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1.
J Biochem ; 175(3): 253-263, 2024 Mar 04.
Article in English | MEDLINE | ID: mdl-37948630

ABSTRACT

Cardiac glycosides (CGs) have been used for decades to treat heart failure and arrhythmic diseases. Recent non-clinical and epidemiological findings have suggested that CGs exhibit anti-tumor activities. Therefore, CGs may be repositioned as drugs for the treatment of cancer. A detailed understanding of the anti-cancer mechanisms of CGs is essential for their application to the treatment of targetable cancer types. To elucidate the factors associated with the anti-tumor effects of CGs, we performed transcriptome profiling on human multiple myeloma AMO1 cells treated with periplocin, one of the CGs. Periplocin significantly down-regulated the transcription of MYC (c-Myc), a well-established oncogene. Periplocin also suppressed c-Myc expression at the protein levels. This repression of c-Myc was also observed in several cell lines. To identify target proteins for the inhibition of c-Myc, we generated CG-resistant (C9) cells using a sustained treatment with digoxin. We confirmed that C9 cells acquired resistance to the inhibition of c-Myc expression and cell proliferation by CGs. Moreover, the sequencing of genomic DNA in C9 cells revealed the mutation of D128N in α1-Na/K-ATPase, indicating the target protein. These results suggest that CGs suppress c-Myc expression in cancer cells via α1-Na/K-ATPase, which provides further support for the anti-tumor activities of CGs.


Subject(s)
Cardiac Glycosides , Humans , Cardiac Glycosides/pharmacology , Cell Line , Cell Proliferation , Gene Expression Profiling , Adenosine Triphosphatases
2.
FEBS Lett ; 596(4): 465-478, 2022 02.
Article in English | MEDLINE | ID: mdl-35076962

ABSTRACT

The c-Myc oncoprotein is frequently overexpressed in human cancers and is essential for cancer cell proliferation. The dysregulation of ubiquitin-proteasome-mediated degradation is one of the contributing factors to the upregulated expression of c-Myc in human cancers. We herein identified USP17 as a novel deubiquitinating enzyme that regulates c-Myc levels and controls cell proliferation and glycolysis. The overexpression of USP17 stabilized the c-Myc protein by promoting its deubiquitination. In contrast, the knockdown of USP17 promoted c-Myc degradation and reduced c-Myc levels. The knockdown of USP17 also suppressed cell proliferation and glycolysis. Collectively, the present results reveal a novel role for USP17 in the regulation of c-Myc stability and suggest its potential as a therapeutic target for cancer treatment.


Subject(s)
Endopeptidases/genetics , Glycolysis/genetics , Proteasome Endopeptidase Complex/metabolism , Proto-Oncogene Proteins c-myc/genetics , Animals , COS Cells , Cell Line, Tumor , Cell Proliferation/genetics , Chlorocebus aethiops , Endopeptidases/metabolism , Epithelial Cells/metabolism , Epithelial Cells/pathology , Gene Expression Regulation, Neoplastic , Glucose/metabolism , Humans , Lactic Acid/metabolism , Proteolysis , Proto-Oncogene Proteins c-myc/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Signal Transduction
3.
Sci Rep ; 11(1): 9528, 2021 05 04.
Article in English | MEDLINE | ID: mdl-33947921

ABSTRACT

The unfolded protein response (UPR) controls protein homeostasis through transcriptional and translational regulation. However, dysregulated UPR signaling has been associated with the pathogenesis of many human diseases. Therefore, the compounds modulating UPR may provide molecular insights for these pathologies in the context of UPR. Here, we screened small-molecule compounds that suppress UPR, using a library of Myanmar wild plant extracts. The screening system to track X-box binding protein 1 (XBP1) splicing activity revealed that the ethanol extract of the Periploca calophylla stem inhibited the inositol-requiring enzyme 1 (IRE1)-XBP1 pathway. We isolated and identified periplocin as a potent inhibitor of the IRE1-XBP1 axis. Periplocin also suppressed other UPR axes, protein kinase R-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF6). Examining the structure-activity relationship of periplocin revealed that cardiac glycosides also inhibited UPR. Moreover, periplocin suppressed the constitutive activation of XBP1 and exerted cytotoxic effects in the human multiple myeloma cell lines, AMO1 and RPMI8226. These results reveal a novel suppressive effect of periplocin or the other cardiac glycosides on UPR regulation, suggesting that these compounds will contribute to our understanding of the pathological or physiological importance of UPR.


Subject(s)
Cardiac Glycosides/pharmacology , Saponins/pharmacology , Unfolded Protein Response/drug effects , Cell Line , Cell Line, Tumor , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum/metabolism , HEK293 Cells , Humans , Periploca/chemistry , Plant Extracts/pharmacology , RNA Splicing/drug effects , Signal Transduction/drug effects , Small Molecule Libraries/pharmacology , X-Box Binding Protein 1/metabolism
4.
J Biol Chem ; 294(44): 16429-16439, 2019 11 01.
Article in English | MEDLINE | ID: mdl-31533987

ABSTRACT

Su(var)3-9, Enhancer-of-zeste, and Trithorax (SET) domain-containing protein 8 (SET8) is the sole enzyme that monomethylates Lys-20 of histone H4 (H4K20). SET8 has been implicated in the regulation of multiple biological processes, such as gene transcription, the cell cycle, and senescence. SET8 quickly undergoes ubiquitination and degradation by several E3 ubiquitin ligases; however, the enzyme that deubiquitinates SET8 has not yet been identified. Here we demonstrated that ubiquitin-specific peptidase 17-like family member (USP17) deubiquitinates and therefore stabilizes the SET8 protein. We observed that USP17 interacts with SET8 and removes polyubiquitin chains from SET8. USP17 knockdown not only decreased SET8 protein levels and H4K20 monomethylation but also increased the levels of the cyclin-dependent kinase inhibitor p21. As a consequence, USP17 knockdown suppressed cell proliferation. We noted that USP17 was down-regulated in replicative senescence and that USP17 inhibition alone was sufficient to trigger cellular senescence. These results reveal a regulatory mechanism whereby USP17 prevents cellular senescence by removing ubiquitin marks from and stabilizing SET8 and transcriptionally repressing p21.


Subject(s)
Cyclin-Dependent Kinase Inhibitor p21/metabolism , Endopeptidases/metabolism , Histone-Lysine N-Methyltransferase/metabolism , Aging/metabolism , Animals , COS Cells , Cell Cycle/physiology , Cell Line , Cell Proliferation/physiology , Chlorocebus aethiops , HCT116 Cells , Histones/metabolism , Humans , MCF-7 Cells , Methyltransferases/metabolism , Protein Processing, Post-Translational , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/metabolism , Ubiquitination/physiology
5.
Molecules ; 24(17)2019 Aug 27.
Article in English | MEDLINE | ID: mdl-31461933

ABSTRACT

In response to cellular stresses, activating transcriptional factor 4 (ATF4) regulates the expression of both stress-relieving genes and apoptosis-inducing genes, eliciting cell fate determination. Since pharmacological activation of ATF4 exerts potent anti-tumor effects, modulators of ATF4 activation may have potential in cancer therapy. We herein attempted to identify small molecules that activate ATF4. A cell-based screening to monitor TRB3 promoter activation was performed using crude drugs used in traditional Japanese Kampo medicine. We found that an extract from Sophora flavescens roots exhibited potent TRB3 promoter activation. The activity-guided fractionation revealed that kurarinone was identified as the active ingredient. Intriguingly, ATF4 activation in response to kurarinone required PKR-like endoplasmic reticulum kinase (PERK). Moreover, kurarinone induced the cyclin-dependent kinase inhibitor p21 as well as cytostasis in cancer cells. Importantly, the cytostatic effect of kurarinone was reduced by pharmacological inhibition of PERK. These results indicate that kurarinone triggers ATF4 activation through PERK and exerts cytostatic effects on cancer cells. Taken together, our results suggest that modulation of the PERK-ATF4 pathway with kurarinone has potential as a cancer treatment.


Subject(s)
Activating Transcription Factor 4/metabolism , Cell Cycle Proteins/genetics , Flavonoids/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Repressor Proteins/genetics , Sophora/chemistry , eIF-2 Kinase/metabolism , Activating Transcription Factor 4/genetics , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Drug Screening Assays, Antitumor , Gene Expression Regulation, Neoplastic/drug effects , HEK293 Cells , HeLa Cells , Humans , Phosphorylation , Promoter Regions, Genetic/drug effects , Protein Serine-Threonine Kinases/genetics , eIF-2 Kinase/genetics
6.
Biol Pharm Bull ; 42(3): 481-488, 2019.
Article in English | MEDLINE | ID: mdl-30828079

ABSTRACT

Lysine-specific demethylase 1 (LSD1/KDM1A) is a histone demethylase and specifically catalyzes the demethylation of mono- and di-methylated histone H3 lysine 4 (H3K4). The LSD1-mediated demethylation of H3K4 promotes the assembly of the c-Myc-induced transcription initiation complex. Although LSD1 and c-Myc are both strongly expressed in human cancers, the mechanisms by which their activities are coordinated remain unclear. We herein demonstrated that LSD1 is a direct target gene of c-Myc. The knockdown of c-Myc decreased the expression of LSD1 in several cancer cell lines. We identified two non-canonical E-boxes in the proximal promoter region of the LSD1 gene. A chromatin immunoprecipitation assay showed that c-Myc bound to these E-boxes in the LSD1 promoter. Importantly, LSD1 mRNA expression correlated with c-Myc expression in human acute myeloid leukemia (AML), glioblastoma, stomach adenocarcinoma, and prostate adenocarcinoma. The present results suggest that LSD1 is induced by c-Myc and forms a positive feedback mechanism in transcription reactions by c-Myc.


Subject(s)
Histone Demethylases/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Cell Line, Tumor , Databases, Factual , Histone Demethylases/genetics , Humans , Neoplastic Stem Cells , Proto-Oncogene Proteins c-myc/genetics , RNA Interference
7.
Molecules ; 23(6)2018 Jun 08.
Article in English | MEDLINE | ID: mdl-29890668

ABSTRACT

The p53 tumor suppressor plays critical roles in cell cycle regulation and apoptotic cell death in response to various cellular stresses, thereby preventing cancer development. Therefore, the activation of p53 through small molecules is an attractive therapeutic strategy for the treatment of cancers retaining wild-type p53. We used a library of 700 Myanmar wild plant extracts to identify small molecules that induce p53 transcriptional activity. A cell-based screening method with a p53-responsive luciferase-reporter assay system revealed that an ethanol extract of Oroxylum indicum bark increased p53 transcriptional activity. Chrysin was isolated and identified as the active ingredient in the O. indicum bark extract. A treatment with chrysin increased p53 protein expression and the p53-mediated expression of downstream target genes, and decreased cell viability in MCF7 cells, but not in p53-knockdown MCF7 cells. We also found that chrysin activated the ATM-Chk2 pathway in the absence of DNA damage. Hence, the inactivation of the ATM-Chk2 pathway suppressed p53 activation induced by chrysin. These results suggest the potential of chrysin as an anti-cancer drug through the activation of p53 without DNA damage.


Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Ataxia Telangiectasia Mutated Proteins/metabolism , Bignoniaceae/chemistry , Checkpoint Kinase 2/metabolism , Flavonoids/pharmacology , Tumor Suppressor Protein p53/metabolism , DNA Damage , Humans , MCF-7 Cells , Plant Extracts/pharmacology , Transcription, Genetic/drug effects , Tumor Suppressor Protein p53/genetics
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