ROS play an important role in ATPR inducing differentiation and inhibiting proliferation of leukemia cells by regulating the PTEN/PI3K/AKT signaling pathway

BACKGROUND: Acute myeloid leukemia (AML) is an aggressive and mostly incurable hematological malignancy with frequent relapses after an initial response to standard chemotherapy. Therefore, novel therapies are urgently required to improve AML clinical outcomes. 4-Amino-2-trifluoromethyl-phenyl retinate (ATPR), a novel all-trans retinoic acid (ATRA) derivative designed and synthesized by our team, has been proven to show biological anti-tumor characteristics in our previous studies. However, its potential effect on leukemia remains unknown. The present research aims to investigate the underlying mechanism of treating leukemia with ATPR in vitro. METHODS: In this study, the AML cell lines NB4 and THP-1 were treated with ATPR. Cell proliferation was analyzed by the CCK-8 assay. Flow cytometry was used to measure the cell cycle distribution and cell differentiation. The expression levels of cell cycle and differentiation-related proteins were detected by western blotting and immunofluorescence staining. The NBT reduction assay was used to detect cell differentiation. RESULTS: ATPR inhibited cell proliferation, induced cell differentiation and arrested the cell cycle at the G0/G1 phase. Moreover, ATPR treatment induced a time-dependent release of reactive oxygen species (ROS). Additionally, the PTEN/PI3K/Akt pathway was downregulated 24 h after ATPR treatment, which might account for the anti-AML effects of ATPR that result from the ROS-mediated regulation of the PTEN/PI3K/AKT signaling pathway. CONCLUSIONS: Our observations could help to develop new drugs targeting the ROS/PTEN/PI3K/Akt pathway for the treatment of AML.

The proteomics research of 4-amino-2-trifluoromethyl-phenyl retinate on human leukemia K562 cells / 中国药理学通报

Aim To explore the proteomics mechanism of the differentiation induction effect of 4-amino-2-trif-luoromethyl-phenyl retinate(ATPR)on human leukemi-a K562 cells. Methods Human leukemia K562 cells were incubated with the same concentration (1 × 10 - 6 mol·L - 1 ) of ATPR or ATRA for 48 hours. The total cell proteins were collected, purified and digested by trypsin, solid phase extraction, and the peptides were detected by ESI-LC-MS / MS. The difference of the pro-tein expression between the cells treated with ATPR and ATRA was compared by using the Discoverer Pro-teome 1. 2 software, and the molecular function, the biological process and other information of those pro-teins were analyzed based on the DAVID, KEGG, STRING databases. Results 120 specific proteins were identified only in the ATPR group, 143 only in the ATRA group, and 422 other proteins in both groups. Results of DAVID analysis showed that ATPR-induced specific proteins were mainly involved in 39 biological processes of proteins and macromolecules metabolism, protein transport and localization and so on. Results of KEGG analysis revealed that ATPR-in-duced proteins participated in signal pathways, mainly metabolic pathways, PI3K-Akt signal pathway, TGF-beta signal pathway and other pathways in cancer. String protein interaction network analysis displayed that ATPR-induced proteins, like EIF3A, EIF6, RPL3, RPL8, RPL13, RPL7A, RPL21, RPS3, RPS14, NACA, BTF3, NHP2L1, PPP2CA proteins had direct interactions with more than or equal to 10 associated proteins. Conclusion The differentiation induction effect of ATPR on K562 cells might be as-cribed to the ATPR-induced proteins interaction net-work and the specific central proteins it induced, which are involved in the regulation of cell prolifera-tion, differentiation and apoptosis.

Inducing effect of 4-Amino-2-Trifluoromethyl-Phenyl Retinate on differentiation of human breast cancer MDA-MB-231 cell and its possible mechanisms / 中国药理学通报

Aim To investigate the effect of 4-Amino- 2-Trifluoromethyl-Phenyl Retinate on human breast cancer cells MDA-MB-231 and the possible mecha-nisms. Method Human breast cancer MDA-MB-231 cells were incubated with different concentrations of ATPR in vitro. MTT assay was performed to measure the proliferation of MDA-MB-231 . Cell growth curves were made by counting cells and morphologic changes were observed by Wright-Giemsa staining. The differ-entiation marker mucin-1 ( MUC-1 ) was measured by enzyme linked immunosorbent assay ( ELISA ) . Cell cycle was examined by Flow cytometry ( FCM ) . The expression of retinoic acid receptors ( RARs) and reti-noid X receptors ( RXRs ) were detected by Western blot and Quantitative real-time PCR (q-RT-PCR),re-spectively. Results Compared with solvent group, ATPR could inhibit the proliferation of MDA-MB-231 cells in a time-and dose dependent manner and induce the maturing and normality of morphology. The express of MUC-1 was significantly decreased, and the progres of cell cycle was blocked in the G0/G1-phase. The ex-pression of RARγ was decreased. Conclusions AT-PR could inhibit proliferation and induce differention of MDA-MB-231cells, it′s associated with RARγ.

4-amino-2-trifluoromethyl-phenyl retinate induced K562 cell differentiation and cell cycle influence / 中国药理学通报

Aim To explore the effect of 4-amino-2-trifluoromethyl-phenyl retinate(ATPR)on proliferation,differentiation activity in K562 cell line,and to research the mechanisms.Methods Cell proliferation was assessed by MTT assay.Cell differentiation index was analyzed by NBT reduction test.Morphologic changes were observed by Wright's staining in the light microscope. Cell cycle was determined by FCM.The mRNA expression of Cyclin D1,Cyclin E,CDK2,CDK4,CDK6,P21cip1,P27kip1,P57kip2,PCNA mRNA were detected by RT-PCR.While the protein expression of cyclin D1 and CDK4 was detected by Western blot.Results The growth of K562 cells was inhibited in a dose-dependent manner.NBT reduction test indicated that the ATPR could induce differentiation of K562 cells and increase the positive cell ratio.Morphologic changes were observed after Wright's staining using inverted phase contrast microscope.The proportion of cells in G0/G1 phase increased while S phase cells decreased.Cell cycle progression was blocked in the G1 phase.The expression of Cyclin E,cyclin D1,CDK2,CDK4,CDK6 mRNA decreased,while PCNA,P21 cip1,P27 kip1 change was not obvious,but P57 (kip2) mRNA expression was increased.Cyclin D1 and CDK4 protein expressions were reduced as well.Conclusions ATPR inhibits the growth of K562 cells and induces differentiation.P57 kip2 plays a key role in differentiation.Moreover,high level of P57kip2 is regulated via inhibiting its degradation through reducing proteasome-dependent proteolysis,and ATPR plays a role in cell cycle arrest.