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Study on antitumor, antioxidant and immunoregulatory activities of the purified polyphenols from pinecone of Pinus koraiensis on tumor-bearing S180 mice in vivo
Accepted Manuscript Title: Study on Antitumor, Antioxidant and Immunoregulatory activities of the Purified Polyphenols from Pinecone of Pinus koraiensis on Tumor-bearing S180 mice in vivo Author: Juanjuan Yi Hang Qu Yunzhou Wu Zhenyu Wang Lu Wang PII: DOI: Reference:
S0141-8130(16)31097-2 http://dx.doi.org/doi:10.1016/j.ijbiomac.2016.10.071 BIOMAC 6651
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
2-8-2016 21-9-2016 17-10-2016
Please cite this article as: Juanjuan Yi, Hang Qu, Yunzhou Wu, Zhenyu Wang, Lu Wang, Study on Antitumor, Antioxidant and Immunoregulatory activities of the Purified Polyphenols from Pinecone of Pinus koraiensis on Tumorbearing S180 mice in vivo, International Journal of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2016.10.071 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Study on Antitumor, Antioxidant and Immunoregulatory activities of the Purified Polyphenols from Pinecone of Pinus koraiensis on Tumor-bearing S180 mice in vivo Juanjuan Yi a, Hang Qu a, Yunzhou Wu b, Zhenyu Wang a∗, Lu Wang a∗ a
Harbin Institute of Technology, 73 Huanghe Road, Nangang District, Harbin 150090,
PR China b
Northeast Agriculture University, Mucai Street 59, Xiangfang District, Harbin, China
*Corresponding author: Zhenyu Wang, Lu Wang Postal address: School of Chemical Engineering, Harbin Institute of Technology, 73 Huanghe Road, Nangang District, Harbin, 150090, PR China. E-mail address: [email protected] (Zhenyu Wang); [email protected] (Lu Wang). Fax numbers: Tell numbers: 0451-86282909, Fax numbers: 0451-86282909.
Highlights
An efficient purification method of polyphenols of Pinus koraiensis pinecone is developed.
•In vivo tumor
antitumor
activities of
the PPP-40 against xenograft Sarcoma 180
cells were evaluated firstly.
•The mechanism of the antitumor activities of the PPP-40 were investigated by TUNEL technique and immunohistochemical method for apoptosis-related proteins Bcl-2, Bax and Caspase-3.
•Pinecone polyphenols are bioactive dietary constituents that enhance health and help prevent and treat cancer through improving antioxidant and immunomodulatory activities.
•Pinus koraiensis pinecone was proved to be a potential antitumor resource of polyphenols.
ABSTRACT Pinecone polyphenols are bioactive dietary constituents that enhance health and help prevent and treat cancer through improving antioxidant and immunoregulatory activities. This study was designed to investigate the antitumor, antioxidant and immunoregulatory activities of the 40% ethanol eluent of polyphenols from pinecone of pinus koraiensis (PPP-40) in Sarcoma 180 (S180)-bearing mice models in vivo. The results of antitumor activity indicated that PPP-40 significantly inhibited S180 tumor growth and the dose of 150 mg/kg exhibited the highest antitumor activity. Moreover, TdT-mediated dUTP nick end labeling (TUNEL) assay results further confirmed the apoptosis of S180 tumor cells. In addition, PPP-40 could obviously promote the expressions of Bax protein and inhibit the Bcl-2 protein, accordingly improve the expressions of activated Caspase-3 as well, which resulted in the activation of mitochondrial apoptotic pathway of tumor cells in S180 mice eventually. The results of antioxidant activity showed that the S180 mice treated with PPP-40 had the higher superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities, the more glutathione (GSH) content, and the lower malondialdehyde (MDA) level in plasma comparing with non-treated control group. Moreover, the administration with PPP-40 (150 mg/kg) significantly accelerated the proliferation of splenocytes (p < 0.01) and increased the monocyte phagocytosis activity in vivo simultaneously. These results revealed that PPP-40 exerts an effective antitumor activity by activating the mitochondrial apoptotic pathway and improving the antioxidant and immunoregulatory activities.
Nonstandard Abbreviations: Pinus koraiensis: P. koraiensis; polyphenols of P. koraiensis pinecone: PPP; Sarcoma 180: S180; TdT-mediated dUTP nick end labeling: TUNEL; Superoxide anions: O2-; Hydrogen peroxide: H2O2; Superoxide dismutase: SOD; Glutathione
peroxidase:
GSH-Px;
Glutathione:
GSH;
Malondialdehyde:
MDA;
Cyclophosphamide: CTX; Phagocytic index: PI.
Key Words: Pinus koraiensis; Pinecone polyphenols; Antitumor activity; Antioxidant activity; Immunoregulatory activity.
1. Introduction Epidemiology proved the active substances of plant were associated with lower incidence of cancers [1,2], partly due to their antitumor, antioxidant and immunoregulatory activities [3-5], which attract more and more attention in natural antitumor agents from plants [6-9]. Moreover, some studies in vitro and in vivo have confirmed that many phenolic constituents had potential antitumor activity without obvious side effects [10-12], which could inhibit the proliferation of cancer cells through many different mechanisms including mitochondrial pathway of apoptosis, termination of cell cycle , PI3K/AKT pathway, immune responses, improving the antioxidant
activities and so on [13-16]. In normal physiological metabolism, a small amount of free radicals play an important role in electron transfer and molecular signal transduction [17]. It has been recently discovered that malignant cells generate excessive free radicals via an autocrine mechanism including superoxide anions (O2-) and hydrogen peroxide (H2O2), which may disturb the redox dynamic equilibrium [18]. Once the normal equilibrium is disrupted, the redundant free radicals will
react with biological substances quickly, which result in protein carbonylation, lipid peroxidation, and particularly genetic toxicity [19]. However, the excessive free radicals can be eliminated by intracellular antioxidant enzymes, such as superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) [20]. Cell survival depends on the balance of intracellular free radicals and antioxidative metabolism, and this sort of balance reflects the redox status in vivo [21,22]. The modulation of immune cell activities by bioactive substances derived from natural plants is an interest in the area of developing therapies against inflammation, autoimmunity, and cancer [23]. Investigating the effects of substances that promote macrophage and lymphocyte response was a potent mean to study immunomodulation and develop new drug [24]. Krifa et al. reported that an aqueous extract of Limoniastrum guyonianum gall mainly containing flavonoids, polyphenols, and tannins, exhibited significant immunoregulatory effect by inducing splenocyte proliferation and stimulating macrophage activation [25]. Moreover, the pretreatment with polyphenols extracted from the fruits of Malus baccata (Linn.) Borkh. (MBP3b) at a dose of 150 mg/kg.bw could also significantly enhance immunoregulatory activity of mice [26]. In addition, previous studies have also shown that a variety of polyphenols can act as immunomodulators, stimulating the immune system such as phagocytic activity and affecting the enzymatic system in order to eliminate tumors and foreign invaders more effectively [27,28]. Pinup koraiensis (P. koraiensis), a member of Pinaceae plants, is widely distributed throughout the world, especially in Manchuria in northeast China [29]. P. koraiensis pinecone has been demonstrated by our laboratory that it possesses abundant biological active substances [30,31].
Our previous studies have also reported the primary components of the purified polyphenol from P. koraiensis pinecone and their biological activities including antitumor and antioxidant activities in vitro [32,33]. The objective of this study was further to explore how the purified polyphenol from P. koraiensis pinecone modulates antioxidant activities and immune responses to exert a potential antitumor activities in S180-bearing mice in vivo.
2. Materials and methods 2.1 Materials and reagents The dried pinecones of P. koraiensis were provided by Yichun Hongxing District Forestry Bureau (Yichun, China). The superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), reduced glutathione (GSH), malondialdehyde (MDA) and protein quantization measurement kits were purchased form Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Tunel commercial kits was purchased from Boster Bio-Engineering Limited Company (Wuhan, China). All other chemicals were of analytical grade purchased from local suppliers. PPP-40 was prepared according to our previous method and enriched with the chromatographic column of D101 macroporous resins. Identified by HPLC, its main ingredients were catechin and taxifolin.
2.2 Animals Male KM mice of SPF-level (6 – 8 weeks old, 22 – 25 g each) were housed in a mouse room at temperature (22 ± 2 °C), light (12 h light/dark cycles) and humidity (50 ± 10 %) and were provided with rodent laboratory chow pellets and tap water for a week to adapt to the environment of mouse room. The experimental protocol was approved by Institutional Animal Ethical committee.
2.3 Tumor inhibition rate The antitumor activity of PPP-40 was further detected using a tumor-bearing S180 mice model. Briefly, all health male mice were injected subcutaneously in the left axilla with S180 tumor cells (1.0×106 cells/mouse) except the normal control group [34]. 24 h later, mice except normal control (non-tumor-inoculated) group were randomly divided into five groups of 12 mice each group. PPP-40 (50, 150 and 300 mg/kg.d)
and positive control cyclophosphamide
(CTX, 20 mg/kg.d) were given by oral administration. The normal control group and model control group were given with the equal volume of normal saline every day. And all animals were executed and tumors of treated mice were harvested and precisely weighed. The tumor inhibition rate was expressed according to the following formula: (the mean tumor weight of control group − the mean tumor weight of treated group) / the mean tumor weight of control group × 100.
2.4 Apoptosis detection of tumor tissues by TUNEL staining Apoptotic cells were detected in situ using TUNEL method according to the manufacturer’s instructions [35]. The mice were sacrificed and the tumors were fixed intact in 4% paraformaldehyde fixative for 24 h. The tumor was cut into six segments, embedded vertically, and sectioned to provide transverse sections. Two micrometer sections were then taken and placed on slides. The slides were rinsed twice with PBS and treated with proteinase K and 3 % H2O2, labeled with fluorescein dUTP in a humid box for 1 h at 37 °C, stained with DAB and counterstained with methyl green. The reaction was visualized using a light microscope (SDS1B, Photoelectric Instrument Co. Ltd., Chongqing, China). And the extent of apoptosis was evaluated by counting the TUNEL-positive cells. The apoptotic index was determined as a
number of TUNEL-positive cells/total number of cells in 5 randomly selected high power fields (magnification × 400).
2.5 Immunohistochemistry Representative paraffin blocks were serially cut at two-micrometer thick, deparaffinized in xylene, rehydrated in graded ethanol and washed with phosphate-buffered saline three times. The sections were boiled into 10 mM citrate buffer (pH 6.0) for antigen retrieval. Endogenous peroxidase was blocked through incubation with 3 % hydrogen peroxide in methanol for 30 min, and incubated with primary rabbit monoclonal antibodies overnight at 4 °C. After several rinses in phosphate-buffered saline, the sections were incubated in the biotinylated secondary antibodies. Bound antibodies were detected according to the instructions of Bax, Bcl-2 and Caspase-3 commercial kits (Boster Bio-Engineering Limited Company, Wuhan, China). Slides were rinsed in PBS, exposed to diaminobenidine, and then counterstained with Mayer's hematoxylin. The negative control was made without addition of the primary antibody during the staining process. The positive cells were counted under a light microscope (SDS-1B, Photoelectric Instrument Co. Ltd., Chongqing, China). and photographed (magnification × 400).
2.6 Flow cytometric analysis of cell cycle After the mice sacrificed, the recovered tumor cells from tumor blocks were fixed with cold 70 % ethanol and stored at 4 °C for at least 24 h, subsequently subjected to PI labeling (PI/RNase Staining Buffer), and the cells were analyzed by flow cytometry (Becton Dickinson, San Jose, CA, USA) [36].
2.7 Determination of the SOD, GSH-Px, GSH and MDA The activities of SOD, GSP-Px and the contents of GSH and MDA in the plasma were
determined using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The SOD and GSH-Px activities were expressed in U/mL, the contents of GSH was expressed in mg/L, and the content of MDA was expressed in nmol/mL.
2.8 Assay of splenocyte proliferation in vivo After the mice sacrificed, the spleens collected from tumor-bearing S180 mice under aseptic conditions were grinded into small pieces and passed through sterilized meshes (200 meshes) to obtain a homogeneous cell suspension at the room temperature. The red blood cells were removed by hemolytic red blood cell lysis solution. Recovered splenocytes were washed twice, then re-suspended in RMPI-1640 complete medium containing 5 % FBS, with 1×106 cell/mL cell concentration [37,38]. The cell was seeded in a 96-well plate with or without ConA (7.5 μg/mL). After incubation for 72 h at 37 °C in a humidified 5 % CO2 incubator, the number of cells was determined by MTT assay using a microplate reader (Model 680, Bio-Rad, Hercules, CA, USA).
2.9 Phagocytosis of monocyte assay The phagocytosis function of monocyte was determined. After 11 days of oral administration, 25 % (v/v) India ink according to 0.2 mL/kg body weight was injected by a tail intravenous injection. A total of 20 μL of blood was collected through eye orbit after 2 min (t1) and 10 min (t2), and added to 2 mL 0.1 % Na2CO3. The absorbance at 600 nm of blood after 2 min (A1) and 10 min (A2) were measured, and the absorbance of normal control group of blood was set as zero. The mice were sacrificed by decapitation, and then the liver and spleen were weighed. Clearance index (K) and phagocytic index (α) were calculated as follows [39]:
K=
lg A1 -lgA 2 t2 -t1
α= K1/3 × body weight/(liver weight + spleen weight)
2.10 Statistical analysis All statistical analyses employed SPSS for Windows, Version 18.0. Data were expressed as means ± standard deviation (SD) of three independent measurements. Statistical analyses were performed by one-way ANOVA. Differences at p < 0.05 and p < 0.01 were considered statistically significant by Duncan’s new multiple-range test.
3. Results and discussion 3.1 Effect of PPP-40 on tumor inhibition rate The potent antitumor activity of PPP-40 in vivo was further validated in tumor-bearing S180 mice. The antitumor activities of CTX and PPP-40 were summarized in Table 1. The inhibition rates of tumor growth of CTX and three different doses of PPP-40 treatments (50, 150, and 300 mg/kg) were 59.02 %, 12.68 %, 48.29 %, and 32.20 %, respectively. The results showed that PPP-40 significantly inhibited tumor growth, and the dose of 150 mg/kg exhibited the highest antitumor activity of three doses of PPP-40 treatments.
3.2 Effect of PPP-40 on cell apoptotic rate To identify whether the reduced tumor growth rate following PPP-40 treatment in tumorbearing S180 mice was due to the cells apoptosis, TUNEL assay was used to characterize apoptosis in S180 tumor sections. As seen in Fig. 1, the results explicitly showed that more apoptotic cells were induced by PPP-40 in tumor sections than that model control group. And the apoptotic rate increased in a dose-dependent manner from 2.12 ± 0.19 % in the model control group (Fig. 1a) to 23.65 ± 1.12 % (p < 0.01) in the experimental group that was treated with 50 mg/ kg PPP-40 (Fig. 1c). The apoptotic rates were both approximately 30 % in the groups of 150 mg/kg and 300 mg/kg (Fig. 1d-e). All the results significantly suggested that
PPP-40-mediated inhibition of tumor growth in tumor-bearing S180 mice was close correlated with more S180 cells apoptosis.
3.3 Effect of PPP-40 on immunohistochemistry staining To clarify whether the reduced tumor growth rate following PPP-40 treatment in tumor-bearing S180 mice was due to the apoptotic signal transduction pathway, we investigated the alterations in the expressions of apoptosis-related proteins Bax, Bcl-2 and Caspase-3 in S180 tumor treated by PPP-40. Fig. 2 and Fig. 3 showed a significant difference in expressions of the proteins Bax and Bcl-2 in tumor-bearing S180 model. In the model group, Bax was not detected (Fig. 2a), whereas the expression of Bcl-2 was promoted in S180 tumor (Fig. 3a). Compared with the model group, the mice treated with PPP-40 exhibited significantly decreased expression of Bcl2 (Fig. 3c-e) and increased expression of Bax (Fig. 2c-e). Moreover, Fig. 4 showed PPP-40 significantly increased the expression of activated caspase-3 comparing with the model group.
3.4 Effect of PPP-40 on Apoptosis of sub-G1 hypodiploid cells To identify whether PPP-40 increased the proportion of apoptotic sub-G1 hypodiploid cells in S180 tumor, PI staining assay was used. As indicated in Fig. 5, the formation of apoptotic DNA in sub-G1 peak was dramatically increased in positive control group (CTX), which was up to 31.51 ± 1.41 %, compared to model control group (3.97 ± 0.78 %) (Fig. 5a). Whereas the groups treated by PPP-40 at the dose of 50, 150 and 300 mg/kg were 12.10 ± 1.17 %, 21.56 ± 1.83 %, 16.24 ± 1.22 % ( Fig. 5c-e) respectively. Among three doses of PPP-40, the dose of 150 mg/kg showed the highest percentage of cells in apoptotic peak (Fig. 5f). These results indicated that PPP-40 promoted S180 tumor cells apoptosis by promoting the formation of apoptotic DNA of tumor tissue.
3.5 Effect of PPP-40 on SOD, GSH-Px, GSH and MDA As showed in Table 2, prominent enhancement of MDA level and significant reduction of antioxidant enzymes SOD, GSP-Px and non-enzymes GSH in model control group were clearly observed in comparison with untreated normal control group (p < 0.05). However, after the administration of PPP-40 (150 and 300 mg/kg), the MDA content in the plasma was significantly reduced, and the levels of SOD, GSP-Px and GSH were increased. The results above showed that PPP-40 could effectively increase the contents of antioxidant enzymes and non-enzymes and lower the level of MDA in the tumor-bearing S180 mice.
3.6 Effect of PPP-40 on splenocytes proliferation T-lymphocyte is an important immunological active cell and plays an important role in enhancing the immune function of organisms [40,41]. As Con A was the mitogen for T cell, the comitogenic activity of PPP-40 with Con A on mice splenocytes was investigated further. The data showed that PPP-40 could enhance specific immune response by exhibiting significant comitogenic activity in Con A-induced splenocytes in S180-bearing mice (Fig. 6). Specifically, the medium-dose of PPP-40 (150 mg/kg) with Con A could significantly stimulate splenocytes proliferation compared with the model group (p < 0.01), with the better effect than the group of 50 mg/kg and the positive control group. The splenocytes proliferation response was related to immunity improvement. The results indicated that PPP-40 possessed a definite and clear synergistic effect on splenocytes proliferation by combining with Con A.
3.7 Effect of PPP-40 on phagocytosis of monocyte Monocyte is the most important phagocyte and plays an important role in immune response. The phagocytosis of monocytes was reflected by the test of carbon clearance [42]. Fig. 7
showed the effect of PPP-40 on the phagocytosis in tumor-bearing S180 mice. The phagocytic index (PI) of model group decreased significantly compared with normal group, from 9.34 ± 0.14 to 3.12 ± 0.19 (p < 0.01). After administration of PPP-40, the phagocytic indexes were all increased. The index in medium dose (150 mg/kg) group was the highest among all treated groups including positive control group. No significant difference was found between low dose group and high dose group (p > 0.05). The results above revealed that PPP-40 could significantly improve the phagocytosis of monocyte in tumor-bearing S180 mice.
3.8 Discussion It was well known that natural polyphenols act as dietary antioxidants which were rich in fruits, vegetables, cereals, red wine and tea. The polyphenols were of great practical effects in counteracting some important pathologies such as cardiovascular disease, Alzheimer’s disease, tooth decay or different infections [43-45]. Accumulating evidences also supported the conclusion that dietary polyphenols protected against some types of cancers [46-48]. Our previous study firstly clarified the structural characterization and predicted the 3D model of the obtained polyphenols from P. koraiensis pinecones [30]. Moreover, it was also proved that the purified polyphenols from P. koraiensis pinecones (PPP-40) had antitumor activity in vitro and could induce apoptosis in LOVO cells through the caspase activation pathway [32]. In addition, our laboratory also reported that the purified polyphenols from P. koraiensis pinecones using a novel multi-channel parallel–serial chromatographic system possessed significant antioxidant activity in vitro [33]. In the present study, we investigated the antitumor, antioxidant and immunoregulatory activities of PPP-40 in S180-bearing mice by analyzing tumor growth, apoptosis, antioxidant enzymes, splenocyte proliferation and monocyte phagocytosis activity
in vivo for the first time. Our data provided important evidences that the PPP-40-mediated antioxidative response and immunomodulation activity were close correlated with PPP-40induced tumor apoptosis in S180-bearing mice. Apoptosis, also known as programmed cells death, has been known as one of the most potent strategies to counter the cancerous growth [49]. A number of studies in vitro and in vivo reported plant polyphenols mediated apoptosis as a consequence of modulation in expression of proteins involved in apoptosis [10-12]. In the presnt study, PPP-40 did show significant tumor-inhibiting activity in S180-bearing mice , and the inhibitory rate was up to 48.29 % at the dose 150 mg/kg (Table 1). Previously, PPP-40 mediated increases in the levels of cytochrome c, caspase-3 and -9, followed by apoptosis in LOVO human colon cells in vitro. PPP-40 also promoted the activation of caspase-8 leading to apoptosis by extrinsic pathway. In the present study, PPP-40 increased levels of caspase-3 (Fig. 4) and pro-apoptotic Bax (Fig. 2) and decreased that of antiapoptotic Bcl-2 (Fig. 3) proteins, followed by apoptosis proved by TUNEL assay (Fig. 1) in S180-bearing mice. These results suggested that the PPP-40 induced apoptosis of S180 tumor cells probably by the endogenous mitochondrial apoptosis pathway by up-regulating expression of Bax to counteract the effect of Bcl-2 and increasing the level of the activated caspase-3. In addition to involvement of cells apoptosis, this dietary agent was also known to arrest cell cycle at certain check points [50,51]. A number of studies have reported polyphenols mediated arrest of the cell cycle, as a result of modulation of cell cycle regulators [52-54]. In our study, the formation of apoptotic DNA in sub-G1 peak was dramatically increased in PPP-40 (150 mg/kg) treated group, which was up to 21.56 ± 1.83 % compared to model group (3.97 ± 0.78 %) (Fig. 5), as a consequence of arresting the cell cycle at G2/M phase and induced apoptosis.
Therefore, plant polyphenols regulated a number of cellular targets involved of cell cycle and it could be an interesting therapeutic agent against a variety of cancers. An unbalanced redox state was believed to be responsible for the initiation and promotion of inflammatory-related diseases, including cancer [55]. Tumor micro-environment resulted in the intracellular accumulation of high levels of reactive oxygen species (ROS) free radical that induced oxidative damage in DNA and other cellular biomolecule. In response to oxidative stress, ROS was able to be eliminated by intracellular antioxidant enzymes [20]. Polyphenols have been intensively investigated for their antioxidant properties. Our results also revealed that PPP-40 could significantly increase the activities of SOD and GSP-Px, GSH content, and decrease the MDA level in the plasma of S180-bearing mice compared to the model group (Table 2). It could also be observed a positive correlation between the levels of intracellular antioxidant enzymes of PPP-40 detected in the plasma of S180-bearing mice and the antitumor capacity in vivo. The immune system played an important role in antitumor effect. The cellular immune response by T cells played a central role in the generation and regulation of the immune response to tumor antigens. Commonly, the progressive tumor growth was frequently accompanied by an immunosuppression regardless of tumor location and etiology [56,57]. Therefore, we investigated the effects of PPP-40 on immunity to elucidate its antitumor mechanism, specifically in tumor-bearing S180 mice. It was well-known that Con A stimulated T cells proliferation. However, the proliferation ability of lymphocytes in the tumor-bearing S180 mice was lower than that in normal mice. This indicated that tumor growth might impair spleen lymphocytes’ function. PPP-40 repaired the damage and significantly promoted the
proliferation ability of Con A-stimulated splenocytes compared with the model control group. However, the positive control CTX, with a high tumor inhibitory rate (Table 1), had an immunosuppressive effect on splenocytes proliferation (Fig. 6). The results indicated that PPP40 significantly increased the activation of T cells and enhanced humoral-mediated immunity specifically in these tumor-bearing S180 mice to counteract the immunosuppression of tumor micro-environment. Macrophages were the most important phagocytes, which played a pivotal role in the host defense against any type of invading cells, including tumor cells [58]. Our results also revealed that PPP-40 displayed strong monocyte phagocytosis activity in vivo (Fig. 7). Taking into consideration of above results, our data suggested that PPP-40 exerted an effective antitumor activities by activating the mitochondrial apoptosis pathway and improving the antioxidant and immunoregulatory activities in S180-bearing mice in vivo.
4. Conclusions In summary, we investigated the antitumor, antioxidant and immunoregulatory activities of the purified polyphenol from P. koraiensis pinecone (PPP-40) in vivo in a S180-bearing mice model. Based on our study, we proposed a model for exploring mechanism of PPP-40-induced apoptosis of tumor cells in S180-bearing mice in vivo (Fig. 8). It provided further evidences that the antitumor activities of PPP-40 in S180-bearing mice were partly due to the activation of mitochondrial-mediated apoptosis pathway, cell cycle arrest and improvement of antioxidant and immune activities. All the results indicated that this functional plant extract could be developed as a potential antitumor agent.
Acknowledgments The authors thank Professor Lu Wei-Hong and Professor Ma Ying from the Chemical
Engineering, Harbin Institute of Technology, for their helpful suggestions and assistance.
Conflict of Interest The authors have declared no conflict of interest.
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Figure captions Fig. 1 The effect of PPP-40 on tumor apoptosis in tumor-bearing S180 mice using a TUNEL technique (magnification×400). The arrows showed the positive expression of apoptosis cells, a: Model group; b: Positive control group (CTX 20 mg/kg); c: Low concentration of PPP-40 group (50 mg/kg); d: Medium concentration of PPP-40 group (150 mg/kg); e: High concentration of PPP-40 group (300 mg/kg); f: the apoptotic rate was calculated as apoptotic cell number/total cell number (mean ± SD, n = 6). Bars with no letters in common are significantly different (p < 0.05). Fig. 2 The effect of PPP-40 on Bax expression of tumor tissue in tumor-bearing S180 mice (magnification×400). The arrows showed the positive expression of Bax, a: Model group; b: Positive control group (CTX 20 mg/kg); c: Low concentration of PPP-40 group (50 mg/kg); d: Medium concentration of PPP-40 group (150 mg/kg); e: High concentration of PPP-40 group (300 mg/kg); f: Quantity analysis (mean ± SD, n = 6). Bars with no letters in common are significantly different (p < 0.05). Fig. 3 The effect of PPP-40 on Bcl-2 expression of tumor tissue in tumor-bearing S180 mice (magnification×400). The arrows showed the positive expression of Bcl-2, a: Model group; b: Positive control group (CTX 20 mg/kg); c: Low concentration of PPP-40 group (50 mg/kg); d: Medium concentration of PPP-40 group (150 mg/kg); e: High concentration of PPP-40 group (300 mg/kg); f: Quantity analysis (mean ± SD, n = 6). Bars with no letters in common are significantly different (p < 0.05).
Fig. 4 The effect of PPP-40 on Caspase-3 expression of tumor tissue in tumor-bearing S180 mice (magnification×400). The arrows showed the positive expression of Caspase-3, a: Model group; b: Positive control group (CTX 20 mg/kg); c: Low concentration of PPP-40 group (50 mg/kg); d: Medium concentration of PPP-40 group (150 mg/kg); e: High concentration of PPP40 group (300 mg/kg); f: Quantity analysis (mean ± SD, n = 6). Bars with no letters in common are significantly different (p < 0.05). Fig. 5 Effects of PPP-40 on cell cycle phase distribution of S180 tumor cells, a: Model group; b: Positive control group (CTX 20 mg/kg); c: Low concentration of PPP-40 group (50 mg/kg); d: Medium concentration of PPP-40 group (150 mg/kg); e: High concentration of PPP-40 group (300 mg/kg)；f: Quantity analysis (mean ± SD, n = 3). Bars with no letters in common are significantly different (p < 0.05). Fig. 6 The proliferation of splenic lymphocyte by treatments of different concentrations of PPP40. Values are mean ± SD (n = 6). (*p < 0.05 compared with the normal group; ** p < 0.01 compared with the normal group;
#
p < 0.05 compared with the model group;
##
p < 0.01
compared with model group). Fig. 7 Effects of PPP-40 on the phagocytosis of monocyte in tumor-bearing S180 mice. Values are mean ± SD (n = 6). Bars with no letters in common are significantly different (p < 0.05). Fig. 8 Proposed a model for mechanism of PPP-40-induced apoptosis of tumor cells in tumorbearing S180 mice in vivo.
Fig.1
a
b
20μm
20μm
d
c
e 2
2
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Apoptotic cells (%)
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c 25 20 15 10 5
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Model
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150 PPP-40 (mg/kg)
Fig.2
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The number of Bax postive cells
70
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Fig. 3
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The number of Bcl-2 postive cells
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Fig. 5
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Fig.7 10
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Fig.8
PPP-40
M G1
G2 S Caspase-3
APOPTOSIS
Table 1 Antitumor activity of PPP-40 against S180 solid tumor grown in mice Samples Dose (mg/kg) Tumor weight (g) Model CAT PPP-40 PPP-40 PPP-40
-20 50 150 300
Tumor inhibition rate
2.05±0.23 0.84±0.08** 1.79±0.19*## 1.06±0.12**# 1.39±0.11**##
-59.02** 12.68**## 48.29**# 32.20**##
* p < 0.05 compared with model group; ** p < 0.01 compared with the model group; # p < 0.05 compared with the control group; ## p < 0.01 compared with the control group. Table 2 Effect of PPP-40 on SOD, GSH-Px, GSH and MDA in the plasma of tumor-bearing S180 mice SOD (U/mL)
GSH-Px (U/mL)
GSH (mg/L)
MDA (nmol/mL)
Grops
Dose (mg/kg)
Normal
--
376.63±10.38
898.55±19.12
8.28±1.12
5.47±0.12
Model
--
307.11±11.47*
356.52±7.23**
2.93±0.23**
17.3±0.23**
CAT
20
369.67±9.54##
762.32±5.78*#
7.63±0.23*##
15.8±0.08**#
PPP-40
50
314.77±7.73*
315.94±7.14**
3.53±0.08**#
16.5±0.14**
PPP-40
150
349.17±14.10*#
504.17±2.10*#
4.36±1.14**#
10.1±0.10**##
PPP-40
300
353.24±8.21*#
571.25±6.12*#
3.79±0.12**#
13.2±0.12**#
* p < 0.05 compared with the normal group; ** p < 0.01 compared with the normal group; # p < 0.05 compared with the model group; ## p < 0.01 compared with the model group.
S0141-8130(16)31097-2 http://dx.doi.org/doi:10.1016/j.ijbiomac.2016.10.071 BIOMAC 6651
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
2-8-2016 21-9-2016 17-10-2016
Please cite this article as: Juanjuan Yi, Hang Qu, Yunzhou Wu, Zhenyu Wang, Lu Wang, Study on Antitumor, Antioxidant and Immunoregulatory activities of the Purified Polyphenols from Pinecone of Pinus koraiensis on Tumorbearing S180 mice in vivo, International Journal of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2016.10.071 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Study on Antitumor, Antioxidant and Immunoregulatory activities of the Purified Polyphenols from Pinecone of Pinus koraiensis on Tumor-bearing S180 mice in vivo Juanjuan Yi a, Hang Qu a, Yunzhou Wu b, Zhenyu Wang a∗, Lu Wang a∗ a
Harbin Institute of Technology, 73 Huanghe Road, Nangang District, Harbin 150090,
PR China b
Northeast Agriculture University, Mucai Street 59, Xiangfang District, Harbin, China
*Corresponding author: Zhenyu Wang, Lu Wang Postal address: School of Chemical Engineering, Harbin Institute of Technology, 73 Huanghe Road, Nangang District, Harbin, 150090, PR China. E-mail address: [email protected] (Zhenyu Wang); [email protected] (Lu Wang). Fax numbers: Tell numbers: 0451-86282909, Fax numbers: 0451-86282909.
Highlights
An efficient purification method of polyphenols of Pinus koraiensis pinecone is developed.
•In vivo tumor
antitumor
activities of
the PPP-40 against xenograft Sarcoma 180
cells were evaluated firstly.
•The mechanism of the antitumor activities of the PPP-40 were investigated by TUNEL technique and immunohistochemical method for apoptosis-related proteins Bcl-2, Bax and Caspase-3.
•Pinecone polyphenols are bioactive dietary constituents that enhance health and help prevent and treat cancer through improving antioxidant and immunomodulatory activities.
•Pinus koraiensis pinecone was proved to be a potential antitumor resource of polyphenols.
ABSTRACT Pinecone polyphenols are bioactive dietary constituents that enhance health and help prevent and treat cancer through improving antioxidant and immunoregulatory activities. This study was designed to investigate the antitumor, antioxidant and immunoregulatory activities of the 40% ethanol eluent of polyphenols from pinecone of pinus koraiensis (PPP-40) in Sarcoma 180 (S180)-bearing mice models in vivo. The results of antitumor activity indicated that PPP-40 significantly inhibited S180 tumor growth and the dose of 150 mg/kg exhibited the highest antitumor activity. Moreover, TdT-mediated dUTP nick end labeling (TUNEL) assay results further confirmed the apoptosis of S180 tumor cells. In addition, PPP-40 could obviously promote the expressions of Bax protein and inhibit the Bcl-2 protein, accordingly improve the expressions of activated Caspase-3 as well, which resulted in the activation of mitochondrial apoptotic pathway of tumor cells in S180 mice eventually. The results of antioxidant activity showed that the S180 mice treated with PPP-40 had the higher superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities, the more glutathione (GSH) content, and the lower malondialdehyde (MDA) level in plasma comparing with non-treated control group. Moreover, the administration with PPP-40 (150 mg/kg) significantly accelerated the proliferation of splenocytes (p < 0.01) and increased the monocyte phagocytosis activity in vivo simultaneously. These results revealed that PPP-40 exerts an effective antitumor activity by activating the mitochondrial apoptotic pathway and improving the antioxidant and immunoregulatory activities.
Nonstandard Abbreviations: Pinus koraiensis: P. koraiensis; polyphenols of P. koraiensis pinecone: PPP; Sarcoma 180: S180; TdT-mediated dUTP nick end labeling: TUNEL; Superoxide anions: O2-; Hydrogen peroxide: H2O2; Superoxide dismutase: SOD; Glutathione
peroxidase:
GSH-Px;
Glutathione:
GSH;
Malondialdehyde:
MDA;
Cyclophosphamide: CTX; Phagocytic index: PI.
Key Words: Pinus koraiensis; Pinecone polyphenols; Antitumor activity; Antioxidant activity; Immunoregulatory activity.
1. Introduction Epidemiology proved the active substances of plant were associated with lower incidence of cancers [1,2], partly due to their antitumor, antioxidant and immunoregulatory activities [3-5], which attract more and more attention in natural antitumor agents from plants [6-9]. Moreover, some studies in vitro and in vivo have confirmed that many phenolic constituents had potential antitumor activity without obvious side effects [10-12], which could inhibit the proliferation of cancer cells through many different mechanisms including mitochondrial pathway of apoptosis, termination of cell cycle , PI3K/AKT pathway, immune responses, improving the antioxidant
activities and so on [13-16]. In normal physiological metabolism, a small amount of free radicals play an important role in electron transfer and molecular signal transduction [17]. It has been recently discovered that malignant cells generate excessive free radicals via an autocrine mechanism including superoxide anions (O2-) and hydrogen peroxide (H2O2), which may disturb the redox dynamic equilibrium [18]. Once the normal equilibrium is disrupted, the redundant free radicals will
react with biological substances quickly, which result in protein carbonylation, lipid peroxidation, and particularly genetic toxicity [19]. However, the excessive free radicals can be eliminated by intracellular antioxidant enzymes, such as superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) [20]. Cell survival depends on the balance of intracellular free radicals and antioxidative metabolism, and this sort of balance reflects the redox status in vivo [21,22]. The modulation of immune cell activities by bioactive substances derived from natural plants is an interest in the area of developing therapies against inflammation, autoimmunity, and cancer [23]. Investigating the effects of substances that promote macrophage and lymphocyte response was a potent mean to study immunomodulation and develop new drug [24]. Krifa et al. reported that an aqueous extract of Limoniastrum guyonianum gall mainly containing flavonoids, polyphenols, and tannins, exhibited significant immunoregulatory effect by inducing splenocyte proliferation and stimulating macrophage activation [25]. Moreover, the pretreatment with polyphenols extracted from the fruits of Malus baccata (Linn.) Borkh. (MBP3b) at a dose of 150 mg/kg.bw could also significantly enhance immunoregulatory activity of mice [26]. In addition, previous studies have also shown that a variety of polyphenols can act as immunomodulators, stimulating the immune system such as phagocytic activity and affecting the enzymatic system in order to eliminate tumors and foreign invaders more effectively [27,28]. Pinup koraiensis (P. koraiensis), a member of Pinaceae plants, is widely distributed throughout the world, especially in Manchuria in northeast China [29]. P. koraiensis pinecone has been demonstrated by our laboratory that it possesses abundant biological active substances [30,31].
Our previous studies have also reported the primary components of the purified polyphenol from P. koraiensis pinecone and their biological activities including antitumor and antioxidant activities in vitro [32,33]. The objective of this study was further to explore how the purified polyphenol from P. koraiensis pinecone modulates antioxidant activities and immune responses to exert a potential antitumor activities in S180-bearing mice in vivo.
2. Materials and methods 2.1 Materials and reagents The dried pinecones of P. koraiensis were provided by Yichun Hongxing District Forestry Bureau (Yichun, China). The superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), reduced glutathione (GSH), malondialdehyde (MDA) and protein quantization measurement kits were purchased form Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Tunel commercial kits was purchased from Boster Bio-Engineering Limited Company (Wuhan, China). All other chemicals were of analytical grade purchased from local suppliers. PPP-40 was prepared according to our previous method and enriched with the chromatographic column of D101 macroporous resins. Identified by HPLC, its main ingredients were catechin and taxifolin.
2.2 Animals Male KM mice of SPF-level (6 – 8 weeks old, 22 – 25 g each) were housed in a mouse room at temperature (22 ± 2 °C), light (12 h light/dark cycles) and humidity (50 ± 10 %) and were provided with rodent laboratory chow pellets and tap water for a week to adapt to the environment of mouse room. The experimental protocol was approved by Institutional Animal Ethical committee.
2.3 Tumor inhibition rate The antitumor activity of PPP-40 was further detected using a tumor-bearing S180 mice model. Briefly, all health male mice were injected subcutaneously in the left axilla with S180 tumor cells (1.0×106 cells/mouse) except the normal control group [34]. 24 h later, mice except normal control (non-tumor-inoculated) group were randomly divided into five groups of 12 mice each group. PPP-40 (50, 150 and 300 mg/kg.d)
and positive control cyclophosphamide
(CTX, 20 mg/kg.d) were given by oral administration. The normal control group and model control group were given with the equal volume of normal saline every day. And all animals were executed and tumors of treated mice were harvested and precisely weighed. The tumor inhibition rate was expressed according to the following formula: (the mean tumor weight of control group − the mean tumor weight of treated group) / the mean tumor weight of control group × 100.
2.4 Apoptosis detection of tumor tissues by TUNEL staining Apoptotic cells were detected in situ using TUNEL method according to the manufacturer’s instructions [35]. The mice were sacrificed and the tumors were fixed intact in 4% paraformaldehyde fixative for 24 h. The tumor was cut into six segments, embedded vertically, and sectioned to provide transverse sections. Two micrometer sections were then taken and placed on slides. The slides were rinsed twice with PBS and treated with proteinase K and 3 % H2O2, labeled with fluorescein dUTP in a humid box for 1 h at 37 °C, stained with DAB and counterstained with methyl green. The reaction was visualized using a light microscope (SDS1B, Photoelectric Instrument Co. Ltd., Chongqing, China). And the extent of apoptosis was evaluated by counting the TUNEL-positive cells. The apoptotic index was determined as a
number of TUNEL-positive cells/total number of cells in 5 randomly selected high power fields (magnification × 400).
2.5 Immunohistochemistry Representative paraffin blocks were serially cut at two-micrometer thick, deparaffinized in xylene, rehydrated in graded ethanol and washed with phosphate-buffered saline three times. The sections were boiled into 10 mM citrate buffer (pH 6.0) for antigen retrieval. Endogenous peroxidase was blocked through incubation with 3 % hydrogen peroxide in methanol for 30 min, and incubated with primary rabbit monoclonal antibodies overnight at 4 °C. After several rinses in phosphate-buffered saline, the sections were incubated in the biotinylated secondary antibodies. Bound antibodies were detected according to the instructions of Bax, Bcl-2 and Caspase-3 commercial kits (Boster Bio-Engineering Limited Company, Wuhan, China). Slides were rinsed in PBS, exposed to diaminobenidine, and then counterstained with Mayer's hematoxylin. The negative control was made without addition of the primary antibody during the staining process. The positive cells were counted under a light microscope (SDS-1B, Photoelectric Instrument Co. Ltd., Chongqing, China). and photographed (magnification × 400).
2.6 Flow cytometric analysis of cell cycle After the mice sacrificed, the recovered tumor cells from tumor blocks were fixed with cold 70 % ethanol and stored at 4 °C for at least 24 h, subsequently subjected to PI labeling (PI/RNase Staining Buffer), and the cells were analyzed by flow cytometry (Becton Dickinson, San Jose, CA, USA) [36].
2.7 Determination of the SOD, GSH-Px, GSH and MDA The activities of SOD, GSP-Px and the contents of GSH and MDA in the plasma were
determined using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The SOD and GSH-Px activities were expressed in U/mL, the contents of GSH was expressed in mg/L, and the content of MDA was expressed in nmol/mL.
2.8 Assay of splenocyte proliferation in vivo After the mice sacrificed, the spleens collected from tumor-bearing S180 mice under aseptic conditions were grinded into small pieces and passed through sterilized meshes (200 meshes) to obtain a homogeneous cell suspension at the room temperature. The red blood cells were removed by hemolytic red blood cell lysis solution. Recovered splenocytes were washed twice, then re-suspended in RMPI-1640 complete medium containing 5 % FBS, with 1×106 cell/mL cell concentration [37,38]. The cell was seeded in a 96-well plate with or without ConA (7.5 μg/mL). After incubation for 72 h at 37 °C in a humidified 5 % CO2 incubator, the number of cells was determined by MTT assay using a microplate reader (Model 680, Bio-Rad, Hercules, CA, USA).
2.9 Phagocytosis of monocyte assay The phagocytosis function of monocyte was determined. After 11 days of oral administration, 25 % (v/v) India ink according to 0.2 mL/kg body weight was injected by a tail intravenous injection. A total of 20 μL of blood was collected through eye orbit after 2 min (t1) and 10 min (t2), and added to 2 mL 0.1 % Na2CO3. The absorbance at 600 nm of blood after 2 min (A1) and 10 min (A2) were measured, and the absorbance of normal control group of blood was set as zero. The mice were sacrificed by decapitation, and then the liver and spleen were weighed. Clearance index (K) and phagocytic index (α) were calculated as follows [39]:
K=
lg A1 -lgA 2 t2 -t1
α= K1/3 × body weight/(liver weight + spleen weight)
2.10 Statistical analysis All statistical analyses employed SPSS for Windows, Version 18.0. Data were expressed as means ± standard deviation (SD) of three independent measurements. Statistical analyses were performed by one-way ANOVA. Differences at p < 0.05 and p < 0.01 were considered statistically significant by Duncan’s new multiple-range test.
3. Results and discussion 3.1 Effect of PPP-40 on tumor inhibition rate The potent antitumor activity of PPP-40 in vivo was further validated in tumor-bearing S180 mice. The antitumor activities of CTX and PPP-40 were summarized in Table 1. The inhibition rates of tumor growth of CTX and three different doses of PPP-40 treatments (50, 150, and 300 mg/kg) were 59.02 %, 12.68 %, 48.29 %, and 32.20 %, respectively. The results showed that PPP-40 significantly inhibited tumor growth, and the dose of 150 mg/kg exhibited the highest antitumor activity of three doses of PPP-40 treatments.
3.2 Effect of PPP-40 on cell apoptotic rate To identify whether the reduced tumor growth rate following PPP-40 treatment in tumorbearing S180 mice was due to the cells apoptosis, TUNEL assay was used to characterize apoptosis in S180 tumor sections. As seen in Fig. 1, the results explicitly showed that more apoptotic cells were induced by PPP-40 in tumor sections than that model control group. And the apoptotic rate increased in a dose-dependent manner from 2.12 ± 0.19 % in the model control group (Fig. 1a) to 23.65 ± 1.12 % (p < 0.01) in the experimental group that was treated with 50 mg/ kg PPP-40 (Fig. 1c). The apoptotic rates were both approximately 30 % in the groups of 150 mg/kg and 300 mg/kg (Fig. 1d-e). All the results significantly suggested that
PPP-40-mediated inhibition of tumor growth in tumor-bearing S180 mice was close correlated with more S180 cells apoptosis.
3.3 Effect of PPP-40 on immunohistochemistry staining To clarify whether the reduced tumor growth rate following PPP-40 treatment in tumor-bearing S180 mice was due to the apoptotic signal transduction pathway, we investigated the alterations in the expressions of apoptosis-related proteins Bax, Bcl-2 and Caspase-3 in S180 tumor treated by PPP-40. Fig. 2 and Fig. 3 showed a significant difference in expressions of the proteins Bax and Bcl-2 in tumor-bearing S180 model. In the model group, Bax was not detected (Fig. 2a), whereas the expression of Bcl-2 was promoted in S180 tumor (Fig. 3a). Compared with the model group, the mice treated with PPP-40 exhibited significantly decreased expression of Bcl2 (Fig. 3c-e) and increased expression of Bax (Fig. 2c-e). Moreover, Fig. 4 showed PPP-40 significantly increased the expression of activated caspase-3 comparing with the model group.
3.4 Effect of PPP-40 on Apoptosis of sub-G1 hypodiploid cells To identify whether PPP-40 increased the proportion of apoptotic sub-G1 hypodiploid cells in S180 tumor, PI staining assay was used. As indicated in Fig. 5, the formation of apoptotic DNA in sub-G1 peak was dramatically increased in positive control group (CTX), which was up to 31.51 ± 1.41 %, compared to model control group (3.97 ± 0.78 %) (Fig. 5a). Whereas the groups treated by PPP-40 at the dose of 50, 150 and 300 mg/kg were 12.10 ± 1.17 %, 21.56 ± 1.83 %, 16.24 ± 1.22 % ( Fig. 5c-e) respectively. Among three doses of PPP-40, the dose of 150 mg/kg showed the highest percentage of cells in apoptotic peak (Fig. 5f). These results indicated that PPP-40 promoted S180 tumor cells apoptosis by promoting the formation of apoptotic DNA of tumor tissue.
3.5 Effect of PPP-40 on SOD, GSH-Px, GSH and MDA As showed in Table 2, prominent enhancement of MDA level and significant reduction of antioxidant enzymes SOD, GSP-Px and non-enzymes GSH in model control group were clearly observed in comparison with untreated normal control group (p < 0.05). However, after the administration of PPP-40 (150 and 300 mg/kg), the MDA content in the plasma was significantly reduced, and the levels of SOD, GSP-Px and GSH were increased. The results above showed that PPP-40 could effectively increase the contents of antioxidant enzymes and non-enzymes and lower the level of MDA in the tumor-bearing S180 mice.
3.6 Effect of PPP-40 on splenocytes proliferation T-lymphocyte is an important immunological active cell and plays an important role in enhancing the immune function of organisms [40,41]. As Con A was the mitogen for T cell, the comitogenic activity of PPP-40 with Con A on mice splenocytes was investigated further. The data showed that PPP-40 could enhance specific immune response by exhibiting significant comitogenic activity in Con A-induced splenocytes in S180-bearing mice (Fig. 6). Specifically, the medium-dose of PPP-40 (150 mg/kg) with Con A could significantly stimulate splenocytes proliferation compared with the model group (p < 0.01), with the better effect than the group of 50 mg/kg and the positive control group. The splenocytes proliferation response was related to immunity improvement. The results indicated that PPP-40 possessed a definite and clear synergistic effect on splenocytes proliferation by combining with Con A.
3.7 Effect of PPP-40 on phagocytosis of monocyte Monocyte is the most important phagocyte and plays an important role in immune response. The phagocytosis of monocytes was reflected by the test of carbon clearance [42]. Fig. 7
showed the effect of PPP-40 on the phagocytosis in tumor-bearing S180 mice. The phagocytic index (PI) of model group decreased significantly compared with normal group, from 9.34 ± 0.14 to 3.12 ± 0.19 (p < 0.01). After administration of PPP-40, the phagocytic indexes were all increased. The index in medium dose (150 mg/kg) group was the highest among all treated groups including positive control group. No significant difference was found between low dose group and high dose group (p > 0.05). The results above revealed that PPP-40 could significantly improve the phagocytosis of monocyte in tumor-bearing S180 mice.
3.8 Discussion It was well known that natural polyphenols act as dietary antioxidants which were rich in fruits, vegetables, cereals, red wine and tea. The polyphenols were of great practical effects in counteracting some important pathologies such as cardiovascular disease, Alzheimer’s disease, tooth decay or different infections [43-45]. Accumulating evidences also supported the conclusion that dietary polyphenols protected against some types of cancers [46-48]. Our previous study firstly clarified the structural characterization and predicted the 3D model of the obtained polyphenols from P. koraiensis pinecones [30]. Moreover, it was also proved that the purified polyphenols from P. koraiensis pinecones (PPP-40) had antitumor activity in vitro and could induce apoptosis in LOVO cells through the caspase activation pathway [32]. In addition, our laboratory also reported that the purified polyphenols from P. koraiensis pinecones using a novel multi-channel parallel–serial chromatographic system possessed significant antioxidant activity in vitro [33]. In the present study, we investigated the antitumor, antioxidant and immunoregulatory activities of PPP-40 in S180-bearing mice by analyzing tumor growth, apoptosis, antioxidant enzymes, splenocyte proliferation and monocyte phagocytosis activity
in vivo for the first time. Our data provided important evidences that the PPP-40-mediated antioxidative response and immunomodulation activity were close correlated with PPP-40induced tumor apoptosis in S180-bearing mice. Apoptosis, also known as programmed cells death, has been known as one of the most potent strategies to counter the cancerous growth [49]. A number of studies in vitro and in vivo reported plant polyphenols mediated apoptosis as a consequence of modulation in expression of proteins involved in apoptosis [10-12]. In the presnt study, PPP-40 did show significant tumor-inhibiting activity in S180-bearing mice , and the inhibitory rate was up to 48.29 % at the dose 150 mg/kg (Table 1). Previously, PPP-40 mediated increases in the levels of cytochrome c, caspase-3 and -9, followed by apoptosis in LOVO human colon cells in vitro. PPP-40 also promoted the activation of caspase-8 leading to apoptosis by extrinsic pathway. In the present study, PPP-40 increased levels of caspase-3 (Fig. 4) and pro-apoptotic Bax (Fig. 2) and decreased that of antiapoptotic Bcl-2 (Fig. 3) proteins, followed by apoptosis proved by TUNEL assay (Fig. 1) in S180-bearing mice. These results suggested that the PPP-40 induced apoptosis of S180 tumor cells probably by the endogenous mitochondrial apoptosis pathway by up-regulating expression of Bax to counteract the effect of Bcl-2 and increasing the level of the activated caspase-3. In addition to involvement of cells apoptosis, this dietary agent was also known to arrest cell cycle at certain check points [50,51]. A number of studies have reported polyphenols mediated arrest of the cell cycle, as a result of modulation of cell cycle regulators [52-54]. In our study, the formation of apoptotic DNA in sub-G1 peak was dramatically increased in PPP-40 (150 mg/kg) treated group, which was up to 21.56 ± 1.83 % compared to model group (3.97 ± 0.78 %) (Fig. 5), as a consequence of arresting the cell cycle at G2/M phase and induced apoptosis.
Therefore, plant polyphenols regulated a number of cellular targets involved of cell cycle and it could be an interesting therapeutic agent against a variety of cancers. An unbalanced redox state was believed to be responsible for the initiation and promotion of inflammatory-related diseases, including cancer [55]. Tumor micro-environment resulted in the intracellular accumulation of high levels of reactive oxygen species (ROS) free radical that induced oxidative damage in DNA and other cellular biomolecule. In response to oxidative stress, ROS was able to be eliminated by intracellular antioxidant enzymes [20]. Polyphenols have been intensively investigated for their antioxidant properties. Our results also revealed that PPP-40 could significantly increase the activities of SOD and GSP-Px, GSH content, and decrease the MDA level in the plasma of S180-bearing mice compared to the model group (Table 2). It could also be observed a positive correlation between the levels of intracellular antioxidant enzymes of PPP-40 detected in the plasma of S180-bearing mice and the antitumor capacity in vivo. The immune system played an important role in antitumor effect. The cellular immune response by T cells played a central role in the generation and regulation of the immune response to tumor antigens. Commonly, the progressive tumor growth was frequently accompanied by an immunosuppression regardless of tumor location and etiology [56,57]. Therefore, we investigated the effects of PPP-40 on immunity to elucidate its antitumor mechanism, specifically in tumor-bearing S180 mice. It was well-known that Con A stimulated T cells proliferation. However, the proliferation ability of lymphocytes in the tumor-bearing S180 mice was lower than that in normal mice. This indicated that tumor growth might impair spleen lymphocytes’ function. PPP-40 repaired the damage and significantly promoted the
proliferation ability of Con A-stimulated splenocytes compared with the model control group. However, the positive control CTX, with a high tumor inhibitory rate (Table 1), had an immunosuppressive effect on splenocytes proliferation (Fig. 6). The results indicated that PPP40 significantly increased the activation of T cells and enhanced humoral-mediated immunity specifically in these tumor-bearing S180 mice to counteract the immunosuppression of tumor micro-environment. Macrophages were the most important phagocytes, which played a pivotal role in the host defense against any type of invading cells, including tumor cells [58]. Our results also revealed that PPP-40 displayed strong monocyte phagocytosis activity in vivo (Fig. 7). Taking into consideration of above results, our data suggested that PPP-40 exerted an effective antitumor activities by activating the mitochondrial apoptosis pathway and improving the antioxidant and immunoregulatory activities in S180-bearing mice in vivo.
4. Conclusions In summary, we investigated the antitumor, antioxidant and immunoregulatory activities of the purified polyphenol from P. koraiensis pinecone (PPP-40) in vivo in a S180-bearing mice model. Based on our study, we proposed a model for exploring mechanism of PPP-40-induced apoptosis of tumor cells in S180-bearing mice in vivo (Fig. 8). It provided further evidences that the antitumor activities of PPP-40 in S180-bearing mice were partly due to the activation of mitochondrial-mediated apoptosis pathway, cell cycle arrest and improvement of antioxidant and immune activities. All the results indicated that this functional plant extract could be developed as a potential antitumor agent.
Acknowledgments The authors thank Professor Lu Wei-Hong and Professor Ma Ying from the Chemical
Engineering, Harbin Institute of Technology, for their helpful suggestions and assistance.
Conflict of Interest The authors have declared no conflict of interest.
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Figure captions Fig. 1 The effect of PPP-40 on tumor apoptosis in tumor-bearing S180 mice using a TUNEL technique (magnification×400). The arrows showed the positive expression of apoptosis cells, a: Model group; b: Positive control group (CTX 20 mg/kg); c: Low concentration of PPP-40 group (50 mg/kg); d: Medium concentration of PPP-40 group (150 mg/kg); e: High concentration of PPP-40 group (300 mg/kg); f: the apoptotic rate was calculated as apoptotic cell number/total cell number (mean ± SD, n = 6). Bars with no letters in common are significantly different (p < 0.05). Fig. 2 The effect of PPP-40 on Bax expression of tumor tissue in tumor-bearing S180 mice (magnification×400). The arrows showed the positive expression of Bax, a: Model group; b: Positive control group (CTX 20 mg/kg); c: Low concentration of PPP-40 group (50 mg/kg); d: Medium concentration of PPP-40 group (150 mg/kg); e: High concentration of PPP-40 group (300 mg/kg); f: Quantity analysis (mean ± SD, n = 6). Bars with no letters in common are significantly different (p < 0.05). Fig. 3 The effect of PPP-40 on Bcl-2 expression of tumor tissue in tumor-bearing S180 mice (magnification×400). The arrows showed the positive expression of Bcl-2, a: Model group; b: Positive control group (CTX 20 mg/kg); c: Low concentration of PPP-40 group (50 mg/kg); d: Medium concentration of PPP-40 group (150 mg/kg); e: High concentration of PPP-40 group (300 mg/kg); f: Quantity analysis (mean ± SD, n = 6). Bars with no letters in common are significantly different (p < 0.05).
Fig. 4 The effect of PPP-40 on Caspase-3 expression of tumor tissue in tumor-bearing S180 mice (magnification×400). The arrows showed the positive expression of Caspase-3, a: Model group; b: Positive control group (CTX 20 mg/kg); c: Low concentration of PPP-40 group (50 mg/kg); d: Medium concentration of PPP-40 group (150 mg/kg); e: High concentration of PPP40 group (300 mg/kg); f: Quantity analysis (mean ± SD, n = 6). Bars with no letters in common are significantly different (p < 0.05). Fig. 5 Effects of PPP-40 on cell cycle phase distribution of S180 tumor cells, a: Model group; b: Positive control group (CTX 20 mg/kg); c: Low concentration of PPP-40 group (50 mg/kg); d: Medium concentration of PPP-40 group (150 mg/kg); e: High concentration of PPP-40 group (300 mg/kg)；f: Quantity analysis (mean ± SD, n = 3). Bars with no letters in common are significantly different (p < 0.05). Fig. 6 The proliferation of splenic lymphocyte by treatments of different concentrations of PPP40. Values are mean ± SD (n = 6). (*p < 0.05 compared with the normal group; ** p < 0.01 compared with the normal group;
#
p < 0.05 compared with the model group;
##
p < 0.01
compared with model group). Fig. 7 Effects of PPP-40 on the phagocytosis of monocyte in tumor-bearing S180 mice. Values are mean ± SD (n = 6). Bars with no letters in common are significantly different (p < 0.05). Fig. 8 Proposed a model for mechanism of PPP-40-induced apoptosis of tumor cells in tumorbearing S180 mice in vivo.
Fig.1
a
b
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d
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A
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Fig.2
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Fig. 3
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The number of Bcl-2 postive cells
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Fig.7 10
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Fig.8
PPP-40
M G1
G2 S Caspase-3
APOPTOSIS
Table 1 Antitumor activity of PPP-40 against S180 solid tumor grown in mice Samples Dose (mg/kg) Tumor weight (g) Model CAT PPP-40 PPP-40 PPP-40
-20 50 150 300
Tumor inhibition rate
2.05±0.23 0.84±0.08** 1.79±0.19*## 1.06±0.12**# 1.39±0.11**##
-59.02** 12.68**## 48.29**# 32.20**##
* p < 0.05 compared with model group; ** p < 0.01 compared with the model group; # p < 0.05 compared with the control group; ## p < 0.01 compared with the control group. Table 2 Effect of PPP-40 on SOD, GSH-Px, GSH and MDA in the plasma of tumor-bearing S180 mice SOD (U/mL)
GSH-Px (U/mL)
GSH (mg/L)
MDA (nmol/mL)
Grops
Dose (mg/kg)
Normal
--
376.63±10.38
898.55±19.12
8.28±1.12
5.47±0.12
Model
--
307.11±11.47*
356.52±7.23**
2.93±0.23**
17.3±0.23**
CAT
20
369.67±9.54##
762.32±5.78*#
7.63±0.23*##
15.8±0.08**#
PPP-40
50
314.77±7.73*
315.94±7.14**
3.53±0.08**#
16.5±0.14**
PPP-40
150
349.17±14.10*#
504.17±2.10*#
4.36±1.14**#
10.1±0.10**##
PPP-40
300
353.24±8.21*#
571.25±6.12*#
3.79±0.12**#
13.2±0.12**#
* p < 0.05 compared with the normal group; ** p < 0.01 compared with the normal group; # p < 0.05 compared with the model group; ## p < 0.01 compared with the model group.