| Propofol has been widely used in clinical anesthesia as intravenous anesthetics,. Studies have shown that propofol can significantly increase the activity of cytotoxic T lymphocytes and is a positive adjustment factor for endoplasmic reticulum stress and JNK signaling pathway of tumor H460 cells Propofol can inhibit tumor’s invasion and metastasis such as lung cancer, ovarian cancer, prostate cancer,osteosarcoma, and exist antitumor effects.The Wnt signaling pathway plays an important role in the process of tumor metastasis,. It is a complex protein network, including three signaling pathway:1.the canonical Wnt pathway, also called Wnt/β-catenin signaling pathway, which activates target genes in the nucleus.2.the cell polarity pathway (Wnt/PCP signaling pathway), which mainly relates to N-terminal kinase and cytoskeletal remodeling.3.the Wnt/Ca2+ signaling pathway, which mainly regulates cell movement and cell adhesion. It can regulates cell biological processes, including cell differentiation, cell polarity formation, cell migration and proliferation. Moreover, the canonical Wnt pathway is also involved in the development and progression of tumors. As an important part of the Wnt/p-catenin signaling pathways, The E-cadherin/catenin (E-cadherin connected with intracellular domain of β-catenin) is an important part of cell adhesion molecules, and play an extremely important role in inhibiting tumor invasion and metastasisE-cadherin is main molecular involved in cell adhesion connection, and play to maintain the cell polarity and the integrity of organizational structure. The antitumor effect of E-cadherin is mainly through complex with P-catenin mediated between homogeneous cell adhesion and inhibiting tumor cell migration and invasive, meanwhile the E-cadherin competitive binding with β-catenin reduce free-floating type of P-catenin in intracellular, participation in the Wnt signaling pathways to inhibit cell proliferation gene expression. The E-cadherin/catenin (E-cadherin connected with intracellular domain of β-catenin) whether E-cadherin changes itself or its components can weaken the cell adhesion ability and enhancement the sports ability of tumor cells, leading to tumor cells easier to escape, invasion and metastasis. In the absence of Wnt signal stimulation, β-catenin combined with degradable compounds composed of GSK-3 through phosphorylation and pan protein and other process occurs degradation to keep theβ-catenin in cytoplasmic at lower levels. Studies have found that expression of P-catenin is closely related to tumor metastasis and enhancement of cell transfer ability, it close with low differentiation in tumor tissue and lymph node metastasis and the variety of human tumor metastasis, such as breast cancer, stomach cancer, prostate cancer.Our previous studies have found that propofol can suppress pulmonary metastasis of intravenous injected MADB106 tumor cells and down-regulating MTA1 and Wntl expressions in the metastatic tumor tissue, and indicate that propofol can inhibit the tumor metastasis and proliferation by the Wnt/β-catenin pathway. At present, the effect of propofol on E-cadherin and β-catenin is unclear. The study is to investigate the effects of different doses of propofol on pulmonary metastasis of intravenous injected MADB106 tumor cells and the expression of E-cadhrin and β-catenin in the metastatic tumor tissue in rats.Material and Methods1.Animal perparetion and reagentsForty adult male Fischer344 rats,12-14 weeks old, weighting 200-220g(purchased from Beijing Charles River experimental animal technical co., LTD).Rat E-cadherin monoclonal antibody, Rat β-catenin monoclonal antibody.(purchased from ProteintechGroup, Inc.); propofol (batch number:JK222, 200mg/20ml,AstraZeneca)2. MADB106 tumor cells cultureMADB106, which purchased from China Center for Type Culture Collection, Wuhan University, is a highly selected variant cell line obtained from a pulmonary metastasis of mammary adenocarcinoma induced in Fischer344 rat. After intravenous inoculation, the tumor metastasizes only to the lungs. MADB106 tumor cells were cultured in complete medium, containing 90% RMPI1640,10% fetal bovine serum, 0.1% penicillin and streptomycin. Cultured cells were maintained in an incubator at 37℃ in 100% humidity atmosphere containing 5% CO2. Cells passage were digestioned by 0.25% trypsin.3. Animal perparetion and groupingForty adult male Fischer344 rats were randomly divided into four groups: normal saline group (group S), intralipid group (group F), propofol 30 mg/kg group (group P1 and 50 mg/kg group (group P2, each group of ten rats. Intraperitoneally injected with 50mg/kg dose of 1% sodium pentobarbital. after reached the appropriate depth of anesthesia, then pumped into the normal saline (5ml/kg), intralipid (5ml/kg) and propofol (30mg/kg and 50mg/kg) 1 hour,respectively.The rats keep the rectal temperature at 37-38℃. Counting respiratory rate and heart rate, respiratory rate is maintained at about 60 beats/min, heart rate is remained at about 320-340 beats/min. One hour after the infusion, MADB106 tumor cells (2×105) were injected intravenously in the rats. Then removed femoral vein catheter and sutured the incision. After the experiment, rats were fed in the standard animal laboratory.4. Sample collection and Pulmonary metastasis countAfter 3 weeks, intrapertioneal injection of 1% sodium pentobarbital and then killed all rats. Rats were inserted endotracheal tube and fixed, then quickly opened the chest and separated lung tissues. Let the rats in the head high, slowly injected 4% paraformaldehyde fixative through the endotracheal tube until 3/4 of the whole lung expansion, stopped injecting fixative, removed the whole lung and washed lung surface with saline. The whole lung in 4% paraformaldehyde fixative fixed 24 hours. Fixed good lung tissue placed on the filter paper to blot 4% paraformaldehyde fixative. Two personnel, who did not participate in the experimental, directly counting the number of pulmonary metastasis and statistics.5. Immunohistochemical stainingSpecimens were embedded in paraffin, four microns sections were cut from the paraffin-embedded specimens and affixed to glass slides. The specimens were dewaxed in xylene and rehydrated in a gradient of ethanol and washed 5 min in distilled water. After the hot fix antigen, specimens were natural cooling, and then washed in PBS 3 times,5 min/times. Endogeneous peroxidase activity was eliminated by soaking the specimens in 3% hydrogen peroxide for 5-10 min, then the the specimens were washed in PBS 3 times,5 min/time. With normal goat serum sealed specimens at room temperature 20-30 min and shook off excess liquid, followed by incubation with the primary antibodies at 4℃ over night, then rewarming 30 min at 37℃, after specimens were washed in PBS 3 times,5 min/time, they were incubated with biotinylated antibody at room temperature 30 min. Specimens were washed in PBS 3 times,5 min/time. Visualization of the immunochemical reaction was accomplished using DAB chromogenic reagent, the reaction was stopped by the addition of distilled water. Specimens were counterstained with hematoxylin and then dehydration, tansparent and mounted.6. Detection of indicators6.1 Count the foci of plmonary metastasis tumor and metastatic inhibitory rateThe fischer344 rats raised 3 weeks after death, resection of pulmonary tumor tissue and in 4% of formaldehyde fixed 24h, after 70% ethanol to wash off.Then, the pulmonary metastasis tumor foci and metastatic inhibitory rate were observed in the metastatic tumor tissue were detected by immunohistochemistry three weeks later. Specimens were embedded in paraffin, four microns sections were cut from the paraffin-embedded specimens and affixed to glass slides. Metastatic inhibitory rate=(1-The reatment group on average of metastatic tumor foci/The control group on average of metastatic tumor foci)×100%6.2 interpretation of the resultsIn normal lung tissue, β-catenin and E-cadherin positioning in the cell membrane and positive reaction were tan or yellow fine granular shading of immunohistochemical. Butβ-catenin and E-cadherin is mainly expressed in the nucleus, immunohistochemical positive reaction for yellow or tan fine granular and cell membrane color reduction of metastatic tumor tissu. In the 400 times higher magnification, five visual fields were randomly selected from bronchioles or alveolar epithelial positive staining by Olympus DP70CCD. Image-Pro Plus 10.0, the special-purpose system for quantitative measurement of medical images, was used to analyse the results quantitatively. A positive cells in the picture as the basis, and then determination and analysis of the whole picture of positive cells mean optical density (OD) values.7. Statistical methodsAll measurement data were expressed as mean±standard deviation (x±s) and were analyzed with the SPSS 13.0 statistical software. Statistical analysis between groups was assessed by one-factor ANOVA. The correlation analysis was estimated using Pearson correlation coefficient. P values less than 0.05 were considered to be statistically significant.Results1. Pulmonary metastasis tumor foci and metastatic inhibitory rateThe metastasis tumor foci and the metastatic inhibition rate in lung showed no significant different between normal saline group and intralipid group(p>0.05). Compared with group S and group F, P1 and P2 group decreased Numbers of pulmonary metastases (p<0.01), metastatic inhibition rate increased (p<0.01). Compared with P1 group, P2 group decreased Numbers of pulmonary metastases (p< 0.01), metastatic inhibition rate increased (p<0.01).The pulmonary metastasis tumor foci of group F, P1 and P2 were 9.63%,48.15%,68.89%, P2 group obviously higher than that of group F. The number of pulmonary metastasis is negatively related to the dose of propofol, Pearson correlation coefficient is-0.879 (P<0.01).2. Expression of E-cadhrin andp-cateninThe expression of E-cadherin in the tumor tissue were staining deep and quantity, the OD values showed no significant different between normal saline group and intralipid group(p>0.05); Compared with group S, the expression of E-cadherin in the tumor tissue decreased and staining shallow, the OD values showed a significantly difference (P<0.05) between P1 group and P2 group; Compared with group P1, the expression of E-cadherin in the tumor tissue decreased and staining shallow, the OD values showed a significantly difference (P<0.05) of P2 group; The expression of E-cadherin is negatively related to the dose of propofol, Pearson correlation coefficient is -0.755 (p<0.01)The expression of p-catenin in the tumor tissue were staining deep and quantity, the OD values showed no significant different between normal S group and F group(p>0.05); Compared with group S, the expression of β-catenin in the tumor tissue decreased and staining shallow, the OD values showed a significantly difference (P<0.05) between P1 group and P2 group; Compared with group P1, the expression ofp-catenin in the tumor tissue decreased and staining shallow, the OD values showed a significantly difference (P<0.05) of P2 group; The expression ofβ-catenin is negatively related to the dose of propofol, Pearson correlation coefficient is-0.693 (p<0.01)ConclusionPropofol can dose-dependently suppress pulmonary metastasis of intravenous injected MADB106 tumor cells by inhibiting the Wnt/β-catenin pathway and down-regulating the expressions of E-cadherin and β-catenin in the metastatic tumor tissue. |
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