Modification And Reconstruction Studies Of Anticancer Peptide Lycosin-Ⅰ

Cancer is one of the major diseases that threaten human health.Despite substantial progress in the clinical treatment of cancer,the development of high-efficiency,specific and low-toxic anti-cancer drugs still have significant clinical needs.As an important part of anti-cancer drugs development,anticancer peptides（ACPs）have the potential to develop into high-quality anticancer drugs due to their rapid broad-spectrum anticancer activity,low tolerance and flexible design properties.Therefore,they have received widespread attention from the scientific and pharmaceutical communities.However,the clinical application of most ACPs is limited due to the narrow therapeutic index and low tumor targeting.The ACP Lycosin-I is an amphiphilicα-helix peptide which identified from the venom of Lycosa singoriensis.Our previous study indicated Lycosin-I have potential for the drug development.In this study,glycosylation and amino acid substitution strategies were performed to improve the anticancer activity,selectivity and tumor targeting of Lycosin-I,which lays the foundation for its development into a highly effective and low toxicity anticancer drug.Firstly,based on the high expression of glucose transporters in cancer cells,and using glycosylation modification strategies,a series of glycopeptides were designed.Previous studies have shown that R-lycosin-Ⅰ which was obtained by replacing the lysine in the sequence of Lycosin-I with arginine has higher anticancer performance.Therefore,we choose the R-lycosin-Ⅰ as a template in glycosylation modification study.Glucose,galactose,glucosamine,arabinose,and mannose derivatives were covalently coupled to the N-terminus of R-lycosin-Ⅰ through glutaric anhydride,which yield 5 high-purity glycopeptides.Only the glucose modified peptide（R-Glc）showed significant improvement in cytotoxicity and selectivity.R-Glc can be diffusely distributed in the A549 cytoplasm,presenting clusters in the nucleus and showing stronger cell uptake ability.According to design principle,the GLUT1 inhibitors and protein knockdown experiments were performed which showed that GLUT1 with high expression in A549 cells was approved to be involved in the cytotoxicity and selectivity enhancement of R-Glc.In the mechanism,R-Glc can disrupt the morphology of cells and promote the release of LDH,indicating that it has membrane lytic activity;R-Glc also can co-localization with mitochondria,decrease mitochondrial membrane potential,increase mitochondrial membrane permeability and release cytochrome C,which caused caspase-3 activation and then promoted cell apoptosis.In the A549 tumor spheroids,R-Glc demonstrated stronger tumor spheroid penetrability and cytotoxicity than that of R-lycosin-Ⅰ.Compared to R-lycosin-Ⅰ,the lung tumor xenograft model of nude mice injected with FITC-R-Glc showed strong fluorescent signals around tumor.By the way of intratumoral injection,3 mg/kg of R-Glc could significantly inhibit nude mice tumor growth.Unlike R-lycosin-Ⅰ,intraperitoneal injection of 9 mg/kg of R-Glc can also significantly reduce the tumor volume and induce numerous tumor cell apoptosis.The administration process had no significant effect on body weight and had no obvious toxicity to the main organs of nude mice.Secondly,according to the p H characteristics of the tumor microenvironment（p H=6.5-6.8）,amino acid substitution strategy was performed to reconstructe the Lycosin-I,which yeild a p H-triggered peptide p HTP-1.Under the culture conditions of p H=6.0,6.6 and 7.4,the IC₅₀values of p HTP-1 against A549 cells were 9,37 and>200μM,respectively.Alanine scanning analysis of p HTP-1 demonstrated that the I10A mutant had the highest cytotoxicity at p H=6.6 and named it p HTP-2.At p H=6.0 and 7.4,the IC₅₀values of p HTP-2 against A549 cells were 15and>200μM,respectively.Due to the best performance,p HTP-2 was choose as a research object in the following experiment.Systematic biological activity tests indicated that p HTP-2 had broad-spectrum,time-concentration dependent and p H conversion in treating cancer cells.In addition,p HTP-2 induced cell death by direct membrane lysis.Due to its weak cytotoxicity at 7.4,the HC₅₀value against erythrocyte and IC₅₀value against cardiomyocytes of p HTP-2 were all above 500μM.In order to expound the highly selective mechanism of p HTP-2,we performed a series of physicochemical parameters tests including zeta potential,particle size and secondary structure and parsed the mechanism as follows:At 6.6,histidines in the p HTP-2 sequence were protonated,which increased the positive charge of the peptide,strengthened the peptides repulsion and decreased the peptide particle size.In PBS and SDS,p HTP-2 presented random coil andα-helix conformations,respectively.Thus,the increased positive charge andα-helix conformation of peptide enhanced its membrane binding and insertion ability,which exerted membrane lysis.At 7.4,p HTP-2 demonstrated weakly protonation andβ-sheet conformation in PBS,which caused peptides to aggregate though intermolecular hydrogen bonding and hydrophobic interaction.The state of aggregation decreased the binding and interaction ability between peptide and cell membrane,showing low cytotoxicity.In the in vivo anticancer study,tail vein injection of 20mg/kg of p HTP-2 showed a significant inhibitory effect on melanoma,and the administration process had no significant effect on the body weight of mice,indicating that p HTP-2 had low side effects.Finally,considering that the preclinical cancer model is an important bridge connecting prospective drug development and clinical treatment,and has important effects on drug discovery,pharmacodynamic evaluation and pharmacological research,we try to establish a tumor organoid model.In recent years,compared to cell lines,more and more studies have shown that the authenticity between organoid models and tumor tissues.Current cancer research models develop in the order of cell lines-3D cell line spheroids-organoids.Based on the successful construction of mouse small intestine and colon organoids,we successfully constructed human colon and colon cancer organoids through tissue separation,selective addition of factors and three-dimensional culture.Human colonic organoids demonstrated a cystic structure that can proliferate and differentiate into crypt and villous domains.Human colon cancer organoids showed dense and irregular spheroids and the histological morphology was similar to that of tumor tissues.After cryopreservation and then resuscitation,colon cancer organoids still grow well.Using colon cancer organoids,we performed a preliminary screening and exploration of ACPs in animal venom.The successful construction of a colon cancer organoid model provides a new method for our laboratory,which will greatly improve the efficiency and level of ACPs research.In summary,the glycosylation modification enhanced the selectivity,cytotoxicity and tumor targeting of R-lycosin-Ⅰ and this improvement was mediated by glucose transporter;the amino acid substitution strategy endowed Lycosin-I with p H triggered characteristic,which greatly enhanced the peptide selectivity and also had significant anticancer activity in vivo.The systemic estabalishment of organoid model has enriched our understanding of preclinical cancer models and promoted the precise development of anticancer drugs.Those peptide design strategies promote the druggability of Lycosin-I and also provide support for the ACPs optimization.