Reactivation Of Mutant P53 To Wild-type-like P53 By Butein

Cancer is one of the major threats to human health.Although a significant breakthrough is made in the treatment of tumors,oncology drug development still face severe challenges.Mutations in the TP53 gene are found in more than 50%of cancer patients.Moreover,mutation of the TP53 gene often result in the gain of oncogenic functions such as promote tumorigenesis and development.Hitherto,mutant p53 has been proven as a target for antitumor drugs,and reactivation of mutp53 by small molecule compounds is an effective tumor treatment strategy.Butein is a member of flavonoid polyphenols family,has been proven to possess various beneficial biological properties such as anti-inflammatory,antibacterial,antiviral,antitumor,protecting the cardiovascular system,protecting liver and kidney,and eliminating free radicals,etc.However,in regard to butein exerts tumor suppressing function by reactivating wild-type activity to mutp53,has not been reported so far.Firstly,we explored the effect of Butein on the conformation of p53R273H and p53R175H.Immunofluorescence and Immunoprecipitation experiments with conformation specific antibodies（PAb1620 and PAb240,which specifically recognize the wild type and mutant p53 respectively）were performed.It was found that the expression of mutp53 significantly decreased in HT29（p53R273H）and SK-BR-3（p53R175H）cells treated with Butein,while the expression of wtp53 increased significantly,indicated that Butein can restore the wild-type conformation of p53R273H and p53R175H.Owing to the close relation of DNA binding activity and transcription function of p53 with its conformation,and the alteration of p53 often result in loss of p53 DNA binding ability and transcription activation function.Therefore,we examine the DNA binding activity of p53R273H and p53R175H through electrophoretic mobility shift assay.The results showed the amount of DNA specifically binding to p53 increased in HT29（p53R273H）and SK-BR-3（p53R175H）cells treated with Butein,implyed that Butein can restore the DNA binding activity of p53R273H and p53R175H.Then we explored whether Butein further restored the transcriptional activity of p53R273H and p53R175H.Through chromatin immunoprecipitation experiment,it was found that the p53 in HT29（p53R273H）and SK-BR-3（p53R175H）cells treated with Butein had increased binding to the PUMA promoter.In addition,Butein could upregulate the m RNA and protein levels of p53target genes such as PUMA and NOXA in HT29（p53R273H）and SK-BR3（p53R175H）cells,indicated that Butein can restore the transcriptional activation function of p53R273H and p53R175H.In addition,we further explored whether Butein up-regulated of p53 target gene is caused by mutp53 reactivation.After suppressing p53 in HT29（p53R273H）and SK-BR-3（p53R175H）cells by RNA interference p53 or combination with p53transcription inhibitor PFT-α,it was found that the up-regulation of p53 target genes by Butein was significantly inhibited,indicated that Butein up-regulates the expression level of p53 target genes through reactivating mutp53.Moreover,other members of p53 families,TAp63 and TAp73,can also exert transcription function like p53.Therefore,TAp73 was knock down by RNA interference,the results showed that in SK-BR-3 cells,the upregulation of p53 target genes by Butein did not change after TAp73 knock down.This phenomenon suggested that Butein up-regulate the p53 target genes in a TAp73 non-dependent manner.Further,we explored the underlying molecular mechanism of Butein reactivation of mutp53.In view of the key role of molecular chaperones on the conformation and activity changes of mutp53,and molecular chaperones Hsp90/Hsp70/Hsp40 can form a stable complex with mutp53 leading to the stabilization of mutp53 and the inhibited ubiquitination degradation of mutp53 by CHIP and MDM2.We firstly explored the potential effects of molecular chaperones Hsp90 and Hsp70 on the remodeling of wild-type conformation and function of Mutp53 by Butein.The results showed that the interaction between Hsp90 and mutp53 was weakened in HT29 and SK-BR-3 cells treated with Butein.Meantime,the acetylation of Hsp90 evidently increased,but ATPase activity of Hsp90 did not change.Additionly,given that the key role of Hsp90 in the stability and conformation of mutp53,we further overexpressed Hsp90 exogenously,and found that the reduced expression of mutp53and the increased expression of wtp53 and p53 target genes in HT29 and SK-BR-3cells by Butein was significantly inhibited after overexpression of Hsp90.This indicateed that Butein may affect the wild-type function of mutp53 by regulating the activity of Hsp90.In addition,owing to the redox state of the cell is related to the conformation of p53,and the binding ability of wtp53 to targeted DNA depends on the reducing environment.We also tested whether Butein can inhibit tumors by inducing oxidative stress,and found that the ROS level in HT29（p53R273H）and SK-BR-3（p53R175H）tumor cells was increased 1.5 and 5.8 times respectively after treatment by Butein（10,20μM）for 3,6,and 12 hours,indicated that Butein can induce oxidative stress.In addition,the DBD of p53 contains 10 Cys,among them,Cys182,Cys229,Cys242,and Cys227 are located on the surface of the core structure of p53.These Cys will expose and more easily to covalent modification once p53 mutation.Butein is anαandβunsaturated ketone with active double bonds,hence possess the possiblity to covalent modification with sulfhydryl groups.We further examined the effect of redox balance and thiol modification on reactivation of mutp53 by Butein.The DTNB experiment was carried out to detect the reaction between Butein and sulfhydryl.The results showed that after 1 hour of incubation with Butein and NAC,Butein had Sulfhydryl-binding activity.In addition,through combination between Butein（10,20μM）and NAC in HT29（p53R273H）and SK-BR-3（p53R175H）cells for 48h,no matter the restoration of transcriptional activation function to mutp53,or the reduced expression of mutp53 and the increased expression of wtp53 by Butein,were obviously reversed.It seem that Butein may directly interact with mutp53through covalent modification,thereby reactivating the wild-type function of mutp53.In conclusion,we found that the wild-type conformation,DNA binding activity and transcriptional activation of DNA-contact mutations p53^R273H and structural mutations p53^R175H were restored by Butein,and Butein may affect the wild-type conformation of mutp53 by regulating the activity of Hsp90.In addition,Butein reactivate wild-type function of mutp53 may through directly interacting with mutp53by covalent modification.This study provide certain directive significance for developing the anti-tumor effect of Butein in mutant p53 tumor.