Enzyme-Instructed Self-Assembled Peptides For Enhancement Of Antitumor Activity By Histone Deacetylase Inhibitors

It has been a major challenge to increase the selectivity of antitumor drugs and reduce their toxic side effects in cancer research.Reducing the radiotherapy resistance of tumor tissues and improving the effect of radiotherapy are also major problems to be overcome in cancer treatment.Enzyme-instructed self-assembly(EISA)is an emerging cancer-selective strategy.During the process of EISA,precursor molecules are catalyzed at positions where the specific enzyme is highly expressed,and then form nanostructures through self-assembly,which in turn exerts the anticancer effect.However,high concentration of EISA moleculars is needed to kill tumors,which is difficult to realize in clinical application.Studies have shown that covalent attachment of small molecule radiotherapy sensitizers to self-assembled peptides can further enhance the radiosensitization effect.Tyroservaltide(YSV)has potential radiosensitizing effect as a novel histone deacetylase inhibitor(HDACi).However,YSV is a kind of short peptide drug and easily biodegradable in vivo.It also requires a high concentration to exert antitumor activity.Therefore,new strategies are needed to maximize the anticancer efficiency of EISA moleculars while maintaining their tumor selectivity.To this end,this paper designed and synthesized a novel peptide-based prodrug NapGDFDFpYSV(compound 1)by combining EISA peptide with anticancer short peptide YSV.In this study,peptide prodrug molecules were synthesized by standard solid phase synthesis.In vitro experiments showed that compound 1 could self-assemble into hydrogel after being catalyzed by alkaline phosphatase(ALP).The TEM results showed that the hydrogel was a dense network formed by nanofibers.The circular dichroism results indicated that the hydrogel had a P-folded secondary structure.The conversion efficiency of compound 1 by ALP(3 and 10 U/mL for 30 min at 4?)was approximately 100%,exhibiting its excellent EISA properties.MTT assays indicated that compound 1 had selective cytotoxicity.It showed up to 3-10 times cytotoxicity to ALP highexpressing cells than to ALP lowexpressing control tumor cells(including normal cells).In addition,it showed 8-fold cytotoxicity than control peptide bearing no YSV(compound 3).At the same time,the IC50 value of compound 1 to ALP highexpressing cells was far below its critical assembly concentration.In vitro cell uptake experiments showed that compound 1 had higher ALP catalytic conversion efficiency and cell uptake efficiency in ALP highexpressing HeLa cells.Western blot assays showed that the histone acetylation level of HeLa cells was significantly higher than that of A549 cells with low ALP expression,indicating that compound 1 induced cell apoptosis by inhibiting histone deacetylase(HDAC)and then showed the anticancer efficacy.In addition,the colony formation experiments showed that compound 1 had good radiosensitizing effect to HeLa cells,with the SER10 value of 1.433 and 1.660 at 10?M and 20 ?M,respectively.The sensitization effect of compound 1 by in situ catalysis was more effective than that of ALP catalysis in vitro.According to the results of comet assay,?-H2AX immunofluorescence analysis,cell cycle and apoptosis analysis,the good radiosensitizing effect of compound 1 maybe due to that compound 1 significantly enhanced the DNA breakage of HeLa cells by y-ray,blocked the irradiated cells in G1 phase and increased the proportion of apoptotic cells.Western blot assays indicated that compound 1 was able to inhibit HDAC activity and to increase the cleavaged PARP in-HeLa cells after irradiation.In summary,this study successfully constructed a novel prodrug small molecule based on peptide EISA.After phosphorylation,the drug activity was greatly reduced,which helped to reduce the side effects of drugs.On the surface of cancer cells that overexpress ALP,prodrug molecules were converted to monomeric molecules with parent drug activity by ALP in situ catalysis,further self-assembled into form nanofibers.The nanofibers were then uptaken by cells and showed anticancer activity by inhibiting HDAC activity.The prodrug exhibited enhanced tumor selectivity and antitumor activity.Further more,prodrug molecules can exert their HDACi activity at a lower concentration by EISA in HeLa cells,and thereby enhance the sensitivity of HeLa cells to ?-ray.This study provides a new strategy for improving the selectivity and biological activity of antitumor drugs,increasing the antitumor efficiency of EISA moleculars,and designing new radiosensitizers.