Cd24Induces Survival Autophagy Through Activation Of NF-κB Signaling Pathway In Colorectal Cancer Cells

ObjectiveCD24is a small heavily glycosylated mucin-like glycosylphosphospha tidylinositol-anchored surface protein with divergent glycosylation patterns within cells. Many studies have reported that CD24is overexpressed in various malignancies. It is tightly correlated with tumorgenesis, carcinogenesis and tumor progression. Interfering with CD24by using CD24monoclonal antibodies and CD24specific siRNAs may influence tumor cell growth. However, Alteration of the expression of CD24protein has no impact on cell proliferation and apoptosis in both HCT116and HT29colorectal cancer cell lines.Hsp90-mediated inactivation of NF-κB switchs autophagy to apoptosis through activation of becn1transcriptional inhibition in selenite-induced NB4cells.We have been proved that CD24interacts with Hsp90via performing co-immunoprecipitation assay. Administration of Hsp90inhibitor causes the degradation of CD24protein. Taken together, we further verified the effect of alteration of the expression of CD24protein on cell proliferation and apoptosis in HCT116and HT29colorectal cancer cells, and determined whether CD24could induce survival autophagy and tended to clarify its underlying mechanisms, which could provide a new insight of therapy targeting on CD24and the feasibility of combined treatments.Material and MethodsRegents and materialsHCT116,HT29colorectal cell lines, pcDNA3.1(+)-CD24plasmid, pcDNA3.1(+) vector, CD24siRNA, Atg5siRNA, GFP-LC3B plasmid(Addgene), RPMI-1640culture medium(Hyclone), LipofectamineTM2000(Invitrogen), Opti-MEM (Invitrogen), Cell counting kit-8(Beyotime), Hochest staining33258(Beyotime),3-Methyladenine(3-MA)(Sigma), BAY11-7082(Beyotime), β-actin(ZSJQ), CD24(a generous gift from Professor Peter at the Institute of Cancer Research in Germany), p62antibody, Beclin-1antibody, LC3A antibody, LC3B antibody, Atg5antibody, Atg3antibody, Atg7antibody, cleaved caspase-3antibody, cleaved-PARP antibody,IκBα antibody, NF-κBp65antibody, phospho-NF-κBp65antibody (all purchased from Cell Signaling Technology), FITC-antibody.Cell cultureThe human colon carcinoma cell lines HCT116and HT29were obtained from ATCC and cultured in RPMI1640containing10%fetal bovine serum(FBS) in a humidified incubator in a5%CO2atmosphere at37℃.Transfection or cotransfection with siRNA and/or plasmid siRNA oligos targeting CD24was:5’-UCUCUCUUCUGCAUCUUUAdTdT-3’, control nonsilencing siRNA was:5’-UUC UCC GAA CGU GUC ACG UTT-3’. siRNA oligos targeting Atg5was:5’-GUCCAUCUAAGGAUGCAAUTT-3’, Control nonsilencing siRNA was:5’-UUC UCC GAA CGU GUC ACG UTT-3’. CD24over-expressed plasmid was constructed in our previous study.HCT116cells were transiently transfected with either CD24over-expressed plasmid or empty vector, while HT29cells were transiently transfected with either CD24siRNA or scilencer negative control, Both cells were transiently transfected with Atg5siRNA or control siRNA and co-transfected plasmids with siRNAs,plasmids with plasmids,siRNAs and siRNAs.All transfection and cotransfection were using LipofectamineTM2000(Invitrogen) according to the manufacturer’s instructions. Cells were then incubated for48h prior to Western blot analysis,cell viability assay and Hoechst33258staining.Extraction of cytoplasmic and nuclear proteinsAfter48h transfection, cells were harvested and washed three times with cold phosphate-buffered saline (PBS).The cytoplamic and nuclear proteins were extracted using the nuclear and cytoplasmic extraction reagents (Beyotime) according to the manufacturer’sprotocol. The protein concentration was determined by BCA Protein Assay Kit (Beyotime).Western blot analysisCells were washed twice in ice-cold PBS, lysed in RIPA buffer for30min on ice, centrifuged at12,000g for15min at4℃, and the supernatants were collected. The protein concentrations were determined by BCA assay. After normalization, equivalent amounts of proteins (20-30ug) were fractionated onto8-15%SDS-PAGE gels and then transferred to Immobilon-polyvinyldifluoride(PVDF) membranes, blocked in5%nonfat milk in Tris-buffered saline-Tween20(TBST) and probed with primary antibodies followed by horseradish peroxidase-label secondary antibodies. The bound antibodies were detected using an enhanced chemiluminescence kit (Gibico, USA) following the manufacturer’s instructions. Anti-β-actin antibody was used as loading controls.GFP-LC3expressionp-EGFP-LC3B expression plasmid was purchased from Addgene (11546; USA),colorectal cancer HCT116and HT29cells were grown on12-well plates. HCT116cells were transiently co-transfected GFP-LC3B plasmid with either CD24over-expressed plasmid or empty vector. HT29cells were transiently co-transfected GFP-LC3B plasmid with either CD24siRNA or negative control siRNA. Both were using LipofectamineTM2000(Invitrogen) according to the manufacturer’s instructions.Images were recorded using inverted phase contrast fluorescence microscope. Autophagosomes were counted in50cells for each cell type.Cell proliferation assayProliferation of cells was measured with Cell Counting Kit-8(CCK-8). Cells after transfection were seeded in a96-well plate at a density of10000cells/well in complete medium and culture for another48h,72h with or without autophagy inhibitors or Atg5siRNA transfection. Each condition in each experiment was studied in six replicate wells. On the day of growth measurement,100ul fresh media containing10%CCK-8was replaced with the spent media, and the cultures were incubated at37℃for additional3hours. Absorbance was measured at450nm using a microplate reader. The results were plotted as the mean±SD of three separate experiments with three determinations per experiment for each experimental conditions.Hoechst33258stainingCell apoptosis was eaxmined using Hoechst33258staining. colorectal cancer HCT116and HT29cells were plated on12-mm glass coverships in culture dishes. After different transfection or cotransfection and treatments,cells were fixed in4.0%paraformaldehyde, then were stained with Hoechst33258for20minutes according to the manufacturer’s instructions. Images were recorded using fluorescence microscopy or laser confocal microscopy. The number of nuclei of condensation and fragmentation were calculated in200cells for each cell type.Autophagy and NF-κB signaling pathway inhibitionAutophagy inhibitor3-methyladenine(3-MA), a phosphatidylinositol3kinase inhibitor that inhibits the formation of autophagosome, and NF-κB inhibitor Bay11-7082were used. Cells were grown in6-well plates for24hours after transfection and then incubated with10mM3-MA for another24hours or5uM Bay11-7082for another12hours prior to Western blot, cell proliferation assay, Hoechst33258staining and LC3staining. Statistical analysisAll experiments were repeated at least three times. The data were expressed as means±SD. Statistical analysis was performed using SPSS13.0for windows. Differences between two groups were analyzed using the two-tailed Student’s t-test and groups of three or more were analyzed using one-way ANOVA mutiple comparision. A statistically significant difference was set at p<0.05.ResultsCD24induces autophagy in colorectal cancer cellsOur preliminary studies have shown that colon carcinoma cell line HCT116expresses very low levels of CD24while HT29with high levels of CD24expression, therefore we examined the effect of CD24expression on induction of autophagy using the twins approach of forced expression and knockdown in these two cell lines by showing respectively high and low CD24expression. Immunoblotting was used to confirm the transfection efficiency and the expression of autophagic associated proteins. CD24knockdown increased the expression of autophagic associated proteins while CD24over-expressed decreased the expression level. Furthermore, GFP-LC3tagged cells showed punctated or diffused pattern after alteration of CD24expression in both cells, which was consistent with the result of immunoblotting.CD24have no apparent impact on cell proliferation and apoptosis in colorectal cancer cellsSome previous studies have shown that treatment with CD24antibodies or CD24specific siRNA in human cancer cells significantly inhibit cell proliferation and cause more cell death. however, Mohamed A.H. Ahmed reported that neither forced expression of CD24in HCT116cells nor reduction of CD24expression in HT29cells has effect on either cell proliferation or apoptosis. This discrepancy prompted us to investigate whether CD24expression influence cell proliferation and apoptosis in HCT116and HT29cells. Cell proliferation was tested using cell count kit-8(CCK-8) after alteration of CD24expression for48hours and72hours. Our data indicated that there was no statistically significant difference in OD value with absorbance at450nm among different groups.We next evaluated apoptosis by performing western blot and Hoechst33258staining, which both revealed that alteration of CD24expression have neither an effect on apoptotic proteins or apoptotic bodies, which were further confirmed what Mohamed A.H. Ahmed had found.CD24-mediated autophagy is a survival process in colorectal cancer cellsAutophagy is a dynamic process that begins with the generation of autophagosomes and terminates with their degradation in lysosomes and corresponds to the autophagic flux.P62is a vital protein which is incorporated into the completed autophagosome and degraded in autolysosomes. To examine the autophagic flux regulated by CD24, we detected the expression of p62after transient transfection of CD24specific siRNA or over-expressed plasmid in HT29cells and HCT116cells. Our results showed that p62was degraded in HT29cells and up-regulated in HCT116cells from24h, and at72h reached the peak, suggesting that CD24underwent functional autophagic flux. To determine whether CD24induced function autophagy, we detected autophagy marker proteins in the presence of3-Methyladenine(3-MA), an inhibitor of class Ⅲ phosphatidylinositol3-kinase(PtdIns3K), which blocked the early steps of autophagy, indicated that 3-MA decreased autophagy marker proteins expression. Our data demonstrated that a complete execution of autophagic pathway, reinforcing our hypothesis that CD24induce functional autophagy. We next evaluated the impact of CD24-mediated autophagy on cell survival. After pharmacological inhibition of autophagy by adding3-MA and genetical abrogation of autophagy by siRNA targeting Atg5, cell counting Kit-8assay, immunoblotting, Hoechst33258were performed among different groups. Cell counting assay indicated that reduction of CD24expression plus adding3-MA or knockdown of Atg5caused more cell death in HT29cells, whereas more cells were viable in HCT116cells, which were consistent with what have found in Hoechst33258assay and immunoblotting. Altogether, these data demonstrated that CD24-induced autophagy promoted survival in HCT116and HT29cells, CD24confered HCT116and HT29cells evading more cell death by inducing survival autophagy.CD24-induced survival autophagy is partly regulated by NF-κB signaling pathway.A growing body of evidences suggested that NF-ＫB signaling pathway was involved in induction of autophagy. To investigate the underlying mechanisms of CD24-induced survival autophagy, we evaluated the activities of NF-κB signaling pathway molecules including IκBα,NF-κBp65and phospho-NF-κBp65by performing western blotting analysis in whole cell lysates, cytoplasmic extractions and nuclear extractions. Additionally, incubation with BAY11-7082, an inhibitor of IκBα phosphorylation, which inhibited NF-κBp65translocation, disrupted basal autophagy and CD24-induced autophagy in both cells, as proved by autophagy marker proteins expression detected by western blotting analysis,and further confirmed by displaying GFP-LC3diffuse pattern. In toto, CD24-induced survival autophagy underwent at least in part via the mechanism of activation of NF-κB signaling pathway.ConclusionIn the present study, we unveiled that CD24induced autophagy and verified that there was no apparent effect on cell proliferation and cell apoptosis by altering the expression of CD24protein in HCT116and HT29colorectal cancer cell lines in vitro. In addition, after pharmacological inhibition and geneticalabrogation of autophagy, we found that CD24-mediated autophagy was a survival process, which was at least partly regulated by NF-κB signaling pathway. Our findings showed that CD24-induced survival autophagy might contribute to the resistance mechanism of therapy targeting CD24, which indicated that such therapy might work on specific individuals. Moreover, These findings provided a new insight on combined therapies with targeting CD24and autophagic inhibitors or interfering with NF-κB signaling pathway, which caused more cancer cell death.