Differentiation Induction Effect Of4-amino-2-trifluoromethyl-phenyl Retinate On Human Breast Cancer MCF-7Cell And Its Possible Mechanisms

As one of the differentiation inducing agents, all-trans retinoic acid （ATRA） hasgenerally been applied to cure acute promyelocytic leukemia successfully, with thegood function of inducing tumor cancer cells to a higher degree of mature.However,the side-effects of ATRA including retinoic acid syndrome and RA resistance limitedits further application in the clinical.4-Amino-2-Trifluoromethyl-Phenyl Retinate（ATPR） is one of the retinoid derivatives designed and synthesized in our team withindependent intellectual property right （Patent number: CN101591280,CN102008443A）, and its anti-proliferation and differentiation induction effects havebeen demonstrated in our previous studies. With high expression of retinoic acidreceptors（RARs）, human breast cancer cell MCF-7was choosed in the present studyto further investigate the anti-tumor effect of ATPR and explore the possiblemechanisms.Objective: To investigate the proliferation inhibition and differentiation inductionactivities of4-amino-2-trifluoromethyl-phenyl retinate （ATPR） on human breastcancer MCF-7cells and its possible mechanisms.Methods:1. MCF-7cells were incubated without （control） or with ATPR (10^-4、10^-5、10^-6、 10^-7、10^-8、10^-9mol/L) or ATRA (10^-5mol/L) for72hours, cell proliferations wereassessed by3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyl tetrazolium bromide （MTT）assay. Cell growth curves were made by counting cells. Morphologic changes wereobserved using inverted phase contrast microscope following Wright-Giemsa staining.The differentiation maker mucin1（MUC-1） was measured by enzyme linkedimmunosorbent assay （ELISA）. The cell cycles were analyzed by flow cytometry.2. MCF-7cells were devided into control group （adding volume DMEM）, alcoholgroup （containing0.05%ethanol）, ATRA group (10^-5mol/L, positive control), andATPR group (10^-5、10^-6、10^-7、10^-8、10^-9mol/L). After the treatment with ATPR orATRA on MCF-7cells for72h, the mRNA expression of retinoic acid receptors（RARα、RARβ、RARγ）, estrogen receptors （ERα and ERβ） and RRIG1were detectedby RT-PCR, the protein expression of retinoic acid receptors （RARα、RARβ、RARγ）were measured by western blotting.3. RRIG1gene of MCF-7cell was silenced by RNA interference technique, ATPRwas added with the final concentration10^-5mol/L6h later, and the expression of ERs、ERK1/2and p38were tested using western blotting..Results:1. The growth of MCF-7cells 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 appeared at48hours andcome to climax in72hours. The inhibitory effect was enhanced with the doseincreasing. The inhibitory efficiency of ATPR (10^-4mol/L) was significantly higherthan other groups and the cells in this group were shrunken and appeared to apoptosis.MCF-7cells treated with ATPR (10^-5、10^-6、10^-7、10^-8、10^-9mol/L) showed higherdegrees of mature and differentiation than control group. Compared with the alcoholgroup, the concentration of MUC-1was significantly decreased in ATPR (10^-5、10^-6、10^-7、10^-8、10^-9mol/L) groups. G0/G1-phase cells were significantly increased while S-phase cells were decreased in ATPR group with the concentration ranging from10^-9mol/L to10^-5mol/L, and the progression of cell cycle was blocked in theG0/G1-phase.2. Compared with that in the alcohol group, the mRNA expression of RARβ, ERβ andRRIG1were increased （P<0.05） while the mRNA expression of RARγ was decreased（P<0.05） in MCF-7cells treated with ATPR (10^-5、10^-6、10^-7、10^-8、10^-9mol/L) for72h. There was no significant difference of RARα and ERα mRNA expressionbetween groups. The results of western blotting also showed that compared with thealcohol group, the expression of RARβ （P<0.05） increased while the expression ofRARγ was decreased （P<0.05） after treatment of ATPR. There was no significantdifference of RARα protein expression between groups in MCF-7cells after thetreatment with ATPR (10^-5mol/L).3. Six hours after RRIG1gene targeted silenced, MCF-7cells were treated withATPR (10^-5mol/L) or ATRA (10^-5mol/L) for72h. RT-PCR results showed that theERβ mRNA expressions of siRNA+ATPR group was significantly decreased （P<0.05）than ATPR group while there was no significant difference of ERα mRNA expressionbetween groups. Western blotting results showed that compared with the ATPR group,the protein expression of p-ERK1/2was increased （P<0.05） while the proteinexpression of p-p38was decreased （P<0.05） in the siRNA+ATPR group.Conclusion:1. ATPR(10^-4、10^-5、10^-6、10^-7、10^-8、10^-9mol/L) could inhibit the proliferation andinduce differentiation in some degree in breast cancer MCF-7cells in vitro.2. The mechanism of the anti-proliferation and differentiation induction effects ofATPR might be involved with its function in mediating the balance between RARsand ERs, and up-regulating the expression of RRIG1. 3. The effect of RRIG1on the expression of ERs might be involved with mediatingthe MAPK signal transduction pathway.