| Background and purposeTrichloroethylene(TCE),as an organic pollutant in the production environment,has been classified as human level I carcinogen,indicating that it may increase the risk of suffering liver cancer.In order to research the malignant transformation effect induced by TCE and its potential mechanism,we applied normal human liver hepatocytes as a model,and according to the selected target molecules,a new curcumin derivative B5 was designed and synthesized to further explore the reversal effect of B5 on malignant transformation,which is expected to supply a theoretical basis for the prevention and control of TCE occupational exposure.Research methodsCell morphology,growth kinetics,proliferation and migration ability and genomic differences were studied at the cell level.The tumorigenic potential,neobiological histopathology and molecular biology(cytokeratin and Ki-67)of THLE-2-TCE cell xenografts in BALB/C nude mice were observed at the animal level.Then,the generation of ROS and its regulation on the process of cell malignant transformation were discussed by oxidative stress inhibitor N-acetyl-L-cysteine(NAC).On this basis,combined with the quantitative results of Label free,the possible molecular mechanism of TCE induced malignant transformation was explored by using pathway protein inhibitor and si RNA lentivirus interference.Finally,according to the screened mechanism targets(AKT,NFκB),a new curcumin derivative B5 was synthesized by the chemical method of twin drug design,and the effect of B5 on reversing cell malignant transformation was comprehensively evaluated,and the screened target molecular mechanism was verified again by molecular docking and molecular dynamics simulation.Research results(1)Biological characterization related to malignant transformation of THLE-2-TCE cells:(1)Morphological characterization.Some THLE-2-TCE cells growing overlapped with different sizes and closely arranged,disordered,polygonal and long protrusions.Multinuclear giant cells were obvious,and the phenomenon of cells morphology dissimilation was distinct.(2)Biological function characterization.THLE-2 cells did not form new organisms.The doubling time of THLE-2-TCE cells(49.92 ± 0.49 hours)was significantly shorter than that of the control group(75.06 ± 1.71 hours).After four weeks cultivation,a macroscopic clonal cell mass was formed,which had a certain ability to anchor independent growth cells.The average 24-hour migration rate(76.67%)and the number of transmembrane cells(525 ± 13.04)were evidently higher than those in the control group(20.83% and 114 ± 6.78).The above differences were statistically meaning(P<0.05).Some EMT features appeared,such as increased expression of N-cadherin and decreased expression of E-cadherin.(3)Biological phenotypic changes.The quantitative results of label free protein displayed that the genome of THLE-2-TCE cells was unstable.The expression of positive correlation proteins(IGF2R,EIF4EBP1,FAM195 B,ZNF185,BCAT1)of cell proliferation,differentiation,migration and invasion and anti-apoptosis were increased significantly.Some proto-oncogenes,for instance,AKAP8,BCL2L1,AXL,JUND and AKT2 were highly expressed,while the expression of some cancer suppressor genes such as BCAR1,SAMD9,BCAR3,NFXL1 and UIMC1 were decreased obviously.The functions of differential proteins were mainly concentrated in oxidative phosphorylation,biological macromolecules,heterocyclic compounds and ion binding,common pathways involved in signal networks,such as oxidative phosphorylation,cell survival and cell cycle.These signaling pathways was abnormally activated,such as TGFβ/SMAD,MAPK,PI3K/AKT/m TOR and NFκB.The above analysis results indicated that THLE-2-TCE cells had biological activities related to malignant transformation.(4)Tumorigenesis experiment in nude mice.The new biology formation rate was 40.00%,and the end volume was 406.17 ± 76.98mm~3 in THLE-2-TCE cells.HE staining of new biology tissues showed that the cells were closely arranged and irregular in shape,with a small amount of stroma,deep staining of nuclei and obvious atypia.Cytokeratin was positively expressed in IHC,which belongs to the source of epithelial cells.The proportion of Ki-67 positive cells was high,which was closed to the Hep G2 group,and the cell proliferation ability was stronger.The establishment of THLE-2-TCE cell nuclear tumor model in nude mice showed that the cell had a certain tumorigenic potential at animal level.(2)Potential molecular mechanisms of malignant transformation in THLE-2-TCE cells.(1)The ROS,gp91,phox-m RNA transcription and protein levels in THLE-2-TCE cells were increased during long-term exposed in TCE.The levels of MDA and SOD increased,and the expression of KEAP1 descended continuously,however,the expression of Nrf2 and HO-1 increased in the first place and then declined,showing that the antioxidant system was activated.The cell migration rate(32.33 ± 6.13%),invasion number(352 ± 41.41)and clone formation rate(230.67 ± 12.04%)of NAC pretreatment were respectively lower than those of TCE induction group(47.67 ± 6.13%),(644 ± 39.23)and(484 ± 16.57%).In nude mice tumor taken shape experiment,the tumor rate of NAC pretreatment(20.00%)was lower than that of TCE induction group(40.00%),and the tumor growth rate and end volume(173.54 ± 26.39 mm~3)were also lower than that of TCE induction group(406.17 ± 76.99 mm~3).The number of Ki-67 immunohistochemical positive cells was lower than that in TCE induced group as well.(2)Protein quantification and KEGG analysis showed that TGFβ,AKT,NFκB and other molecules were significantly up-regulated in THLE-2-TCE cells.The invasion and migration ability of NFκB Si-RNA cells were decreased.The NFκB interference resulted in decreased expression of Cyclin D1.Furthermore,after treatment with AKT inhibitor MK-2206 or TGFβR inhibitor LY364947,the protein expression level of AKT and NFκB was declined obviously contrast with the untreated group.The expression level of TGFβ/AKT/NFκB pathway molecules was decreased significantly in NAC group.These results suggested that ROS may through regulate TGFβ/AKT/NFκB signaling pathway to participate in TCE induced malignant transformation in cells.(3)The binding free energy(-55.02 ± 3.22 Kcal/mol)of curcumin derivative B5 is twice that of curcumin(-27.06 ± 3.22 Kcal/mol),and its affinity with AKT(-6.28 Kcal/mol)is also stronger than that of curcumin(-3.57 Kcal/mol).The morphology of THLE-2-TCE cells was induced to change from long spindle and star shape to full polygon,and the cell protuberances were significantly reduced and shortened by B5.The clone formation rates of group 0,2,4 and 8 μM were 98.67 ± 3.40%,51.47 ± 6.69%,37.82 ±4.85% and 17.00 ± 6.68%,respectively.The cell invasion rate was 96.67 ± 7.72%,55.00± 9.80%,34.00 ± 6.53% and 11.33 ± 3.40% in B5 groups(0,5,10 and 15 μM),respectively.The G2/M phase arrest ratio was 18.29%,38.87%,57.21% and 60.73%.The fluorescence strength of ROS was declined in a concentration dependent way and Nrf2/HO-1 was activated.The expression levels of TGFβ,AKT,NFκB were decreased.These results indicated that B5 may eliminate ROS and inhibit AKT/NFκB to exert the effects of anti-proliferation,anti-migration and reverse malignant transformation of THLE-2-TCE cells.Conclusion(1)TCE long-term exposure can induce malignant transformation of THLE-2 cells,enhance the ability of cell migration,invasion and clone formation,and result in tumorigenesis in nude mice.(2)TCE exposure can induce the sustained production of ROS,and TGFβ/AKT pathway can be activated by ROS,whereafter promotes NFκB nuclear transcription,and induces the expression of cyclin,to enable cells to obtain the ability of malignant proliferation.The results manifested that ROS/TGFβ/AKT/NFκB signal axis may be one of the important toxicological mechanisms of TCE exposure induced malignant transformation of human hepatocytes.(3)B5 can eliminate ROS in THLE-2-TCE cells,exert antioxidant effect through Nrf2/HO-1 pathway,and then inhibit the expression of AKT/NFκB to reverse the malignant transformation of THLE-2 cells induced by TCE.TCE exposure induced ROS and the activation of TGFβ/AKT/NFκB may be one of the important toxicological mechanisms inducing the malignant transformation of THLE-2 cells.A novel curcumin derivative B5 was synthesized through twin drug design and chemical synthesis.Computer simulation showed that B5 could clear intracellular ROS and target AKT to reverse malignant transformation of cells.It can be used as a candidate drug for precise targeted prevention and treatment of TCE occupational liver malignant transformation.This research provides a new experimental basis for the potential malignant transformation of hepatocytes caused by TCE exposure,and provides research thought for further clarifying the pathogenic mechanism of malignant transformation of hepatocytes caused by TCE,which has a certain value in the prevention and treatment of occupational liver diseases.Figure 38 Table 28 Reference 225... |
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