Role Of P62/RIP1-NFκB Signaling Pathway In Sulfasalazine-induced Apoptosis Of Human Glioma Cells

Sulfasalazine (Sulfalsalazine, SAS) has been used in a clinical study of glioma, becauseit can inhibit the proliferation of malignant glioma cells. Although studies have shown thatthe anti-tumor mechanism of SAS is mainly related to its inhibition of NF-κB signalingpathways, and researches have shown that p62is an important regulatory factor of NF-κBactivation in the process of tumorigenesis, but its regulatory mechanism is not fullyunderstood. Studies suggest that autophagy may also be involved in the anti-tumor effect ofSAS. Current studies consider that autophagy is a double-edged sword, according todifferent cell type, drug type and duration and dose of drugs, there are obvious differences inthe mechanisms of autophagy involved in cell survival or death, p62as an autophagyregulatory protein is closely related in NF-κB signaling pathways.Objective:In this study, we used human glioma U251cells as the object of this study, investigatedthe mechanism of SAS in inhibiting NF-κB signaling pathway and inducing apoptosis ofU251cells, analyzed from the point of view that p62involved in the activation of NF-κBsignaling pathway.Methods:①The survival and apoptosis rates of U251cells treated with SAS were detected byMTT, Western blot analysis and Hoechst33258staining; The transcriptional activity ofNF-κB of U251cells treated with SAS was detected by NF-κB luciferase reporter geneanalysis; The expression of IκBα, p65and p50in U251cells treated with SAS were detectedby Western blot, immunofluorescence, and other methods in protein levels as well asmorphological aspects;②We applied RNAi technology, transfected with si-p62plasmid tostudy the impact of p62knockdown on the apoptosis rate and NF-κB transcriptional activityof U251cells;③The expression of p62and LC3in U251cells treated with SAS weredetected by Western blot, immunofluorescence, and other methods in protein levels andmorphological aspects; The impact of inhibiting autophagy on p62protein levels wasanalyzed by autophagy specific inhibitor3-MA;④The impact of increased expression ofp62protein on the apoptosis rate and NF-κB transcription activity of U251cells wereanalyzed by autophagy specific inhibitor3-MA;⑤We transfected with si-p62plasmid to study the impact of inhibiting autophagy on the apoptosis rate and NF-κB transcriptionactivity of U251cells analyzed by autophagy specific inhibitor3-MA;⑥We usedco-immunoprecipitation experiments to further study the mechanism of p62involved in theactivation of NF-κB signaling pathway, to research whether p62involved in the activation ofNF-κB signaling pathway by interacting with RIP1.Results:①The MTT results showed that SAS made the survival rate of U251cells decreased indose and time dependent manners; Western blot, Hoechst33258staining results suggest thatSAS made the apoptosis rate of U251cells increased in dose and time dependent manner;Western blot results showed that SAS can make the expression of p-IκBα protein increased;NF-κB luciferase assay results showed that SAS significantly inhibit the NF-κBtranscriptional activity of U251cells; The indirect immunofluorescence results showed thatSAS decreased p65and p50into the nuclear;②After RNAi technology interfere with theexpression of p62gene, MTT, flow cytometry, Hoechst33258staining results showed thatthe apoptosis rate of U251cells treated with SAS was more significantly increased; NF-κBluciferase Assay and Western blot results showed more pronounced inhibition of the NF-κBtranscriptional activity of U251cells treated with SAS;③Western blot results showed thedecreased expression of p62protein and the increased expression of LC3II protein by SAS;The indirect immunofluorescence results showed that the distribution of p62and LC3changed, mainly in the cytoplasm, and punctate aggregation appeared; Western blot resultsshowed that the autophagy inhibitor3-MA made the decreased expression of p62proteininduced by SAS increased in U251cells;④NF-κB luciferase Assay, Western blot resultsshowed that the autophagy inhibitor3-MA made the inhibition of NF-κB transcriptionalactivity induced by SAS weaker in U251cells; MTT, flow cytometry, Hoechst33258staining results showed that the autophagy inhibitor3-MA made the decreased apoptosis rateinduced by SAS less in U251cells;⑤After RNAi technology interfere with the expressionof p62gene, the autophagy inhibitor3-MA also made inhibition of NF-κB transcriptionalactivity induced by SAS weaker and the decreased apoptosis rate induced by SAS less inU251cells;⑥The co-immunoprecipitation results showed that SAS can reduce thecombination of p62and RIP1.Conclusion: In summary, p62may involve in the regulation of NF-κB signaling pathway by SAS,the inhibition of p62expression can increase SAS-induced inhibition of NF-κB signalingpathway and U251cells apoptosis. In addition, SAS induced autophagy and therefore causedthe decrease of p62protein.The inhibition of autophagy can decrease SAS-induced inhibitionof NF-κB signaling pathway and U251cells apoptosis. P62may interact with RIP1to form asignaling complex, involve in the activation of NF-κB signaling pathway. Therefore, the p62protein level plays an important role in SAS-induced inhibition of NF-κB signaling pathwayand U251cells apoptosis. Targeting inhibiting p62may be a new strategy to improve theanti-tumor effect of SAS.