| BackgroundCervical cancer is the fourth most common tumor of cancer-related deaths worldwide;Nevertheless,the precise mechanism remains unclear and needs to be further elucidated.Most cervical cancers are thought to be caused by persistent infection with the oncogenic high-risk human Papillomavirus(hr-HPV),yet the viral infection,there are complex etiopathogenesis.Therefore,it is important to understand the biological functions and molecular mechanisms of the initiation,progression and prevention of the uterine cervix.Currently,no specific and high-sensitivity screening biomarkers have been found in cervical cancer.The carcinoma survival rate is less than 17 months.Currently,the most widely used platinum-based chemotherapy regimens have an effective rate of only 20%to 30%.As to the tumor cells,it has the ability to escape death and is resistant to chemotherapeutic drugs;At the same time,the cytotoxic effects of chemotherapy drugs and radiotherapy on patients should not be underestimated.Therefore,there is an urgent need to develop new low-toxicity anticancer drugs or more effective treatment strategies for drug-resistant patients and patients with advanced refractory tumors.Cancer suppressive peptides derived from natural compounds in animals,plants and microorganisms bring hope for the prevention and replacement of cancer,especially the active peptides and other components that are screened from human,animal serum and tissue.These cell physiological responses and physiological immune responses have shown their potential as specific biomarkers and anti-cancer effects in vitro and in vivo.Our group identified proteomics technology from plasma to identify polypeptide m/z 6455.5Da as biomarkers and potential tumor suppressors.There is evidence that the key genes of Apolipoprotein C1(APOC1)involved in this peptide have prognosis with cervical cancer,which suggests that the mechanism of action of the peptide may be related to autophagy,which may provide a new therapeutic targets.In order to further study the biological function and underlying molecular mechanism of polypeptide m/z 6455.5Da in cervical cancer,this study mainly based on cervical cancer as a model to design in vitro experiments and animal model experiments.The biological functions such as proliferation,apoptosis,autophagy,and combination with specific chemotherapeutics are discussed,and how to play the corresponding molecular biological mechanism is clarified.The main purpose and content are divided into two parts as follows:Part Ⅰ:The role and mechanism of polypeptide m/z 6455.5Da on the biological behavior of cervical cancer cells;Part Ⅱ:Molecular mechanism of polypeptide m/z 6455.5Da exacerbating cervical cancer cell death through ROS/AMPK axis-mediated autophagy flux inhibition;ObjectiveTo identify the effect of polypeptide m/z 6455.5Da on the proliferation,clonal colony formation,invasion ability and apoptosis of cervical cancer cells HeLa and CaSki in vitro;to investigate the effect of m/z 6455.5Da peptide-mediated ROS/AMPK axis on apoptosis,autophagy and mitochondrial dysfunction of cervical cancer cells,exploring the anti-cancer mechanism of m/z 6455.5Da and the mechanism of autophagy involved in the regulation of cervical cancer cell apoptosis.Part Ⅰ The mechanism of polypeptide m/z 6455.5Da inhibiting the proliferation of cervical cancer cellsMaterials and Method1.The polypeptide m/z 6455.5Da was synthesized by chemistry in vitro,identified by high performance liquid chromatography(HPLC)and mass spectrometry(MS)for subsequent;2.The proliferation activity function assays were performed to detect the effects of polypeptide m/z 6455.5Da in in normal epithelial cells(HerEpiC and HaCat)and cervical cancer cell lines(HeLa,CaSki and SiHa),including live cells dynamic monitoring,Cell Counting Kit-8(CCK-8)assay,the 5-ethynyl-2’-deoxyuridine glycoside(EDU)proliferation assay and colony formation assay.3.The mobility of HeLa and CaSki were detected by transwell assay after treated with polypeptide m/z 6455.5Da;4.The localization of polypeptide m/z 6455.5Da in HeLa and CaSki were detected by FITC-modified;5.Western blot were used to evaluate Cleaved Caspase 8,Bax,Cleaved Caspase 7,and Cleaved PARP protein levels in HeLa and CaSki cell lines;6.Flow cytometry was used to evaluate the apoptosis rate in HeLa and CaSki after treated with polypeptide m/z 6455.5Da;7.To investigate the changes of cell membrane in HeLa and CaSki after polypeptide m/z 6455.5Da induced,the lactate dehydrogenase test(LDH)was used;8.CCK-8 assay was used to detect changes of proliferation activity after co-treated with polypeptide m/z 6455.5Da or death inhibitors(Z-VAD-FMK,CQ,necro statin-1);9.To observe the formation of autophagosomes in HeLa and CaSki after polypeptide m/z 6455.5Da induced,transmission electron microscope was used;10.Statistical analysis:Data were obtained from at least three independent experiments and presented as mean ± standard deviation(x ± s).Statistical analysis was performed using SPSS(21.0)statistical software.Comparisons between the two groups were performed using independent sample t-tests.The Student’s t-test was used to measure the differences between the two groups.One-way analysis of variance(ANOVA)was used to for comparisons between multiple sets of quantitative data.Classification data were compared between different groups.χ2 test.With α=0.05 as the test level,P<0.05 was considered statistically significant,otherwise the difference is not considered statistically significant.Results1.Live cell dynamic imaging and CCK8 results showed that polypeptide m/z 6455.5Da inhibited the proliferation of HeLa and CaSki cell lines in a time-and concentration-dependent manner,the difference was statistically significant(P<0.01);meanwhile,polypeptide m/z 6455.5Da was not inhibit the proliferation of HerEpiC and HaCat lines(P>0.05);2.EDU and cell clone colony formation assay results showed that a significant decrease of cell proliferation activity in IC25 group,IC50 group,and IC75 group,when compared to the control(P<0.001);3.The migration ability of HeLa and CaSki cell lines was decreased in the IC25 group and the IC50 group(P<0.001);4.The localization of polypeptide m/z 6455.5Da(FITC fluorescent)in HeLa and CaSki showed that it may affect the biological function of HeLa and CaSki by interacting with the cell membrane or cytoplasm;5.Cleaved Caspase 8,Bax,Cleaved Caspase 7,and Cleaved PARP were upregulated in IC25 group,IC50 group,and IC75 group,when compared to the control(P<0.05);6.A significant increase of apoptosis rate were observed in IC25 group and IC50 group,when compared to the control(P<0.01);7.LDH assay results showed that cell death rates were increased in the IC10 group,IC25 group,IC50 group,and IC75 group,which has a significant increase compared with control(P<0.01);8.The inhibition of proliferation activity of IC50+CQ group was increased compared with IC50 group(P<0.05);The IC50+Z-VAD-FMK group was decreased compared to the IC50 group(P<0.05);The IC50+necrostatin-1 group had no significant change(P>0.05);9.The number of autophagosomes in the IC50 group was increased.Summary1.Polypeptide m/z 6455.5Da is a low toxicity tumor suppressor peptide,which can significantly inhibit the proliferation of cervical cancer cells and induce apoptosis;2.Polypeptide m/z 6455.5Da can cause mitochondrial dysfunction to activate endogenous apoptotic signal and induce apoptosis;3.Polypeptide m/z 6455.5Da-induced autophagy may be involved in the regulation of cervical cancer cell apoptosis.Part Ⅱ The mechanism of polypeptide m/z 6455.5Da exacerbating cervical cancer cell death through ROS/AMPK axis-mediated autophagy flux inhibitionMaterials and Methods1.RNA-seq was performed on RNA samples from cells treated with polypeptide m/z 6455.5Da and control cells,and GSEA analysis of differential genes was performed to further understand the effects on signaling pathways in cervical cancer cells.Regulation2.Western blot was used to detect the expression of p-AMPK,p-mTOR,LC3BⅠ/Ⅱ,ATG 7,p62,Beclin-1 in cervical cancer cells treated with polypeptide m/z 6455.5Da;3.Observe and detect the level of f ROS in cervical cancer cells treated with polypeptide m/z 6455.5Da by fluorescence microscope and flow cytometry;consider the effect of polypeptide m/z 6455.5Da on cervical cancer cell death combined with ROS inhibitor(NAC);4.The ATP in cervical cancer cells treated with polypeptide m/z 6455.5Da was detected by ATP detection kit polypeptide m/z 6455.5Da;5.The mRFP-GFP-LC3 adenovirus and p62 mRNA expression levels were used to detect the autophagy flux in cervical cancer cells treated with polypeptide m/z 6455.5Da;6.Acridine orange(AO)staining was used to detect changes in lysosomal pH;7.Western blot was used to detect the expression of Rab7,LAMP1,and LAMP2,and the fusion of autophagosomes and lysosomes in cervical cancer cells was analyzed in cervical cancer cells treated with polypeptide m/z 6455.5Da;8.The effects of cancer cell death treated with polypeptide m/z 6455.5Da was combined with the early autophagy inhibitor 3-MA or later autophagy inhibitor CQ,the abnormality of autophagy flux was detected by Cell Counting Kit-8(CCK-8)assay,,cloning formation assay,TUNEL assay and Western blot.;9.Combination with cisplatin,the polypeptide m/z 6455.5Da was used in the proliferation of cervical cancer cell,which was observed by CCK8 and cell dynamic monitoring;10.Constructed tumor transplantation model of nude mice,the growth,autophagy,and apoptosis of the polypeptide m/z 6455.5Da,combination with cisplatin were detected by Western blot and immunohistochemical;11.Statistical analysis:Data were obtained from at least three independent experiments and presented as mean ± standard deviation(x-±s).Statistical analysis was performed using SPSS(21.0)statistical software.Comparisons between the two groups were performed using independent sample t-tests.The Student’s t-test was used to measure the differences between the two groups.One-way analysis of variance(ANOVA)was used to for comparisons between multiple sets of quantitative data.Classification data were compared between different groups.χ2 test.With α=0.05 as the test level,P<0.05 was considered statistically significant,otherwise the difference is not considered statistically significantResult1.GSEA analysis results showed that HeLa cells in the IC50 group could be enriched for differential genes with low expression in MYC TARGETS,IL2-STAT5,Notch,mTORC1,and KRAS pathways;2.The intracellular ATP measurement results showed that the intracellular ATP levels in the cell lines of IC25 group,IC50 group,and IC75 group decreased,and the TUNEL test results showed that:compared with the IC50 group,the apoptosis rate of the IC50+3-MA group was weakened,and the apoptosis rate of the IC50+CQ group was increased,the difference was statistically significant,P<0.05;WB results showed that:Compared with IC50 group,the expression of LC3B-Ⅱ/Ⅰ in IC50+3-MA group was down-regulated,the difference was statistically significant,P<0.05;there was no significant change in LC3B-Ⅱ/Ⅰ in IC50+CQ group.7.After combined application of polypeptide m/z 6455.5Da and cisplatin(CDDP),CCK8 results show that m/z 6455.5Da peptide can enhance the lethality of low-dose CDDP(2.5μM),lethality was dose-dependent,the difference was statistically significant,P<0.05.The dynamic monitoring results showed that IC25 concentration could significantly enhance the killing effect of low-dose cisplatin(2.5 μM)on HeLa.8.In nude mice in vivo tumor formation model experiments,it was shown that the polypeptide m/z 6455.5Da and the combined group can significantly inhibit the cervical cancer cell proliferation,induce tumor cell apoptosis.Immunohistochemical results showed that Ki-67,PCNA,and Bcl-2 were weakly positive in the tumor tissue of the polypeptide m/z 6455.5Da group and the combined group,while the control group was strongly positive;meanwhile,Bax,LC3B,and P62 were strongly positive.The control group was weakly positive;WB results showed that compared with the control group,the expression of Bax in the treatment group was up-regulated,and the difference was statistically significant,P<0.05.Summary1.Polypeptide m/z 6455.5Da is a targeted autophagy regulator with anti-cancer effect,which inhibites autophagy flux by blocking the fusion of autophagosomes and lysosomes;2.Polypeptide m/z 6455.5Da promotes the initiation of autophagy through ROS-AMPK aix,and by inhibits the maturation of late endosomes and lysosomal membrane protein activity,the accumulation of non-fused autophagosomes exacerbates cervical cancer death;3.Polypeptide m/z 6455.5Da improve the killing effect of low-dose cisplatin on difference was statistically significant,P<0.05;Western blot results showed that p-AMPK expression was up-regulated,p62,ATG7,Beclin-1,and LC3B-Ⅱ/Ⅰ were up-regulated,and p-mTOR was down-regulated in the IC25,IC50,and IC75 cell lines.The differences were statistically significant,P<0.05;3.The ROS measurement results showed that the results of fluorescence microscope and flow cytometry showed that the ROS level in the IC50 group increased.Compared with the IC50 group,the ROS level in the IC50+NAC group decreased,the difference was statistically significant,P<0.05;Compared with the IC50 group,the cell survival rate increased,the difference was statistically significant,P<0.05;Western blot results showed that compared with the IC50 group,the IC50+NAC group had p-AMPK,LC3B-II/I,Cleaved Caspase 8,Cleaved PARP expression was down-regulated,the difference was statistically significant,P<0.05;4.The mRFP-GFP-LC3 adenovirus autophagy localization results showed that the number of autophagosomes in the IC50 group was significantly increased compared with the control group and the positive control(rapamycin-induced autophagy),the difference was statistically significant,P<0.01;There was no significant increase in autolysosomes,P>0.05;p62 mRNA expression levels were slightly up-regulated,but the difference was not statistically significant,P>0.05;5.Acridine orange(AO)staining showed no significant changes in AO staining in the IC50 group compared with the control group,and the difference was not statistically significant,P>0.05;Western blot results showed that LAMP1,LAMP2,and RAB7 expression in the IC25 group,IC50 group,and IC75 group were down-regulated,The difference is statistically significant,P<0.05;6.After the polypeptide m/z 6455.5Da was combined with autophagy inhibitors(3-MA and CQ),CCK8 results showed that compared with the IC50 group,the proliferation inhibition effect of the IC50+3-MA group was weakened,and the difference was statistically significant.P<0.05;IC50+CQ group’s proliferation inhibition was enhanced,and the difference was statistically significant,P<0.05.EDU test results showed that:compared with the IC50 group,the cell proliferation activity of the IC50+3-MA group was enhanced;the cell proliferation activity of the IC50+CQ group was weakened,the difference was statistically significant,P<0.05.cervical cancer cells,and it is a potential adjuvant for chemotherapy.Conclusions1.The polypeptide m/z 6455.5Da inhibits the proliferation of HeLa and CaSki cells in a concentration-dependent manner in vitro and in vivo,and causes cell mitochondrial dysfunction to induce apoptosis,and the polypeptide has small side effects on normal cytotoxicity;2.The m/z 6455.5Da peptide promotes the initiation of autophagy through ROS-induced AMPK activation,and by inhibiting the maturation of later endosomes and lysosomal membrane protein activity,the accumulation of non-fused autophagosomes exacerbates HeLa and CaSki cells Death.3.The polypeptide m/z 6455.5Da is a new type of autophagy inhibitor with anti-cancer effect.It blocks autophagosome and lysosome fusion,thereby inhibiting autophagy flux.4.The polypeptide m/z 6455.5Da can enhance the killing effect of low-dose cisplatin on HeLa and CaSki cells and reduce the toxicity of cisplatin by inhibiting autophagy flux.It is a potential adjuvant for chemotherapy. |
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