A Chemo-photodynamic Cascade Strategy To Kill Cancer Cells Based On P53 Upregulation

Chemotherapy,a popular treatment,is facing great challenges.For example,multidrug resistance can directly lead to treatment failure.There are many reasons for multidrug resistance,including apoptosis signal pathway mediated resistance.At present,many chemotherapeutic drugs are cytotoxic drugs.When they act on cancer cells,they can cause DNA damage.At this time,some genes will respond in cancer cells,p53,As a transcription factor,it will regulate the expression of downstream pro apoptotic genes and promote cell apoptosis.However,at the same time,it is also possible to up regulate the expression of anti apoptotic genes,so as to produce drug resistance and enable cancer cells to survive.It can be seen that the complexity of cell signal pathway increases the difficulty of controlling the process and final result of chemotherapy.Therefore,if in the process of chemotherapy,avoiding the complex apoptosis signal pathway and killing cancer cells directly,it will help to improve this problem.To realize this process,the key lies in which node and how to avoid the signal pathway in the whole process of chemotherapeutic drugs.To solve the problems,we propose a cascade strategy,that is,based on the up regulation of p53 induced by chemotherapeutic drugs to activate photosensitizers,chemotherapy and photodynamic killing cancer cells.Two aspects of research work have been completed:1.5-fluorouracil(5-FU)up regulates p53 mRNA expression and triggers a chemo-photodynamic cascade strategy to kill cancer cells.In order to realize the cascade strategy,a functionalized DNA tetrahedron DT-ACe6 was constructed,that is,the aptamer AS 1411 was modified on one vertex of the DNA tetrahedron to specifically recognize the highly expressed nucleolin on the cancer cell membrane.The other three vertices were coupled with the photosensitizer Ce6,and quenched with the DNA strand labeled with quenching group as the response unit of p53 mRNA.The assembly process of the functionalized DNA tetrahedron,its response to p53 gene,it can specific target to cancer cells and p53 can release of photosensitizer were verified by electrophoresis,fluorescence spectroscopy,laser confocal imaging and flow cytometry.MTT assay showed that after A549,A549/DDP,HepG2 and HepG2/ADM cells were incubated with 0.08 mM 5-FU,the up-regulated p53 mRNA in cancer cells could activate DT-ACe6,release photosensitizer and realize chemo-photodynamic cascade killing.With the increase of DT-ACe6 concentration,the survival rate of cancer cells decreased significantly(<30%).Based on this strategy,we can avoid the influence of subsequent complex signal pathways in the interaction between chemotherapeutic drugs and cancer cells,and directly kill cancer cells through photodynamic effect.This method is universal and shows good killing effect on non drug resistant cells and drug resistant cells.2.Functionalized DNA tetrahedron loaded with doxorubicin and photosensitizer at the same time is used to study the killing of cancer cells by chemo-photodynamic cascade strategy.In the previous work,the up regulation of p53 mRNA in cells after the action of 5-FU was not particularly significant,and the dosage of photosensitizer was large.On the one hand,it may be that the concentration of free drugs in cells is low,resulting in the insufficient expression of p53 mRNA in cells;On the other hand,the process of 5-FU inducing cells to produce p53 mRNA and triggering photodynamic process is carried out in two steps,with a long interval,while the life of p53 mRNA is short,and the number of photosensitizers released by activating DT-ACe6 is less.Therefore,in order to improve the killing effect of chemo-photodynamic cascade strategy on cancer cells,we constructed a functionalized DNA tetrahedron DT(Dox)-ACe6 loaded with chemotherapeutic drugs and photosensitizers at the same time.After DT(Dox)-ACe6 enters cancer cells through endocytosis,it can release a large amount of Dox,so as to promote the expression of p53 mRNA in cells.Then,p53 mRNA immediately hybridizes with the DNA strand marked with quenching group on DT(Dox)-ACe6,and competes from DT(Dox)-ACe6 to make BHQ2 away from Ce6.Under 660 nm laser irradiation,Ce6 on DT(Dox)-ACe6 can produce cytotoxic reactive oxygen species,so as to exert the photodynamic Killing effect.The results of laser confocal imaging and real-time fluorescence quantitative PCR showed that Dox could increase the drug content in cells and induce the significant up regulation of p53 mRNA,which was higher than that stimulated by free DOX.At the same time,Dox and Ce6 were linked through p53 mRNA to construct a cascade killing strategy,which significantly reduced the survival rate of cancer cells,both less than 25%.This cascade killing strategy proposed by us can effectively avoid the complex signal pathways in chemotherapy,help to ensure drug safety,overcome the problem of tumor drug resistance,and provide a new idea for the combined application of cancer chemotherapy and other therapies.