The Expression Of ADAM-17 In Renal Clear Cell Carcinoma And The Molecular Mechanism In Specific Effect Of Targeted Inhibition Of ADAM-17 Through Notch Pathway

BackgroundRenal cell carcinoma is the most common renal malignant solid tumor, which is the malignant tumor originating in renal urinary tubule. renal cell carcinoma is about 2%-3% in all of adult malignant tumors. the typical kidney triad: hematuria, lumbago, and abdominal mass has less than 6%-10% in current clinic, In renal cell carcinoma patients is about 30% during advanced cancer, metastatic renal cell carcinoma is in approximately 30% of patients after surgery. At present, the main methods of treatment for localized renal cell carcinoma is operation resection, however, the treatment effect is not satisfied for metastatic renal cell carcinoma which is advanced tumor before diagnosis. The present treatment to metastatic renal cell carcinoma includes chemotherapy and immune therapy and molecular targeted therapy, the majority of pathological types of renal cell carcinoma is clear cell carcinoma, as a kind of adenocarcinoma, accounting for about 60%-85%. As for the radiotherapy, chemotherapy is not sensitive, and targeting of clinical use of drugs, significantly improved the survival of patients.The current targeted drugs such as sorafinib, sunitinib etc. based on selective inhibiting VEGFR2,3 or KIT, VEGFR-1 and other effects on the renal cell carcinoma cells to exert antitumor effects, the most important one is the Notch signal pathway. The current research focus on how to improve the inhibition efficiency of inhibiting the Notch pathway, at present, targeting inhibition of the Notch pathway is mostly based on the inhibition of the Notch pathway receptor and ligand synthesis, however, this kind of inhibitor has limited influence for three key sites of the Notch pathway. As the report of the foreign research for Notch pathway activation, the S3 site was mostly reported, a representative for inhibitors of gamma secretase inhibitors ADAM-17 also known as TACE as an early detection of TNF-alpha inhibitors, based on last researches, in leukemia Notchl protein release depends on the ADAM-17 for the Notch signal pathways, which reveals the important role of ADAM-17 in the Notch pathway such as ADAM-17 plays a key role of the enzyme In the S2 site of the Notch pathway Marimastat, is the only one which can inhibit TACE of the metalloprotease family. The main effect of Marimastat is connected to the basement membrane closely, inhibition of TNF-alpha converting enzyme, inducting programmed death by stabilizing the cell surface TNF-receptor alpha. Due to it against TACE (ADAM-17) function, through the Notch signal pathway on tumor has attracted wide attention.The designation of our experiment is that compared with the cutting effect of ADAM-17 on extracellular S2 sites, the S3 site where is the Notch gamma secretion enzyme cutting is located in the transmembrane region, the transmembrane region can be affected by multiple signaling pathways. As the tumor cell signal pathway is a complicated three-dimensional structure. The mutual influence of each signal path has a number of known, unknown effect, so the inhibition of the gamma secretase influences on other signaling pathway is inevitable;, however, to S2 site the possibility of affected than the S3 site is more smaller; so, inhibition of S2 site should be specific than S3 site, in the other words, iinhibition of AD AM-17 can achieve better effect in Notch pathway, it could provide new ideas for target inhibition in renal cell carcinoma in the future.ObjectiveThe mechanism of the occurrence and development of renal tumors is not clear, the Notch signaling pathway is to play an important role in it. We research on Notch pathway key enzyme ADAM-17 (TACE) expression in renal cell carcinoma and the specific effect of two inhibitors of Notch pathway in proliferation, cell invasion in renal cell carcinoma 786-0 cell and OS-RC-2 cellMathod1. Expression of ADAM-17 in renal cell carcinoma1.1 Extraction of renal cell carcinomaSixty-seven pairs of clear cell renal carcinoma (CCRCC) tissues and 10 adjacent normal kidney tissues were collected at the Department of Urology of the Shandong Provincial Hospital of China. All RCC cases were confirmed clinically and pathologically to be of the clear cell type. All tumor specimens were staged based on the 2002 AJCC TNM classification of malignant tumors (Table 1). The samples were snap-frozen in liquid nitrogen and stored at-80℃ until analysis. Prior written informed consent was obtained from all patients and the study was approved by the Protection of Human Subjects Committee of the hospital.1.2 Immunohistochemical detection of ADAM-17 in renal cell carcinomaWe collected renal cell carcinoma tissues and normal renal tissue specimens, used Rabbit anti human ADAM-17 polyclonal antibody, Dewaxing hydration, antigen, IHC repair kit experiment, counterstain, DAB color, dehydration transparent and microscopic imaging were strictly in accordance with the method of Super Polymer-two step IHC Kit. The positive expression of ADAM-17 is mainly concentrated in the surrounding membrane, red brown staining, however, renal cell arrangement standardized in normal renal tissues, no mitotic and no such staining around the periphery of the cell expression. At last, we got the numbers of positive cases.1.3 Western blot assay to detect the expression of ADAM-17We collected the renal cell carcinoma in different groupes according to the clinical stages. Renal cell carcinoma tissue clinical stage I and clinical stage Ⅱ as a group, III and IV as a group, the normal kidney tissues as a group, all of them were randomly selected 3 cases to extracted total protein and analyzed the expression of ADAM-17 protein by Western blot assay. Using GAPDH as an internal reference, with the ratio of ADAM-17 and GAPDH express the level of ADAM-17 protein in each group. To calculated for each protein band optical density value of IOD (Integral Optical Density). We used the IPP 6.0 software, then calculate the ratio between each protein bands and corresponding with the reference band gray value1.4 Flow cytometry analyze the cell cycle of ADAM-17 positive renal cell carcinoma.According to the results of immunohistochemical assay, we campared the stage III and IV stage ADAM-17 positive expression and negative renal cell carcinoma. We used flow cytometry to analyze the cell cycle of AD AM-17 positive renal cell carcinoma.2. The study of specific effect with inhibition of ADAM-17 through the Notch pathway2.1 Cell culture and reagentsThe CCRCC cell lines 786-0 and OS-RC-2 were purchased from Chinese Academy of Medical Sciences and tumor cell library. The cells were cultivated in RPMI 1640 medium and Dulbecco’s modified Eagle’s medium (Aidlab Biotechnologies Co. Beijing, China), respectively, and supplemented with 10% fetal calf serum in a humidified incubator at 37℃ with a mixture of 95% air and 5% CO2.2.2 Cell treatment and groupingWe treated the 786-0 and OS-RC-2 cells with ADAM-17 inhibitor, Marimastat (Tocris, UK) at concentrations of 1 μmol/L,2 μmol/L and 3 μmol/L diluted in 1640 medium to a final volume of 2 ml, or the same concentrations of the y-secretase inhibitor, DAPT (EMD bioscience, CA) for 24 hours. The control group was provided by cells incubated with 2 ml of 1640 medium alone. Afterwards cells were collected for further testing.2.3 Western blot assay786-0 cells and OS-RC-2 cells were lysed in radio-immunoprecipitation assay buffer and equal amounts of the protein extracts (30 μg per lane) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were then transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA) for western blotting. The primary antibodies against NOTCH 1 (activated Notch intracellular domain), HES-1 (Abeam, Cambridge, MA), and (3-actin (Aidlab Biotechnologies Co., Beijing, China) were incubated with membranes overnight at 4℃. After 3 washes, for 15 min each, in Tris-buffered saline supplemented with 0.1% Tween 20, membranes were incubated with peroxidase-conjugated goat anti-mouse/rabbit IgG antibodies (Aidlab Biotechnologies Co. Beijing, China) for 1 h at room temperature. The bound anti-bodies were visualized by an enhanced chemi luminescence detection system using medical X-ray films.2.4 Comparative inhibition of proliferation analysis with CCK-8 assayCells were seeded in a 96-well plate at approximately 8×104 in a volume of 100μl/well. Wells were also prepared that contained known numbers of four kinds of cells to be used to create a calibration curve. To measure apoptosis,10 μl of the CCK-8 solution (Dojindo, Japan) was carefully added to each well of the plate. The plate was incubated for 1-4 h in the incubator during which time the absorbance was measured at 450 nm using a microplate reader at 30,60, and 90 min.2.5 Transwell assay for cell invasionCell invasive ability was determined using the Transwell test kit (Corning, NY, USA). Briefly, matrigel was mixed with 1640 medium at a ratio of 1:7 and 100 μl was added to each upper-transwell then placed into the incubator for 1 hour for the mixture to set. Then,786-0 cells were serum-starved for 12 h in pre-warmed 1640 media alone to eliminate the effects of serum. Twenty-four hours after the application of matrigel,600 μ1 of 10% FBS solution was added to the lower transwell. The serum starved cells were resuspended to a density of 2.5 ×105 in 1640 solution without FBS in a final volume of 1 ml, with or without Marimastat or DAPT. From this,100μl was added to each transwell (2.5×104). After 48h in the incubator, the transwell casters were purged into PBS to remove the non-adherent cells, and then submerged it in 4% paraformaldehyde for 10 min for fixation, and finally replaced in PBS. After the membrane was dried, cells were observed and counted under a microscope (400x).2.6 Flow cytometry and cell cycle apoptosis1×106 cells were plated in 100 ml culture flasks and allowed to proliferate until 70-80% confluence was attained. Cells were then treated with Marimastat (1μmol/L or 3μmol/L), DAPT (1μmol/L or 3μmol/L), or DMSO (15μl) as control. After 24 h, cells were washed then resuspended in PBS. To measure apoptosis, the Annexin-FITC Apoptosis Detection Kit (KAIJI BIOTECH, Nan Jing, CN) was used according to its instructions. Briefly, fresh cells were labeled with 1:500 diluted Annexin Ⅴ-biotin conjugated with FITC followed by incubation with 1:1000 diluted PI. Annexin Ⅴ-PI expression levels were measured by FACS Calibur (BD Science, NY, USA) and analyzed by Modfit Software.2.8 Statistical analysisAll data were analyzed using the SPSS statistical software package (SPSS Inc., Chicago, IL) All data were expressed as mean ± standard deviation (SD) unless otherwise specified. Intergroup differences for two variables were assessed by unpaired t-test. Differences in parameters between groups were evaluated by ANOVA followed by unpaired t test with Bonferroni correction for multiple comparisons. P<0.05 was considered statistically significant.Results1ADAM-17 is over expressed in renal carcinoma tissuesThrough immunohistochemical staining assay we found that ADAM-17 was highly expressed in renal carcinoma tissues. Specifically, we observed 43 positive cases among a total of 67 cases (64.18%). The expression rate in the T1-T4 stages were 21.43%,63.67%, 84.00% and 83.33%, respectively. ADAM-17 was highly expressed as the tumor stage increased, in the stage Ⅰ,only 3/14 tissues were ADAM-17 positive but in the stage Ⅲ and Ⅳ, the ADAM-17 positive tissue were increased to 21/25 and 5/6. To evaluate these results, we found that the positive expression rate of ADAM-17 was greater in the high tumor stage than low tumor stage (x2 = 16.39 P＜0.01). In contrast, it was hardly expressed in non-renal carcinoma tissues. Indeed, from a total of 67 samples, only one sample was positive, resulting in a positive expression rate of 1.49% (P<0.05). In phase Ⅰ, Ⅱ, Ⅲ and Ⅳ, ADAM-17 expression in renal cell carcinoma.we used the western blot assay to found that the relative optical density value in every group were 0.67 ± 0.11,0.73 ± 0.31,0.79 ± 0.25,0.89±0.09 respectively. In ADAM-17 positive expression in renal cell carcinoma, with the increasing stages of renal cell carcinoma, ADAM-17 expression also increased (t=18.74 P<0.01) and the expression of ADAM-17 in stage IV renal cell carcinoma has a significant increase (0.89±0.09 vs 0.79±0.25).In the flow cytometry assay to analyze the cell cycle of ADAM-17 positive renal cell carcinoma, we found that phase III and phase IV whatever positive or negative ADAM-17 expression in renal cell carcinoma, the cell cycle in G2 phase were higher proportion (0.31 vs 0.22). It shows that the higher stage of renal cell carcinoma, the higher degree of malignancy, meanwhile, the positive expression of ADAM-17 in renal cell carcinoma tissue is obviously up-regulated on the cell cycle of G2 phase ratio, the difference was statistically significant (x2 =20.76 P<0.01).2. The study of specific effect with inhibition of ADAM-17 through the Notch pathwayAfter treatment with either Marimastat or DAPT, We found that regardless of whether cells were treated by Marimastat or DAPT, expression of Notch 1 and HES-1 proteins was considerably decreased (P＜0.05). Thus, the expression of Notch 1 and HES-1 proteins was more readily decreased in the Marimastat treated renal carcinomas than in those treated by DAPT. Notably, the same concentrations of each inhibitor were used for treatments.In the CCk-8 assay, we found the ODs were readily decreased in both cell lines when compared with the DMSO treated control. Moreover, we found that the mean OD value of Marimastat-treated 786-0 cells was lower than that for cells treated with the same dose of DAPT, statistical analysis revealed that the two inhibitors both significantly decreased the invasive ability (P<0.05, P＜0.05). However, under the same dose conditions, Marimastat rendered a greater impact on the two types of renal carcinoma cell lines than did DAPT (P<0.05).We tested the invasive capacity of the renal carcinoma cells,786-0, treated with either Marimastat or DAPT at different concentrations by Transwell assay. Treatment with either Marimastat or DAPT reduced the number of 786-0 invasive cells in a dose-dependent manner when compared with the non-treated control group. Notably, the drug-induced reduction in invasive cell number was significantly more potent with Marimastat treatment than with DAPT (p<0.05).Analysis of Annexin V-PI staining showed apoptotic rates of 3.4% and 5.4% for 786-0 after DAPT treatment with 1 μmol/L and 3 μmol/L, respectively, and 4.5% and 7.7% following Marimastat treatment with the same doses. Lower levels of apoptosis (2.8%) were detected in the control group. The following statistical analysis showed that the apoptosis rates of 786-O after Marimastat treatment was greater than that attained after treatment with DAPT at the same concentrations (P<0.05).Conclusions1、ADAM-17 is over expression in renal cell carcinoma, the positive expression of AD AM-17 is increased with the tumor staging increases2、For the high stage of renal cell carcinoma, positive expression of ADAM-17 increases the cell proliferation and effects the invasion and development of renal cell carcinoma.3、Inhibition of ADAM-17 expression may become a new target treatment sites in renal cell carcinoma.4、Marimastat and DAPT can reduce the Notch 1 protein and Hes-1 protein expression, reduce the cell proliferation in 786-0 cell line in order to decrease the its invasion and increase the rate of apoptosis.5、Compared with y-secretase, inhibition of ADAM-17 expression more effectively inhibits Notch pathway-mediated renal cancer cell proliferation and invasion.