| [Introduction]Pancreatic cancer is a digestive tract tumor that develops extremely quickly. Its morbidity and mortality rate has been increasing in recent years. There is no effective method in modern medicine to treat Pancreatic cancer。 However, we found that Jia Wei Wu Mei Wan (JWWMW) has a good curative effect on pancreatic cancer from clinical observations. Preliminary experiments confirmed that JWWMW can inhibit pancreatic cancer sw1990 cells xenografts growth. It’s Inhibitory rate is lower than GEM. The prescription combined with GEM has synergistic effect. We want to research the optimal dose of JWWMW, and research its inhibitory mechanism by comparative proteomics. We also want to explore new ideas for pancreatic cancer treatment.[Methods]1) 50 tumor-bearing mice were divided into four groups, namely model group, low dose group (JWWMW), middle dose group (JWWMW), high dose group (JWWMW). After the murine model of bearing human pancreatic carcinoma was established, the mice in model group received sterile water orally 0.4ml/d for 20 days. The mice in low dose group received JWWMW decoction orally 0.4mg/0.4ml /d. The mice in middle dose group received JWWMW decoction orally 0.8mg/0.4ml/d. The mice in high dose group received JWWMW decoction orally 1.6mg/0.4ml/d.2) Measurements and calculations of tumor volume in the mice was done once every 5 days during the 20 days experiment, and at the same time the tumor growth curve was drawn.3) After treatment of 20 days, the mice were put to death. The weight of the tumors was evaluated and the rate of suppression of tumor was calculated.4) Cell apoptosis was detected by flow cytometry5) The apoptotic indices were examined with TUNEL method and flow cytometry analysis.6) Four groups of differential proteins of transplanted tumor samples were identified and analyzed by comparative proteomics using iTRAQ technology. Our standards for a significant differential protein is if its confidence level is higher than 95%, when compared with the model group it has over 1.5 times up-regulation or down-regulation, it has two or more unique peptides, and its quantitative frequency is greater than or equal to 3.7) The significant differential proteins were given GO annotations, KEGG pathway analysis, analysis of the of significant differential protein position in a given cell, molecular function and biological processes and its involvement in metabolic pathways.8) The significant differential proteins were then validated again using Western blotting and immunohistochemistry methods to prove the reliability of proteomics results.[Results]1) NOD/SCID mice model containing xenograft human pancreatic cancer cell line SW1990 was successfully established.2) When the experiment was carried out for 15 days, three mice in high dose group died.So,the data of group D was missed.3) At 7 days (when the tumor had just formed), the tumor volume in each group had no significant difference (P> 0.05). As time went by, the tumor volume increased significantly in each group. However, the tumor growth rate in group C (middle dose group) and its transplanted tumor volume was significantly less than that of the other groups. Compared with group A (untreated group), the difference was statistically significant (P<0.05). When collecting data, groupC (middle dose group) had the smallest tumor volume, compared with group A (untreated group). The difference was statistically significant (P<0.05).4) At the beginning, the weight differences among all the groups were not statistically significant (P> 0.05). After drugs were administered for 20 days, the data collected for the weight of the mice minus their tumor weight of each group showed no significant difference (P> 0.05).5) The transplanted tumor weight of Group A was (0.97±0.14) g. The transplanted tumor weight of GroupB was (0.96±0.24) g. The transplanted tumor weight of Group C was (0.76±0.18) g. Compared with group A (untreated group), GroupC (middle dose group) and GroupB (low dose) had lighter tumor weight than Group A. The comparison between Group C and A was statistically significant (P<0.05), with a tumor inhibitory rate of 22%. Group B’s tumor weight was slightly lighter compared with Group A’s. The difference was not statistically significant.6) Flow cytometry results showed that the early apoptotic rate (%) of Groups A, B, C were 17.02±2.75,24.03±1.70,29.68±0.76 respectively. The middle dose group had the highest rate of apoptosis, at about 30 percent, far higher than the other three groups. When compared with the other three groups, the difference was statistically significant (P<0.05).7) TUN EL assay test results show the apoptotic index (%) of Groups A,B,C were 19.2 ±7.4,48.3±11.3,59.4±19.3 respectively. The tumor tissue apoptosis rate of all groups with treatment was significantly higher than the model group (P<0.01). The middle dose group had the highest apoptosis index.7) We identified significant differential proteins by comparative proteomic approach based on iTRAQ technology. We compared GroupC (JWWMW middle dose group) with the model group and got a result of over±1.5 times proteins and 56 types of differential proteins. On this basis, we identified 17 kinds of proteins with more than two unique peptides and possessing a high quantification number (greater than or equal to 3). Compared to the model group, there were 15 down-regulated proteins, and two up-regulated proteins. They involve cytoskeleton-associated proteins, tumor associated nutrient supply proteins, effectors, and other trace elements etc.8) GO analysis revealed the cellular components of differential proteins were mainly cell parts, cells; molecular functions were mainly binding, catalytic activity; biological processes were mainly cellular processes and metabolic processes.9) KEGG pathway enrichment analysis showed the metabolic pathways of differential proteins was associated with anabolic nutrients, intracellular signal transduction pathways, and cytoskeleton-associated proteins.10) Protein Western blotting and immunohistochemistry were revalidated for some differential proteins. The expression of myosin regulatory light chain 2 (MYLII) and low carbonic anhydrase 3 (CAⅢ) in GroupC (JWWMW middle dose group) was lower than that of Group A (untreated group), and the expression of high mobility protein 17 (HMG-17) expression in group C (JWWMW middle dose group) was higher than that of Group A (untreated group), consistent with the findings of proteomics.[Conclusion]1) JWWMW alone can inhibit pancreatic cancer sw1990 cells xenografts growth. The inhibitory rate can reach 22%, followed by the low-dose group. The high dose group showed a certain amount of toxicity. JWWMW likely induces apoptosis in pancreatic cancer cells and plays an early inhibitory effect on pancreatic cancer growth2) Through down-regulating the expressions of MLCⅡ, sarcoplasmic/endoplasmic reticulum calcium ATPase, CAIII, and FABP4 differential proteins while up-regulating the expressions of selenoprotein and HMG-17 differential proteins to induce anti-tumor effect. We hypothesized that these six proteins are the targets of JWWMW.3) The main functions of differential protein molecules are binding, catalytic activity; the biological processes involved are mainly cellular processes and metabolic processes. The KEGG pathways involved are mainly nitrogen metabolism, Arginine and pro line metabolism, PPAR signaling pathways, and calcium signaling pathways.4) The results of protein immunization blotting and immunohistochemistry verifies that the proteomics results are reliable...
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