Study On The Mechanism Of The Difference Of Tolerance Stressed By Zinc Ions Between Cervical Cancer Cells And Normal Epithelial Cells

Background Zinc could be used as biodegradable medical material,and is gaining more attention now.Zinc is a transition metal element.Two electrons in the outer layer of its atomic orbital are easily captured by oxygen to form ROS.After malignant transformation of normal cells,hypoxia environment and Warburg effect metabolism features enable cancer cells to have relatively high ROS level basis,so as to adapt to malignant characteristics such as unlimited proliferation,invasion and metastasis of tumor cells.However,ROS is also a double-edged sword.When ROS exceeds the tolerance limit,it can cause cell death.At present,many chemotheraputic drugs utilize ROS to eradicate tumor cells.The final degradation products of zinc-based biomaterials implanted into human body are zinc ions,which can cause the increase of active oxygen free radicals and then kill cancer cells,which is of great significance for expanding the clinical application range of zinc-based biomaterials.Objective To study the biological mechanism of the difference of tolerance to zinc ions stress between tumor cells and normal cells,providing the basic theory for the potential clinical application of zinc-based biomaterials.Methods Firstly,the CCK8 assays were used to compare and analyze the activity changes of different tumor cells and normal cells lines under the intervention of exogenous zinc salts.Zinc ions were established with the phenotype of killing various tumor cells in human body.Secondly,cervical cancer cells was selected as the research object,a correlation analysis of Zn2+-ROS-Regulated Cell Death was established through zinc ion fluorescent probe FluoZinTM-3,ROS fluorescent probe DCFHDA,flow cytometry.Thirdly,AnnexinV PI FITC was adopted to detect the apoptosis of cells and real-time fluorescence quantitative PCR(qPCR)was used to observe the molecular changes of zinc transporter family,redox system as well as mRNA level of candidate genes related to cell death,speculating the possible ways and causes of zinc-induced cell death.Fourthly,the key indicators of oxidative stress such as redox regulation molecule,antioxidant enzymes,ROS and their ability to inhibit ROS were detected to analyze the mechanism for the soaring ROS in cervical cancer cells induced by zinc ions.Lastly,through transcriptome sequencing analysis,the hypothesis of Zn2+-ROS-Ferroptosis signaling pathway was proposed.With a variety of inhibitors intervention,qPCR and Western blot(WB),immunohistochemistry(IHC)and RNA interference experiments,key molecules of zinc-induced ferroptosis were identified,furtherly exploring the main signaling pathway of ferroptosis in cervical cancer cells stressed by zinc ions.ResultsPart ? Phenotype changes of cervical cancer cells and normal epithelial cells under the stress of zinc ions1.Under the stress of zinc ions(160?M),cell death was induced in 12 kinds of cancer cells derived from human reproduction,digestion and respiratory system.2.The basal level of ROS in cervical cancer cells is high.Under the intervention of exogenous zinc ions,the ROS level of cancer cells increased significantly with the time of zinc ions,compared with no significant change in normal cells.3.Zinc ions enhanced the mRNA transcription of Zip4 in cervical cancer cells,the mRNA level of ZnT1 was up-regulated,ZnT7 was down-regulated,and in normal cervical epithelial cells,all of the mRNA level of Zipl,Zip4,Zip6,Zip7,Zip9,Zip 10,Zip14,ZnT1,ZnT4 and ZnT7 showed a significant increase.4.In the process of malignant transformation of normal cervical epithelial cells,which refers to the progress of precancerous lesions and invasive cancer,the IHC results of clinical cervical tissue samples suggested that the expression of Zip4 level was diminished and the ZnT1 level was decreased in malignant transformed cells,indicating that the dependence of the malignant transformed cervical epithelial cells on zinc was increased.Part ? Oxidative stress changes of cervical cancer cells and normal epithelial cells under the stress of zinc ions1.Zinc ions led to the continuous depletion of GSH in cervical cancer cells,while normal cells showed continuous increase of GSH under the stress of zinc ions,indicating that zinc ions are the trigger to the continuous increase of ROS in cervical cancer cells.2.The activity of antioxidant enzymes such as manganese superoxide dismutase(SOD2),glutathione peroxidase(Gpx),catalase(CAT)and thioredoxin reductase(TxrR)in cervical cancer cells are significantly lower than normal epithelial cells.Under the stress of zinc ion,the activity levels of SOD2,CAT and TrxR in cervical cancer cells were increased,which indicated that zinc ions may stimulate the increase of reactive oxygen radicals such as O2·-and H2O2 in cervical cancer cells,enhancing activity of antioxidant enzymes to neutralize and eliminate ROS produced by cervical cancer cells.3.Under the stress of zinc ions,cervical cancer cells showed a significant decrease in the ability to inhibit OH·-(P<0.01),and such OH·-inhibiting ability was half of normal epithelial cells,suggesting that the increase in OH·-induced by zinc ions was the important mechanism of cervical cancer cell death.4.The IHC results of clinical cervical tissue samples suggested Keapl expression gradually increased and Nrf2 decreased in cervical cancer cells,indicating that the cervical cancer cells had reduced antioxidant capacity.Part ? Transcriptome sequencing analysis of cervical cancer cells and normal epithelial cells as well as the molecular mechanism of ferroptosis1.In the absence of exogenous zinc stress,the results of transcriptomics sequencing showed that the mRNA transcription levels of the related ferroptosis molecules such as COX2,ALOX15,and ACSL4 in cervical cancer cells were 2 times higher than that of normal cervical epithelial cells.Keapl increased by nearly 50 times,the membrane iron transport protein FPN1 decreased by nearly 10 times,comparing with normal cevical cells.2.Under the stress of zinc ions,KEEG enrichment pathway analysis showed that the key molecules of ferroptosis signaling pathway such as HO-1,SLC7A11,TF,Ferritin,GCL and ACSL4 were significantly up-regulated,among which HO-1 was at 4,8h up-regulated by approximately 50 and 30 times respectively.It was suggested that HO-1 is a key signaling molecule for zinc ion to induce ferroptosis in cervical cancer cells.3.The addition of iron chelating agent(DFO),ferroptois inhibitor(Fer-1),and vitamin E could significantly inhibit cell death.When adding necrosis inhibitor(Nec1),cell death was partially inhibited,suggesting that some cells died through the necrosis pathway under the stress of zinc ions.If apoptosis inhibitor(Z-VAD),or autophagy inhibitor(Apocynin)were added,cell death was not inhibited.This suggests that the death of cervical cancer cells stressed by zinc ions may be mainly through ferroptosis.4.QPCR and WB results suggested that zinc ions significantly enhance TF,HO-1,NOX1,COX2,ALOX15 and other ferroptosis molecules transcription expression levels,while FPN1 transcription expression was diminished.These markers may be the important molecules triggering ferroptosis in cervical cancer cells.5.The results of RNA interference assay showed that after HO-1 was interfered,the ROS level under zinc ion stress was significantly reduced,which indicated that the HO-1 gene played a key role in the zinc ion-induced increase of ROS in cancer cells.6.The IHC results of clinical cervical tissue specimens showed that during the malignant transformation of normal cervical epithelial cells,the expression of the ferroptosis signaling molecules ALOX15,ALOXE3 and COX2 increased;the expression of TF increased,and the expression of FPN1 decreased,suggesting that malignant transformation of cervical epithelial cells have an increased dependence on iron;The increase of SLC7A11 and Gpx4 expression,suggested that malignant transformed cells could enhance antioxidant capacity to adapt to their own higher levels of oxidative stress.Conclusion Cervical cancer cells-Siha and normal epithelial cells-HcerEpic had different tolerances stressed by zinc ions.Zinc ions caused GSH depletion and increased ROS production in cervical cancer cells,which was the key mechanism of inducing ferroptosis through Zn2+-Keap-Nrf2-HO-1 pathway.