Effect Of Propofol On Pulmonary Metastasis And Expression Of MTA1and Wnt1in Tumor-burdened Rats

Propofol (2,6-diisopropylphenol) has been the most common general anesthesia as an induction and maintenance agent, which is widely used as a sedative agent in the intensive care unit (ICU). In addition to the anesthetic effect, it has been reported that propofol exerts a number of non-anesthetic effects, such as antioxidant, neuroprotective, immune regulation and antiemetic. In recent years, as the tumor incidence is higher and higher, explore the effect of propofol on tumor metastasis has become a hot spot. Propofol can promote the differentiation of T lymphocytes, enhancing the antitumor immunity. In addition, propofol can activate gamma-aminobutyric acid receptors, inhibit the migration and invasion of colon carcinoma cells. In breast cancer cells, propofol reduces the level of matrix metalloproteinases by inhibiting NF-kB activity, which restrained migration and invasion of tumor cells. It also can reduce the expression of matrix metalloproteinases in lung cancer. In other word, propofol for most tumor cells, characterized by inhibition.Metastasis-associated gene family is closely related to the tumor occurrence and development. It contains three different genes (MTA1, MTA2, MTA3) and six reported isoforms(MTA1, MTAls, MTA1-ZG29p, MTA2, MTA3and MTA3L). Among them, MTA1, MTA2and MTA3are considered to be a part of Nucleosomeremodeling and histone deacetylation complex. The fundamental function of MTA family members seem to be affected the state of chromatin to adjust DNA replication and regulated histone deacetylation process. MTA1, the first member found in this family, is located on human chromosome14q32.3, for a total length of2.2Kb. It contains a single open reading frame. The initiation codon is located at the97-99position of nucleotides and the termination codon at nucleotide position2206-2208. MTA1protein has two src-homology (SH)-binding motifs at its carboxy terminal region, which are not only involved in the regulation of the interaction between protein and protein in signal transduction pathway, but also be composed of cytoskeletal. MTA1protein also include several domain structures, which are involved in protein and protein, protein and DNA interactions, such as BAH (bromo-adjacent homology) domain, SANT (SWI, ADA2, N-CoR, TFIIIB-B) domain and GATA zinc finger motif. These structures may be involved in invasion and metastasis of malignant tumor. MTA1was over expressed in breast cancer, ovarian cancer, esophageal cancer, lung cancer, gastric cancer, colorectal cancer and other tumors. The study found, it can influence tumor metastasis through various wanys:①MTA1can repress the transcription of some tumor suppressor genes by affecting chromosome replication and regulating histone deacetylation process, which is resulted in tumor metastasis.②MTA1can enhance the transcriptional activity and stability of hypoxia-inducible factor (HIF)-la protein by recruiting it to histone deacetylase1(HDAC1) and deacetylating it, which increased angiogenesis and enhanced the metastatic potential of tumor cells.③Tumor suppressor p53is deacetylated by MTA1protein, resulting in inhibition of apoptosis induced by p53 gene.㎝TA1can significantly alter the assembly of the cytokeratin filament system and the localization of the actin cytoskeleton protein, and thus make cells obtain an invasive and metastatic phenotype.⑤MTA1as important upstream modifiers of Wntl transcription and translation, which can increase expression of Wntl transcription and translation and initiate Wnt signaling pathway by directly inhibiting the epression of Six3, allowing derepression of Wntl. Blocking or decreasing the expression of MTA1can inhibit the ability of invasion and metastasis of tumor cells. Therefore, MTA1can be used as a target for tumor therapy.Wnt signaling pathway is a complex protein network, including at least three signaling pathway:①the canonical Wnt pathway, also called Wnt/β-catenin signaling pathway, which activates target genes in the nucleus;②the cell polarity pathway (Wnt/PCP signaling pathway), which mainly relates to N-terminal kinase and cytoskeletal remodeling; and③the Wnt/Ca2+signaling pathway, which mainly regulates cell movement and cell adhesion. Wnt signaling pathway 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. Wntl, the first member found in this family, is one of the main members of the canonical Wnt signaling pathway. In the Wnt signaling pathway:when Wntl protein signaling is present, Wntl can act on its transmembrane receptor consisting of Frizzled and low-density-lipoprotein receptor-related protein5/6(LRP5/6), β-catenin escapes from degradation and gradually accumulates in the cytoplasm, then the accumulated β-catenin is translocated to the nucleus, where it binds to the transcription factor T cell factor (TCF)/lympophoid enhancer factor (LEF) DNA-binding protein, and stimulates the expression of various downstream target genes, such as c-Myc, cyclin D1and Slug, and inhibits the expression of E-cadherin, and thereby enhances the ability of invasion and metastasis of tumor cells. In the absence of Wntl protein signaling, β-catenin forms a complex with APC and Axin, which tesults in the degradation of β-catenin in an ubiquitination-dependent manner. Corepressor can bind to the TCF/LEF DNA-binding protein and inhibit the transcription of target genes, when there was no (3-catenin accumulation in nucleus. In breast cancer, gastric cancer, liver cancer, colorectal cancer, prostate cancer and other tumors, Wntl was over expressed, and associated with tumor stage, grade and lymph node metastasis. Thereby, Wntl is one of the important factors in tumor invasion and metastasis.MTA1and Wntl are closely associated with tumor development. Blocking or decreasing the expression of MTA1and Wntl can inhibit tumor metastasis. At present, the effect of propofol on MTA1and Wntl is unclear. We used the rat pulmonary metastasis model of MADB106tumor cells to detect the effect of propofol on pulmonary metastasis and expression of MTA1and Wntl, each group of ten rats.Material and Methods1Animal perparetion and groupingForty adult male Fischer344rats,12-14weeks old, weighting200-220g, were randomly divided into four groups:normal saline group (group S), intralipid group (group F), propofol30mg/kg group (group P30) and50mg/kg group (group P50), each group of ten rats. Before and after the experiment, the rats were fed in standard animal laboratory, the room temperature was maintained at25-27℃and the room humidity was maintained at50%-70%under at8/8h light/dark regimen, each cage of five rats. Food and water were provided adlibitum.2. Experiment cells and 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 Fischer344rat. After intravenous inoculation, the tumor metastasizes only to the lungs. MADB106tumor cells were cultured in complete medium, containing90%RMPI1640,10%fetal bovine serum,0.1%penicillin and streptomycin. Cultured cells were maintained in an incubator at37℃in a humidified atmosphere containing5%CO2. Cells were cultured in a cell dish, replaced the culture medium every two or three days, and detached them from cell dish by using0.25%trypsin, cell passage ratio was about1:2to1:3, probably passaged1-2times a week, the number of cell passage was controlled in less than20generations.3. Experimental implementation processIntraperitoneally injected with50mg/kg dose of1%sodium pentobarbital, after reached the appropriate depth of anesthesia which means their righting reflex and corneal reflex disappeared, animals were inserted a catheter in femoral vein. Then pumped into the normal saline (5ml/kg), intralipid (5ml/kg) and propofol (30mg/kg and70mg/kg)1hour,respectively.Heating blanket is used during anesthesia in rats, which kept the rectal temperature at37-38℃. Counting respiratory rate and heart rate, respiratory rate is maintained at about60beats/min, heart rate is remained at about320-340beats/min.After pumped into drugs for30min, with0.25%trypsin detached MADB106tumor cells and counted them under an inverted microscope, then with PBS solution diluted to a concentration of2×105/ml. To be pumped into drugs1h, each rats were injected0.5ml MADB106tumor cells via the femoral vein. Then removed femoral vein catheter and sutured the incision, intramuscular injection of penicillin20000U/kg. After the experiment, rats were fed in the standard animal laboratory.4. Sample collectionAfter3weeks, intrapertioneal injection of1%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 injected4%paraformaldehyde fixative through the endotracheal tube until3/4of the whole lung expansion, stopped injecting fixative, removed the whole lung and washed lung surface with saline. The whole lung in4%paraformaldehyde fixative fixed24hours.5. Pulmonary metastasis countFixed good lung tissue placed on the filter paper to blot4%paraformaldehyde fixative. Two personnel, who did not participate in the experimental, directly counting the number of pulmonary metastasis and statistics.6. 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 washed5min in distilled water. After the hot fix antigen, specimens were natural cooling, and then washed in PBS3times,5min/times. Endogeneous peroxidase activity was eliminated by soaking the specimens in3%hydrogen peroxide for5-10min, then the the specimens were washed in PBS3times,5min/time. With normal goat serum sealed specimens at room temperature20-30min and shook off excess liquid, followed by incubation with the primary antibodies at4℃over night, then rewarming30min at37℃, after specimens were washed in PBS3times,5min/time, they were incubated with biotinylated antibody at room temperature30min. Specimens were washed in PBS3times,5min/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.7. Immunohistochemiacal image analysisIn the400times higher magnification, five visual fields were randomly selected by Olympus DP70CCD. Image-Pro Plus10.0, 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.8. Statistical methodsAll measurement data were expressed as mean±standard deviation (x±s) and were analyzed with the SPSS13.0statistical software. Statistical analysis between groups was assessed by one-factor ANOVA. The correlation analysis was estimated using Pearson correlation coefficient. P values less than0.05were considered to be statistically significant.Results1. Pulmonary metastasisThe number of pulmonary metastasis in group S and group F were13.50±2.63and12.20±2.15, there was no statistically significant difference (P>0.05). Compared with group S and group F, the number of pulmonary metastasis in group P30and P50were7.00±1.05and4.20±1.55, the number of pulmonary metastasis were decreased in this two groups and the difference was statistically significant (P<0.01). 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 MTA1and WntlMTA1is mainly expressed in the nucleus of tumor cells. The OD values of group S and group F were1.33±0.14and1.27±0.83, the expression of MTA1in this two groups showed no significant difference (P>0.05). In group P30and P50, the OD values were0.93±0.91and0.53±0.13, it was found a significant difference in two groups, the expression of MTA1were significantly lower than group S and group F (P<0.01) and negatively correlated with the dose of propofol, Pearson coefficient is-0.980(P<0.01).Wntl is mainly expressed in the cytoplasm of tumor cell. The OD values of group S (0.67±0.90) and group F (0.63±0.73) had no significant difference. In group P30and P50, the OD values were0.35±0.30and0.24±0.3, which showed a significantly difference (P<0.01) and were evidently lower than group S and group F (P<0.01). There is a negative correlation between the expression of Wntl and the dose of propofol, Pearson coefficient is-0.916(P<0.01).There was a positive correlation between MTA1and Wntl expression, Pearson coefficient is0.902(P<0.01).ConclusionPropofol can suppress the expression of MTA1and Wntl in MADB106tumor tissues by a dose-dependent manner in tumor-burdened rats, and thus inhibit tumor metastasis.