Study On The Synergistic Mechanism Of Anticancer Peptide TAT-KLA And Paclitaxel In Inducing Apoptosis Of HepG2 Cells

Over the past fifty years,humans have achieved unprecedented success in medicine,but cancer remains one of the most serious threats to human survival.Current cancer treatments include traditional surgery,chemotherapy,radiation,biological and hormonal treatments,of which chemotherapy plays an important role.However,the strong toxicity of chemotherapy drugs to normal tissues has also become a major obstacle to the implementation of chemotherapy.In addition,if simply using one treatment method or drug will make patients resistant to specific drugs based on the complexity of cancer.The combination of multiple anti-tumor drugs could reduce the dose of drugs while also can reduce the toxicity to normal cells and delay drug resistance.Therefore,the clinical treatment of combined drugs has gradually received attention.Anticancer peptide?ACP?has a broad-spectrum of anticancer activity,low immunogenicity,low-cost manufacturability,excellent tissue permeability,and it is easily modified to enhance the stability and biological activity in vivo.These advantages make ACP have the potential to be developed into a new generation of antitumor drugs.Some peptides could increase the susceptibility of cancer cells to anticancer drugs by altering the permeability of cell membranes to increase the uptake of conventional anticancer drugs.The peptide _D?KLAKLAK?₂ with all D-amino acids,hereinafter referred to as KLA.KLA is an anti-cancer peptide which can induce cancer cell apoptosis.But its ability to enter cells is weak,so chemical modification is essential for improving the uptake rate.In the previous work,we have designed and synthesized a hybrid peptide KLA-TAT by attaching cell penetrating peptide TAT to the C-terminal of KLA.The results showed that the coupling of two peptides converted into a membrane-active peptide which imparted a new property.The hybrid peptide could pass through the cell membrane through two mechanisms:membrane breaking and clathrin-dependent endocytosis.The anticancer activity in vitro increased aroun 24 times compared with KLA.In this topic,we compared the anticancer activity of KLA-TAT and TAT-KLA which were modified at different positions,and then studied the anticancer activity and mechanism of the combination of peptides and commonly used clinical chemotherapy drugs with liver cancer cells as models.Firstly,the peptide KLA-TAT and TAT-KLA were synthesized by Fmoc solid-phase synthesis method,and purified by HPLC.The purity of the peptide was above95%and it was confirmed by the mass spectrum.Secondly,the anti-proliferation effects of KLA-TAT and TAT-KLA were detected by MTT screening and flow cytometry.After comparison we chose TAT-KLA combined with paclitaxel to detect the combined anticancer activity on HepG2 cells.The results showed that the co-administration group greatly improved the effect of each drug alone,and the two drugs showed significant synergistic effect.Further studies such as flow cytometry,fluorescence microscopy and Western Blotting have also confirmed that these two drugs enter cells through membrane disruption and induce apoptosis through the mitochondrial pathway to achieve anticancer efficacy.The above results showed that the combination of TAT-KLA and paclitaxel could not only effectively overcome the resistance of tumor cells to paclitaxel,but also reduced the toxicity and side effects.This strategy of covalently coupling modification and co-administration with paclitaxel provides valuable theoretical support for clinical medication.