UM-6 Induces Autophagy And Apoptosis Via The Hippo-YAP Signaling Pathway In Cervical Cancer

Background:Although cervical cancer is almost completely preventable through HPV vaccination and screening,cervical cancer remains an important public health problem affecting women's health worldwide.The yearly number of new instances of cervical cancer in China was as high as one-quarter of the global total,and the five-year survival rate of advanced cervical cancer is less than 20%.Although surgery combined with adjuvant therapy(chemotherapy,radiotherapy,or a combination of both)has a satisfactory local control rate in early-stage cervical cancer,the prognosis is extremely poor when the tumor progresses to an advanced or metastatic stage,and the outcome of conventional radiotherapy is not satisfactory.Melittin,the most researched amphiphilic cationic peptide with excellent cytolytic characteristics and no drug resistance,is the major peptide component of bee venom.By breaking cell membranes,melittin causes tumor cells necrosis and apoptosis in cell culture and animal models.Melittin's therapeutic application is limited by its non-specific cytotoxicity,poor bioavailability,and strong hemolytic action.As a result,we designed UM-6,a novel fusion peptide based on melittin,and preliminary results showed that UM-6 not only retained its toxic effect on tumor cells but also avoided the defects of melittin,such as hemolysis.However,the specific anti-cervical cancer mechanism of UM-6 and the safety of its in vivo application need to be further investigated.Objective:To investigate the mode of cervical cancer cell death caused by the novel fusion peptide UM-6 and to clarify the specific mechanism by which it exerts its anti-cancer effects.To further investigate the safety and efficacy of UM-6 under different modes of administration in vivo,and to lay the foundation for preclinical studies of UM-6.Methods:1.UM-6 hemolytic and cellular uptake assay.Melittin and UM-6 hemolysis rates were compared using a hemolysis assay at the same molar concentrations.Cervical cancer cells were treated with FITC-labeled UM-6,and the subcellular localization and cellular integrity of UM-6 were studied using confocal imaging at various time points.2.The effect of UM-6 on cervical cancer cell growth.MTT assay was used to determine cell viability in Hela,Caski,Si Ha,ME180,and C33A cervical cancer cell lines,and the half-inhibitory concentration(IC50)was calculated.The influence of UM-6 on colony formation in the above cervical cancer cell lines was determined using plate colony assay.The effect of UM-6 on cervical cancer cell migration and invasion.Hela and Caski cells were treated with 0-30(?)g/ml UM-6,and the migration and invasion were assessed using the Transwell assay and the 3D invasion assay;the expression of epithelial-mesenchymal transition(EMT) markers was assessed using Immunoblotting.3.The effect of UM-6 on apoptosis of cervical cancer cells.Hela and Caski cells were treated with 0-30(?)g/ml UM-6.TUNEL assay was used to detect genomic DNA breaks in late stages of apoptosis;flow cytometry was used to detect phosphatidylserine(Annexin V)outgrowth on the cell membrane during apoptosis; Immunoblotting was used to detect the expression of Bax,Bcl-2,Cleaved-Caspase9,Cleaved-Caspase8,Cleaved-Caspase3,and Cleaved-PARP1.4.The effect of UM-6 on autophagic flux in cervical cancer cells.In Hela and Caski cells,GFP-LC3 and m Cherry-GFP-LC3 adenoviruses were transfected for 48 hours.The number of LC3 punctate aggregates was quantified under confocal fluorescence microscopy after treatment with 30(?)g/ml UM-6 for another 24 h.The autophagy early-stage inhibitor Wortmannin(WM),as well as the autophagy late-stage inhibitors chloroquine(CQ)and bafilomycin A1(Baf A1),were co-administered with UM-6,and immunofluorescence was used to assess the colocalization of LC3,p62,and LAMP1.Immunoblotting was used to detect the autophagy-associated markers LC3,p62,and LAMP1 expression.Using small interfering RNA(si RNA)to silence ATG5 and ATG7,the role of autophagy in cell death caused by UM-6 was explored.5.The expression and subcellular location of YAP were determined using a tissue microarray.Immunoblotting was used to detect YAP expression in clinical cervical carcinoma tissues.The c Bio Portal web tool was used to look at YAP genetic changes in diverse cancers.6.The effect of UM-6 on the Hippo pathway.Hela and Caski cells were treated with UM-6,and the expression of total protein LATS1,phospho-LATS1(Thr1079 and Ser909),total protein YAP,phospho-YAP(Ser127 and Ser397)were detected by Immunoblotting,and cytoplasmic and nuclear protein was extracted separately to detect the expression of YAP.The subcellular localization of YAP was detected by immunofluorescence.The effect of UM-6 on YAP transcriptional activity was detected by dual-luciferase reporter gene assay and q RT-PCR.The ubiquitination level of YAP was detected by immunoprecipitation.The expression of YAP was inhibited by si RNA and the expression of Cleaved-Caspase9,Cleaved-PARP1,LC3 and p62 was detected by immunoblotting to investigate the effect of YAP on apoptosis and autophagy in cervical cancer cells.7.UM-6 in vivo administration safety assay and in vivo therapeutic dose exploration. A subcutaneous xenograft model was constructed using Hela cells in nude mice, which were randomly divided into five groups(five in each group):a)PBS;b)IFN;c)cisplatin(CDDP,25mg/kg);d)UM-6(4 mg/kg);e)UM-6(8 mg/kg);f)UM-6 (12 mg/kg).The above drugs were injected intraperitoneally every two days while tumor growth was monitored for a total of nine treatments.To investigate the toxicity of UM-6 administered in vivo,serum levels of creatinine(Cr),Blood Urea Nitrogen(BUN),Myocardial enzyme,and estrogen(Estrogen),glutamic-pyruvic transaminase(ALT)and glutamic oxalacetic transaminase(AST)were assessed using an enzyme-linked immunosorbent assay(ELISA).The therapeutic effect was also used to determine the effective therapeutic dose.8.The safety and efficacy of UM-6 in mice model.Nude mice were used to create a subcutaneous xenograft model,and UM-6(8 mg/kg)or saline was injected intraperitoneally every two days for nine courses,with tumor growth monitored. Tumors were collected and weighed at the conclusion of treatment,then paraffin-embedded and subjected to immunohistochemistry(IHC),immunofluorescence detection in frozen sections,and tissue protein extraction for immunoblotting to detect autophagy,apoptosis,and Hippo pathway proteins expression.9.The effect of UM-6 inhibits cervical cancer metastasis in vivo.Abdominal metastasis models were constructed in nude mice using Hela cells(10~6cells per mouse),and UM-6(8 mg/kg)or saline was injected into the tail vein every two days for seventeen treatments.The mice were examined for intra-abdominal tumor burden at the end of treatment,and all metastases were photographed and weighed, as well as liver metastases for pathological examination.Results:1.The hemolysis of UM-6 was lower than that of Melittin,and no significant hemolysis was observed when the concentration of UM-6 reached 64(?)M,while 50%of erythrocytes were completely lysed at a concentration of 2(?)M of Melittin. Cytoplasmic enrichment was observed after 5 minutes of treatment with UM-6, indicating that UM-6 can be taken up by cells while reducing hemolysis.2.UM-6 inhibited the proliferation and clone formation of cervical cancer cell lines Hela,Caski,Si Ha,ME180,and C33A in vitro,and UM-6 was less toxic to human keratinocyte Ha Ca T and normal epithelial cell line MCF-10A.3.UM-6 inhibits the migration and invasion of cervical cancer cell lines Hela and Caski in vitro and suppresses the epithelial-mesenchymal transition(EMT).4.UM-6 promotes apoptosis(type I cell death)in cervical cancer cells.UM-6 treatment increases mitochondrial outer membrane permeability(MOMP),activates the Caspases,and activates intrinsic and extrinsic apoptotic pathways.5.UM-6 causes autophagosome accumulation in cervical cancer cells,boosts autophagosome-lysosome fusion,and promotes autophagic flux.Increased autophagic flux promotes autophagy-dependent cell death(type II cell death),and pharmacological inhibition of autophagy or interference with autophagy-related genes ATG5 and ATG7 can partially reverse the UM-6-induced cell death.6.YAP is overactivated in cervical cancer,and moderate/strong positive signals for YAP were detected in the cytoplasm and nucleus of cervical cancer samples;notably,the rate and intensity of immunosignal positivity for cytosolic YAP was significantly higher.Bioinformatic analysis suggested that YAP amplification occurs frequently in different types of cancers.7.UM-6 activates the Hippo pathway,promotes YAP protein degradation,and inhibits YAP transcriptional activity.UM-6 inactivates YAP oncoprotein through at least four mechanisms:(?)Ser127 phosphorylation-induced cytoplasmic retention; (?)Ser397 phosphorylation-induced degradation;and(?)increased autophagic flux-mediated YAP lysosomal dependent degradation.(?)UM-6 decreases YAP-TEAD interactions by inhibiting YAP nuclear localization.These four mechanisms act synergistically to inhibit YAP oncogenic activity.8.No significant toxicity was observed with the in vivo application of UM-6.The main biochemical indicators Cr,BUN,ALT,AST,Myocardial enzyme,and estrogen levels were not significantly different between the groups.When UM-6 was treated at a dose of 8 mg/kg,the tumor suppression rate was comparable to that of Cisplatin.9.UM-6 was able to inhibit the proliferation of xenograft tumors in nude mice by a mechanism related to activation of apoptosis,autophagy and the Hippo pathway.10.In nude mice models with intraperitoneal implantation of Hela cells,tail vein injection of UM-6 inhibited tumor burden and metastasis and suppressed the size and extent of metastatic nodules in the liver.Conclusions:1.UM-6 inhibited cervical cancer development while lowering hemolysis,and it had no significant toxic side effects in vivo administration.2.UM-6 suppressed cervical cancer proliferation both in vitro and in vivo,with the main mechanisms being apoptosis(type I cell death)and autophagy-dependent cell death activation(type II cell death).The mitochondrial apoptotic pathway and the caspase-dependent apoptotic pathway were both activated in UM-6-treated cervical cancer cells;increased autophagic flux was also observed,including increased autophagosome synthesis and accumulation of autophagolysosomes;and the increased autophagic flux acted synergistically with the apoptotic pathway to promote the death of cervical cancer cells.3.UM-6 inhibited cervical cancer cell invasion and metastasis in vitro while suppressing EMT.In a mouse model of intraperitoneal implantation,UM-6 treatment was able to reduce the tumor burden and metastasis extent.4.YAP is moderately/strongly expressed in cervical cancer samples.In vitro and in vivo,UM-6 triggers the Hippo pathway,promotes YAP protein degradation and cytoplasmic retention in a phospho-LATS1(Thr1079 and Ser909)dependent manner,and suppresses YAP nuclear localization and transcriptional activity.