ABSTRACT
Ridaifen-G (RID-G), a tamoxifen analog that we previously synthesized, has potent growth inhibitory activity against various cancer cell lines. Tamoxifen is an anticancer drug known to act on an estrogen receptor (ER) and other proteins. However, our previous studies interestingly suggested that the mechanism of action of RID-G was different from that of tamoxifen. In order to investigate the molecular mode of action of RID-G, we developed a novel chemical genetic approach that combined a phage display screen with a statistical analysis of drug potency and gene expression profiles in thirty-nine cancer cell lines. Application of this method to RID-G revealed that three proteins, calmodulin (CaM), heterogeneous nuclear ribonucleoproteins A2/B1 (hnRNP A2/B1), and zinc finger protein 638 (ZNF638) were the candidates of direct targets of RID-G. Moreover, cell lines susceptible to RID-G show similar expression profiles of RID-G target genes. These results suggest that RID-G involves CaM, hnRNP A2/B1, and ZNF638 in its growth inhibitory activity.
Subject(s)
Antineoplastic Agents/chemistry , Tamoxifen/analogs & derivatives , Antineoplastic Agents/metabolism , Antineoplastic Agents/pharmacology , Calmodulin/antagonists & inhibitors , Calmodulin/metabolism , Cell Line, Tumor , Cell Survival/drug effects , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/metabolism , Drug Screening Assays, Antitumor , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/antagonists & inhibitors , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/genetics , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/metabolism , Humans , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/metabolism , Patch-Clamp Techniques , Peptide Library , Phosphorylation , Protein Binding , RNA-Binding Proteins , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Tamoxifen/chemistry , Tamoxifen/metabolism , Tamoxifen/pharmacology , Transcription Factors , Transcriptome/drug effectsABSTRACT
Autophagy is a self-proteolysis process in eukaryotic cells that results in the sequestering of intracellular proteins and organelles in autophagosomes. Activation of autophagy progress continued growth of some tumors, instead extensive autophagy induces cell death. In a previous study, we synthesized a novel tamoxifen derivative, Ridaifen (RID)-B. RID-B induced mitochondria-involved apoptosis even in estrogen receptor (ER)-negative cells. Since tamoxifen induces autophagy other than apoptosis, we treated ER-negative Jurkat cells with RID-B in the present study. RID-B treatment induced apoptosis and LC3 and lysosome colocalization, which results in the formation of autolysosomes. Western blotting revealed that LC3 was converted to LC3-I to LC3-II with RID-B treatment, suggesting that RID-B induced autophagy without ER involvement. Moreover, overexpression of the anti-apoptotic protein Bcl-2 suppressed the RID-B-induced cell death, but not the induction of autophagy. These results presumed that RID-B-induced autophagy is independent of Bcl-2, making RID-B-induced autophagy different from RID-B-induced apoptosis. Since Beclin 1 level is unchanged during RID-B treatment, RID-B induced autophagy pathway is Bcl-2/Beclin1 independent noncanonical pathway.
Subject(s)
Autophagy/drug effects , Proto-Oncogene Proteins c-bcl-2/metabolism , Pyrrolidines/administration & dosage , Receptors, Estrogen/metabolism , Tamoxifen/analogs & derivatives , Antineoplastic Agents, Hormonal/administration & dosage , Humans , Jurkat Cells , Tamoxifen/administration & dosageABSTRACT
In a survey of nonpeptide noncovalent inhibitors of the human 20S proteasome, we found that a novel tamoxifen derivative, RID-F (compound 6), inhibits all three protease activities of the proteasome at submicromolar levels. Structure-activity relationship studies revealed that a RID-F analog (RID-F-S*4, compound 25) is the smallest derivative compound capable of inhibiting proteasome activity, with a potency similar to that of RID-F. Kinetic analyses of the inhibition mode and competition experiments involving biotin-belactosin A (a proteasome inhibitor) binding indicated that the RID-F derivatives interact with the protease subunits in a different manner. Culturing of human cells with these compounds resulted in accumulation of ubiquitinated proteins and induction of apoptosis. Thus, the RID-F derivatives may be useful lead chemicals for the generation of a new class of proteasome inhibitors.
Subject(s)
Proteasome Inhibitors/chemistry , Proteasome Inhibitors/pharmacology , Tamoxifen/analogs & derivatives , Tamoxifen/pharmacology , Apoptosis/drug effects , Cell Line, Tumor , HEK293 Cells , HeLa Cells , Humans , Molecular Docking Simulation , Proteasome Endopeptidase Complex/chemistry , Proteasome Endopeptidase Complex/metabolism , Structure-Activity RelationshipABSTRACT
Tamoxifen is an anticancer agent widely used for treatment of estrogen receptor (ERα)-positive breast cancer. We previously developed a novel synthesis of tamoxifen and its derivatives, named Ridaifens (RIDs). Some of them, including RID-SB8, exhibited a stronger anticancer activity than tamoxifen in ERα-positive MCF-7 cells while having lost the affinity for ERα, suggesting an ERα-independent anticancer mode of action. In this study, we investigated the underlying mechanism by which RID-SB8 exerts anticancer activity. As expected, anticancer activity of RID-SB8 was not influenced upon knockdown of ERα expression in MCF-7 cells. RID-SB8 exerted similar anticancer effects on thirteen ERα-negative cancer cell lines including human gliosarcoma SF539 cells. In SF539 cells, RID-SB8 triggered loss of mitochondrial membrane potential (ΔΨ(m)) and progression of apoptosis accompanied by activation of caspases and translocation of apoptosis-inducing factor (AIF) to the nucleus. Furthermore, it induced reactive oxygen species (ROS), and a ROS scavenger, N-acetylcysteine (NAC), canceled loss of ΔΨ(m) and progression of apoptosis triggered by RID-SB8. Using fifteen human cancer cell lines, we demonstrated a significant correlation between RID-SB8 concentration required for ROS production and that required for cytotoxic effect across these cell lines, but such correlation was not observed for tamoxifen. Finally, the selective induction of ROS and cytotoxic effect on cancer cells by RID-SB8 were confirmed. From these results, we concluded that RID-SB8 exerts an anticancer effect via a mode of action distinct from tamoxifen, and that RID-SB8 could become a promising anticancer lead compound which selectively induces ROS formation and apoptosis in cancer cells.