In Vitro Study On The Differentiation Mechanism Of NB₄Cells Induced By TanshinoneⅡA And Comparative Study With ATRA And As₂O₃

BackgroundLeukemia is the most common type of children malignant tumor. Acutepromyelocytic leukemia (APL) is a distinctive subtype of acute myeloid leukaemia(AML), which represents approximately10%of acute myelogeneous leukemias. Withthe clinical application of all-tran retinoid acid (ATRA) and arsenic trioxide (As203),the complete remission rate of APL has been improved greatly. But the adverseeffects and drug resistance cause an abroad attention. It is necessary to find asubstitute with more safety and less adverse effects. TanshinoneⅡA (TanⅡA), analcohol-extracted product from Salvia miltiorrhiza, can induce differentiation of manytumer cells and should be considered as a potential anti-leukemia reagent.ObjectiveTo study the differentiation and growth inhibition of APL cells line NB4cellsinduced by TanⅡ A in vitro，and compared that with ATRA and As203．To provide atheoretical basis of TanⅡ A for clinical application.MethodsNB4cells were divided into seven groups treated with tanshinoneⅡA，ATRAand As2O3in different concentration. These cells were observd at12，24，48，72，96,and120hours respectively.We observd NB4cell growth conditions, morphologicalchanges,detected cell surface differentiation antigens (CD11band CD33) by using flow cytometry (FCM). The PML-RARα fusion protein and STAT1protein, STAT2protein,STAT3protein, STAT5a protein and STAT5b protein were assayed by Westernblotting，the PML/RARα mRNA and stat1mRNA, stat2mRNA,stat3mRNA andstat5ab mRNA were assayed by real-time PCR.Nuclear bodies were observd byindirect immnnofluotesent method．Results1. TanⅡA, ATRA and As2O3inhibited the growth and induced differentiation ofNB4cells. The degree of differentiation and growth inhibition of NB4cells wasincreasing with the dosage and treatment time of TanⅡA.2.55umol/L of TanⅡA isthe optimal concentration to inhibit the NB4cell proliferation.2. TanⅡA, ATRA and As2O3up-regulated CD11bexpression and down-regulatedCD33expression.2.55umol/L is the optimal concentration of Tan ⅡA.3. TanⅡA, ATRA and As2O3didn’t affect PML/RARα mRNA level, but theyinduced the degeneration of PML-RARα protein and release of PML proteinfrom NBs into its normal position in NB4cell nuclei.4. TanⅡA and ATRA up-regulated Stat1and Stat2mRNA level and enhancedSTAT1and STAT2protein expression. As2O3didn’t affect Stat1, Stat2mRNA andSTAT1, STAT2protein.5. TanⅡA，ATRA and As2O3didn’t affect stat3,stat5ab mRNA. TanⅡA，ATRAand As2O3didn’t affect STAT3、STAT5a、STAT5b protein.ConclusionsTanⅡA can inhibite the growth of NB4cells and induce significantdifferentiation in time-and dose-dependent manners.2.55umol/L is the optimalconcentration of TanⅡA to inhibit the NB4cell proliferation. The effect of2.55umol/L TanⅡA was similar to that of ATRA and As2O3at concentration of1.0umol/L. Furthermore, TanⅡA can induce the degeneration of PML-RARα protein,induce release of PML protein from NBs into its normal position in NB4cellnuclei, and restore the normal structure of nuclear bodies. TanⅡA, ATRA and As2O3 didn’t affect PML/RARα mRNA level.TanⅡA can up-regulate Stat1, Stat2mRNAand enhance STAT1, STAT2protein expression. TanshinoneⅡA may be consideredas a potential anti-leukemia reagent.