The Mechanism Of Natural Buxus Alkaloids Downregulation Acetylation Of Mutant P53

TP53 is one of the most frequently mutated genes in human cancer(50%).In tumor cells,the posttranslational modifications(PTMs)and/or mofccular chaperones that participates in stabilizing mutant p53,so that these can inhibit ubiquitylation-proteasome and/or autophagy degradation,thus contribute to mutant p53 acquire oncogenic gain of functions(GoFs)leading to increased metastasis and tumor progression,transactivation of new target genes or inappropriate interactions with other cellular proteins,and drug resistance.Therefore,mutant p53 is an attractive druggable target for cancer therapy.Natural buxus alkaloids KBA01 is a kind of triterpenoid alkaloids,it also can be used as traditional Chinese medicine.In previous study,we found that KBA01 also has antitumor activity.And the KBA01 may degrade endogenous and exogenous mutations of p53R273H protein,and switch the conformation of mutant pS53R2731H to the conformation of wild-type p53 and activate the expression of the downstream target gene of wild-type p53.However,weather the alkaloid KBA01 only inhibit growth of tumor cells with p53R213H mutation remain to be further explored.Therefore,we selected tumor cell lines with different p53 backgrounds to explore the mechanism of the alkaloid KBA01 downragulation mutant p53.In this study,we first selected tumor cell lines with with different p53 backgrounds,and we detecte the expression of SIRT1,p-SIRT1,p53 and Acety1-p53 in mutant pS53R273H and P53R280K tumor cell lines after treated with KBA01(10?M)at 12h,24h and 48h.The results showed that the expression of SIRT1 and p-SIRT1 in HT29,MDA-MB-468,SW480 and MDA-MB-231 cells was significantly upregulated,and the expression of p53 and Acetyl-p53 downregulation after treated with KBA01 at 48h.We also selected mutant p53R175H and p53L194F tumor cell lines,such as breast cancer SK-BR-3 and T-47D cells,the expression of SIRT1,p-SIRT1,p53 and Acetyl-p53 were not affected by the KBA01.In addition,we used the exogenous introduced mutant p53R273H cells(H1299-R27310 to verify the previous experimental results.We acquire the same experimental results as the endogenous mutant P53R273H after treated the cells with KBA01.At the same time,we also selected the wild-type p53 and p53-deficient tumor cells,such as colon cancer HCT116(wt p53)cells and non-small cell lung cancer H1299 p53-/-(p53 null)cells,to verify whether KBA01 directly inhibit mutant p53 or through regulating the activity of wild-type p53.The results showed that the expression of SIRT1,p53 and the related proteins was not ehanged after the cells treated with KBA01 in HCT116 cells.However,the expression of SIRT1 and p-SIRT1 did not change after treated with KBA01,and no expression of p53 and Acetyl-p53 was detected in H1299 p53-/-cells.All together,these results indicated that the natural buxus alkaloids KBA01 could specifically upregulate the expression of SIRT1 and p-SIRT1 in mutant P53R273H and p53R280K cells,and decrease the expression of p53 and Acetyl-p53.Next,we validate the natural buxus alkaloids KBA01 wether leads to reduce the expression of acetylation of mutant p53 and mutant p53 by upregulating SIRT1.Thus,the expression of p53 and Acetyl-p53 was detected in HT29 cells treated with SIRT1 inhibitor Suramin(30?M)or combined with KBA01(10?M)at 24h,48h and 72h.The results showed that the expression of mutant p53 was upregulated and accompanied by the upregulation of acetylated mutant p53,and the downregulation of acetylation of mutant p53 and mutant p53 was resumed after the combination with KBA01.The results showed that KBA01 is elevated by SIRT1,and lead to reduce the expression of acetylation of mutant p53 and mutant p53.The expression ofacetyltransferase p300 was detected in colorectal cancer HT29 cells treated with KBA01(10?M)for 12h,24h and 48h.The results showed that the expression of p300 decreased at 48h.These results indicate that acetylation plays an important role in the stability of mutant p53,and that KBA01 may break the acetylation and deacetylation balance of mutant p53,inhibit the acetylation of mutated p53,and inhibit the tumor-promoting function of mutant p53.At the same time,we also explored whether the upstream factors AMPK,JNK and mTORC1 were involved in upregulation of SIRT1 expression The expression of AMPK,P-AMPK,JNK,p-JNK,mTORC1,mTOR,p-mTOR,p70S6K and p-p70S6K were detected in HT29 cells after treatment with KBA01(10?M)for 12h,24h and 48h.The results showed that the expression of these proteins decreased at 24h and 48h.However,the KBA01 did not upregulate AMPK and JNK expression.We hypothesized that the KBA01 inhibits tumor growth by reducing the expression of mTORC1,and upregulates SIRT1,inhibits phosphorylation of p70S6K,then inhibits the growth of tumors.At the same time,we used PAb240 which specifically recognizes mutant p53,and PAb1620 antibody that recognizes wild-type p53 to verify whether KBA01 transfer other mutations in tumor cells while recovering the function of DNA binding acticity and transcriptional activity of mutant p53.Thus,we treated mutant p53R273H and p53R280K tumor cells with KBA01(10?M)for 48h,and the expression of PAb240 and PAbl620 in these cells was detected.The results showed that KBA01 can reduce the expression of PAb240 and increase the expression of PAbl620 in these cells at 48h.We used KBA01(10?M)to treat breast cancer SK-BR-3 cells with mutant p53R175H for 48h.The results showed that the expression of PAb240 did not change in the cells treated with KBA01,and the expression of PAb1620 was not detected.In addition,we used KBA01(10?M)to treat H1299-R273H cells with exogenous mutant P53R273H to verify whether the same results were obtained as the endogenous mutant p53R273H cell line.The results showed that KBA01 treated cells for 48h could decrease the expression of PAb240 and increase the expression of PAbl620 in H1299-R273H cells.At the same time,we also selected wild-type p53 colon cancer HCT116 cells as the control group,the results showed that the use of KBA01 treatment of cells 48h,no detection of PAb240 expression,and PAbl620 expression did notehange.At the same time,we also treated HT29 cells with KBA01(10?M)for 12h,24h,48h,then extracted the nuclear protein,and used biotin labeled DN.sequences that could specifically bind to wild-type p53 to verify whether KBA01 could recover mutant p53R273H DNA binding activity.The results showed that with the increase of time gradient,the amount of DNA binding to p53 increased,which indicated that KBA01 could restore the DNA binding activity of mutant p53R273H.We also used KBA01(10?M)to treat different p53 mutant tumor cells for 12h,24h and 48h,the p53 target genes PUMA and p21 were detected in these tumor cells.The results showed that the expression of protein and mRNA of PUMA and p21 was upregulated in these cells with mutations of p53R273H and p53R280K.Whereas in cells with mutations of p53R175H and p53L194F,the expression of protein and nRNA of PUMA and p21 did not change treated by the KBA01.In tumor cells with wild-type p53 and p53 deletions,the expression of PUMA and p21 did not change treated by the KBA01.These results suggest that KBA01 can restore the conformation of mutant p53R273H and p53R280K and active its DNA binding and transcriptional activity.However,KBA01 has no effect on mutant p53R175H and wad-type p53.In conclusion,we investigated the anti-tumor mechanism of KBA01 in various mutant p53 tumor cells.The natural buxus alkaloids KBA01 specifically promotes the expression of SIRT1 and p-SIRT1 in mutant p53R2/3H and p53R280K tumor cells,and induce the downregulation of p53 and Acetyl-p53.It also inhibits the expression of mTORC1,then inhibit the phosphorylation of p70S6K by upregulating the eypression of SIRT1,induce tumor cell death.While restoring the conformation of mutant p53 in these cells,and recovering the DNA binding activity and transcriptional activity of mutant p53.