The Co-administration/Targeted Modification To Anticancer Peptide And The Mechanism Study

During the past two decades,the development of cancer treatment has evolvedfrom nonspecifc cytotoxic agents to selective,mechanism-based therapeutics,such as chemotherapeutics,targeting agents,monoclonal antibodies and other targeted therapeutics.However,the effecy of most anticancer drugs is limited due to the narrow therapeutic index,signifcant toxicity and frequently acquired resistance.Thus,the development of strategies to improve targeting ability of anticancer drugs is greatly needed.Cation anticancer peptides(ACPs)have been considered as novel therapeutic candidates due to their unique mechanism,broad-spectrum anticancer activity,low immunogenicity,and low tolerance.However,the relatively low selectivity between cancer cells and normal cells was the primary obstacle for these peptides to be developed as anticancer drugs.So,in this study,the cation membrane active peptide HPRP-A1 and pro-apoptotic pepide(PAP)kla were used to be the model peptide,the tumor homing/penetrating peptide(THP)iRGD as well as cell penetrating peptide(CPP)TAT were selected to chemically conjugate and/or co-administration with the two anticancer peptides,to improve the anticancer activity,peamiability and tumor targeting ability.1.Co-administration of iRGD with peptide HPRP-A1 to improve anticancer activity and membrane penetrabilityiRGD peptide is widely recognized as an efficient cell membrane penetration peptide targeting to?_v?₃ integrins and neuropilin-1(NRP-1)receptors,which show high expression in many tumor cells.The anticancer activity,cancer specificity and penetration activity in vitro and in vivo of the co-administered peptides were examined on 2D monolayer cells,3D multi-cellular spheroids(MCS)and xenograft nude mice.Co-administration of iRGD and HPRP-A1 exhibited stronger anticancer activity and tumor specificity against A549 Non-Small Cell Lung Cancer(NSCLC)cells with NRP-1 receptor overexpression compared with HPRP-A1 alone.A549 cells showed uptake of the peptides combination and destruction of the integrity of the cell membrane,as well as adherence to the mitochondrial net,resulting in induction of apoptosis by a caspase-dependent pathway.The i RGD peptide dramatically increased the penetration depth of HPRP-A1 on A549 MCS and anticancer efficacy in an A549xenograft mouse model.Our results suggest that the co-administration strategy of anticancer and penetrating peptides could be a potential therapeutic approach for cancer treatment in clinical practice.2.The targeted modification to cation anticacer peptide HPRP-A1 and the mechanism studyIn this study,a novel chimeric peptide HPRP-A1-iRGD,composed of a tumor homing/penetration domain iRGD and a cationic anticancer peptide domain HPRP-A1 by chemically conjugation,was utilized to study the targeted modification to enhance peptide specificity,penetration and tumor accumulation abilities.iRGD exhibit tumor-targeting and tumor-penetrating activities by specifically binding to the targeted NRP-1 receptor,as the homing/penetration domain,it attributes to enhance the tumor selectivity,permeability and targeting of HPRP-A1 by targeted receptor dependence.Meanwhile,as an anticancer active domain,HPRP-A1 kills cancer cells by disrupting the cell membrane and inducing apoptosis.The in vitro membrane selectivity against cancer cells such as A549 and MDA-MB-23 and HUVEC normal cells,the penetrability assessment on A549 3D MCS model and the in vivo tumor tissue accumulation test on A549 xenograft model indicated that HPRP-A1-iRGD exhibited significant increases on the selectivity to the high NRP-1 expression membrane,penetration distance to 3D MCS and accumulation in tumor tissues by intravenous injection,compared with HPRP-A1 alone.The mechanism of the targeting ability enhancement of HPRP-A1-iRGD was demonstrated by the pull-down assay and biolayer interferometry(BLI)test,that is,the chimeric peptide could specifically bind to the NRP-1 protein with high affinity.We believe that the chemical conjugation with iRGD to increase the specificity,penetration and tumor tissue accumulation of HPRP-A1 would be an effective and promising approach for the targeted modification of peptides as anticancer therapeutics.3.kla-TAT co-administration with HPRP-A1 and iRGD separately to improve the anticancer activity/targeting ability and the mechanism studyThe peptide _D(KLAKLAK)₂ with all D-amino acids,also referred to as _D(KLA)₂ or kla,is a well-known PAP that is positively charged and that locates to mitochondria,where it induces cancer cell apoptosis through mitochondrial swelling.However,the apoptosis induction was dependent on the internalization of cancer cells,so,it is the core issue to modify the kla peptide to increase the cancer cell uptake rate.In this study,we designed and synthesized a hybrid peptide,kla-TAT,by attaching the TAT peptide to the C-terminus of kla which is different from the reported TAT-kla.Interestingly,the hybrid peptide internalized into A549 cells through two transmembrane mechanisms:endocytosis mediated by the clathrin-mediated endocytosis route and the rapid membrane disruption mechanism.The anticancer activity of the kla peptide increased around 24 times after conjugation with TAT peptide.Aothough the TAT modification to kla increased the anticancer activity significantly;the lower dosage used the lower toxicity to normal cells.HPRP-A1,as an ACPs with high anticancer activity and well synergistic effect with other agents was selected to co-administration with kla-TAT then.To enhance more kla peptides pass through cell membrane,a double improvement strategies were designed by chemically conjugation with cell penetration peptide TAT as well as co-administration with cationic membrane active peptide HPRP-A1,and the double anticancer mechanism of the kla-TAT peptide and HPRP-A1 including membrane disruption and apoptosis induction was verified through in vitro experiments.Not only conjugation kla with TAT but also co-administration with HPRP-A1 could not improve the cancer cell selectivity.Previous study has demonstrated that iRGD could increase the anticancer activity and targeting ability of HPRP-A1 by co-administration.So,in this study,the iRGD was selected to co-administate with kla-TAT peptide.The penetration of the combination peptides to A549 monolayer cell and 3D MCS were detected,at the same time,the peptides collection in tumor tissue was tested on xenograft mice model.The result demonstrated the co-administration of iRGD strengthened the permeability of kla-TAT peptide against A549 2D and 3D sphere and dramatically enhanced the accumulation of kla-TAT peptide in tumor tissue on the xenograft mice model.We believe the chemical conjugation plus co-administration approach may provide a promising way for cancer treatment in clinical practices.In a conclusion,in this study,the cationic membrane active peptide HPRP-A1 and tumor pro-apoptotic peptide kla were selected as the model peptides,both chemical modification and co-administration with tumor targeting peptide and cell penetrating pepitde could significantly increase the anticancer activity in vitro/in vivo as well as the targeting ability.This study would provide theoretical foundation for the targeted modification to ACPs in clinical use.