Differentiation Induction And Action Mechanisms Of 4-amino-2-trifluoromethyl-phenyl Retinate In Human NB4 Promyelocytic Leukemia Cells

Malignant tumor(commonly known as cancer) is one of the major diseases with threats to human health.Differentiation therapy has become a new breakthroughs among tumor therapeutics which makes the tumour cells reversely differentiate into normal cells or nearly under the effect of the differentiation inducers.Retinoid is the representative of differentiation inducers.We reformed the polar group at the end of the carbon chain and modified the cyclohexene ring with all-trans retinoic acid(ATRA) as the lead compound, synthesized a series of new retinoic acid derivatives which had been depurated and identified.After screening of the derivatives with structure-activity, 4-amino-2-trifluoromethyl-phenyl retinate(ATPR) has powerful activity in inhibiting proliferation and inducing cell differentiation,suggesting that it is expected to become a novel differentiation inducer with intellectual property rights.This rearch was to investigate the proliferation inhibition and differentiation induction activities of the novel retinoic acid derivate ATPR in NB4 cells and investigate the expression of the compoments along the transforming growth factorβ1(TGF-β1) signaling pathway, c-myc oncogene and human telomerase reverse transcriptase(hTERT) to elucidate the mechanisms of ATPR partly. Objective: To investigate the proliferation inhibition and differentiation induction activities of the novel retinoic acid derivate ATPR in NB4 cells and explore the mechanisms of ATPR.Methods:1. The changes of growth curves induced by different concentrations of ATPR were analysed by cell counting.The extent of functional differentiation induced by different concentrations of ATPR were analysed by NBT reduction test.The expression of the maturation specific cell surface markers CD11b, CD13 and the distribution of cell cycle were determined by FCM analysis.2. The expression of TGF-β1, TGF-βⅠreceptor (TβRⅠ), TGF-βⅡreceptor (TβRⅡ), Smad2, Smad3, Smad4 and Smad7 mRNA along the TGF-β1 signaling pathway were detected by RT-PCR.The effect of specific TβRⅠinhibitor SB431542 on the defferentiation induced by ATPR were measured by Wright's stain,NBT reduction test and the detection of the surface differentiation antigen.3. The expression of c-myc, hTERT mRNA and protein of NB4 cells were detected by RT-PCR and Western Blot respectively.Results:1. ATPR inhibited the proliferation of NB4 cells in a dose- and time-dependent manner.The NBT positice cell ratio and the expression level of the surface differentiation antigen CD11b of NB4 cells were increased while the expression level of CD13 were decreased. The proportion of cells in G0/G1 phase was increased while S phase cells decreased.Cell cycle progression was blocked in the G1 phase.2. After the treatment with ATPR,the expression of TGF-β1, TβRⅠ, TβRⅡ, Smad2, Smad3 mRNA along TGF-β1 signaling pathway had no statistically significant differences at 24h.They were most significantly increased after 2d-3d individually and reached their peaks at 4d-5d individually when cells were induced by ATPR.The mRNA level of Smad4 started to increase from 4d treatment and reached its peak at 5d while Smad7 significantly began to increase after 5d treatment.The TβRⅠspecific inhibitor SB431542 could suppress the effect of ATPR on NB4 cell differentiation determined by Wright's stain,NBT reduction test and the detection of the surface differentiation antigen.3. After the treatment with ATPR,the mRNA and protein expression levels of c-myc and hTERT were significantly reduced in a time-dependent maner.Conclusions:1. ATPR has powerful activity in inhibiting proliferation and inducing differentiation. The further research on the anti-tumor mechanisms will make for developing novel effective anti-cancer drugs.2. Up-regulation of the TGF-β1 signaling pathway is one of the important mechanisms of the defferentiation effcts of ATPR.3. ATPR probably through upregulating TGF-β1 signaling pathway to inhibit the activation of c-myc and hTERT,thus induce differentiation of NB4 cells. But its exact mechanisms and the relationship of signal transduction pathways need to be furtherly confirmed.