Functional Properties Of The Novel Antitumor Agents:NS,TAT-camptothecin Conjugates TAT-CPT/2CPT And[Cys-CPT^2,9]penetratin In Antitumor Applications

In cancer treatment,the constant use of traditional chemotherapeutical agents causes a dramatic increase in the emergence of multidrug resistant tumors and many chemotherapeutical agents are becoming increasingly ineffective,which seriously threaten public health and life safety.Therefore,development of alternative agents becomes more and more critical to fight against resistant tumors.The antimicrobial peptides(AMPs)play an essential part in the innate immune response.Nowadays,they are considered as a novel class of antitumor candidate with characteristics including potent activity against tumor cells and low susceptibility to conventional mechanisms.In addition,cell-penetrating peptides(CPPs)share the common structural features with AMPs.They are widely used to deliver various molecular into cells because of their membrane translocating ability.In cancer therapy,they also could play a promising role in improving therapeutic efficiency of chemotherapeutical agents.Therefore,AMPs and CPPs can be regarded as ideal templates for the development of better antitumor drugs with potent activity.In this paper,we synthesized a series of new antitumor agents,including novel antimicrobial peptide NS,TAT-CPT/2CPT conjugates constructed by combining camptothecin with cell penetrating peptides TAT,and[Cys-CPT2,9]penetratin.In order to evaluate their potential in antitumor application,we systematically studied their antitumor activity and functional properties.1.Multiple functional properties of novel antimicrobial peptide NS in antitumor application.In this study,we evaluated its cytotoxicity on tumor cells and studied the possible mechanism of action.The results showed that NS could efficiently kill tumor cells by disrupting membrane and inhibiting DNA synthesis.In addition,it was found that NS could efficiently deliver plasmids into cell after N-terminal stearyl modification,like many reported cell-penetrating peptides.Stearyl-NS at 3:1 and 4:1 N/P ratios exhibited several orders of magnitude higher transfection efficiency than plasmid only,suggesting that NS could work as a potential vector for tumor gene therapy.Taken together,the ability of NS to kill tumor cells and deliver plasmid indicates that it has the potential to develop into a new antitumor agent to defend against multidrug-resistant tumor.2.Antitumor activity of conjugates TAT-CPT/2CPT.In this part,we introduced CPT group to the N-terminal of TAT via a releasable disulfide carbonate linker and synthesized TAT-CPT and TAT-2CPT conjugates.Additionally,their antitumor activity and detailed action mode were further determined and analysized.It has been shown that the linked TAT could enhance antitumor activity of CPT;the conjugate TAT-2CPT dispiayed more effective killing activity against tumor cells.Interestingly,conjugates could effectively kill tumor cells via rapid membrane disruption,similar to antimicrobial peptides.In addition,they could also exhibit antitumor activity via higher level of activation of caspase3/7.Furthermore,the results of molecular dynamics(MD)simulations showed that conjugates could lead to obvious orientation changes of saturated SN1 chain(+0.0043,+0.0143),further clarifying the perturbation of conjugates and membrane bilayers.The results reveal that membrane targeting and intracellular interaction make conjugates highly promising for future antitumor application.3.Design of[Cys-CPT2,9]penetratin with high membrane penetrating ability and antitumor activity.Based on the previous research,a analogue of penetratin,[Cys-CPT2,9]penetratin,was designed and synthesized by substitution of Gln2 and Asn9 residue with Cys-CPT.We hoped to develop a more intriguing cell penetrating peptide with potent membrane penetrating ability and effective antitumor activity by enlargiing hydrophobic patch.Cell uptake results indicated that the internalization of[Cys-CPT2,9]penetratin was mainly mediated by clathrin and macropinocytosis-mediated endocytosis.Its translocating efficiency was 5 times higher in comparison with the level of penetratin.In addition,[Cys-CPT2,9]penetratin could effectively inhibit and kill tumor cells,exhibiting remarkable antitumor activity.Thus,[Cys-CPT2,9]penetratin has more appealing potential in future antitumor therapy because of its efficient penetrating ability and higher antitumor activity.In the present study,we systematically studied the functional properties of NS,TAT-CPT/2CPT conjugates and[Cys-CPT2,9]penetratin,and evaluated their potential to be used as promising antitumor candidates.The findings obtained from our study would contribute to the establishment of novel strategy for tumor treatment.