) Before
After
18.92 19.20 18.52 19.45
23.54 24.85 24.72 26.07
Increase of body weight (g)
4.62 5.65a 6.20a 6.62a
TV (mm3 ) Before
After
235 ± 90 245 ± 97 268 ± 102 254 ± 98
1765 ± 456 1225 ± 335 985 ± 312 848 ± 212
RTV
TIR (%)
7.5 5.0 3.7 3.3
/ 33.3 50.7a 56.0a
Data are presented as mean ± SD (n = 5). a P < 0.05 significantly different from control group.
TUNEL-positive cells from control and TP-1 administrated animals were shown in Fig. 3D. The number of TUNEL-positive cells in tumors was significantly greater in TP-1 treatment group compared to the control group (P < 0.05 or P < 0.01), suggesting that TP-1 could induce apoptosis in tumor tissue. To monitor the effect of TP-1 on tumor angiogenesis, we examined the intratumoral MVD by immunostaining with anti-CD34 antibody. The number and size of blood vessel profiles were demonstrated by CD34 antigen which is considered to be a marker of capillary endothelial cells [21]. A significant decrease in MVD was evident in the TP-1treated groups compared to the control group (P < 0.05 or P < 0.01), particularly in the high-dose group that had very few stained microvessels (Fig. 4A and B), suggesting a significant decrease in new vessel formation.
3.4. Effect of TP-1 on HIF-1alpha, VEGF, AUF-1 and AEG-1 protein expression in nude mice We next examined the expressions of an array of pro-tumor mediators related to angiogenesis and tumorigenesis, including HIF-1alpha, VEGF, AUF-1 and AEG-1, in tumors from control and TP1-treated mice. As shown in Fig. 5, TP-1 administration significantly repressed the expression of HIF-1alpha and VEGF in HCC transplanted tumors. Consistently, substantial decreases in the protein levels of AUF-1 and AEG-1 could be observed in SMMC-7721 tumor xenograft from PBS or TP-1 treated mice. These data suggested the capacities of TP-1 in suppressing tumor growth and distant metastasis in vivo might be at least partially through down-regulating the protein expression of HIF-1alpha, VEGF, AUF-1 and AEG-1.
Fig. 3. (A) Effect of TP-1 on cell proliferation mark PCNA in tumors as determined by immunohistochemical analysis. The photographs are representative images taken at a magnification of 200× (B) Quantification of immunohistochemical staining for PCNA in tumors. (C) Effect of TP-1 on the protein expression of PCNA in tumors. Lane 1: Control; Lane 2: 0.5 mg/kg TP-1; Lane 3: 1 mg/kg TP-1 and Lane 4: 2 mg/kg TP-1. (D) TUNEL-positive cells in tumors upon TP-1 treatment. Data are presented as mean ± SD (n = 5). * P < 0.05, ** P < 0.01 significantly different from control group.
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Fig. 4. (A) Effect of TP-1 on the tumor microvessel density (MVD) in tumors. The photographs are representative images taken at a magnification of 200×. (B) Quantification of immunohistochemical staining for CD34 in tumors.
proliferation, induction of tumor cell apoptosis and angiogenesis), which are at least in part mediated through inhibition of HIF1alpha/VEGF and AUF-1/AEG-1 signaling pathways. In the future, preclinical and clinical developments are needed to explore its possible clinical uses in cancer therapy. Acknowledgements This research is funded by National Natural Science Foundation of China (grant No. 81472328), Liver disease AIDS Foundation of You An Hospital, Capital Medical University (BJYAH2011-034), National Science and Technology Support Project (2012BAI15B08), and Key Project of National Communicable Disease (2012ZX10002015-002). References Fig. 5. Western blot analysis of HIF-1alpha, VEGF, AUF-1 and AEG-1 protein expression in tumors. Lane 1: Control; Lane 2: 0.5 mg/kg TP-1; Lane 3: 1 mg/kg TP-1 and Lane 4: 2 mg/kg TP-1.
4. Conclusions Angiogenesis occurs in both physiological and pathological conditions for new blood vessel formation and is a necessary event for tumor growth and metastasis [22]. VEGF is a tumor-secreted cytokine with grave importance in tumor angiogenesis and invasion [23], which acts as an angiogenic factor in neo-angiogenesis by promoting the proliferation and migration of endothelial cells, as well as the formation of new blood vessels [24,25]. Recent studies have demonstrated that HCC is generally considered to be a hypervascular tumor and that inhibition of angiogenesis via downregulation of VEGF has become a promising approach for the treatment of HCC [26–28]. Furthermore, lager amount of evidence has shown that HIF-1alpha, AFU-1 and AEG-1 play an important role in the growth of highly vascularized tumors, such as HCC [29,30,17,31,32]. Therefore, the combination regimens targeting HIF-1alpha/VEGF and AUF-1/AEG-1 might be an effective therapeutic strategy in the prevention as well as treatment of human HCC. In conclusion, our work provides novel in vivo evidence that TP-1 attenuates tumor growth and pulmonary metastasis by exerting a range of prominent antitumor effects (inhibition of cell
[1] J.S. Lee, I.S. Chu, J. Heo, D.F. Calvisi, Z. Sun, T. Roskams, A. Durnez, A.J. Demetris, S.S. Thorgeirsson, Hepatology 40 (2004) 667–676. [2] B.A. Cahill, D. Braccia, Clin. J. Oncol. Nurs. 8 (2004) 393–399. [3] H.B. El-Serag, K.L. Rudolph, Gastroenterology 132 (2007) 2557–2576. [4] S. Coulon, F. Heindryckx, A. Geerts, C. Van Steenkiste, I. Colle, H. Van Vlierberghe, Liver Int. 31 (2011) 146–162. [5] V. Granci, Y.M. Dupertuis, C. Pichard, Curr. Opin. Clin. Nutr. Metab. Care 13 (2010) 417–422. [6] S.S. Verbridge, N.W. Choi, Y. Zheng, D.J. Brooks, A.D. Stroock, C. Fischbach, Tissue Eng. A 16 (2010) 2133–2141. [7] J.A. Chen, M. Shi, J.Q. Li, C.N. Qian, Hepatol. Int. 4 (2010) 537–547. [8] M. Varela, M. Sala, J.M. Llovet, J. Bruix, Cancer Treat. Rev. 29 (2003) 99–104. [9] D. Aoki, S. Ueno, F. Kubo, T. Oyama, T. Sakuta, K. Matsushita, I. Maruyama, T. Aikou, Anticancer Res. 25 (2005) 133–138. [10] J. Folkman, J. Natl. Cancer Inst. 82 (1990) 4–6. [11] J. Folkman, Y. Shing, J. Biol. Chem. 267 (1992) 10931–10934. [12] M.P. Barr, D.J. Bouchier-Hayes, J.J. Harmey, Int. J. Oncol. 32 (2008) 41–48. [13] C. Tzao, S.C. Lee, H.J. Tung, H.S. Hsu, W.H. Hsu, G.H. Sun, C.P. Yu, J.S. Jin, Y.L. Cheng, Dis. Markers 25 (2008) 141–148. [14] A. Basu, A.G. Contreras, D. Datta, E. Flynn, L. Zeng, H.T. Cohen, D.M. Briscoe, S. Pal, Cancer Res. 68 (2008) 5689–5698. [15] C. Lin, R. McGough, B. Aswad, J.A. Block, R. Terek, J. Orthop. Res. 22 (2004) 1175–1181. ¨ G. Finkenzeller, B. Wiedenmann, S. [16] Z. von Marschall, T. Cramer, M. Hocker, Rosewicz, Gut 48 (2001) 87–96. [17] Y. Yang, P. Kang, J. Gao, C. Xu, S. Wang, H. Jin, Y. Li, W. Liu, X. Wu, Tumor Biol. 35 (2014) 2747–2751. [18] A.M. Staub, Methods Carbohydr. Chem. 5 (1965) 5–6. [19] N. Weidner, J.P. Semple, W.R. Welch, J. Folkman, N. Engl. J. Med. 324 (1991) 1–8. [20] M.M. Bradford, Anal. Biochem. 72 (1976) 248–254. ˜ [21] N.G. Ordónez, Adv. Anat. Pathol. 19 (2012) 281–295. [22] J.L. Masferrer, K.M. Leahy, A.T. Koki, B.S. Zweifel, S.L. Settle, B.M. Woerner, D.A. Edwards, A.G. Flickinger, R.J. Moore, K. Seibert, Cancer Res. 60 (2000) 1306–1311.
120
C. Li et al. / International Journal of Biological Macromolecules 75 (2015) 115–120
[23] R.S. Finn, A.X. Zhu, Expert Rev. Anticancer Ther. 9 (2009) 503–509. [24] A.E. Frankel, P.S. Gill, Leuk. Res. 28 (2004) 675–677. [25] M.H. Oak, J. El Bedoui, V.B. Schini-Kerth, J. Nutr. Biochem. 16 (2005) 1–8. [26] D. Ribatti, B. Nico, D. Mangieri, V. Longo, D. Sansonno, A. Vacca, F. Dammacco, Histol. Histopathol. 22 (2007) 285–289. [27] I.T. Chiang, Y.C. Liu, W.H. Wang, F.T. Hsu, H.W. Chen, W.J. Lin, W.Y. Chang, J.J. Hwang, In Vivo 26 (2012) 671–681. [28] J.A. Chen, M. Shi, J.Q. Li, C.N. Qian, Hepatol. Int. 29 (2010) 537–547.
[29] H. Tanaka, M. Yamamoto, N. Hashimoto, M. Miyakoshi, S. Tamakawa, M. Yoshie, Y. Tokusashi, K. Yokoyama, Y. Yaginuma, K. Ogawa, Cancer Res. 66 (2006) 11263–11270. [30] S. Yasuda, S. Arii, A. Mori, N. Isobe, W. Yang, H. Oe, A. Fujimoto, Y. Yonenaga, H. Sakashita, M. Imamura, J. Hepatol. 40 (2004) 117–123. [31] J. Zheng, C. Li, X. Wu, Y. Yang, M. Hao, S. Sheng, Y. Sun, H. Zhang, J. Long, C. Hu, Tumour Biol. 35 (2014) 2265–2269. [32] J. Zheng, C. Li, X. Wu, M. Liu, X. Sun, Y. Yang, M. Hao, S. Sheng, Y. Sun, H. Zhang, J. Long, Y. Liang, C. Hu, Int. J. Biol. Macromol. 64 (2014) 106–110.