Effects And Mechanism Of Periplaneta Americana Polypeptides PAE₂ Reverses Multidrug Resistance In HepG2/ADM Cells

Objective:To investigate the effect and mechanism of Periplaneta americana polypeptide PAE₂ on reversing the Multi-drug resistance（MDR）of human hepatocellular carcinoma drug-resistant cell line HepG2/ADM.Methods:The effects and mechanism of PAE₂ monomer of Periplaneta americana to reverse liver cancer MDR were determined by using the following methods in vitro and in vivo.（1）HepG2,resistant ADM cell line HepG2/ADM and Balb/c-nude nude mice were used as the research subjects;（2）MTT method to detect MDR of HepG2/ADM cells;Determine the reverse dose of Periplaneta americana polypeptides PAE₂ ,CⅡ-3 and skim cream;（3）Observe the accumulation of AMD in HepG2/ADM cells by laser confocal microscopy;（4）The effect of PAE₂ on the apoptosis-related proteins Bax,Bcl-2,Caspase-9,PARP in HepG2/ADM cells was detected by Western Blot;（5）To clarify the mechanism of PAE₂ to reverse MDR,the relative expression levels of m RNA and protein of P-gp,MRP,LRP,BCRP,PKC,GST-π,Topo II in HepG2/ADM cells were detected by immunocytochemical staining and RT-qPCR;（6）Immunocytochemical staining was used to text the relative expression of VEGF in HepG2/ADM cells and determine the effect of PAE₂ on the angiogenic capacity of HCC;（7）In Balb/c-nude nude mice,establish an axillary xenograft model of HepG2/ADM cells;（8）Observe the general status of axillary xenografts,immune organs,liver and kidney function;（9）Use the same method as cell experiments observe tumor growth inhibition and tumor growth.Induces apoptosis of tumor cells,regulates multidrug resistance-related proteins and related enzymes in tumor tissues,affects tumor angiogenesis;（10）Observe the tumor histopathological examination changes by HE staining.Results:（1）In the results of MTT,HepG2/ADM cells showed different RI to 5-FU,ADM,CTX,VCR and OXP,The RI was between 2 and 5,showing cross-resistance;（2）Determine the dose of PAE₂ by MTT method.The IC₅（3.21±1.09μg/m L）、IC₁₀（22.19±7.10μg/m L）、IC₂₀（183.19±64.38μg/m L）of PAE₂ on HepG2/ADM cells for 48 h was calculated by SPSS.And using they as Low,medium and high dose;（3）The high-dose PAE₂ combined with 5-FU,AMD,VCR,CTX and OXP could reverse the MDR of HepG2/ADM to different chemotherapy drugs.The RF were 1.15,1.16,1.25,1.94 and 1.39,respectively,which proved that PAE₂ reversed the MDR effect;（4）The results of laser confocal experiments proved that PAE₂ can promote the accumulation of ADM in HepG2/ADM cell lines,reduce the efflux effect of MDR cells on drugs and the cross-resistance of HepG2/ADM cell;（5）Western Blot experiments showed that PAE₂ mainly induces the apoptosis of HepG2/ADM cell line by activating the expression of apoptotic proteins through signal pathways involved in Bax/Bcl-2 heterodimer;Caspase-9 and PARP protein-mediated apoptotic pathways also have a certain effect;（6）Through the immunocytochemical staining,we found that compared with HepG2 cells,the relative expression levels of P-gp,LRP,BCRP,VEGF,PCK,GST-π,Topo II in HepG2/ADM cell lines were significantly up-regulated,there was a statistical difference,and MDR.PAE₂ has a significant inhibitory effect on the expression of P-gp,LRP,BCRP,VEGF,PCK,GST-π,Topo II protein.（7）RT-qPCR results showed that HepG2/ADM cell line produced MDR and was highly expressed with MDR1,LRP,BCRP,GST-πand Topo II,resulting in drug efflux and insufficient uptake.The abnormal metabolism of drugs in cells is related to PAE₂ ,which significantly inhibits the expression of MDR-related proteins and related enzymes m RNAd,which has statistical significance;（8）In experiments related to tumor formation in Balb/c-nude nude mice,after observe the viscera index,Cr,BUN,AST,ALT test,shows that the relative safety of Periplaneta americana extract,main organs of nude mouse non-toxic side effects,and can reduce the renal injury of transplanted tumor mice,protect heart cells,the effect of intravenous drug delivery way’s role in this may be due to oral medicine group;（9）Different routes of administration and doses of PAE₂ could inhibit the growth of MDR tumors.Periplaneta americana polypeptide PAE₂ in intravenous administration group had the best tumor inhibition effect;（10）In Western Blot experiments,it was observed that the effect of PAE₂ on tumor apoptosis in Balb/c-nude nude mice was mainly mediated by signal pathways related to Bax/Bcl-2 and PARP molecules;（11）In the immunohistochemical staining experiments,the relative expression levels of P-gp,LRP,BCRP,GST-π,PKC,Topo II and VEGF in the group of most resistant strains were higher than those of the sensitive strains.It has been shown that the MDR production of HCC is related to the high expression of the above factors in vivo;PAE₂ Could reduces the expression of LRP,BCRP,PKC,GST-π,Topo II,VEGF in vivo;（12）RT-qPCR experiments proved that the relative expression levels of MRP,BCRP in the groups of most resistant strains were higher than those of the sensitive strains,PAE₂ could reduces the m RNA levels of MDR1,MRP,LRP,BCRP,PKC GST-π,Topo II,VEGF in vivo;（13）In the pathological HE staining observation,the tumor tissue of Balb/c-nude nude mice after PAE₂ administration reduced the volume of tumor cells,the arrangement tended to be sparse,tumors showed a large area of coagulative necrosis and nuclear fragmentation,the malignancy of the tumor decreased,suggesting that PAE₂ can still play a good antitumor effect in vivo;Conclusion:（1）HepG2/ADM cells showed multi-drug resistance characteristics.Compared with HepG2,HepG2/ADM cells also showed MDR characteristics after subcutaneous transplantation into tumor in Balb/c-nude nude mice.（2）The CⅡ-3,skim cream and polypeptide PAE₂ extracted by Periplaneta americana could reverse the multi-drug resistance of human hepatocellular carcinoma cells HepG2/ADM in vitro and in vivo.（3）The Periplaneta americana polypeptide PAE₂ reversing MDR of HepG2/ADM cells role better than CⅡ-3 and skim cream.（4）Periplaneta americana polypeptide PAE₂ might affect the MDR in multi-drug resistant HepG2/ADM cells of human liver cancer by affecting the expression of apoptosis-related proteins and related proteins and enzymes that mediate multi-drug resistance,reduce drug effusion,promote intracellular drug accumulation and cell apoptosis.