A Study On Zinc-iron Nanomaterials Promoting The Degradation Of Mutant P53 Protein In Tumor Cells

p53,a protein encoded by TP53 gene,is a critical tumor suppressor that plays an important role in inhibiting tumorigenesis.However,TP53 mutations occur in more than 50% human cancers in which they steadily accumulate in cancer cells.Not only dose mutated p53 lose tumor suppressive activities possessed by wild-type p53,but gains new functions.Such gain-of-functions have a vital impact on tumorigenesis,such as promoting growth and translocation of cancer cells and their resistance to drugs.There have been some small molecular compounds applied for the treatment of cancers expressing mutated p53,mainly through restoration of wild-type p53 activities and degradation of mutated p53 protein.Unfortunately,these compounds have limited effects and lack certain specificity.Nanomaterials have been widely utilized in life sciences due to their unique physical and chemical properties.Given the facts above,we wonder whether engineered nanomaterials could regulate the stability of mutated p53.Up to now,there has no relevant report on nanomaterials that can achieve it.Based on the fact that zinc ions can regulate the stability of mutant p53 protein,we hypothesized that synthesis of zinc-containing nanomaterials could regulate the homeostasis of mutant p53 protein in tumor cells.In this work,zinc-iron nanomaterials were synthesized through oil-phase one-pot method by adopting metal doping strategy.First,zinc-iron nanomaterials were characterized with TEM,DLS and surface charge,confirming that the synthesized nanomaterials were uniformly spherical,with a size of about 10 nm,and their surfaces were with negatively charge.At the same time,MRI trials in vitro showed that the zinc-iron nanomaterials still retained a good MRI imaging function,which provides a good reference for precise treatment and surgical navigation in the future.Second,the effects of zinc-iron nanomaterials were evaluated in mutant p53 cell lines containing different mutation sites and wild-type p53 cell lines,and it was found that the nanomaterials had good clear effect on both conformational and DNA-contact mutant p53 proteins,as well as exogenously expressed mutated p53,but not its wild-type counterpart.Meanwhile,it was proven that the scavenging effect of zinc-iron nanomaterials on mutant p53 was mainly achieved through K48 ubiquitin-proteasome pathway without affecting the transcription level of p53 gene.In addition,we discovered that the nanoparticles entered tumor cells mainly through endocytosis before releasing zinc ions in the lysosomal acidic environment.What's more,it was revealed that zinc-iron nanomaterials were competent to cause the rise of intracellular reactive oxygen species after their treatment on tumor cells,while the degradation of mutant p53 caused by the nanomaterials could be relieved by zinc ion chelators and reactive oxygen inhibitors.Therefore,the elevation of intracellular zinc ions and the rise of reactive oxygen species are the main factors for zinc-iron nanomaterials to induce mutant p53 degradation.Next,the influence induced by zinc-iron nanomaterials on cell fate of the cells expressing mutant p53 was assessed.The results showed that zinc-iron nanomaterials could specifically kill mutant p53 tumor cells,but had less toxicity to wild-type p53 cells and normal cells at the same treatment concentration.Therefore,the nanomaterials had good safety for non-mutant p53 cells.Besides,cell cycle arrest,as well as inhibition of the proliferation and migration of tumor cells,occurred in cancer cells with mutated p53 after the cells were treated with the nanoparticles,with the gain-of-functions of mutant p53 tumor cells relieved.Finally,the antitumor activity of zinc-iron nanomaterials and the effect of their combination with chemotherapeutic drugs were evaluated in vitro and in vivo.Cisplatin is an efficient antitumor drug used in the treatment of a wide variety of tumors,such as ovarian cancer,prostate cancer,lung cancer and other solid tumors,but drug resistance often occurs during the treatment.The nanoparticles were capable of significantly increasing the killing effect of chemotherapeutic agent cisplatin on tumor cells with p53 mutations,which showed that zinc-iron nanomaterials increased the sensitivity of tumor cells to chemotherapeutic drugs after inducing the degradation of mutant p53 protein in them.It was also discovered in vivo that,at the safe dose where zinc-iron nanomaterials had no significant effect on mouse body weight,the nanoparticles exhibited a certain ability to inhibit the growth of ES-2(containing S241 F p53 mutation)subcutaneous tumors and PDX in situ breast cancer(containing Y220 C p53 mutation),and could significantly inhibit tumor growth in mice after combined treatment with cisplatin,consistent with the results of chemosensitization in vitro.At last,the tumor tissue sections after treatment were analyzed,and the results illustrated that the levels of p53 protein were significantly decreased and apoptotic tumor cells were significantly increased in the zinc-iron nanomaterial-treated group and the combined treatment group with cisplatin.Zinc-iron nanomaterials were firstly used in this study to degrade mutant p53 and effectively improve the sensitivity of tumors with p53 mutations to chemotherapeutics,providing important reference values for clinical treatment and application in the future.