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Antitumor and antiangiogenic activity of Schisandra chinensis polysaccharide in a renal cell carcinoma model
International Journal of Biological Macromolecules 66 (2014) 52–56
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Antitumor and antiangiogenic activity of Schisandra chinensis polysaccharide in a renal cell carcinoma model Hai-Ming Qu a,1 , Shi-Jian Liu b,1 , Chun-Ying Zhang a,∗ a b
Department of Urology, Section 2, The Second Affiliated Hospital of Harbin Medical University, Harbin 150040, China Department of Nephrology, Section 1, The Second Affiliated Hospital of Harbin Medical University, Harbin 150040, China
a r t i c l e
i n f o
Article history: Received 9 January 2014 Received in revised form 4 February 2014 Accepted 9 February 2014 Available online 18 February 2014 Keywords: Schisandra chinensis Polysaccharide Renal cell carcinoma (RCC) Vascular endothelial growth factor (VEGF) Antitumor and antiangiogenic activities
a b s t r a c t The aim of this study was to determine the antitumor and antiangiogenic effects of the Schisandra chinensis polysaccharides (SCP) in selected renal cell carcinoma (RCC) cells and evaluate its potential mechanism of action. In vitro, endothelial growth factor (VEGF) secretion by Caki-1 was blockaded in response to SCP treatment for 48 h. In vivo, a significant tumor growth inhibition effect was observed after SCP administration for 4 weeks. Moreover, SCP treatment decreased the level of VEGF, CD31 and CD34 in RCC tumor tissues. Further analysis of the tumor inhibition mechanism indicated that the number of apoptotic tumor cells increased significantly; the expression of Bax and p53 increased; and the expression of Bcl-2 decreased dramatically in transplanted tumor tissues following SCP administration. These results indicated that the potential mechanisms involved by which SCP exerted its antitumor and antiangiogenic activity might be associated with the up-regulation of Bax and p53, downregulation of Bcl-2, as well as the reduction of VEGF, CD31 and CD34 in xenografted tumors. These findings demonstrated that the SCP is a potential antitumor agent for RCC treatment. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Renal cell carcinoma (RCC) is the most common malignancy of the adult kidney, and annual estimates of newly diagnosed cases have been steadily increasing [1,2]. Although nephrectomy is the most effective treatment at an early stage, therapeutic options for unresectable and/or metastatic RCC are limited due to an inherent tumor resistance to conventional chemotherapy and radiotherapy [3,4]. Because RCC is characterized by a lack of specific clinical signs that allow the diagnosis at an early stage, a high proportion of patients will have metastasis at the time of first diagnosis and confront with an often unpredictable course [5]. In view of these conditions, the discovery of new strategies or molecular targeting therapies for metastatic RCC remains a priority. In recent years, novel agents for RCC targeting cancer-specific pathways have been developed based on the precise understanding of molecular mechanisms underlying the progression of RCC [6,7]. As a result of the hypervascularity in RCC, several new agents targeting the vascular endothelial growth factor (VEGF) pathway have demonstrated significant activity in patients with advanced RCC, such as sunitinib, sorafenib and pazopanib [8]. Although they are
currently being used with some success in patients with advanced RCC, the effect is insufficient, and it is therefore necessary to discover new antiangiogenic targets for the treatment of RCC [9,10]. Schisandra chinensis is a traditional Chinese medicine and has been officially listed in the Chinese Pharmacopoeia as a tonic and sedative agent [11]. Chemical investigations of the extracts of S. chinensis have revealed the presence of amino acid, polysaccharide, sesquiterpene, vitamin, organic acid, volatile oil, especially bioactivie components lignan and triter-penenoid [12]. Current researches have revealed that polysaccharides from S. chinensis possess a large variety of beneficial effects including immunomodulating [13,14], anticancer [15,16], antioxidant [17], anti-diabetic [18] and anti-aging [19], etc. However, the anti-tumor and antiangiogenic activities of polysaccharides from S. chinensis on RCC have not been reported. Therefore, the aim of this study is to determine the antitumor and antiangiogenic action of SCP toward RCC, and gained insights into the mechanisms involved through which SCP exerts its effects.
2. Materials and methods 2.1. Materials
∗ Corresponding author. Tel.: +86 451 86605579; fax: +86 451 86605579. E-mail address: [email protected] (C.-Y. Zhang). 1 Contributed equally to this paper. http://dx.doi.org/10.1016/j.ijbiomac.2014.02.025 0141-8130/© 2014 Elsevier B.V. All rights reserved.
The ethanol-insoluble residue generated during lignans industrial production from S. chinensis via 75% ethanol extraction was
H.-M. Qu et al. / International Journal of Biological Macromolecules 66 (2014) 52–56
purchased from Zhongxing Pharmaceutical Co., Ltd., Zhenjiang, Jiangsu Province, China. 2.2. Polysaccharide extraction After the dried ethanol-insoluble residue of S. chinensis was rushed or ground into powder with a blender, the powders (500 g) were extracted three times with distilled water (3 h/time). The whole extract was concentrated under reduced pressure at 50 ◦ C and added by ethanol (final concentration 75% (v/v)) to precipitate the crude polysaccharide at 4 ◦ C overnight. The precipitate was collected by centrifugation (6000 rpm, 20 min) and redissolved in distilled water. Finally, the supernatant was lyophilized to give 53 g of crude S. chinensis polysaccharides (SCP). 2.3. Cell lines and cell culture Human RCC cell line Caki-1 was purchased from American Type Culture Collection. They were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 2 mM l-glutamine, penicillin (100 U/ml) and streptomycin (100 g/ml). Cells were incubated at 37 ◦ C in a humidified atmosphere with 5% CO2 and subcultured after trypsinization. 2.4. Detection of VEGF secretion produced by Caki-1 cells Caki-1 cells were seeded at 2 × 104 cells/well in 96-well culture plates in triplicates. Next day, the supernatants were discarded and the cells were washed twice with PBS. Thereafter cells were cultured in the presence/absence of SCP (100, 200 and 400 g/ml) under hypoxic or normoxic conditions for 24 h. After treatment, the amounts of VEGF in each supernatant was determined using Quantikine human VEGF Immunoassay kits (R&D Systems, Minneapolis, MN) and were normalized versus total protein content as determined using the Bradford assay.
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(BD Bioscence). Protein content was measured by the Bradford assay using bovine serum albumin (BSA) as a standard. Equal amount of protein (30 g) were resolved by 10–12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (Bio-Rad) by electroblot analysis. Nitrocellulose blots were blocked with 5% (w/v) nonfat dried-milk and incubated with the indicated primary antibody against p53 (1:10,000, Sigma, Alcobendas, Spain), Bax (1:200, Santa Cruz Biotechnology, Santa Cruz, CA) and Bcl-2 (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA) in Tris-buffered saline (TBS) overnight at 4 ◦ C, and then the blots were washed and stained with the appropriate horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at room temperature. Immunostained proteins were visualized on Xray film using the enhanced chemiluminescence detection system (Amersham–Pharmacia Biotech, Piscataway, NJ). A -actin antibody (Merck, Darmstadt, Germany) (1:10,000) was used for loading control. 2.7. RNA extraction and reverse transcriptase–polymerase chain reaction (RT-PCR) Total RNA was extracted from tumor samples using TRI® reagent (Biotech Labs, Houston, TX, USA) and RT-PCR was performed as described previously [21]. cDNA fragments were amplified by polymerase chain reaction using the following VEGF-specific primers: sense 5 -AGGAGGGCAGAATCATCACG-3 and anti-sense 5 -CAAGGCCCACAG GGATTTTCT-3 . 2.8. Determination of VEGF VEGF levels in tumor cytosolic extracts were determined using Quantikine human VEGF Immunoassay kits (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. Data were normalized by the protein concentration in each sample.
2.5. In vivo xenograft model
2.9. Immunohistochemistry
Seven-week-old male athymic BALB/c nude (nu/nu) mice were purchased from Experimental Animal Center of Harbin Medical University. Animals were housed in a specific pathogen-free facility under controlled temperature and humidity. The procedures involving animals and their care were conducted in accordance with institutional guidelines for Laboratory Animal Care of Experimental Animal Center, Harbin Medical University. Early passage Caki-1 cells were harvested and 5 × 106 cells were implanted subcutaneously into both flanks of each mouse. Treatment was initiated after 15 days when the tumors reached 100–150 mm3 in volume. The animals were randomized into normal control and SCP-treated groups (5 mice per group). Group-I received vehicle only (5 ml/kg) via intra-peritoneal injection (i.p.) served as a normal control. Group-II received SCP at a dose of 400 mg/kg i.p. every third day for 4 weeks, respectively. Tumor volume (TV) was measured every 6 days by measuring two perpendicular dimensions (long and short) using a caliper and calculated as TV = (a × b2 )/2, where a is the larger and b is the smaller dimension of the tumor [20]. Four weeks later, mice were sacrificed and the tumors were collected. A portion was fixed in10% formalin and embedded in paraffin to prepare the block for immunohistochemistry, and the other portions were frozen in liquid nitrogen and maintained at −80 ◦ C.
Serial sections (5-m-thick) were cut from each paraffin block and deparaffinized in xylene and rehydrated through a graded ethanol concentrations. Then, they were incubated with 3% hydrogen peroxide for 20 min at room temperature to inactivate endogenous peroxidase and heated in 0.1 M citrate buffer (pH 6.0) for 5 min in a microwave to retrieve antigens. After rinsing in TBS, non-specific sites binding to the first antibody were blocked with a blocking solution containing 2.5% bovine serum albumin (Sigma-Aldrich, St Louis, MO) and 2% normal goat serum (Vector Laboratories, Burlingame, CA) in TBS (pH 7.4) for 1 h. Afterwards, the sections were incubated overnight at 4 ◦ C with the primary antibody against CD-31 (1:200 dilution in blocking solution; Santa Cruz Biotechnology, Santa Cruz, CA) and CD-34 (1:100 dilution in blocking solution; Santa Cruz Biotechnology, Santa Cruz, CA). Sections of tumor samples processed identically, but incubated with diluents (blocking solution) in the absence of any primary antibodies, were used as negative control. Then, the sections were washed in TBS and incubated for 20 min with the appropriate biotinylated secondary antibodies (Dako LSAB® System-HRP, Dako, Glostrup, Denmark) at a 1:500 dilution for 1 h. After an extensive wash in TBS, the sections were incubated with avidin–biotin–horseradish peroxidase complex (Dako) for 20 min at room temperature to localize bound antibodies. Staining of the protein was visualized by incubating sections with 3,3 -diaminobenzidine tetrahydrochloride (DAB) and lightly counterstaining them with hematoxylin. The different sections were analyzed per xenograft tumor. For histological assessment, CD31-positive and CD34-positive micro vessels were viewed under Nikon 80i microscope at magnifications of ×600 and
2.6. Western blotting Tumor tissue was homogenized in order to extract the cytosolic and nuclear contents with a commercially available kit
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examined using a Microcomputer Imaging Device model 4 image analysis system. The expressions of vessel densities were measured by counting number of vessel profiles (identified by CD31 and CD34 staining) per square millimeter in each image. 2.10. Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) assay
2.11. Statistical analysis All determinations were done at least 3 times and results are presented as the mean ± SD of three experiments. Significance was analyzed by Student’s t test with P < 0.05 considered significant. 3. Results and discussion 3.1. Effect of SCP on VEGF secretion by Caki-1 cells To examine the inhibitory effect of SCP on angiogenesis, we measured the amounts of VEGF secreted by human RCC Caki-1 cells upon SCP treatment under hypoxic or normoxic conditions. As shown in Fig. 1, SCP significantly suppressed VEGF production by Caki-1 cells at 24 h compared with that of the cells treated with hypoxia alone, with the increase in the concentrations. These results demonstrate that SCPs inhibits hypoxia-induced VEGF secretion, indicating its antiangiogenic activity. 3.2. Effect of SCP on xenografted tumor growth Given our previous results showing the inhibitory effects of SCP on signaling and proliferation in RCC Caki-1cells, we examined the ability of SCP to inhibit tumor growth in vivo using human tumor models xenografted in athymic mice. Compared with the vehicletreated group, SCP significantly inhibited tumor growth in Caki-1 xenografted athymic mice (Fig. 2), and the difference become significant after one weeks’ SCP treatment (P < 0.05). Furthermore, there was no substantial body weight change in the SCP-treated group relative to vehicle-treated group during the experiment, which could be considered as the antitumor activity of SCP taking precedence over the toxicity on tumor-bearing mice.
Fig. 1. Effect of SCP on VEGF production by Caki-1 cells. The quantitative data are presented as the mean ± S.D. of three independent experiments. *** Denotes P < 0.001 versus the control supernatants from cells cultured under normoxic conditions. ### Denotes P < 0.001 versus the control supernatants from cells cultured under hypoxic conditions.
Tumor volume (TV, cm3)
The tumor tissues were examined by the TUNEL assay for the detection of apoptotic cells in situ using an Apop Tag Plus Peroxidase in situ apoptosis detection kit from Chemicon (Temecula, CA), with the following modifications [22]. Briefly, the nuclei of tissue sections were stripped of protein by incubation with proteinase K (20 g/ml in PBS) for 15 min at room temperature and immediately fixed in 40% paraformaldehyde at room temperature for 5 min. The slides were then washed with 0.5% Triton X-100 (Sigma Chemical Co., St. Louis, MO) in PBS for 5 min at room temperature and incubated in a 1% glycine-PBS solution. After being rinsed with distilled water, the slides were equilibrated for 10 min with terminal deoxynucleotidyl transferase (TdT) buffer (30 mM Tris, pH 7.2, 140 mM sodium cacodylate, 1 mM cobalt chloride), and incubated in a TdT mix containing 100 U TdT (Gibco BRL, Gaithersburg, MD) and 0.5 l biotin-16-dUTP (Boehringer Mannheim) in TdT buffer in a humid atmosphere at 37 ◦ C for 2 h. Biotin-16-dUTP labeled in 3-OH of DNA was detected by streptavidin-HRP followed by color development with a solution containing DAB and hydrogen peroxide and hematoxylin was used for counterstaining. The cells whose nucleus turned a distinct brown color were considered as positive cells and the cells whose nucleus turned green were negative cells. For the quantification of TUNEL expression, the numbers of positive cells were counted in 20 random fields at ×200 and the mean number was calculated.
3000
Vehicle SCP (400mg/ml)
2000
1000 *
*
*
29
36
42
*
0 15
22
Days Fig. 2. Effect of SCP on Caki-1 xenografted human tumor growth. The quantitative data are presented as the mean ± S.D. of five independent experiments. * Denotes P < 0.05 versus the control.
3.3. Effect of SCP on VEGF expression in xenografted tumors Tumor angiogenesis is a critical step for the growth and metastasis of solid tumors [23,24]. Since VEGF is the most important angiogenic molecule associated with tumor-induced neovascularization [25], we investigated the effects of SCP on VEGF levels in mice using an ELISA kit. As seen from Fig. 3A, the level of VEGF was found to be markedly lower in tumor lysates derived from SCP-treated mice than that in vehicle-treated tumors (P < 0.01). In addition, VEGF mRNA levels almost disappeared in SCP-treated tumors compared with the vehicle-treated tumors (Fig. 3B). Summarizing, our results suggest that the contribution of SCP to inhibit VEGF expression would block tumor growth and angiogenesis in Caki-1 tumor-bearing mice.
3.4. Effect of SCP on CD-31 and CD-34 expression in xenografted tumors To determine whether inhibitory effects of SCP on tumor growth are associated with the suppression of angiogenesis, we examined the immunohistochemical expression of two endothelial markers, CD31 and CD3, in tumor tissues [26,27]. As shown in Fig. 4, an immunohistochemistry approach showed that Caki1 tumors from vehicle-treated mice showed many CD31 and CD34-immunopositive cells, but in contrast, tumor sections from SCP-treated mice showed few or no CD31 and CD34immunoreactive cells. These results indicate that SCP could blockade angiogenesis to fight against tumor progression.
H.-M. Qu et al. / International Journal of Biological Macromolecules 66 (2014) 52–56
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Fig. 6. Effect of SCP on Bcl-2, Bax and p53 protein expression in xenografted tumors.
3.6. Effect of SCP on Bcl-2, Bax and p53 protein expression in xenografted tumors Fig. 3. (A) Effect of SCP on the level of VEGF in tumor cytosolic fractions measured by ELISA; (B) Effect of SCP on the mRNA levels of VEGF in tumor cytosolic fractions measured by RT-PCR. The quantitative data are presented as the mean ± S.D. of five independent experiments. ** Denotes P < 0.01 versus the control.
Fig. 4. Quantifications of CD31 and CD34-immunopositive cells in xenografted tumors by immunohistochemistry. The quantitative data are presented as the mean ± S.D. of five independent experiments. ** Denotes P < 0.01 versus the control.
After confirming that SCP induces apoptosis during RCC carcinogenesis, our next attempt was to determine the impact of SCP on the regulation of the apoptotic signal transduction pathway involved in SCP-induced apoptosis. As is well established that Bcl-2 family proteins are the crucial players in the programmed apoptotic pathway [28], we examined whether or not SCP can affect the expression of proapoptotic protein Bax and antiapoptotic protein Bcl-2 in our mice model. Interestingly, it was observed that the mice administered with SCP revealed a significant decreased expression of Bcl-2 and increased expression of Bax in contrast to those in the control group, and thereby increasing the ratio of Bax to Bcl-2, which is an important factor for the occurrence of apoptosis (Fig. 6). It is well known that the tumor suppressor protein p53 is inactivated in about half of human cancers [29,30]. For this reason, we determined p53 expression in Caki-1 tumors by Western blot. As seen from Fig. 6, p53 expression in tumor mass of SCP-treated mice was markedly higher than that of vehicle-treated mice. Collectively, these findings demonstrate that SCP might trigger apoptosis via the up-regulation of Bax and p53 and downregulation of Bcl-2 gene expression in Caki-1 tumor-bearing mice. 4. Conclusions
Fig. 5. Quantifications of apoptotic cells in xenografted tumors by TUNEL assay. The quantitative data are presented as the mean ± S.D. of five independent experiments. ** Denotes P < 0.01 versus the control.
3.5. The effects of SCP on apoptosis of xenografted tumors To further confirm whether the inhibitory effect of SCP on RCC xenografts growth is via apoptotic mediated mechanisms, the TUNEL assay was conducted for detecting the apoptosis. As presented in Fig. 5, we observed that the average number of TUNELpositive cells was significantly higher in tumors treated with SCP (P < 0.01) than that in the control tumor. These results indicated that SCP displays its antitumor activity via inducing apoptosis of tumor tissue cells.
Recently, a greater emphasis has been given toward researching complementary and alternative medicine in cancer management. Several studies have revealed that many natural health products that inhibit angiogenesis also manifest other anticancer activities, and have been used as potent anticancer drugs [31]. Advances in the understanding of the pathogenesis and molecular biology of RCC have identified angiogenesis as a key factor in the development of the disease [32,33]. In general, RCC is characterized by rich neovascularization and develops a fine vascular network around the cancer cells [34]. Most hypervascularized RCC also express a number of angiogenesis-related factors, in particular VEGF [35,36]. As well, VEGF is highly expressed in many human cancers, but RCC in particular produces remarkably high levels [37]. Therefore control of VEGF is essential for treating patients with RCC. The present work has addressed the antineoplastic and antiangiogenic potential of SCP in RCC. In vitro, exposure of SCP to Caki-1 cells for 24 h resulted in a decrease of VEGF secretion under hypoxic conditions. Furthermore SCP exerted a potent inhibitory activity on the growth of xenografted RCC Caki-1 cells in athymic nude mice. We also observed that the low expression of VEGF level and two endothelial markers, CD31 and CD3, in tumor tissues. Meanwhile the proapoptotic protein Bax and p53 increased, whereas antiapoptotic protein Bcl-2 decreased in a mouse xenograft model after drug
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treatment. Besides, there was no substantial body weight change in SCP-treated mice. Collectively, these results indicated that SCP might be safe and effective for use in the treatment for RCC. Acknowledgment This study was supported by the fund of Heilongjiang Province Science and Technology Plan Project (No. GC05C40701). References [1] R.J. Motzer, N.H. Bander, D.M. Nanus, New England Journal of Medicine 335 (1996) 865–875. [2] A. Jemal, R. Siegel, E. Ward, T. Murray, J. Xu, C. Smigal, M.J. Thun, CA: A Cancer Journal for Clinicians 56 (2006) 106–130. [3] R.J. Amato, Annals of Oncology 16 (2005) 7–15. [4] B.I. Rini, Clinical Cancer Research 13 (2007) 1098–1106. [5] R.H. Weiss, P.Y. Lin, Kidney International 69 (2006) 224–232. [6] J. Bellmunt, C. Montagut, S. Albiol, J. Carles, P. Maroto, A. Orsola, BJU International 99 (2007) 274–280. [7] B.R. Lane, B.I. Rini, A.C. Novick, S.C. Campbell, Urology 69 (2007) 3–10. [8] X. Wang, L. Zhang, A. O’Neill, B. Bahamon, D.C. Alsop, J.W. Mier, S.N. Goldberg, S. Signoretti, M.B. Atkins, C.G. Wood, R.S. Bhatt, British Journal of Cancer 108 (2013) 319–326. [9] M.T. Pirrotta, P. Bernardeschi, G. Fiorentini, Current Medicinal Chemistry 18 (2011) 1651–1657. [10] K.T. Flaherty, I. Puzanov, Biochemical Pharmacology 80 (2010) 638–646. [11] C.J. Liu, S.Q. Zhang, J.S. Zhang, Q. Liang, D.S. Li, Natural Product Research 26 (2012) 2199–2203. [12] Z.S. Wang, H.X. Chen, W.J. Zhang, G.S. Lan, L.K. Zhang, Journal of Medicinal Plant Research 5 (2011) 1207–1216. [13] L.Q. Yang, R.H. Chen, X.Y. Wu, Q.Y. Fan, J.L. Zhao, Food Science 29 (2008) 392–394. [14] Y. Chen, J. Tang, X. Wang, F. Sun, S. Liang, International Journal of Biological Macromolecules 50 (2012) 844–848. [15] T. Zhao, G. Mao, R. Mao, Y. Zou, D. Zheng, W. Feng, Y. Ren, W. Wang, W. Zheng, J. Song, Y. Chen, L. Yang, X. Wu, Food and Chemical Toxicology 55 (2013) 609–616.
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Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Antitumor and antiangiogenic activity of Schisandra chinensis polysaccharide in a renal cell carcinoma model Hai-Ming Qu a,1 , Shi-Jian Liu b,1 , Chun-Ying Zhang a,∗ a b
Department of Urology, Section 2, The Second Affiliated Hospital of Harbin Medical University, Harbin 150040, China Department of Nephrology, Section 1, The Second Affiliated Hospital of Harbin Medical University, Harbin 150040, China
a r t i c l e
i n f o
Article history: Received 9 January 2014 Received in revised form 4 February 2014 Accepted 9 February 2014 Available online 18 February 2014 Keywords: Schisandra chinensis Polysaccharide Renal cell carcinoma (RCC) Vascular endothelial growth factor (VEGF) Antitumor and antiangiogenic activities
a b s t r a c t The aim of this study was to determine the antitumor and antiangiogenic effects of the Schisandra chinensis polysaccharides (SCP) in selected renal cell carcinoma (RCC) cells and evaluate its potential mechanism of action. In vitro, endothelial growth factor (VEGF) secretion by Caki-1 was blockaded in response to SCP treatment for 48 h. In vivo, a significant tumor growth inhibition effect was observed after SCP administration for 4 weeks. Moreover, SCP treatment decreased the level of VEGF, CD31 and CD34 in RCC tumor tissues. Further analysis of the tumor inhibition mechanism indicated that the number of apoptotic tumor cells increased significantly; the expression of Bax and p53 increased; and the expression of Bcl-2 decreased dramatically in transplanted tumor tissues following SCP administration. These results indicated that the potential mechanisms involved by which SCP exerted its antitumor and antiangiogenic activity might be associated with the up-regulation of Bax and p53, downregulation of Bcl-2, as well as the reduction of VEGF, CD31 and CD34 in xenografted tumors. These findings demonstrated that the SCP is a potential antitumor agent for RCC treatment. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Renal cell carcinoma (RCC) is the most common malignancy of the adult kidney, and annual estimates of newly diagnosed cases have been steadily increasing [1,2]. Although nephrectomy is the most effective treatment at an early stage, therapeutic options for unresectable and/or metastatic RCC are limited due to an inherent tumor resistance to conventional chemotherapy and radiotherapy [3,4]. Because RCC is characterized by a lack of specific clinical signs that allow the diagnosis at an early stage, a high proportion of patients will have metastasis at the time of first diagnosis and confront with an often unpredictable course [5]. In view of these conditions, the discovery of new strategies or molecular targeting therapies for metastatic RCC remains a priority. In recent years, novel agents for RCC targeting cancer-specific pathways have been developed based on the precise understanding of molecular mechanisms underlying the progression of RCC [6,7]. As a result of the hypervascularity in RCC, several new agents targeting the vascular endothelial growth factor (VEGF) pathway have demonstrated significant activity in patients with advanced RCC, such as sunitinib, sorafenib and pazopanib [8]. Although they are
currently being used with some success in patients with advanced RCC, the effect is insufficient, and it is therefore necessary to discover new antiangiogenic targets for the treatment of RCC [9,10]. Schisandra chinensis is a traditional Chinese medicine and has been officially listed in the Chinese Pharmacopoeia as a tonic and sedative agent [11]. Chemical investigations of the extracts of S. chinensis have revealed the presence of amino acid, polysaccharide, sesquiterpene, vitamin, organic acid, volatile oil, especially bioactivie components lignan and triter-penenoid [12]. Current researches have revealed that polysaccharides from S. chinensis possess a large variety of beneficial effects including immunomodulating [13,14], anticancer [15,16], antioxidant [17], anti-diabetic [18] and anti-aging [19], etc. However, the anti-tumor and antiangiogenic activities of polysaccharides from S. chinensis on RCC have not been reported. Therefore, the aim of this study is to determine the antitumor and antiangiogenic action of SCP toward RCC, and gained insights into the mechanisms involved through which SCP exerts its effects.
2. Materials and methods 2.1. Materials
∗ Corresponding author. Tel.: +86 451 86605579; fax: +86 451 86605579. E-mail address: [email protected] (C.-Y. Zhang). 1 Contributed equally to this paper. http://dx.doi.org/10.1016/j.ijbiomac.2014.02.025 0141-8130/© 2014 Elsevier B.V. All rights reserved.
The ethanol-insoluble residue generated during lignans industrial production from S. chinensis via 75% ethanol extraction was
H.-M. Qu et al. / International Journal of Biological Macromolecules 66 (2014) 52–56
purchased from Zhongxing Pharmaceutical Co., Ltd., Zhenjiang, Jiangsu Province, China. 2.2. Polysaccharide extraction After the dried ethanol-insoluble residue of S. chinensis was rushed or ground into powder with a blender, the powders (500 g) were extracted three times with distilled water (3 h/time). The whole extract was concentrated under reduced pressure at 50 ◦ C and added by ethanol (final concentration 75% (v/v)) to precipitate the crude polysaccharide at 4 ◦ C overnight. The precipitate was collected by centrifugation (6000 rpm, 20 min) and redissolved in distilled water. Finally, the supernatant was lyophilized to give 53 g of crude S. chinensis polysaccharides (SCP). 2.3. Cell lines and cell culture Human RCC cell line Caki-1 was purchased from American Type Culture Collection. They were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 2 mM l-glutamine, penicillin (100 U/ml) and streptomycin (100 g/ml). Cells were incubated at 37 ◦ C in a humidified atmosphere with 5% CO2 and subcultured after trypsinization. 2.4. Detection of VEGF secretion produced by Caki-1 cells Caki-1 cells were seeded at 2 × 104 cells/well in 96-well culture plates in triplicates. Next day, the supernatants were discarded and the cells were washed twice with PBS. Thereafter cells were cultured in the presence/absence of SCP (100, 200 and 400 g/ml) under hypoxic or normoxic conditions for 24 h. After treatment, the amounts of VEGF in each supernatant was determined using Quantikine human VEGF Immunoassay kits (R&D Systems, Minneapolis, MN) and were normalized versus total protein content as determined using the Bradford assay.
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(BD Bioscence). Protein content was measured by the Bradford assay using bovine serum albumin (BSA) as a standard. Equal amount of protein (30 g) were resolved by 10–12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (Bio-Rad) by electroblot analysis. Nitrocellulose blots were blocked with 5% (w/v) nonfat dried-milk and incubated with the indicated primary antibody against p53 (1:10,000, Sigma, Alcobendas, Spain), Bax (1:200, Santa Cruz Biotechnology, Santa Cruz, CA) and Bcl-2 (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA) in Tris-buffered saline (TBS) overnight at 4 ◦ C, and then the blots were washed and stained with the appropriate horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at room temperature. Immunostained proteins were visualized on Xray film using the enhanced chemiluminescence detection system (Amersham–Pharmacia Biotech, Piscataway, NJ). A -actin antibody (Merck, Darmstadt, Germany) (1:10,000) was used for loading control. 2.7. RNA extraction and reverse transcriptase–polymerase chain reaction (RT-PCR) Total RNA was extracted from tumor samples using TRI® reagent (Biotech Labs, Houston, TX, USA) and RT-PCR was performed as described previously [21]. cDNA fragments were amplified by polymerase chain reaction using the following VEGF-specific primers: sense 5 -AGGAGGGCAGAATCATCACG-3 and anti-sense 5 -CAAGGCCCACAG GGATTTTCT-3 . 2.8. Determination of VEGF VEGF levels in tumor cytosolic extracts were determined using Quantikine human VEGF Immunoassay kits (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. Data were normalized by the protein concentration in each sample.
2.5. In vivo xenograft model
2.9. Immunohistochemistry
Seven-week-old male athymic BALB/c nude (nu/nu) mice were purchased from Experimental Animal Center of Harbin Medical University. Animals were housed in a specific pathogen-free facility under controlled temperature and humidity. The procedures involving animals and their care were conducted in accordance with institutional guidelines for Laboratory Animal Care of Experimental Animal Center, Harbin Medical University. Early passage Caki-1 cells were harvested and 5 × 106 cells were implanted subcutaneously into both flanks of each mouse. Treatment was initiated after 15 days when the tumors reached 100–150 mm3 in volume. The animals were randomized into normal control and SCP-treated groups (5 mice per group). Group-I received vehicle only (5 ml/kg) via intra-peritoneal injection (i.p.) served as a normal control. Group-II received SCP at a dose of 400 mg/kg i.p. every third day for 4 weeks, respectively. Tumor volume (TV) was measured every 6 days by measuring two perpendicular dimensions (long and short) using a caliper and calculated as TV = (a × b2 )/2, where a is the larger and b is the smaller dimension of the tumor [20]. Four weeks later, mice were sacrificed and the tumors were collected. A portion was fixed in10% formalin and embedded in paraffin to prepare the block for immunohistochemistry, and the other portions were frozen in liquid nitrogen and maintained at −80 ◦ C.
Serial sections (5-m-thick) were cut from each paraffin block and deparaffinized in xylene and rehydrated through a graded ethanol concentrations. Then, they were incubated with 3% hydrogen peroxide for 20 min at room temperature to inactivate endogenous peroxidase and heated in 0.1 M citrate buffer (pH 6.0) for 5 min in a microwave to retrieve antigens. After rinsing in TBS, non-specific sites binding to the first antibody were blocked with a blocking solution containing 2.5% bovine serum albumin (Sigma-Aldrich, St Louis, MO) and 2% normal goat serum (Vector Laboratories, Burlingame, CA) in TBS (pH 7.4) for 1 h. Afterwards, the sections were incubated overnight at 4 ◦ C with the primary antibody against CD-31 (1:200 dilution in blocking solution; Santa Cruz Biotechnology, Santa Cruz, CA) and CD-34 (1:100 dilution in blocking solution; Santa Cruz Biotechnology, Santa Cruz, CA). Sections of tumor samples processed identically, but incubated with diluents (blocking solution) in the absence of any primary antibodies, were used as negative control. Then, the sections were washed in TBS and incubated for 20 min with the appropriate biotinylated secondary antibodies (Dako LSAB® System-HRP, Dako, Glostrup, Denmark) at a 1:500 dilution for 1 h. After an extensive wash in TBS, the sections were incubated with avidin–biotin–horseradish peroxidase complex (Dako) for 20 min at room temperature to localize bound antibodies. Staining of the protein was visualized by incubating sections with 3,3 -diaminobenzidine tetrahydrochloride (DAB) and lightly counterstaining them with hematoxylin. The different sections were analyzed per xenograft tumor. For histological assessment, CD31-positive and CD34-positive micro vessels were viewed under Nikon 80i microscope at magnifications of ×600 and
2.6. Western blotting Tumor tissue was homogenized in order to extract the cytosolic and nuclear contents with a commercially available kit
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examined using a Microcomputer Imaging Device model 4 image analysis system. The expressions of vessel densities were measured by counting number of vessel profiles (identified by CD31 and CD34 staining) per square millimeter in each image. 2.10. Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) assay
2.11. Statistical analysis All determinations were done at least 3 times and results are presented as the mean ± SD of three experiments. Significance was analyzed by Student’s t test with P < 0.05 considered significant. 3. Results and discussion 3.1. Effect of SCP on VEGF secretion by Caki-1 cells To examine the inhibitory effect of SCP on angiogenesis, we measured the amounts of VEGF secreted by human RCC Caki-1 cells upon SCP treatment under hypoxic or normoxic conditions. As shown in Fig. 1, SCP significantly suppressed VEGF production by Caki-1 cells at 24 h compared with that of the cells treated with hypoxia alone, with the increase in the concentrations. These results demonstrate that SCPs inhibits hypoxia-induced VEGF secretion, indicating its antiangiogenic activity. 3.2. Effect of SCP on xenografted tumor growth Given our previous results showing the inhibitory effects of SCP on signaling and proliferation in RCC Caki-1cells, we examined the ability of SCP to inhibit tumor growth in vivo using human tumor models xenografted in athymic mice. Compared with the vehicletreated group, SCP significantly inhibited tumor growth in Caki-1 xenografted athymic mice (Fig. 2), and the difference become significant after one weeks’ SCP treatment (P < 0.05). Furthermore, there was no substantial body weight change in the SCP-treated group relative to vehicle-treated group during the experiment, which could be considered as the antitumor activity of SCP taking precedence over the toxicity on tumor-bearing mice.
Fig. 1. Effect of SCP on VEGF production by Caki-1 cells. The quantitative data are presented as the mean ± S.D. of three independent experiments. *** Denotes P < 0.001 versus the control supernatants from cells cultured under normoxic conditions. ### Denotes P < 0.001 versus the control supernatants from cells cultured under hypoxic conditions.
Tumor volume (TV, cm3)
The tumor tissues were examined by the TUNEL assay for the detection of apoptotic cells in situ using an Apop Tag Plus Peroxidase in situ apoptosis detection kit from Chemicon (Temecula, CA), with the following modifications [22]. Briefly, the nuclei of tissue sections were stripped of protein by incubation with proteinase K (20 g/ml in PBS) for 15 min at room temperature and immediately fixed in 40% paraformaldehyde at room temperature for 5 min. The slides were then washed with 0.5% Triton X-100 (Sigma Chemical Co., St. Louis, MO) in PBS for 5 min at room temperature and incubated in a 1% glycine-PBS solution. After being rinsed with distilled water, the slides were equilibrated for 10 min with terminal deoxynucleotidyl transferase (TdT) buffer (30 mM Tris, pH 7.2, 140 mM sodium cacodylate, 1 mM cobalt chloride), and incubated in a TdT mix containing 100 U TdT (Gibco BRL, Gaithersburg, MD) and 0.5 l biotin-16-dUTP (Boehringer Mannheim) in TdT buffer in a humid atmosphere at 37 ◦ C for 2 h. Biotin-16-dUTP labeled in 3-OH of DNA was detected by streptavidin-HRP followed by color development with a solution containing DAB and hydrogen peroxide and hematoxylin was used for counterstaining. The cells whose nucleus turned a distinct brown color were considered as positive cells and the cells whose nucleus turned green were negative cells. For the quantification of TUNEL expression, the numbers of positive cells were counted in 20 random fields at ×200 and the mean number was calculated.
3000
Vehicle SCP (400mg/ml)
2000
1000 *
*
*
29
36
42
*
0 15
22
Days Fig. 2. Effect of SCP on Caki-1 xenografted human tumor growth. The quantitative data are presented as the mean ± S.D. of five independent experiments. * Denotes P < 0.05 versus the control.
3.3. Effect of SCP on VEGF expression in xenografted tumors Tumor angiogenesis is a critical step for the growth and metastasis of solid tumors [23,24]. Since VEGF is the most important angiogenic molecule associated with tumor-induced neovascularization [25], we investigated the effects of SCP on VEGF levels in mice using an ELISA kit. As seen from Fig. 3A, the level of VEGF was found to be markedly lower in tumor lysates derived from SCP-treated mice than that in vehicle-treated tumors (P < 0.01). In addition, VEGF mRNA levels almost disappeared in SCP-treated tumors compared with the vehicle-treated tumors (Fig. 3B). Summarizing, our results suggest that the contribution of SCP to inhibit VEGF expression would block tumor growth and angiogenesis in Caki-1 tumor-bearing mice.
3.4. Effect of SCP on CD-31 and CD-34 expression in xenografted tumors To determine whether inhibitory effects of SCP on tumor growth are associated with the suppression of angiogenesis, we examined the immunohistochemical expression of two endothelial markers, CD31 and CD3, in tumor tissues [26,27]. As shown in Fig. 4, an immunohistochemistry approach showed that Caki1 tumors from vehicle-treated mice showed many CD31 and CD34-immunopositive cells, but in contrast, tumor sections from SCP-treated mice showed few or no CD31 and CD34immunoreactive cells. These results indicate that SCP could blockade angiogenesis to fight against tumor progression.
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Fig. 6. Effect of SCP on Bcl-2, Bax and p53 protein expression in xenografted tumors.
3.6. Effect of SCP on Bcl-2, Bax and p53 protein expression in xenografted tumors Fig. 3. (A) Effect of SCP on the level of VEGF in tumor cytosolic fractions measured by ELISA; (B) Effect of SCP on the mRNA levels of VEGF in tumor cytosolic fractions measured by RT-PCR. The quantitative data are presented as the mean ± S.D. of five independent experiments. ** Denotes P < 0.01 versus the control.
Fig. 4. Quantifications of CD31 and CD34-immunopositive cells in xenografted tumors by immunohistochemistry. The quantitative data are presented as the mean ± S.D. of five independent experiments. ** Denotes P < 0.01 versus the control.
After confirming that SCP induces apoptosis during RCC carcinogenesis, our next attempt was to determine the impact of SCP on the regulation of the apoptotic signal transduction pathway involved in SCP-induced apoptosis. As is well established that Bcl-2 family proteins are the crucial players in the programmed apoptotic pathway [28], we examined whether or not SCP can affect the expression of proapoptotic protein Bax and antiapoptotic protein Bcl-2 in our mice model. Interestingly, it was observed that the mice administered with SCP revealed a significant decreased expression of Bcl-2 and increased expression of Bax in contrast to those in the control group, and thereby increasing the ratio of Bax to Bcl-2, which is an important factor for the occurrence of apoptosis (Fig. 6). It is well known that the tumor suppressor protein p53 is inactivated in about half of human cancers [29,30]. For this reason, we determined p53 expression in Caki-1 tumors by Western blot. As seen from Fig. 6, p53 expression in tumor mass of SCP-treated mice was markedly higher than that of vehicle-treated mice. Collectively, these findings demonstrate that SCP might trigger apoptosis via the up-regulation of Bax and p53 and downregulation of Bcl-2 gene expression in Caki-1 tumor-bearing mice. 4. Conclusions
Fig. 5. Quantifications of apoptotic cells in xenografted tumors by TUNEL assay. The quantitative data are presented as the mean ± S.D. of five independent experiments. ** Denotes P < 0.01 versus the control.
3.5. The effects of SCP on apoptosis of xenografted tumors To further confirm whether the inhibitory effect of SCP on RCC xenografts growth is via apoptotic mediated mechanisms, the TUNEL assay was conducted for detecting the apoptosis. As presented in Fig. 5, we observed that the average number of TUNELpositive cells was significantly higher in tumors treated with SCP (P < 0.01) than that in the control tumor. These results indicated that SCP displays its antitumor activity via inducing apoptosis of tumor tissue cells.
Recently, a greater emphasis has been given toward researching complementary and alternative medicine in cancer management. Several studies have revealed that many natural health products that inhibit angiogenesis also manifest other anticancer activities, and have been used as potent anticancer drugs [31]. Advances in the understanding of the pathogenesis and molecular biology of RCC have identified angiogenesis as a key factor in the development of the disease [32,33]. In general, RCC is characterized by rich neovascularization and develops a fine vascular network around the cancer cells [34]. Most hypervascularized RCC also express a number of angiogenesis-related factors, in particular VEGF [35,36]. As well, VEGF is highly expressed in many human cancers, but RCC in particular produces remarkably high levels [37]. Therefore control of VEGF is essential for treating patients with RCC. The present work has addressed the antineoplastic and antiangiogenic potential of SCP in RCC. In vitro, exposure of SCP to Caki-1 cells for 24 h resulted in a decrease of VEGF secretion under hypoxic conditions. Furthermore SCP exerted a potent inhibitory activity on the growth of xenografted RCC Caki-1 cells in athymic nude mice. We also observed that the low expression of VEGF level and two endothelial markers, CD31 and CD3, in tumor tissues. Meanwhile the proapoptotic protein Bax and p53 increased, whereas antiapoptotic protein Bcl-2 decreased in a mouse xenograft model after drug
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treatment. Besides, there was no substantial body weight change in SCP-treated mice. Collectively, these results indicated that SCP might be safe and effective for use in the treatment for RCC. Acknowledgment This study was supported by the fund of Heilongjiang Province Science and Technology Plan Project (No. GC05C40701). References [1] R.J. Motzer, N.H. Bander, D.M. Nanus, New England Journal of Medicine 335 (1996) 865–875. [2] A. Jemal, R. Siegel, E. Ward, T. Murray, J. Xu, C. Smigal, M.J. Thun, CA: A Cancer Journal for Clinicians 56 (2006) 106–130. [3] R.J. Amato, Annals of Oncology 16 (2005) 7–15. [4] B.I. Rini, Clinical Cancer Research 13 (2007) 1098–1106. [5] R.H. Weiss, P.Y. Lin, Kidney International 69 (2006) 224–232. [6] J. Bellmunt, C. Montagut, S. Albiol, J. Carles, P. Maroto, A. Orsola, BJU International 99 (2007) 274–280. [7] B.R. Lane, B.I. Rini, A.C. Novick, S.C. Campbell, Urology 69 (2007) 3–10. [8] X. Wang, L. Zhang, A. O’Neill, B. Bahamon, D.C. Alsop, J.W. Mier, S.N. Goldberg, S. Signoretti, M.B. Atkins, C.G. Wood, R.S. Bhatt, British Journal of Cancer 108 (2013) 319–326. [9] M.T. Pirrotta, P. Bernardeschi, G. Fiorentini, Current Medicinal Chemistry 18 (2011) 1651–1657. [10] K.T. Flaherty, I. Puzanov, Biochemical Pharmacology 80 (2010) 638–646. [11] C.J. Liu, S.Q. Zhang, J.S. Zhang, Q. Liang, D.S. Li, Natural Product Research 26 (2012) 2199–2203. [12] Z.S. Wang, H.X. Chen, W.J. Zhang, G.S. Lan, L.K. Zhang, Journal of Medicinal Plant Research 5 (2011) 1207–1216. [13] L.Q. Yang, R.H. Chen, X.Y. Wu, Q.Y. Fan, J.L. Zhao, Food Science 29 (2008) 392–394. [14] Y. Chen, J. Tang, X. Wang, F. Sun, S. Liang, International Journal of Biological Macromolecules 50 (2012) 844–848. [15] T. Zhao, G. Mao, R. Mao, Y. Zou, D. Zheng, W. Feng, Y. Ren, W. Wang, W. Zheng, J. Song, Y. Chen, L. Yang, X. Wu, Food and Chemical Toxicology 55 (2013) 609–616.
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