| Purpose:The demethylating drug azacitidine(AZA)plays an important role in the treatment of acute myeloid leukemia(AML),but the efficacy still needs to be further improved.This study was to explore the effects of combining all trans retinoic acid(ATRA)on the biological functions of AML cells on the basis of AZA,and further explore the effects and molecular mechanism of ATRA enhancing AZA inhibiting AML through studies in vitro and in vivo.Methods:AML cell lines(SKM1 and HL60)were treated with AZA in combination with ATRA to explore the effects on cell proliferation,colony formation,cellular morphology,apoptosis,differentiation and cycle by CCK8 assay,methylcellulose substrate,Wright-giemsa staining,Hoechst 33342 staining and flow cytometry.Real-time quantitative PCR(q PCR)and Western Blot(WB)were applied to detecting the m RNA and protein expression levels of related molecules and analyzing their significance.Subcutaneous tumor model were constructed to verify the effect of AZA enhanced with ATRA on inhibiting AML by testing nude mice body weight,subcutaneous tumor volume and weight,and HE staining of subcutaneous tumors.Transcript sequencing to screen out potential target molecules by the anti AML effect of AZA enhanced with ATRA and verified by q PCR and WB.The expression levels of target molecules were modulated in SKM1 cells to evaluate the effects of the modulated target molecules on SKM1 cells treated with the AZA+ATRA group by cell functional assays.Results:1.ATRA enhances the anti AML effect of AZAIn SKM1 and HL60 cells,AZA+ATRA can further inhibit proliferation by23.17%(AZA+ATRA,56.29±2.81%;AZA,73.27±2.36%;p=0.0028),inhibit colony formation by 51.80%(AZA+ATRA,22.33±3.30;AZA,46.33±2.49;p=0.0012),promote apoptosis by 45.53%(AZA+ATRA,33.67±1.70%;AZA,23.13±1.56%;p=0.0030),block G0/G1 phase by 39.65%(AZA+ATRA,56.00±1.63;AZA,40.10±3.10%;p=0.0103);In HL60 cells,AZA+ATRA can further inhibit proliferation by 21.40%(AZA+ATRA,56.33±5.79%;AZA,71.67±4.38%;p=0.0404),inhibit colony formation by 54.12%(AZA+ATRA,29.67±3.30;AZA,64.67±5.73;p=0.0017),promote apoptosis by 70.84%(AZA+ATRA,38.32±3.70%;AZA,22.43±2.11%;p=0.0062),and block G0/G1 phase by 31.21%(AZA+ATRA,59.70±1.26%;AZA,45.50±2.30%;p=0.0063).In SKM1 and HL60 cells,AZA+ATRA further promoted differentiation than AZA and promoted undifferentiated cells to undergo apoptosis,significantly down-regulated the expression of proliferative molecules PCNA 80.68%(p=0.0078),72.09%(p=0.0092),CDC45 42.76%(p=0.0118),74.87%(p=0.0146),MCM7 73.35%(p=0.0123),29.63%(p=0.0222),significantly down-regulated the expression of anti-apoptotic protein BCL-2 by 63.99%(p<0.0001),42.22%(p=0.0048),MCL-1 by 33.40%(p=0.0007),58.52%(p=0.0101),and up-regulated the expression of pro-apoptotic protein BAK by 34.71%(p=0.0314),76.07%(p=0.0120),NOXA by 31.80%(p=0.1547),47.14%(p=0.0063),activated caspase3 by 2.82%(p=0.5149),54.26%(p=0.0252),and significantly down-regulated the expression of cycle regulatory molecules CCNB1 47.37%(p=0.0009),47.06%(p=0.0150),CCND2 58.43%(p=0.0082),63.01%(p=0.0073).2.AZA+ATRA could further inhibit AML development confirmed in vivo experimentATRA promoted the anti AML effect of AZA in vivo,which caused a significant decrease in subcutaneous tumor volume by 32.06%(AZA+ATRA,330.91±79.89mm~3;AZA,487.05±109.17 mm~3,p=0.0274),a significant decrease in subcutaneous tumor weight by 65.48%(AZA+ATRA,0.184±0.009 g;AZA,0.322±0.012 g;p<0.0001),and massive tumor cell necrosis were found by tumor pathology.3.AZA+ATRA potentiates the anti AML effect of AZA by modulating S100A8Transcript sequencing revealed that the S100A8 expression level in AZA treated SKM1 cells was up-regulated by approximately 252.08%compared with that in the NC group.While AZA combining with ATRA could reverse the increase in the S100A8 expression level caused by AZA and down-regulated by approximately422.81%compared with that in the AZA group.Validated by q PCR and WB,the S100A8 m RNA and protein expression level in the AZA group were up-regulated by approximately 52.49%(p=0.0001),18.92%(p=0.0004),respectively,compared with that in the NC group.The S100A8 m RNA and protein expression level in the AZA+ATRA group were down-regulated by approximately 68.56%(p<0.0001),35.68%(p=0.0001),respectively,compared with that in the AZA group.Further analysis using online databases found that high expression level of S100A8 was associated with poor prognosis in AML.Treatment of SKM1 cells with S100A8recombinant protein(the solvent is RPMI 1640)revealed a decrease in apoptosis of approximately 19.60%in the AZA+ATRA group(p=0.0078),but there are no significant differences between the AZA+ATRA group treated with RPMI 1640 in other functional experiments.S100A8 expression in SKM1 cells was knocked down with si RNA and the total apoptosis rate was increased by approximately 61.87%(p=0.0004),140.61%(p<0.0001)in the AZA+ATRA group compared to the AZA and ATRA group,respectively.In SKM1 cells with S100A8 being knockdown,the m RNA expression of BCL-2 and MCL-1 were down-regulated by approximately 24.23%(p=0.0175),32.41%(p=0.0049),and the protein expression were down-regulated by approximately 24.49%(p=0.0218)、45.92%(p=0.0097),respectively.Conclusions:AZA combined with ATRA can inhibit the proliferation and colony formation of leukemia cells,promote apoptosis and differentiation,and cause cells in G0/G1 phase arrested.S100A8 recombinant protein treatment can reverse the apoptosis induced by AZA+ATRA,while knocking down the expression level of S100A8 can further promote the apoptosis induced by AZA+ATRA,suggesting that AZA combined with ATRA enhances the anti leukemia effect of AZA by inhibiting the expression of S100A8. |
PDF Full Text Request