Effects Of Different Concentrations Of Am80 On Proliferation,Differentiation And Chemotherapy Resistance Of Human Neuroblastoma SH-SY5Y Cells

Objective Am80(Tamibarotene)is a synthetic retinoic acid receptor(RAR)agonist.In this study,we investigated the effects of Am80 on the proliferation of human neuroblastoma cell line SH-SY5 Y and the expression of tyrosine kinase receptor TrkA at different concentrations,and further investigated the effect of Am80 on chemoresistance of neuroblastoma.Methods The human neuroblastoma cells SH-SY5 Y cells were routinely cultured,and the inhibition of cell proliferation was detected by Cell Counting Kit-8(CCK-8)at different concentrations of Am80(diluted to different concentrations,0,10,20,40,80,160μmol/L).After treatment with Am80 cells at concentrations of 0,10 and 20 μmol/L for48 h,the expression of TrkA,MYCN mRNA and protein was respectively detected by TR-PCR and Western blot.Well-grown SH-SY5 Y cells were randomly divided into experimental group and blank control group.Cells were treated with different concentractions of Am80(0,10,20,40,80,160μmol/L)for 48 h,and CCK-8 method was used to detect the proliferation rate.The cells was respectively treated with Doxorubicin either alone or in combination with Am80(10,20μmol/L)for 48 h,and their multiplication capacity was measured using CCK-8 assays.The cells were treated with media which concluded concentration of 0,10,20μmol/L Am80 for 48 h,then using media which contained1μmol/L Doxorubicin cultivate them for 1h,after that,using flow cytometry to detect mean fluorescence intensity related to intracellular Doxorubicin.Results As the concentration of Am80 increased,the inhibition rate of SH-SY5 Y cells gradually increased(P<0.05).Among them,when the concentration was 10μmol/L,Am80 had no significant inhibitory effect on SH-SY5 Y cells(3.51±1.68%,Inhibition ratio < 5%),but when the concentration was 20μmol/L,it showed weak inhibition(9.60±1.97%,Inhibition ratio<10%).The inhibition rate of SH-SY5 Y cell proliferation(57.43±4.95 %)was significantly enhanced at Am80 with a concentration of80μmol/L,compared with 10,20μmol/L Am80,the difference was statistically significant(P<0.05).The results of TR-PCR and Western blot showed that after treatment withdifferent concentrations of Am80(0μmol/L,10μmol/L,20μmol/L)for 48 h,the expression of TrkA mRNA and protein increased with the increase of Am80 concentration.The difference was statistically significant(P<0.05).As the concentration of Am80 increased,the expression level of MYCN gradually decreased.When the concentration of Am80 was10μmol/L,the expression of MYCN mRNA was 0.65±0.0458.When the concentration was 20μmol/L,the expression was 0.36±0.0557,the difference was statistically significant(P<0.05).As mentioned above,the different concentrations of tamibarotene could effectively inhibit the proliferation of SH-SY5 Y cells in a dose-dependent manner(P<0.05);After doxorubicin was used in SH-SY5 Y cells for 48 h respectively combined with 10,20μmol/L Am80,the ability of doxorubicin to inhibit the proliferation of SH-SY5 Y cells was increased,and the IC50 value of doxorubicin was decreased from0.977 ± 0.134μmol/L to 0.668 ± 0.129 and 0.463 ± 0.122μmol/L respectively.The intracellular accumulation of doxorubicin also increased.The difference was statistically significant(P<0.05).Conclusion Different concentrations of Am80 were applied to human neuroblastoma SH-SY5 Y cells.The results showed that Am80 could inhibit the proliferation of SH-SY5 Y cells in a dose-dependent manner,and the expression of TrkA mRNA and protein increased and MYCN decreased with the increase of Am80 concentration.Therefore,it is speculated that the underlying mechanism involves with increasing the expression of TrkA by down-regulation of MYCN,thereby exerting a therapeutic effect on neuroblastoma.In addition,Am80 could increase the intracellular concentration of doxorubicin,thereby increasing the chemosensitivity of SH-SY5 Y cells to doxorubicin.It will provide theoretical support for the application of Am80 in the treatment of neuroblastoma and a certain experimental basis for the induction of differentiation in the treatment of neuroblastoma.