P120ctn-1 And P120ctn-3 Regulate Cell Cycle And Cell Proliferation Of Lung Cancer Respectively By Means Of β-catenin And Kaiso

PurposeOur previous study found that p120ctn could affect lung cancer cell invasion and proliferation when E-cadherin was in the presence. We also found that p120ctn-1 can regulate P-catenin, but p120ctn-3 could not. So we speculated that the mechanism of p120ctn-1 and 3 affect lung cancer cell proliferation was likely to be different. p120ctn-1 is likely to affect cell proliferation by means of regulatingβ-catenin activation in the classical WNT pathway, and p120ctn-3 is very likely to affect lung cancer cells by other factors. Although there is an article which has already reported p120ctn-3 can inhibit cell cycle, but the molecular mechanism of p120ctn-3 affecting cell cycle is not really clear. Kaiso which was found as a combination ligand of p120ctn is a nuclear BTB/POZ-ZF transcription inhibitors. Kaiso is able to identify specific DNA promoter sequences TCCTGCnA (Kaiso-binding sites, KBS), and inhibits the corresponding genes. As the promoter sequence of cyclinDl also contains KBS, it is likely to be the potential downstream target gene of kaiso. Interestingly, the zinc finger domain which is the binding site of Kaiso with p120ctn is just the binding site of it with the KBS. Therefore, we assume:p120ctn-3 is likely to combine with Kaiso, and inhibits Kaiso combinates with the KBS, and regulates subcellular localization of Kaiso, thus affecting the transcription of cyclinDl, thereby affecting the cell cycle. Therefore, we overexpressed and interfered p120ctn as well as Kaiso.Respectively study the roles of p120ctn-1 and 3 in lung cancer cell proliferation and cycle. Method1,cell cultureLung adenocarcinoma cell line SPC and A549 were purchased from ATCC. The cell lines were maintained in medium, supplemented with 10% fetal bovine serum (GIBCO Inc., Los Angeles CA, USA). For serum starvation experiments, cells were starved in DMEM contain 0.1% FBS, and then added DMEM contain 10% FBS back for reverse.2,3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) AssayCell proliferation was evaluated each day for four days after the MTT treatment. The absorbance, which is directly proportional to the number of living cells in the culture, was measured at 550 nm using a microplate reader (Model 550, Bio-Rad, Hercules, CA, USA). A blank with dimethyl sulfoxide (DMSO) alone was taken and subtracted from all values.3,Flow cytometry (FCM)After 48 hours of culture, cells from each experimental group were collected and digested with trypsin and fixed with 75% ice-cold ethanol at 4℃overnight. Cells (1×106) were centrifuged at 1500 rpm 5 minutes, and were resuspended with 50μg/ml propidium iodide for 45 minutes in the dark before analysis. The percentages of cells in the different cell cycle phases were determined using a FACSCalibur Flow Cytometer with CellQuest 3.0 software.4,RT-PCRRT-PCR was performed with the RNA PCR Kit (AMV) Version 3.0, according to the manufacturer's instructions.5,Western Blot50μg proteins were separated by SDS-PAGE. After transferring to polyvinylidene fluoride membrane, the membrane was incubated overnight at 4℃with either the mouse monoclonal antibody against p120ctn and Kaiso. After incubation with peroxidase-coupled anti-mouse IgG at 37℃for 2 hours, the proteins were visualized using DAB/ECL.6,Chromatin immunoprecipitation assay(ChIP)We finded out cyclinDl promoter sequence (GenBank:AY439218.1) in GENEBANK, and designed the corresponding promoter sequences containing the KBS fragment primers (TAKARA company was commissioned), upstream: 5'-CCCTCTCATGTAACCACGAA-3',downstream:5'-TGGTTTTGTTGGGGGTGT AG-3'. Chromatin immunoprecipitation assay was performed according to the protocol of ChIP assay kit (Upstate Biotechnology, Lake Placid, NY).7,Immunofluorescent stainingCells grown on glass coverslips were fixed with ice-cold 100% methanol for 15 minutes at-20℃, followed by permeabilization with 0.2% Triton X-100. Kaiso was detected using goat polyclonal antibodies p120ctn was detected using mouse monoclonal antibodies. Primary antibodies were applied overnight at 4℃followed by incubation with secondary antibody conjugated to rhodamine/fluorescein isothiocyanate (FITC)-labeled. The nuclei were counterstained with propidium iodide/4,6 diamidino-2-phenylindole. The cells were examined with an OlympusⅨ51 fluorescent microscope (Olympus, Tokyo, Japan), and images were recorded with a CoolPIX 5400 camera (Nikon, Japan).8,ImmunoprecipitationCells were washed twice with 5 ml of PBS followed by incubation on ice with lysis buffer containing 0.5% NP-40,50 mM Tris,150 Mm NaCl,1 mM phenylmethylsulfonyl fluoride,5 mg/ml leupeptin,2 mg/ml aprotinin,1 mM sodium orthovanadate, and 1 mM EDTA for 5 minutes. Cells were harvested from the plates, and transferred to a 1.5 ml tube. The lysate was centrifuged at 16,000 g for 5 minutes at 4℃and the supernatant transferred to a new tube. Lysates were quantified by Bradford assay and equal amounts of total protein were used for immunoprecipitation with the anti-p120ctn or anti-Kaiso mAb. The immunocomplexes were then subjected to SDS-PAGE.9,Statistical AnalysisAll data were expressed as mean±standard deviation (S.D.) for in vitro experiments performed at least 3 times, and the one-way analysis of variance followed by a least significant different test (LSD) for multiple comparisons was used for statistical analysis via SPSS 13.0 for Windows (SPSS Inc., Chicago, IL, USA). P values less than 0.05 were considered statistically significant.ResultsKnockdown p120ctn of A549 and SPC slowed down cell proliferation. Respectively recover p120ctn-1 and 3 subtypes can effectively return the cell proliferation of A549 and SPC. Knockdown p120ctn of A549 and SPC increased the number of cells in G0/G1 phase and significantly decreased the number of cells in S phase; After recovering p120ctn-1A and 3A respectively, the number of SPC-K2 cells in Go/G1 phase decreased significantly, and the number of cells in S phase increased significantly. In A549 and SPC cells, p120ctn-1 and 3 subtypes can up-regulate the protein and mRNA levels of cyclinD1 and cyclinE; In the SPC-K2 cells, recover p120ctn-1 and 3 subtypes didn't change the protein and mRNA levels of kaiso. After recovering p120ctn-1A, the protein and mRNA levels ofβ-catenin significantly raised, while recover p120ctn-3 can't change the protein and mRNA levels ofβ-catenin significantly. ChIP assay confirmed kaiso can combine with the KBS of cyclinDl promoter region. In A549 and SPC cells, kaiso can effectively regulate the protein and mRNA levels of cyclinDl and cyclinE. Immunoprecipitation showed that in A549 and SPC cells, p120ctn-3 can combine with kaiso, but failed to detect the combination between p120ctn-1 and kaiso; ChIP assay confirmed that in the A549 cells overexpression of p120ctn-1A can't affect the combination of kaiso with cyclinDl promoter region, but overexpression of p120ctn-3 can significantly reduce the combination of kaiso with cyclinDl promoter region. Immunofluorescence and cellular fractionation confirmed p120ctn-3 can effectively regulate the subcellular localization of kaiso, and the nuclear transfer of kaiso depends on the nuclear transfer of p120ctn-3.Conclusions1,p120ctn-1 and 3 subtypes can promote the expression of cyclinDl and cyclinE and affect cell proliferation and cell cycle.2,p120ctn-1 affects cell proliferation and cell cycle of lung cancer by means ofβ-catenin.3,p120ctn-3 affects cell proliferation and cell cycle of lung cancer by means of kaiso.