Differentiation Induction Effect Of 4-Amino-2-Trifluoromethyl-phenyl Retinate On Human Breast Cancer Cells Via PTEN/PI3K/AKT Signaling Pathway

Retinoic acid(RA) plays a spectrum of pleiotropic effects in cell growth and differentiation that are relevant for embryonic development and adult physiology. The RA activity is mediated primarily by members of the retinoic acid receptors.RARs form heterodimers with members of the retinoid X receptors(RXRs) subfamily and act as ligand-regulated transcription factors through binding specific RA response elements(RAREs) located in target genes promoters.All-trans retinoic acid(ATRA) as one of the differentiation inducing agents, has already been applied to cure acute promyelocytic leukemia.However, the side-effects of ATRA including retinoic acid syndrome and RA resistancelimitedits further application in the clinical.4-Amino-2-trifluoromethyl-Phenyl Retinate(ATPR) is one of the retinoid derivatives designed and synthesized in our team.The differentiation induction effects have been approved in human breast cancer cells MCF-7.In general,we divide breast cancer into ER positive breast cancer and ER negative breast cancer.In this study, we chose the ER-negative human breast cancer cells MDA-MB-231 to survey the differentiation induction effect of ATPR.Besides, the relationship between ATPR and its receptors will be summarized in this paper.Objective:To investigate the differentiation induction activities of 4-amino-2-trifluoromethyl-phenyl retinate(ATPR) on human breast cancer cells and itspossible mechanisms.Methods:1. MDA-MB-231 cells were incubated with ATPR(10-4-10-9 mol/L) or ATRA(10-5 mol/L) for 72 hours, cell proliferations were assessed by MTT assay. Cell growth curves were observed by counting cells.Morphologic changes were observed by Wright-Giemsa staining. The differentiation maker mucin 1(MUC-1) was detected by enzyme linked immunosorbent assay(ELISA). The cell cycles were analyzed by flow cytometry.2. MDA-MB-231 cells were incubated with Estradiol(10-6mol/L),ATPR or ATRA combine with E2 and different concentrations of ATPR combine with E2 for 72 h,morphologic changes were observed by wright-giemsa staining.The differentiation maker mucin 1(MUC-1) was detected by enzyme linked immunosorbent assay(ELISA). The cell cycles were analyzed by flow cytometry.3. MCF-7 cells and MDA-MB-231 cells were devided into control group(DMEM), alcohol group(containing 0.05% ethanol), ATRA group(10-5 mol/L, positive control), and ATPR group(10-5mol/L). After treat with ATPR or ATRA on cells for 72 h, the m RNA expression of retinoic acid receptors(RARα、RARβ、RARγ) and retinoid X acid receptors(RXRα、RXRβ、RXRγ) were detected by q-RT-PCR, the protein expression of retinoic acid receptors(RARα、RARβ、RARγ) and retinoid X acid receptor(RXRα、RXRβ、RXRγ) were measured by western blotting.4. The expression of Estorgen receptors(ERα、ERβ)、PTEN、AKT and p-AKT were tested using western blotting.Results:1. The growth of MDA-MB-231 cells was inhibited after the treatment with ATPR(10-4,10-5,10-6,10-7,10-8,10-9mol/L) in vitro. The inhibitory effect come to climax in 72 hours. The inhibitory effect was enhanced with the dose increasing.Compared with the solvent group, The morphologic changes was enhanced with the dose increasing in ATPR group.The concentration of MUC-1 was significantly decreased in ATPR or ATRA alone groups.G0/G1-phase cells were significantly increased while S-phase cells were decreased in ATPR or ATRA groups.2.Differentiation induction effect of ATPR under the stimulation of E2 in MDA-MB-231 cells.Compared with the solvent group, the E2 group were not have change.In the ATPR(10-6~10-9mol/L)+E2 groups,The morphologic changes was enhanced with the dose increasing. The concentration of MUC-1 was no change in E2 group.The MUC-1 was enhanced with the dose decreasing in ATRP+E2 groups.The progression of cell cycle was blocked in the G0/G1-phase in ATPR+E2 groups.3. Compared with the alcohol group,the m RNA expression of RARβ and RXRα were increased while the m RNA expression of RARγ was decreased in MCF-7 cells. There were no difference of RARα, RXRβ and RXRγ.The m RNA expression of RARγ were decreased in MDA-MB-231 cells.There were no difference of RARα, RARβ, RXRα, RXRβ and RXRγ.The results of western blotting also showed the same.4. Compared with the alcohol group,Western-blot results showed that the expression of ERβ were higher,with no significant difference of the expressions of ERα in MCF-7 and MDA-MB-231 cells.5. Compared with the alcohol group,Western-blot results showed that the expression of PTEN evidently increased while the phosphorylation AKT were retardarced in MCF-7and MDA-MB-231 cells.Conclusion:1. ATPR could inhibit the proliferation and induce differentiation in in breast cancer cells MDA-MB-231 in vitro.2. 2.The effect of ATPR on the expression of RARs and RXRs are different in ER different human breast cancer cells.3. ATPR induced markedly changes in PTEN and PI3K/AKT Signaling pathway.