| Background:Cervical cancer is one of the few malignancies for which the etiology is well established.Persistent infection with high-risk human papilloma virus(HPV)is a crucial influence in the development and progression of cervical cancer.Although the introduction of cervical cancer vaccine has changed the global pattern of cervical cancer prevention and control,differences in customs,geography,economy,and national conditions have led to uneven access to cervical cancer vaccine.Currently,there is still no clinically specific treatment for HPV infection,therefore,the development of a drug that can effectively clear the HPV virus has become an important breakthrough point for early prevention and treatment of cervical cancer.Melittin is the main component of bee venom.Studies have shown that melittin can exert anti-tumor effects by inhibiting proliferation,inducing apoptosis,inhibiting metastasis and angiogenesis,and blocking cell cycle,and it also have antiviral and anti-inflammatory effects.However,the hemolytic property of melittin is the biggest obstacle to its clinical application,therefore,the modification and improvement of melittin has become the focus of research.The project team successfully constructed a new patented fusion peptide UM-6(CN201810575627.8)with 34 amino acids and melittin as the main body,in order to inject new opportunities for anti-HPV and cervical cancer treatment.Metabolic reprogramming and elevated ROS levels are the two main features of cancer.The "Warburg Effect":malignant tumor cells preferentially use glycolysis to supply themselves with energy even in an oxygen-rich environment.Studies have shown that many metabolic intermediates of the glycolytic pathway increase significantly after viral infection,even depending on the expression of oncoproteins encoded by viral genes,suggesting that virus-encoded oncoproteins may be important factors in promoting aerobic glycolysis and regulating related molecules,and targeting HPV may become an effective measure to inhibit glycolysis in cervical cancer,but there are currently no clinical drugs that can inhibit this signature of tumor cells.Reactive oxygen species(ROS)are single electron reduction products produced by the combination of oxygen and single electron released from the respiratory chain of cells.Physiological levels of ROS are essential for cell proliferation,migration and angiogenesis,but higher than physiological levels of ROS,or disruption of the balance between ROS and antioxidants,will inevitably lead to massive non-specific oxidation of proteins and subsequent cellular cascade signaling effects,leading to cell cycle arrest,genetic mutations and even death,called"oxidative stress".It is not clear how HPV infection is related to ROS and glycolysis,and what role ROS and glycolysis play in the anti-HPV and proliferation inhibition of cervical cancer by UM-6.Therefore,the purpose of this study is to investigate the effect of UM-6 on the malignant biological behavior of cervical cancer cells,to investigate the mechanism of UM-6 anti-HPV and cervical cancer,to elucidate the important role of oxidative stress and glycolysis in the anti-HPV and inhibition of cervical cancer cell proliferation of UM-6,and to provide a theoretical basis for the clinical translation of the novel fusion peptide UM-6 against cervical cancer.Methods:1.Effect of UM-6 on malignant phenotype and HPVE7 expression in cervical cancer cells①.The effects of UM-6 on the proliferation and apoptosis of cervical cancer cells were investigated by CCK8,clone formation assay,flow cytometry,ROS detection,transmission electron microscopy and other experiments.②.To detect the changes of apoptosis-related proteins Bcl-2 and Bax as well as HPVE7,PI3K,AKT,p-AKT and p-PI3K protein expression after the effect of UM-6 by Western Blot and real-time fluorescence quantitative PCR.③.Application of the pan-casepase inhibitor z-VAD-FMK and the antioxidant NAC(N-acetyl-L cysteine)to explore the mechanism by which UM-6 affects the malignant phenotype of cells.2.Screening for differentially expressed pathways and proteins after UM-6 action and conducting mechanistic studies①.High-throughput proteomics technology was applied to UM-6-treated cervical cancer cell lines and their controls to screen out differentially expressed proteins,and KEGG analysis and clustering analysis of differentially expressed proteins were performed to identify significantly down-regulated proteins and related pathways.Bioinformatics analysis was performed to identify the differentially expressed proteins that were associated with poor prognosis of cervical cancer.②.In vitro experiments were performed to verify the effect of UM-6 on the expression of target proteins.③.Interfere with HPVE7 expression to explore its effect on differentially expressed proteins and PI3K/AKT pathway protein expression.④.Apply PI3K inhibitor LY294002 and agonist 740Y-P to explore their effects on UM-6 and its interrelationship.3.Effect of UM-6 on the radiosensitivity of cervical cancer cells①.The effect of UM-6 on the radiosensitivity of cervical cancer cells was analyzed by giving different doses of radiation treatment to cervical cancer cells after UM-6 treatment.4.In vivo experiments①.To construct a nude mouse transplantation tumor model of human cervical cancer Hela cells,observe the general condition of nude mice,compare the size and weight of transplantation tumors in UM-6 treated and control groups,calculate the tumor inhibition rate,and investigate the safety and tumor inhibition effect of UM-6 in vivo.②.The expression of Ki-67,P16,HPVE7,Bcl2,Bax,AKT,p-AKT,PI3K,p-PI3K,PKM2 and LDHA proteins were detected by immunohistochemistry and Western Blot to further investigate the mechanism of the anti-cervical cancer action of UM-6.Results:1.①The inhibitory effect of UM-6 on the proliferation of cervical cancer cells showed concentration dependence,the IC50 concentration of UM-6 on Hela cells for 12 hours was 30.91 μg/ml,SiHa cells IC50 concentration was 55.74μg/ml,CaSki cells IC50 concentration was 35.69 μg/ml,C33A cells IC50 concentration was 44.03μg/ml.②The number of clone formation in UM-6-treated cervical cancer cells was significantly lower than that in the control group(p<0.05).③After 12 hours of treatment with UM-6,Western Blot showed that the expression level of Bcl-2 protein decreased and Bax increased.④The ROS production of cervical cancer cells increased after UM-6 treatment,and cells showed different degrees of death,mainly early and late apoptosis.⑤ Transmission electron microscopy showed that the mitochondrial cristae of the cells disappeared and the structure was disordered after the effect of UM-6.⑥The antioxidant NAC and the pan-casepase inhibitor z-VAD-FMK were able to partially reverse the effects of UM-6 on proliferation and apoptosis of cervical cancer cells.⑦UM-6 down-regulated HPVE7 expression in cervical cancer cells(p<0.05).NAC was able to partially reverse the down-regulation of UM-6 on HPVE7 expression in cervical cancer.2.①High-throughput proteomics techniques revealed that UM-6 significantly down-regulated cellular glycolytic pathway proteins in cervical cancer cells.Bioinformatics analysis revealed that the expression of lactate dehydrogenase(LDHA)and pyruvate kinase isoenzyme(PKM2)was increased in cervical cancer tissues and was closely associated with poor prognosis of cervical cancer.②UM-6 reduced lactate secretion,decreased glucose uptake,and inhibited PKM2,LDHA,p-AKT,and p-PI3K protein expression in cervical cancer cells.③The expression of p-AKT,p-PI3K,LDHA,and PKM2 proteins were down-regulated after interfering with HPVE7 expression(p<0.05).④The cell proliferation ability decreased after the application of PI3K inhibitor LY294002,and the combined inhibition rate reached 44%with CI=0.699 after the combined effect of 10μg/ml of UM-6 and 10μM of LY294002 on cervical cancer Hela cells,which were synergistic with each other.The combined inhibition rate of 20 μg/ml of UM-6 and 10 μM of LY294002 on cervical cancer SiHa cells reached 35%with CI=0.78,which was synergistic.⑤LY294002 down-regulated LDHA and PKM2 protein expression and decreased lactate production and glucose uptake(p<0.05).⑥PI3K agonist 740Y-P partially reversed the UM-6-induced decrease in glucose uptake and lactate production in cervical cancer cells(p<0.05).3.Graphpad 8.0 single hit multi-target effect model to plot the survival curves and calculated the radiosensitization ratio(SER),SER=1.34,indicating that UM-6 treatment could enhance the sensitivity of cervical cancer cells to radiotherapy.4.In vivo experiments:① The general condition of the nude mice in the experimental and control groups was good.The volume of transplanted tumors in the experimental group of nude mice was smaller than that in the control group(p=0.0257).②The body weight of nude mice in the experimental group before the beginning and after the end of the experiment was not significantly different from that in the control group(p=0.7532),(p=0.4890).③Compared with the control group,the expression of p-PI3K,p-AKT,HPVE7,p16,LDHA and PKM2 protein was decreased in the transplanted tumors of the experimental nude mice,while the expression of AKT and PI3K protein was unchanged.The expression of Ki67 in transplanted tumors of nude mice was detected by immunohistochemistry,and Ki67 expression was down-regulated in transplanted tumors of nude mice in the experimental group(p<0.05).Conclusion:1.UM-6 inhibits cervical cancer cell proliferation and induces apoptosis in a ROS-dependent manner.2.UM-6 inhibits HPV18E7 expression in cervical cancer cells at the transcriptional and translational levels in a ROS-dependent manner.3.UM-6 inhibits cervical cancer cell proliferation through the HPVE7/PI3K/AKT glycolytic pathway.4.UM-6 can enhance the radiosensitivity of cervical cancer cells.5.UM-6 is safe in vivo experiments.UM-6 inhibits the growth of transplanted tumors in nude mice through the HPVE7/PI3K/AKT glycolytic pathway. |
PDF Full Text Request