| Objective:To observe the impact on tumor growth of tumor-bearing nude mice intervened by XiaotanSanjie Recipe, based on the human gastric cancer mice model. Observe and analyze the serum and tumor tissue protein spectrum changes of tumor-bearing mice intervened by XiaotanSanjie Recipe on the platform of proteomics. Screen the different protein spots related with XiaotanSanjie Recipe and identify some of them to investigate possible anti-cancer mechanism of XiaotanSanjie Recipe.Methods:1.Modelling:Fifty-two BALB/c-nu/nu mice were randomly divided into experimental group (n=42), and control group (n=10). MKN-45, a cell line from poor differentiated human gastric cancer, was cultured in vitro to an exponential phase. Then, the cancer cells were injected into the subcutaneous site of BALB/c-nu/nu mice to build tumor-bearing models. When diameters of subcutaneous transplanted tumors grow near 1cm, the mice were sacrificed and active tumor was divided into many smaller pieces with a diameter of 1.5mm. The smaller pieces were inoculated into new BALB/c-nu/nu mice for passage culture in vivo. The cancer cells were activated by the passage culture in vivo 5 times. Then, they were regarded as the original tumor and inoculated again into 42 BALB/c-nu/nu mice to prepare the tumor-bearing models in a large scale with the same method mentioned above. When the volumes of most of transplanted tumors had growed near 80mm3,12 mice models with irregular tumors were excluded.Thirty tumor-bearing mice were remained in the following study.2. Impact of XiaotanSanjie Recipe on tumor-bearing mice and tumor growth:Thirty tumor-bearing mice were randomly divided into model group, positive control group and intervention group, ten for each. All mice were fed freely. Additionally the the model group was fed with saline, the positive control group received Xeloda and the intervention group was administrated with XiaotanSanjie Recipe. All drugs including saline were delivered by intragastric administration with a dosage of 0.4ml every mouse per day. The response of tumor-bearing mice to treatment was observed carefully, the diameter of tumor and body weight of every mouse was measured every 5 days to draw curves of tumor volume and body weight. All mice were sacrificed after 2 weeks treatment. Their blood was collected from the orbital venous plexus. Draw the supernate blood serum for cryopreservation after centrifugalization. The tumor of every nude mouse model was separated from the mouse and weighted. Some of them were fixed with formalin solution for histopathological examination and others were saved by liquid nitrogen freezing.3. Impact of XiaotanSanjie Recipe on blood serum proteome expression of tumor-bearing mice:Collect the serum samples obtained from the Part 1 experiment to process protein quantify, isoelectric focusing (IEF), strips balance, SDS-PAGE, stain bleaching after depletion the high abundance proteins in order to get two-dimensional gel electrophoresis maps. Gel maps were analyzed with PDQUST. Points from control group, model group and intervention group were matched and twice or more different content were considered different points.4. Impact of XiaotanSanjie Recipe on tumor Proteome expression of tumor-bearing mice:Collect the serum samples obtained from the Part 1 experiment to process isoelectric focusing (IEF), strips balance, SDS-PAGE, stain bleaching in order to get two-dimensional gel electrophoresis maps. Gel maps were analyzed with PDQUST. Points from model group and intervention group were matched and 2.5 times or more different content were considered different points.5. Identification and database search of different proteins:Those different proteins obtained from the Part2 Part3 experiment were digested, analyzed with MALDI-TOF/TOF-MS mass spectrograph and searched from protein database.Results:1.Modelling:Tumor-bearing models were successfully established by injecting human gastric cancer MKN-45 injected into the subcutaneously into BALB/c-nu/nu mice. The rate of tumor formation reached 100%. The mice models achieved good homogeneity by excluded the ones bearing irregular tumor, this ensured the following experiments.2. Impact of XiaotanSanjie Recipe on tumor-bearing nude mice:After administration, mice positive control group appears to lose weight, eat and active less, their skin became dull, compared with model group and intervention group. After administrated for 5 days, there was no significant difference of body weight tumor volume and relative tumor volume in model group, positive control group and intervention group (P＞0.05).After administrated for 10 days, body weight of positive control group decreased significantly compared with model group and intervention group (P<0.05), tumor volume and relative tumor volume of positive control group and intervention group decreased significantly compared with model group (P＜0.05). After the experiment, all the nude mice were sacrificed. Inhibition rate of positive control group and intervention group were 48% and 41% respectively.3. Impact of XiaotanSanjie Recipe on serum Proteome expression of tumor-bearing mice:Depletion the high abundance proteins the serum can get protein map with satisfying repeatability and stability. The protein points had approximately same distribution and background. By comparison to control group,25 different protein spots were obtained in model group. In those different proteins, the expressions of 12 proteins were promoted and 9 proteins depressed. By comparison to model group,19 different protein spots were obtained in intervention group. In these different proteins, the expression of 14 proteins was promoted and 5 depressed. There were 9 proteins which had significant difference between intervention group and model group, but had no difference between intervention group and control group. Three of them were promoted in model group comparing with control group, but depressed to the normal level in the intervention group. The other six proteins were depressed in model group comparing with control group, but promoted to the normal level in the intervention group.4. Impact of XiaotanSanjie Recipe on tumor proteome expression of tumor-bearing mice:Two-dimensional gel electrophoresis on samples of tumor tissue had satisfying repeatability and stability. The proteins had a proximately the same distribution and background. By comparison, the model group with the intervention group, a total of 41 different protein spots obtained. In which,8 proteins only appeared in model group,3 only appeared in intervention group. The expression of 30 different proteins reached 2.5 times or more. The expressions of 13 proteins were promoted, and 17 depressed in the intervension group.5. Identification different proteins and retrieval recognition:A total of 21 most differentially expressed proteins were identified, including APO1, SOD, POX1, HSP27, ubiquity protein ligase, haptoglobin, and HSP60, etc. These proteins can be divided according to their functions into 8 species such as cell proliferation, differentiation or apoptosis related,energy metabolism related, lipid metabolism related,cytoskeleton and sports related, oxidative stress related and heat shock related proteins.Conclusion:1. Both Xelod and XiaotanSanjie Recipe inhibited the tumor growth of tumor-bearing nude mice obviously. But Xeloda had certain toxicity in nude mice; XiaotanSanjie Recipe can improve the living condition of nude mice.2. XiaotanSanjie Recipe can promote the expression level of APO1, POX1, SOD and HSP27.It also can depress the expression level of haptoglobin, ubiquity protein ligase, histone methyltransferase, etc.3. The study found that XiaotanSanjie Recipe maybe play an anti-tumor effects through regulating the expression of cell proliferation,differentiation or apoptosis related,energy metabolism related, lipid metabolism related,cytoskeleton and sports related, oxidative stress-related and heat shock related proteins.4. The anti-tumor function of XiaotanSanjie Recipe has the characteristic of multi-target, multi-way, multi-link and multi-level.5. Proteome study may open up new avenues for Efficacy monitoring and mechanism discovery of traditional Chinese herbal compound.
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