Effect And Mechanism Of Antimicrobial Peptide PFR Combined With Chemotherapy Drugs On Proliferation Of Acute Myeloid Leukemia HL60 Cells

Acute myeloid leukemia is a malignant blood cancer with limited treatment.Both anthracyclines and pyrimidines are commonly used chemotherapy drugs for leukemia.The high-dose treatment is effective,but there are some shortcomings such as toxic side effects.Therefore,reducing the side effects of these chemotherapy drugs and improving their efficacy are scientific problems that need to be solved urgently.The antimicrobial peptide PFR has the advantages of small molecular,low synthesis cost,no obvious side effects,which is the first choice for the combined action of drugs.This thesis intends to explore the effect of PFR peptide combined with anthracycline or pyrimidine chemotherapy drugs on the proliferation of acute myeloid leukemia HL60 cells and its potential molecular mechanism.The results of cell proliferation assay and MTT assay showed that PFR peptide combined with anthracycline drug adriamycin could not significantly inhibit the proliferation of HL60 cells,but PFR peptide combined with pyrimidine drug cytarabine significantly inhibited the proliferation of HL60 cells.The synergistic inhibition was found to occur through the necrotic pathway by flow cytometry.The proportion of necrosis in the 120 ?M PFR peptide group was 26.82±0.47% and the 1.8 ?M cytarabine group was 16.2±1.44%.The combined drug group was 51.93±0.40%,and the proportion of HL60 cell necrosis was significantly increased in the combined drug group compared with the single drug group.Further,we also confirmed that the PFR peptide combined with cytarabine synergistically inhibited HL60 proliferation which was mediated through the necrotic pathway detected by PI absorption assay and LDH release assay.The necrosis inhibitor Nec-1 can significantly alleviate the inhibition of HL60 cell proliferation induced by PFR peptide combined with cytarabine.PFR peptide can increase cytosolic calcium flux.Although cytarabine alone can not increase cytosolic calcium flux,cytoplasmic calcium in the combined drug group is significantly higher than that in the PFR peptide alone.Both cytosolic calcium chelating agent BAPTA-AM and calcium-free medium treatment combined with medicated cells significantly attenuated HL60 cell necrosis,suggesting that HL60 necrosis caused by PFR peptide combined with cytarabine is elevated by cytoplasmic calcium flux.The flow cytometry assay showed that PFR peptide combined with cytarabine also caused a significant increase in cytoplasmic reactive oxygen species(ROS).The release of ROS from the control group was 2.20±0.11% at the concentration of 120 ?M PFR peptide,1.75±0.22% at the concentration of 1.2 ?M cytarabine,and 3.18±0.32% in the combined drug group.To further validate the in vivo anti-tumor effect of PFR peptide combined with cytarabine,we conducted animal-level experimental studies in the xenograft nude mouse model.The results showed that the tumor weight was 1.35 ± 0.13 g in the control group,0.97 ± 0.06 g in the PFR peptide group,0.92 ± 0.13 g in the cytarabine group,and 0.61 ± 0.10 g in the PFR peptide and cytarabine group.The tumor weight of the combined drug group was significantly lower than that of the control group,and there was no obvious toxic side effect.In conclusion,this study found that PFR peptide combined with anthracycline adriamycin have no obvious synergistic effect,while PFR peptide combined with pyrimidine cytarabine can significantly inhibit the proliferation of acute myeloid leukemia HL60 cells.The mechanism involved in this synergistic effect is mediated by elevated cytoplasmic calcium flux to induce necroptosis.The conclusion of this study provides a solid theoretical basis and new ideas for the treatment of acute myeloid leukemia.