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17Effect of oleanolic acid on the apoptosis of S180 cell protein in tumor-bearing
mice
Wu Boyan , Guo Yanying, Dong Jing (corresponding author)
1. Heilongjiang university of Chinese medicine, 150040; 2. Harbin city maternal and child health hospital, 150010; 3. Qiqihar medical college, 161006
Abstract: Objective: To investigate the effect of oleanolic acid against tumor associated protein on cell apoptosis pathway of mice S180 .Methods: Mice were inoculated with S180 sarcoma ascites on the right armpit to build, solid tumor model, randomized (saline, cyclophosphamide, oleanolic acid group), continuous administration of 8 days. The mice were sacrificed, stripping the tumor tissue, the expression of Cyt-C and Caspase-3 in tumor tissues was detected by immunohistochemical method. Results: oleanolic acid can significantly increased the expression level of S180 in tumor tissue Cyt-C, Caspase-3, compared with the model group had significant difference (P<0.01), has antitumor effect. Conclusion: Oleanolic acid can increase S180 mice tumor Cyt-C, Caspase-3 expression. The mechanism may be to promote the release of Cyt-C and subsequent activation of Caspase-3, inducing tumor cell apoptosis and anti-tumor effect.
Keywords: oleanolic acid; apoptosis; Cyt-C; Caspase-3; antitumor
Apoptosis is a cell in vivo induction of a specific signal, the death pathway is activated in apoptosis-related genes under the control of the process of programmed cell death, is an active process of cell clearance. Apoptosis pathway has become one of the most effective means of treatment of cancer. Oleanolic acid (OA), also known as Qingsisu, pentacyclic triterpenoids, are widely present in hawthorn, lilac, diffusa, jujube, Prunella, Ligustrum lucidum, leaves, wood natural ingredients such as plants [1]. Oleanolic acid have lower blood sugar, lowering blood pressure, liver, immune regulation, anti-aging, anti-viral, anti-ulcer and other effects [2]. Oleanolic acid also has anti-tumor effect [3]. Can lower the expression of mutant P53 protein within the tumor cells, the cells within the tumor necrosis factor (TNF-a) has obvious induce action [4], can promote tumor cell apoptosis[5].
This paper mainly studies of oleanolic acid on S180 tumor bearing mice tumor tissue Cyt-C, Caspase-3 protein effects. The mechanisms of the apoptosis of oleanolic acid cell tumor, and provide reference data and more thinking for tumor treatment.
Material
1.1 Experimental animal
Kunming mice, weight 18~22g, male and female, provided by experimental animal center of Heilongjiang University Of Chinese Medicine.
1.2 Drugs, reagents and tumor
Oleanolic acid was purchased from Hunan sanwei Biotechnology Co. Ltd.; cyclophosphamide (CTX) was purchased from Shanghai Hualian Pharmaceutical Co. Ltd; two steps PV detection kit and Caspase-3, Cyt-C monoclonal antibody was purchased from Wuhan boster Biological Engineering Co., Ltd.; mouse S180 ascites tumor strain was purchased in Cancer Hospital Affiliated to Harbin Medical University.
2^ Test methods
2.1 mouse S180 sarcoma tumor model
Take 18 ~ 22g , 30mice, will S180 tumor strain diluted 1:3 with saline to prepare 0.2ml each vaccination right anterior axillary solid tumor model.
2.2 Detection of Caspase-3 and Cyt-C.
Take vaccinated mice were randomly divided into three groups: model group, cyclophosphamide (CTX) group, oleanolic acid (OA) group. First dose six hours after inoculation,
the model group with saline 0.2ml / only fed once a day, CTX group received 40mg/kg intraperitoneal injection, 1 day, oleanolic acid group at 4.992mg/kg administered orally, once daily, administered continuously for 8 days. Mice were killed, peel the tumor tissue, the conventional formalin-fixed, paraffin-embedded, sections, HE staining, using PV-step two-step immunohistochemical assay, expression of Caspase-3 N Cyt-C situation. Using Motic Images Advanced 3.2 analysis software to analyze the Cyt-C of tumor tissue positive cells and the average optical density of Caspase-3 positive cells and the average optical density value.
2.3 Statistical Methods
The results were expressed as mean ± standard deviation ( x± s) said, using t-test, SPSS 17.0 software processing.
Table 1 Oleanolic acid on S180 tumor-bearing mice tumor cells of Cyt-C_
Group Dose (mg/kg) N the mean optical
_density value ( x±s)_
Model group - 10 0.1495±0.02048
CTX group 40 10 0.2181±0.01831**
0A group_4.992_10_0.2076±0.009300**_
Note: Compared with model group, ** P <0.01.
Table 2 Oleanolic acid on S180 tumor-bearing mice tumor cells of Caspase-3_
Group Dose (mg/kg) N the mean optical
_density value ( x±s)_
Model group - 10 0.1188±0.01246
CTX group 40 10 0.2384±0.11325**
OA group_4.992_^_0.2250±0.01788**_
Note: Compared with model group, ** P <0.01.
3^ Results
Effect of oleanolic acid on S180 cells in tumor tissue Cyt-C, Caspase-3, stained by immunohistochemistry method, observation of Cyt-C and Caspase-3 expression on the cell membrane and cytoplasm appear yellow or brown granular staining positive cells. In the 10 x 40 high magnification, each specimen were randomly selected five horizons, select all of the positive granules within view of S180 mice tumor tissue Cyt-C and Caspase-3 cells mean optical density analysis. The results are shown in Table 1, Table 2 compared with model group, oleanolic acid group Cyt-C and Caspase-3 average optical density values were significantly increased (P <0.01), the difference was significant, with statistical significance.
4^ Discuss
Inducing tumor cell apoptosis pathway main treatment of cancer, tumor cell death process control tumor cell proliferation rate, play treat tumors. Caspase family in the induction of apoptosis plays an important role, Caspase is the main enzyme execution, it is usually exist as inactive zymogen. In some apoptosis initiation induced factors and gene regulation function, the cell membrane receptor protein cause apoptosis signal to the Caspase, resulting in a waterfall at the activation, cells in morphology change make cells apoptosis eventually, therefore, Caspase play a key role in apoptosis, is the ultimate way to execute apoptosis. Caspase upon activation will result in substrate cleavage, apoptosis may be irreversible. Caspase has become an important symbol of apoptosis[3]..
Caspase-3, is one of the main executor of apoptosis in the Caspase family, is also important in the process of apoptosis effect factors. Caspase-3, the activation is an important symbol of cell apoptosis in irreversible stages [4]. When stimulated by apoptotic signals, endogenous apoptosis in mitochondria. Mitochondrial release of Cyt-C to cytoplasm, Cyt-C, and an original cell apoptosis protease activating factor-binding. In the presence of dATP, through the role of cysteine proteases
recruitment domain, Apaf-1 and Caspase-9 zymogen binding. The Caspase-9 zymogen activation, the activation of downstream effector caspases such as Caspase-2, 3,6,7,8,10, initiate apoptosis [5]. This experiment was observed by immunohistochemistry apoptosis, experimental results show that oleanolic acid can increase S180 mice tumor Cyt-C, Caspase-3 expression and induce tumor cell apoptosis and anti-tumor effect . Oleanolic acid on tumor cell apoptosis induced by multiple factors influence. The induction of tumor cell apoptosis pathway mechanism needs further study, research and development for the clinical treatment of cancer anticancer drugs with low toxicity for reference.
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Foundation item: the scientific research project of Education Department of Heilongjiang Province,
No.: 12531610; Heilongjiang Province Natural Science Fund project, serial number: D200844.
Author unit: Heilongjiang University Of Chinese Medicine Biology Department.
Author introduction: Wu Boyan (1958-), female, Professor, tutor of master, mainly engaged in
traditional Chinese medicine research.
E-mail: wuboyan1958@163.com.
Tel: 13313650384.
Author introduction: Dong Jing (1987-), female, is mainly engaged in the research of anti-tumor. Qiqihar Medical School biology department, zip code: 161006. E-mail: mmmm_tt@163.com Tel: 13303628250
