Abstract
The research investigated the effect of Patrinia heterophylla Bunge (Valerianaceae) polysaccharides (PHB-P1) on U14-bearing mice. The tumor weight of mice treated with PHB-P1 (30, 60 mg/kg body weight) was significantly lower than that of the control group, a decrease of serum lactate dehydrogenase (LDH) activity was observed, and the serum alkaline phosphatase (AKP) level was increased slightly. The number of apoptotic tumor cells was significantly increased in the mice by treatment of PHB-P1 (30, 60 mg/kgbw). Cell cycle analysis showed the accumulation of tumor cells in the G2/M phase and a relative decrease of the S phase. By the immunohistochemical analysis, PHB-P1 (30, 60 mg/kgbw) might up-regulate the expression of p53 and Bax, and significantly inhibited the expression of Bcl-2 in tumor tissues. In conclusion, PHB-P1 could inhibit tumor growth and induce tumor cell apoptosis.
Introduction
In recent years, traditional Chinese herbal remedies have gradually gained considerable attention as a new source of anticancer drugs as well as new chemotherapy adjuvants to enhance the efficacy of chemotherapy and to ameliorate the side effects of cancer chemotherapy. Although the healing mechanisms are still largely unknown, some of the drugs have been used to help cancer patients fight their disease with reduced side effects compared with other treatments (CitationFas et al., 2006). Many medicinal plants are used as antitumor drugs (CitationSuh et al., 2002; CitationJasril et al., 2003; CitationVaramini et al., 2007; CitationManosroi et al., 2007); botanical polysaccharides are recognized as naturally occurring antioxidants and may have antiviral and antitumor activities (CitationSchepetkin & Quinn, 2006; CitationYang et al., 2007). Therefore, plant-derived polysaccharides might provide a unique opportunity for the discovery of novel therapeutic agents and adjuvants.
Some studies show that natural polysaccharides could exert anticancer effects through induction of cell apoptosis and immuno-modulation (CitationOoi & Liu, 2000; CitationLi et al., 2007; CitationShao et al., 2008). However, some polysaccharides were shown to have inhibitory effect on tumor growth as well as stimulative effects (CitationZhang et al., 2005; CitationJin et al., 2007). The induction of tumor cell apoptosis by polysaccharides was considered to be related to change of expression of p53, Bcl-2, Bax genes, which are the important regulators in apoptosis (CitationWei & Ru, 1997; CitationLi et al., 2007).
Patrinia heterophylla Bunge (Valerianaceae), a herbaceous plant found in Asia is used as a folklore medicine against hematocele, typhoid fever, carbuncle and abnormal uterine bleeding (CitationLiu & Peng, 1994), and possesses potent cancer cell cytotoxicity (CitationWang et al., 2001; CitationLu et al., 2007). Polysaccharides are a major bioactive compound from Patrinia heterophylla, so we evaluated the antitumor activity of the polysaccharides from Patrinia heterophylla against U14 cervical carcinoma-bearing mice and attempted to elucidate possible mechanism in this work.
Materials and methods
Chemicals and reagents
Cyclophosphamide (CTX) was from Hengrui Pharmacy (Jiangsu Province, China); hematoxylin, eosin and DEAE-cellulose was purchased from Sigma (St Louis, MO). An in situ cell death detection kit was obtained from Roche Diagnostics (Mannheim, Germany). LDH and AKP kits were supplied from Nanjing Jiancheng Bioengineering Institute (Jiangsu Province, China). Rabbit polyclonal antibody to Bax (P-19), Bcl-2 (N-19) and p53 (FL-393) was from Santa Cruz Biotechnology (Santa Cruz, CA). Other chemicals used were of analytical grade.
Sample preparation
Patrinia heterophylla from Qinhuangdao Limin Pharmaceuticals (Hebei Province, China) was identified by Zhao Jian-Cheng at Hebei Normal University, China, where the voucher specimen was deposited (D307). The plant used was located at the outskirts of Yanshan University in May 2007. The samples were air-dried and then macerated with petroleum ether to remove lipids. Proteins of the water extracts were removed by the Sevag method (CitationLi, 2005). The crude polysaccharides were extracted by 95% alcohol precipitation, dried with a lyophilizer (Thermo Savant, San Jose, CA, USA). Two elution peaks (I, II) were shown by the crude polysaccharides treated with DEAE-cellulose chromatography (), and the collected solutions I and II were concentrated with a rotary evaporator (RE-52AA, Shanghai, China) and purified by dialysis. The two kinds of solution were dried with a lyophilizer; the yields of I and II were 1.62% and 1.05% (w/w), and named as PHB-P1 and PHB-P2, respectively. During the process of DEAE-cellulose chromatography, the polysaccharide content of the column effluent was monitored by an automated microplate reader (Multiskan MK3, Finland) at 490 nm using the sulfuric acid-phenol method (CitationLin et al., 1996).
Figure 1. Elution curve of polysaccharides of Patrinia heterophylla by DEAE- cellulose chromatography.
The qualitative analysis of PHB-P1 and PHB-P2 was carried out by color reaction with Molish, Seliwanoff and Fehling reagents (these three reagents gave positive tests for detecting the presence of carbohydrates) (CitationLi, 2005). The results indicated that PHB-P1 might be a major component of ketose and reducible carbohydrate, composed of terose and pentose. PHB-P2 might mainly contain aldose, made up of triose and terose. The contents of carbohydrate, uronic acids and protein of PHB-P1 and PHB-P2 were measured by colorimetry according to CitationFu et al. (2006), CitationJiao et al. (2005) and CitationWang et al. (2001).
Cell line
The uterine cervical carcinoma (U14) cell line was obtained from the Institute of Medical Material, Chinese Academy of Medical Sciences, and propagated in the abdominal cavity of mice for 8-10 days.
Animals
Female Kunming mice (6-8 weeks old and 18-22 g weight) were from the Experimental Animal Center of Xiehe Medical University. The mice were fed standard pellet diet and water in a controlled condition of temperature and humidity. Animal procedures were performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals, and approved by the Animal Ethics Committee of the University in March 2007.
Effect of PHB-P1 on tumor growth
Animals were randomly divided into groups consisting of 10 mice per group. The U14 cells (8-10 days growth) were injected into the left axilla of the mice subcutaneously with 2 × 106 cells. After 24 h of inoculation, PHB-P1 and PHB-P2 were orally administered at a dose of 30 and 60 mg/kg bw, respectively. The group treated with vehicle alone (sterile physiological saline, p.o.) was taken as the control treatment, and the group treated with CTX (25 mg/kg bw, i.p.) was considered as the standard reference drug. All the groups were treated once daily for the duration of the 14-day studies. On day 15, all animals were executed, and the samples of blood and tumor tissues were collected for the next study. The tumor inhibition ratio was expressed according to the following formula:
Histopathological analysis
The tumor samples from the treated and control mice were immediately fixed in 4% formalin, and then embedded in paraffin. The sections (4 μm thick) were stained with hematoxylin-eosin (HE) for microscopic analysis.
Terminal deoxynucleotide-transferase-mediated digoxigenin triphosphate-biotin nick end-labeling (TUNEL) staining assay
An in situ cell death detection kit was used for the TUNEL staining assay according to the manufacturer’s instructions. The cells were distinguished by the color in the sections of tumor tissue samples as follows: 1) positive cell whose nucleus was a distinct brown; 2) negative cell whose nucleus was green. The number of positive cells was counted with a hemocytometer under a microscope (Nikon, Tokyo, Japan).
Assay for serum LDH and AKP levels
Serum of the blood samples (4°C for 4 h after collection) was obtained by centrifugation at 2,000 g for 10 min. LDH and AKP levels were measured using the kits according to the manufacturer’s instructions. Absorbance was measured by an automated microplate reader (Multiskan MK3, Helsinki, Finland).
Immunohistochemical analysis for the expression of p53, Bcl-2 and Bax
Tumor tissue sections were stained with a standard immunohistochemical streptavidin-peroxidase method and observed with a light microscope (Nikon). The cells whose nucleus (or membrane) turned distinct brown color were considered as positive cells, and the cells whose nucleus (or membrane) turned blue counterstained by hematoxylin were negative cells. The numbers of positive cells were counted and were given in percent of all tumor cells.
Cell cycle analysis
The tumor samples were prepared for analysis following the instruction of the Cycletest™ Plus DNA reagent kit (Becton Dickinson, San Jose, CA). After staining of cells with propidium iodide solution for 30 min, the distribution of cells was analyzed in the different phases of the cell cycle by CellQuest software.
Statistical analysis
The data were expressed as the mean ± SD. One-way analysis of variance (ANOVA) and the Duncan test were used for multiple comparisons (SPSS 12.0); p <0.05 was considered statistically significant.
Results
Effects of PHB-P1 on tumor growth
The quantitative analysis data for PHB-P1 and PHB-P2 are shown in , uronic acids content of PHB-P1 and PHB-P2 was 23.5% and 19.8%, respectively. To reveal the role of PHB-P1 on cervical cancer growth in vivo, the tumor-bearing mice were treated with PHB-P1 and PHB-P2 at two doses (30, 60 mg/kgbw). The tumor weight of mice treated with PHB-P1 was lower than that of the control group (p <0.05). However, there was no significant difference between PHB-P2 and the control group. Moreover, no significant difference of tumor weight was displayed between PHB-P1 treatment group and CTX treatment group. The tumor growth inhibition ratio of low and high dose of PHB-P1 was 34.15% and 43.90%, respectively (). On the basis of these results, PHB-P1 (30, 60 mg/kgbw) was used in the following experiments.
Table 1. Content of carbohydrate, uronic acids and protein of PHB-P1 (%).
Table 2. Effect of PHB-P1 treatment on the tumor growth.
Effects of PHB-P1 on tumor cell morphology
Apoptotic cells were clearly observed in tumors treated with PHB-P1 (30, 60 mg/kgbw) compared to the control group. It showed that PHB-P1-treated tumor cell chromatin was condensed and accumulated at the side of the nucleic membranes. The nucleic shape was irregular and the surface of nucleic membrane was rough, and the broken nucleus was encapsulated by intact membrane, containing intact organelles and apoptotic bodies. At the same time, some necrosis of cells was detected as shown by irregular cell morphology, membranolysis and nucleus fragmentation ().
Figure 2. Pathology sections of tumour tissues of the experimental mice (×200). (a) The control group; (b) The CTX treatment group; (c) PHB-P1 (30 mg/kg b.w.) treatment group; (d) PHB-P1 (60 mg/kg b.w.) treatment group.
Detection of apoptosis of tumor tissue cells by TUNEL assay
Apoptosis was further measured by the TUNEL staining assay in tumor tissue sections. As shown in , the number of apoptotic cells was increased by treatment of PHB-P1 (30, 60 mg/kgbw) compared with the control group (p <0.01). It demonstrated that the apoptosis of tumor tissue cells was induced by the administration of PHB-P1.
Table 3. Detection of apoptotic cells by TUNEL assay.
Effects of PHB-P1 on the level of serum LDH and AKP
The serum LDH level was reduced by 18.92% (30 mg/kgbw) and 23.94% (60 mg/kgbw) in the mice treated with PHB-P1 compared with the control group (p <0.01). On the other hand, the levels of serum AKP were changed slightly among the groups, and no significant difference was found between the experimental groups ().
Table 4. Activity of serum LDH and AKP in U14-bearing mice.
Effects of PHB-P1 on distribution of cell cycle
In comparison with the control group, a higher proportion of cells was displayed in G2/M phase, and a concomitant lower proportion of cells showed in S phase by treatment of PHB-P1 (30, 60 mg/kgbw) (p <0.01). Treatment of CTX (25 mg/kgbw) accumulated cells in the G2/M phase and decreased in the S phase compared with the control group (p <0.01, ).
Table 5. Effect of PHB-P1 on distribution of cell cycle.
Effects of PHB-P1 on Bcl-2, Bax and p53 expression in tumor cells
To analyze whether PHB-P1 inhibited the tumor growth by the expression changes of p53, Bcl-2 and Bax, the expression of p53, Bcl-2 and Bax was assessed by immunohistochemical analysis. As summarized in , PHB-P1 (30, 60 mg/kgbw) augmented the number of p53-positive cells compared with the control group (p <0.01). Meanwhile, compared with 76.32% Bcl-2-positive cells in the control group, Bcl-2-positive cells decreased by 31.85% and 36.18% (p < 0.05, p < 0.01) in the PHB-P1 (30, 60 mg/kgbw) treatment groups, respectively. On the other hand, the number of Bax-positive cells increased by treatment of PHB-P1 groups compared to the control group (p <0.05).
Table 6. Effects of PHB-P1 on the expression of p53, Bax and Bcl-2 of tumor.
Discussion
This study was designed to assess the anticancer effects of the PHB-P1 on cervical cancer, which is the second most common cancer in women worldwide (CitationIwakawa et al., 2007). Many studies reported that some natural polysaccharides were used to treat cancer, such as polysaccharides from Panax ginseng of Araliaceae, Phellinus linteus of Polyporaceae, Ganoderma lucidum of Polyporaceae, and Solanum nigrum of Solanaceae (CitationLee et al., 1997; CitationLi et al., 2004, Citation2008; CitationCao & Lin, 2006). In the present study, we investigated tumor inhibition effect of PHB-P1 on U14-bearing mice. The tumor weight was decreased significantly by treatment of PHB-P1. In addition PHB-P1 reduced the release of LDH in the mouse serum. Here it should be noted that the high level of LDH in the blood was associated with a number of different diseases (CitationNaziroğlu et al., 1999), and the growth of tumor was maintained by serum LDH (CitationKviecinski et al., 2008). One report showed that the level of serum LDH was reduced in 1,2-dimethylhydrazine-induced colon tumor mice by treatment of 0.02% Ulmus davidiana (Ulmaceae) Nakai glycoprotein (CitationLee & Lim, 2007). During the experimental period, no toxic symptoms were observed among the mice, and botanical polysaccharides were generally considered to be non-toxic agents (CitationLi et al., 2007; CitationVinsova & Vavrikova, 2008). In our results there was a slight increase in the level of serum AKP, which served as a useful biomarker enzyme of liver toxicity (CitationSu et al., 2003). Hence, we speculated that there was no obviously adverse effect of PHB-P1 on mice.
Disturbance of the cancer cell cycle is one therapeutic targets for development of new anticancer drugs (CitationCarnero, 2002). In this study, tumor cells accumulated in the G2/M phase by treatment of PHB-P1. Similar results reported that dehydrocostuslactone induced accumulation of cells at the G2/M phase in the human ovarian cancer cells (CitationChoi & Ahn, 2009). In addition, a strong correlation was found between the G2/M arrest and the induction of apoptosis (CitationConcin et al., 2003). Hence, one mechanism of the antitumor effects of PHB-P1 could be an inhibition of the malignant proliferation of tumor cells.
Apoptosis is an important homeostatic mechanism for the host to balance cell division and cell death, and therefore it is employed by the host to maintain the appropriate number of cells in the body. Induction of apoptosis in cancer cells has been considered as one of the strategies for anticancer drug development (CitationZhivotovsky & Orrenius, 2006). The results of TUNEL assay demonstrated that PHB-P1 might induce more apoptotic cells compared with the control group; meanwhile, partially apoptotic and necrotic cells were observed by histological examination. Furthermore, as shown in immunohistochemical analysis, PHB-P1 might down-regulate Bcl-2 expression and up-regulate p53 and Bax expression in the tumor tissues. Similar results were reported that the expression of p53, Bax and Bcl-2 was regulated by botanical polysaccharides in some cancer cell lines (CitationLŭ et al., 2002; CitationLi et al., 2007). A decrease in expression of Bcl-2 was observed in the curcumin-treated human ovarian cancer cells, while the levels of p53 and Bax were increased (CitationShi et al., 2006). The gene p53 played a key role in the regulation of cell cycle arrest in the G2/M phase and was involved in DNA repair through activation of ribonucleotide reductase (CitationLevine, 1997). p53 was known to induce apoptosis through inhibition of Bcl-2, amplification of death signals and activation of caspases (CitationHaupt & Haupt, 2004). Bcl-2 acted as a major inhibitor of cell death, therefore guarding against apoptosis while Bax was a promoter of apoptosis. Apoptosis occurrence always depended on the correct ratio of Bcl-2/Bax (CitationThomadaki & Scorilas, 2006). We inferred that the regulation of p53, Bcl-2 and Bax was strongly associated with PHB-P1-induced tumor cell apoptosis.
Taken together, PHB-P1 might be developed as an adjunct to anticancer drugs, and needs to be further studied in vitro and in vivo.
Acknowledgements
The authors would like to thank Zhao Jian-Cheng who authenticated the Patrinia heterophylla samples used. Geng Guo-Xia and Lu Wen-Zong equally contributed to the work.
Declaration of interest
This study was supported by the Science and Technology Development Fund of Science and Technology Investing Company, Qinhuangdao, China (D08).
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