Reactivation Of Mutant P53 To Wild-Type-Like P53 By Natural Buxus Alkaloids

Cancer is one of the major threats to human health.According to the Global Cancer Statistics 2018 by IARC,the new cancer cases and total annual cancer deaths climb into the 18.1 million and 9.6 million respectively,all over the world.In current,chemotherapy remains one of the main means for tumor treatment.Compared with small molecules by organic synthesis,natural compounds have a lot of advantages,such as wide source,low price and low toxicity.Furthermore,they possess the potential to inhibit tumors by regulating multiple key signaling pathways.Therefore,screening natural compounds with anti-tumor activity and exploring the potential mechanism of anti-tumor can provide a basis for boosting and facilitating the development of their clinical application and utilization of other natural compounds.In tumors,tumor suppressor gene TP53,have high frequency of mutation,the mutation not only result in a loss of function,worsestill,mutp53 gain some characteristics to drive malignant progression of tumor.Owing to the conformational change caused by p53 mutation usually feeble and reversible,reactivation mutp53 by natural compounds is a feasible theary to inhibit tumor harbor mutp53.As the active component of populus,buxus alkaloid has the effect on treating heart,cerebrovascular,fatty liver and other diseases,but seldom reported the function of buxus alkaloid to inhibit tumor.In our preliminary experiment,KBA01,a triterpenoid alkaloid extracted from microphylla,had been observed to inhibit the proliferation of tumor cells.Moreover,wtp53 and its downstream genes such as PUMA,p21 could be significantly up-regulated while mutp53 was degraded by KBA01.It is suggested that KBA01 may be a anti-tumor compound with the function to reactive mutp53.Based on this premise,we further to verify the function of KBA01about mutp53 reactivation and explore the potential mechanism.In our study,the effect of KBA01 on the conformation of p53^R273H were investigated firstly.Immunoprecipitation experiment with conformation specific antibodies（PAb1620 and PAb240,which specifically recognize the wild type and mutant p53 respectively）was carried out.The results showed that the expression of mutp53 was down-regulated and the expression of wtp53 was up-regulated in HT29cells(p53^R273H)treated by KBA01,suggested that the wild-type conformation of p53^R273H was restored by KBA01.Owing to the close relation between conformation and abilities of DNA binding,transcriptional activation of p53,whether DNA binding activity of p53^R273H could be restored by KBA01 was explored.Electrophoretic mobility shift assay was carried out.The results showed that the amount of DNA specifically binding to p53 significantly increased in HT29(p53^R273H)and MDA-MB-231(p53^R280K)treated by KBA01,suggested that the DNA binding activity of p53^R273H,p53^R280Kcould be restored by KBA01.Then,whether the transcriptional activation activity of p53^R273H and p53^R280Kcould be restored by KBA01 was explored.Firstly,by constructing PUMA promoter luciferase reporter gene vector into H1299-p53R175H and H1299-p53R273H cells,it was shown that the expression of luciferase increased obviously in H1299-p53R273H cells treated by KBA01.At the same time,the binding of p53 to PUMA promoter was enhanced in HT29(p53^R273H),H1299(p53^R273H)and MDA-MB-231(p53^R280K)cells after treatment with KBA01.In addition,the protein levels of PUMA,BAX and other p53 target genes in HT29(p53^R273H)were up-regulate by KAB01,suggested that the transcriptional activation activity of p53^R273H and p53^R280K was restored by KBA01.Given the binding of MDM2 and the reduction of Acetyl-p53（Lys382）can lower the stability of p53,whether the stability of mutp53 and wtp53 could be regulated by KBA01 through regulating the interaction of MDM2-p53 and the level of Acetyl-p53（Lys382）was explored.The results showed that after treatment with KBA01,the interaction of MDM2-mutp53 in HT29(p53^R273H)was significantly enhanced,and the interaction of MDM2-wtp53 showed a certain weakening trend;the expression of Acetyl-wtp53（Lys382）was up-regulated,and the expression of Acetyl-mutp53（Lys382）was significantly decreased,suggested that KBA01 the wtp53 potentially was stabilized and the mutp53 was degraded by KBA01 through regulating the interaction between MDM2 and p53 also the level of Acetyl-p53（Lys382）.In addition,whether the up-regulation of p53 target gene by KBA01 caused by mutp53 reactivation was further explored.After inhibition of p53 in HT29(p53^R273H)by RNA interference and transcriptional inhibitor PFT-alpha,the results showed that the up-regulation of p53 target gene by KBA01 was significantly inhibited,suggested that up-regulation of p53 target gene caused by mutp53 reactivation.Meanwhile,it was shown that after treatment with KBA01,the interaction between mutp53 and TAp63/TAp73 in HT29(p53^R273H)was broken and the target genes JAG1 of TAp63and TAp73 was transcriptional activated through Immunoprecipitation and Quantitative Real-time PCR assayes.Next,TAp63 and TAp73 were knocked down by si RNA,but the up-regulation of p53 target genes in HT29(p53^R273H)by KBA01 did not change,suggested that the up-regulation of p53 target genes by KBA01 not attributed by TAp63 and TAp73.On the other hand,whether KBA01 could induce oxidative stress and then inhibit tumors was explored.The results showed that the level of Reactive oxygen species（ROS）in SK-BR-3(p53^R175H),HT29(p53^R273H)and other cancer cells increased after treatment with KBA01,suggested that the oxidative stress in cancer cells was induced by KBA01.In addition,on account of the property of heat shock proteins to drive the refolding of misfolded proteins,the effect of them on the regulation of mutp53 by KBA01 was investigated.The results showed that mutp53 reactivation by KBA01 was not altered after inhibition of Hsp40,Hsp70 and Hsp90,but the degradation of mutp53 was affected,suggested that heat shock proteins involved in the degradation of mutp53 by KBA01.At the same time,whether KBA01 could affect the chaperone activity of Hsp90 was explored.The results showed that the chaperone activity of Hsp90 in HT29(p53^R273H)was enhanced by KBA01 through down-regulating the level of Acetyl-Hsp90（Lys294）.In addition,the effect of KBA01 on heat shock response in HT29(p53^R273H)was also investigated.The results showed that after treatment with KBA01,the protein interaction between mutp53 and Phospho-HSF1（Ser326）was broken,and the m RNA level of HSF1 target genes such as Hsp70 and Hsp90 were down-regulated,suggested that the heat shock response mediated by HSF1 in HT29(p53^R273H)was reduce by KBA01 through breaking the interaction between mutp53 and Phospho-HSF1（Ser326）.Besides,the signaling pathways related to tumor suppressive function of KBA01 was explored by transcriptome sequencing.The results showed that after treatment with KBA01,p53,apoptosis and other signaling pathways in HT29 cells were significantly increased,while the DNA repair and cycle pathways were obviously down-regulated,suggested that KBA01 could inhibit tumors by up-regulating p53 signaling pathway,and possibly through inducing cycle arrest,apoptosis,DNA repair deficiency.In conclusion,we found that the wild-type conformation,DNA binding activity and transcriptional activation of DNA-binding mutations p53^R273H and p53^R280K were restored by KBA01,and wtp53 possibly was stabilized and mutp53 was degraded by KBA01 through regulating the protein interaction between MDM2 and p53 and the level of Acetyl-p53（Lys382）.Also,the results of transcriptome sequencing confirmed that the p53 signaling pathway was indeed up-regulated in HT29(p53^R273H)by KBA01,and possibly through inducing apoptosis,cycle arrest and so on.This study has certain directive significance for developing the anti-tumor effect of KBA01 in mutant p53tumor.