Design Of Novel Peptide Probe And Its Application In Detection Of Prostate Cancer Cells

Background:Prostate cancer is one of the most common tumors in older adults.In clinical practice,most patients are diagnosed with prostate cancer at an advanced stage and lose the chance of complete cure.At present,the screening diagnosis of prostate cancer is mainly based on serum PSA detection and digital rectal examination,but the above detection methods have significant limitations.Therefore,there is an urgent need in identifying new biomarkers for early diagnosis of prostate cancer.The relationship between zinc and copper ions and the occurrence and development of prostate cancer provides a new method for early diagnosis of prostate cancer.By comparing the difference of zinc and copper ion enrichment ability between prostate healthy epithelial cells and prostate cancer cells,normal cells and cancer cells can be distinguished,to achieve the purpose of early diagnosis of prostate cancer.The detection of metal ions in organisms by fluorescence probe technology has become a research hotspot in recent years,and the advantages of visual detection make this technology very suitable for medical research and disease diagnosis.At present,many types of fluorescent probes with excellent performance have been reported,especially peptide probes.Objective:To design a peptide fluorescent probe for specific detection of zinc and copper ions according to different principles.In order to detect the content of zinc and copper ions in healthy prostate epithelial cells and prostate cancer cells using peptide probe imaging which allow early diagnosis of prostate cancer.Methods:1.Zinc ions probes L?H₂L and copper ion probes DP5 were synthesized by solid-phase synthesis;2.The selectivity of the probe for the target ion and its anti-interference ability were detected by fluorescence spectroscopy;3.The binding mode of the probe to the target ion was studied by fluorescence titration and Job working curve method;4.Whether the probe could be applied to the detection of the target ion in the organism was assessed by pH response,cytotoxicity,reversibility test and response time determination;5.The expression of zinc and copper ion-related transporter genes in the cell line was verified by real-time quantitative PCR and Western blot;6.The probe is used to image and detect the target ions in prostate cancer cells and normal prostate epithelial cells,and the cancer cells and normal cells are distinguished by comparing the fluorescence intensity,so as to verify the feasibility of the probe application in the early diagnosis of prostate cancer.Results:1.The probes L and H₂L that can detect zinc ions and DP5 that can detect copper ions were successfully designed and synthesized,and the detection of hydrogen sulfide was realized based on the complex of DP5-Cu²⁺;2.The results of fluorescence spectroscopy showed that the probes L and H₂L could target and recognize zinc ions and had good anti-interference ability;the probe DP5 could target and recognize the detected copper ions and the detection was not interfered by the other metal ions;the complex based on DP5-Cu²⁺could specifically detect hydrogen sulfide without significant fluorescence response to other anionic groups;3.The results of fluorescence titration and Job working curve show that the binding mode of probe L to Zn²⁺is 1:1;the binding mode of H₂L to Zn²⁺is 2:1,and the optimal structures of H₂L and L-Zn-L are obtained by theoretical calculation;the binding mode of DP5 to Cu²⁺is 2:1,and the optimal structures of DP5 and DP5-Cu-DP5 are obtained by theoretical calculation;4.The results of pH response,cytotoxicity,and response time measurements indicate that probes L,H₂L,and DP5 have the advantages of rapid detection,freedom from environmental pH in the organism,and low cytotoxicity,and that H₂L and DP5 also have the advantage of reversible detection of target ions;5.RT-PCR and Western blot results showed that the expression of zinc transporter ZIP1 gene in prostate cancer cells was significantly lower than that in normal prostate epithelial cells?P<0.05?,and the expression of copper transporter regulatory gene Ctr1 in prostate cancer cells was significantly higher than that in normal prostate epithelial cells?P<0.05?,providing an important theoretical basis for cell imaging;6.The results of cell imaging studies indicate that probes L and H₂L can target and recognize intracellular zinc ions and can be accurately quantified;probe DP5 can specifically recognize intracellular copper ions and achieve accurate quantification of copper ion content;accurate identification of prostate cancer cells is achieved by distinguishing the differences in the ability of prostate cancer cells and normal epithelial cells to enrich for Zn²⁺and Cu²⁺,providing a new means for early diagnosis of prostate cancer;complexes based on DP5-Cu²⁺can also achieve the detection of intracellular hydrogen sulfide.Conclusion:1.Zn²⁺and Cu²⁺have the potential to be biomarkers for the early diagnosis of prostate cancer;2.The probes L and H₂L can target and identify intracellular zinc ions,and probe DP5 can achieve the detection of intracellular copper ions and hydrogen sulfide;3.The probes L,H₂L and DP5 are expected to be novel tools for early diagnosis of prostate cancer;4.We found that the progression of prostate cancer may have a close link with changes in zinc and copper content within cancer cells in vitro studies.