ID3 is a novel target gene of p53 and modulates lung cancer cell metastasis.

The tumor suppressor p53 prevents cancer development by regulating dozens of target genes with diverse biological functions. Although numerous p53 target genes have been identified to date, the dynamics and function of the regulatory network centered on p53 have not yet been fully elucidated. We herein identified inhibitor of DNA-binding/differentiation-3 (ID3) as a direct p53 target gene. p53 bound the distal promoter of ID3 and positively regulated its transcription. ID3 expression was significantly decreased in clinical lung cancer tissues, and was closely associated with overall survival outcomes in these patients. Functionally, ID3 deficiency promoted the metastatic ability of lung cancer cells through its effects on the transcriptional regulation of CDH1. Furthermore, the ectopic expression of ID3 in p53-knockdown cells restored E-cadherin expression. Collectively, the present results demonstrate that ID3 plays a tumor-suppressive role as a downstream effector of p53 and impedes lung cancer cell metastasis by regulating E-cadherin expression.

Hypothermia Attenuates Neurotoxic Microglial Activation via TRPV4.

Therapeutic hypothermia (TH) provides neuroprotection. However, the cellular mechanisms underlying the neuroprotective effects of TH are not fully elucidated. Regulation of microglial activation has the potential to treat a variety of nervous system diseases. Transient receptor potential vanilloid 4 (TRPV4), a nonselective cation channel, is activated by temperature stimulus at 27-35 °C. Although it is speculated that TRPV4 is associated with the neuroprotective mechanisms of TH, the role of TRPV4 in the neuroprotective effects of TH is not well understood. In the present study, we investigated whether hypothermia attenuates microglial activation via TRPV4 channels. Cultured microglia were incubated under normothermic (37 °C) or hypothermic (33.5 °C) conditions following lipopolysaccharide (LPS) stimulation. Hypothermic conditions suppressed the expression of pro-inflammatory cytokines, inducible nitric oxide synthase, and the number of phagocytic microglia. AMP-activated protein kinase (AMPK)-NF-κB signaling was inhibited under hypothermic conditions. Furthermore, hypothermia reduced neuronal damage induced by LPS-treated microglial cells. Treatment with TRPV4 antagonist in normothermic culture replicated the suppressive effects of hypothermia on microglial activation and microglia-induced neuronal damage. In contrast, treatment with a TRPV4 agonist in hypothermic culture reversed the suppressive effect of hypothermia. These findings suggest that TH suppresses microglial activation and microglia-induced neuronal damage via the TRPV4-AMPK-NF-κB pathway. Although more validation is needed to consider differences according to age, sex, and specific central nervous system regions, our findings may offer a novel therapeutic approach to complement TH.

AMPK activators suppress cholesterol accumulation in macrophages via suppression of the mTOR pathway.

Atherosclerosis is a persistent inflammatory state that contributes significantly to cardiovascular disease, a primary cause of mortality worldwide. Enhanced lipid uptake by macrophages and their transformation into foam cells play a key role in the development of atherosclerosis. Recent studies using in vivo mouse models indicated that activation of AMPK has anti-atherosclerotic effects by upregulating the expression of cholesterol efflux transporters in foam cells and promoting cholesterol efflux. However, the pathway downstream of AMPK that contributes to elevated expression of cholesterol efflux transporters remains unclear. In this study, we found that activation of AMPK by AICAR and metformin inhibits foam cell formation via suppression of mTOR in macrophages. Specifically, activation of AMPK indirectly reduced the phosphorylation level of mTOR at Ser2448 and promoted the expression of cholesterol efflux transporters and cholesterol efflux. These inhibitory effects on foam cell formation were counteracted by mTOR activators. Metformin, a more nonspecific AMPK activator than AICAR, appears to inhibit foam cell formation via anti-inflammatory effects in addition to suppression of the mTOR pathway. The results of this study suggest that the development of new drugs targeting AMPK activation and mTOR inhibition may lead to beneficial results in the prevention and treatment of atherosclerosis.

Monocyte-Derived miRNA-1914-5p Attenuates IL-1ß-Induced Monocyte Adhesion and Transmigration.

Atherosclerosis can lead to cardiovascular and cerebrovascular diseases. Atherosclerotic plaque formation is promoted by the accumulation of inflammatory cells. Therefore, modulating monocyte recruitment represents a potential therapeutic strategy. In an inflammatory state, the expression of adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) is upregulated in endothelial cells. We previously reported that miR-1914-5p in endothelial cells suppresses interleukin (IL)-1ß-induced ICAM-1 expression and monocyte adhesion to endothelial cells. However, whether monocyte miR-1914-5p affects monocyte recruitment is unclear. In this study, IL-1ß decreased miR-1914-5p expression in a human monocyte cell line. Moreover, miR-1914-5p inhibition enhanced adhesion to endothelial cells with the upregulation of macrophage-1 antigen (Mac-1), a counter-ligand to ICAM-1. Transmigration through the endothelial layer was also promoted with the upregulation of monocyte chemotactic protein-1 (MCP-1). Furthermore, a miR-1914-5p mimic suppressed IL-1ß-induced monocyte adhesion and transmigration in monocytes with Mac-1 and MCP-1 downregulation. Further investigation of miR-1914-5p in monocytes could lead to the development of novel diagnostic markers and therapeutic strategies for atherosclerosis.

The ubiquitin-specific protease USP17 prevents cellular senescence by stabilizing the methyltransferase SET8 and transcriptionally repressing p21.

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.

Development of Cell-Penetration PG-Surfactants and Its Application in External Peptide Delivery to Cytosol.

Recently, development of techniques to deliver pharmacologically active biomacromolecules such as peptides and proteins to cytosol has gained much interest. Here, we applied the peptide gemini (PG)-surfactants to a novel platform to design cell penetration lipopeptides (CP-PGs), which can deliver exogenous peptides and proteins to cytosol. Among the number of candidate CP-PGs having different peptide sequences at the X-, Y-, and Z-positions, we focused on those having two C12 alkyl chains appended to the side chain of two Cys residues, the betaine sequence -Asp-Lys-Asp-Lys- between the alkylated Cys residues (i.e., at the X-position), and having different cationic peptide sequences of oligo-Lys or oligo-Arg at the Y- and/or Z-positions. With respect to cytotoxicity for mammalian cells such as NIH3T3 cells upon 1 h exposure, those having (Lys)3 (K3-DKDKC12 and DKCK12-K3) showed lower cytotoxicity (IC50 = 241 and 198 µM) among those having oligo-Lys, (Lys)n (n = 1, 3, 5; IC50 = 88-197 µM). Similar lower cytotoxicity was also observed for the CP-PG having two (Lys)3 at both N- and C-terminal sides (K3-DKDKC12-K3) (IC50 = 225 µM). In contrast, the CP-PG having (Arg)3 at the N-terminal side (R3-DKDKC12) showed higher cytotoxicity (IC50 = 88 µM). Carrier abilities of the CP-PGs for exogenous peptides were evaluated using the proapoptotic domain (PAD) peptide, which induces apoptosis by disturbing mitochondrial membranes after delivery into cytosol. As a result, the CP-PGs of K3-DKDKC12, DKCK12-K3, K3-DKDKC12-K3, DKCK12-K5, and R3-DKDKC12 exhibited micromolar range carrier ability (the necessary half concentration to induce cell death (EC50) by delivering PAD peptide to cytosol was 10, 6.2, 8.5, 5.8, and 11.5 µM, respectively). Especially, the carrier abilities of DKCK12-K3 and DKCK12-K5 were superior to the well-established cell penetration Arg-rich R8 peptide (EC50 = 6.8 µM). Together, our results indicate that the PG-surfactant molecular framework could be a potential new platform to design efficient cell penetration carrier materials.

An irreversible inhibitor of peptidyl-prolyl cis/trans isomerase Pin1 and evaluation of cytotoxicity.

Pin1 (protein interacting with never in mitosis A-1) is a member of the peptidyl prolyl isomerase (PPIase) family, and catalyzes cis-trans isomerization of pThr/Ser-Pro amide bonds. Because Pin1 is overexpressed in various cancer cell lines and promotes cell growth, it is considered a target for anticancer agents. Here, we designed and synthesized a covalently binding Pin1 inhibitor (S)-2 to target Pin1's active site. This compound inhibited Pin1 in protease-coupled assay, and formed a covalent bond with Cys113 of Pin1, as determined by ESI-MS. The acetoxymethyl ester of (S)-2, i.e., 6, suppressed cyclin D1 expression in human prostate cancer PC-3 cells, and exhibited cytotoxicity. Pin1-knockdown experiments indicated that a target for the cytotoxicity of 6 is Pin1.

Lysine-Specific Demethylase 1 (LSD1/KDM1A) Is a Novel Target Gene of c-Myc.

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.

Kurarinone from Sophora Flavescens Roots Triggers ATF4 Activation and Cytostatic Effects Through PERK Phosphorylation.

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.

Anti-Tumorigenic Activity of Chrysin from Oroxylum indicum via Non-Genotoxic p53 Activation through the ATM-Chk2 Pathway.

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.

Pseudokinase tribbles 1 (TRB1) negatively regulates tumor-suppressor activity of p53 through p53 deacetylation.

Tribbles 1 (TRB1) is one of the mammalian orthologs of Drosophila Tribbles, which regulates development and cell proliferation. TRB1 is suggested to act as a scaffold protein in signaling pathways for important cellular processes. TRB1 has also been identified as a myeloid oncogenic driver and mediates leukemogenesis through the mitogen-activated protein extracellular kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway and CCAAT/enhancer binding protein (C/EBP) transcriptional factors. However, the physiological roles of TRB1 in solid tumors have not been clarified. Here, we show that TRB1 interacts with p53 and suppresses its tumor suppressor activity. TRB1 knockdown enhances transcriptional activity of p53 and decreases cell viability. Interestingly, TRB1 enhances histone deacety lase 1 (HDAC1)-mediated p53 deacetylation and decreases DNA binding of p53. These results suggest that TRB1 is involved in the proliferation of tumor cells by inhibiting the activities of tumor suppressor p53 in solid tumors.

Positive Regulation of Interleukin-2 Expression by a Pseudokinase, Tribbles 1, in Activated T Cells.

Tribbles 1 (TRB1), a member of the Tribbles family, is a pseudokinase that is conserved among species and implicated in various human diseases including leukemia, cardiovascular diseases, and metabolic disorders. However, the role of TRB1 in the immune response is not understood. To evaluate this role, we examined regulation of TRB1 expression and the function of TRB1 in interleukin-2 (IL-2) induction in Jurkat cells, a human acute T cell leukemia cell line. We found that TRB1 was strongly induced by phorbol 12-myristate 13-acetate (PMA) and ionomycin in these cells. IL-2 expression was induced in Jurkat cells activated by PMA and ionomycin; however, knockdown of TRB1 resulted in decreased induction of IL-2. TRB1 null Jurkat cells established using the CRISPR/Cas9 system also showed reduction of IL-2 expression on PMA/ionomycin stimulation. TRB1 knockdown also markedly inhibited IL-2 promoter activation. To determine the mechanism of the stimulatory effect on IL-2 induction, we focused on histone deacetylases (HDACs), and found that HDAC1 preferentially interacts with TRB1. TRB1 suppressed the interaction of HDAC1 with nuclear factor of activated T cells 2 (NFAT2), which is a crucial transcription factor for IL-2 induction. These results indicate that TRB1 is a positive regulator of IL-2 induction in activated T cells.

TGF-ß decreases the stability of IL-18-induced IFN-γ mRNA through the expression of TGF-ß-induced tristetraprolin in KG-1 cells.

We have previously reported that transforming growth factor-ß (TGF-ß) down-regulates interferon-γ (IFN-γ) production in an interleukin-18 (IL-18) treated mouse natural killer (NK) cell line, LNK5E6. In LNK5E6 cells, TGF-ß exhibited no inhibition of the IL-18-induced transcription of IFN-γ, but did stimulate the degradation of IFN-γ mRNA induced by IL-18. In the present study, we investigated the mechanism of the down-regulatory effects of TGF-ß on IFN-γ mRNA expression in a human myelomonocytic cell line, KG-1, which produces IFN-γ in response to IL-18 alone. Interestingly, IL-18 induced the production of the IFN-γ through the stabilization of IFN-γ mRNA, but not the enhanced transcription of IFN-γ gene. The stability of IFN-γ mRNA was regulated by mRNA destabilizing elements in the 3'untranslated region (UTR) of IFN-γ mRNA, especially adenylate-uridylate (AU)-rich elements (AREs) in the 5' half of 3'UTR. Tristetraprolin (TTP), one of the ARE-binding proteins, destabilizes IFN-γ mRNA, and IL-18 repressed the expression of TTP mRNA. Moreover, TGF-ß repressed the IL-18-induced expression of IFN-γ mRNA through the induction of TTP mRNA to destabilize IFN-γ mRNA. Our data is the first to reveal that the crosstalk between IL-18 and TGF-ß through the expression of TTP regulates the production of IFN-γ.

Delivery of external proteins into the cytoplasm using protein capsules modified with IgG on the surface, created from the amphiphilic two helix-bundle protein OLE-ZIP.

This study explores a new method for delivering therapeutic proteins into specific cells using OLE-ZIP capsules that present IgG. OLE-ZIP capsules is a spherical caspules prepared from amphihilic dimetic coiled-coil peptide, OLE-ZIP. Upon presenting cetuximab, these capsules showed preferential uptake in A431 cells and increased cytotoxicity when loaded with RNase A.

Thrombopoietin exerts a neuroprotective effect by inhibiting the suppression of neuronal proliferation and axonal outgrowth in intrauterine growth restriction rats.

Chronic hypoxia in utero causes intrauterine growth restriction (IUGR) of the fetus. IUGR infants are known to be at higher risk for neurodevelopmental disorders, but the mechanism is unclear. In this study, we analyzed the structure of the cerebral cortex using IUGR model rats generated through a reduced uterine perfusion pressure operation. IUGR rats exhibited thinner cerebral white matter and enlarged lateral ventricles compared with control rats. Expression of neuron cell markers, Satb2, microtubule-associated protein (MAP)-2, α-tubulin, and nestin was reduced in IUGR rats, indicating that neurons were diminished at various developmental stages in IUGR rats, from neural stem cells to mature neurons. However, there was no increase in apoptosis in IUGR rats. Cells positive for Ki67, a marker of cell proliferation, were reduced in neurons and all glial cells of IUGR rats. In primary neuron cultures, axonal elongation was impaired under hypoxic culture conditions mimicking the intrauterine environment of IUGR infants. Thus, in IUGR rats, chronic hypoxia in utero suppresses the proliferation of neurons and glial cells as well as axonal elongation, resulting in cortical thinning and enlarged lateral ventricles. Thrombopoietin (TPO), a platelet growth factor, inhibited the decrease in neuron number and promoted axon elongation in primary neurons under hypoxic conditions. Intraperitoneal administration of TPO to IUGR rats resulted in increases in the number of NeuN-positive cells and the area coverage of Satb2. In conclusion, suppression of neuronal proliferation and axonal outgrowth in IUGR rats resulted in cortical thinning and enlargement of lateral ventricles. TPO administration might be a novel therapeutic strategy for treating brain dysmaturation in IUGR infants.

Involvement of cardiac glycosides targeting Na/K-ATPase in their inhibitory effects on c-Myc expression via its transcription, translation and proteasomal degradation.

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.

Corosolic acid attenuates platelet-derived growth factor signaling in macrophages and smooth muscle cells of pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a progressive and life-threatening disease that is characterized by vascular remodeling of the pulmonary artery. Pulmonary vascular remodeling is primarily caused by the excessive proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs), which are facilitated by perivascular inflammatory cells including macrophages. Corosolic acid (CRA) is a natural pentacyclic triterpenoid that exerts anti-inflammatory effects. In the present study, the effects of CRA on the viability of macrophages were examined using monocrotaline (MCT)-induced PAH rats and human monocyte-derived macrophages. Although we previously reported that CRA inhibited signal transducer and activator of transcription 3 (STAT3) signaling and ameliorated pulmonary vascular remodeling in PAH, the inhibitory mechanism remains unclear. Therefore, the underlying mechanisms were investigated using PASMCs from idiopathic PAH (IPAH) patients. In MCT-PAH rats, CRA inhibited the accumulation of macrophages around remodeled pulmonary arteries. CRA reduced the viability of human monocyte-derived macrophages. In IPAH-PASMCs, CRA attenuated cell proliferation and migration facilitated by platelet-derived growth factor (PDGF)-BB released from macrophages and PASMCs. CRA also downregulated the expression of PDGF receptor ß and its signaling pathways, STAT3 and nuclear factor-κB (NF-κB). In addition, CRA attenuated the phosphorylation of PDGF receptor ß and STAT3 following the PDGF-BB simulation. The expression and phosphorylation levels of PDGF receptor ß after the PDGF-BB stimulation were reduced by the small interfering RNA knockdown of NF-κB, but not STAT3, in IPAH-PASMCs. In conclusion, CRA attenuated the PDGF-PDGF receptor ß-STAT3 and PDGF-PDGF receptor ß-NF-κB signaling axis in macrophages and PASMCs, and thus, ameliorated pulmonary vascular remodeling in PAH.

Design of Cell-Adhesive Shellac Derivatives and Endowment of Photoswitchable Cell-Adhesion Properties.

The emergence of new biodegradable cell-adhesion materials is an attractive topic in biomaterial chemistry, particularly for the development of cell incubation scaffolds and drug encapsulation materials used in in situ regenerative therapy. Shellac is a natural resin with unique film-forming properties and high miscibility with various chemicals, in addition to being biodegradable and nontoxic to biological systems. However, since native shellac does not adhere to mammalian cells, there have been no reports of using shellac to develop cell-adhesive biomaterials. In this study, we report on the development of cell-adhesive shellac derivatives through slight chemical modification. Shellac is a mixture of oligoesters that consists of hydroxyl fatty acids and resin acids, and therefore, all oligomers have one carboxylic acid group at the terminal. We discovered that a simple modification of hydrophobic chemical groups, particularly those containing aromatic groups in the ester form, could dramatically improve cell-adhesion properties for mammalian cells. Furthermore, by using photocleavable esters containing aromatic groups, we successfully endowed photoswitchable properties in cell adhesion. Given that shellac is a low-cost, biodegradable, and nontoxic natural resin, the modified shellacs have the potential to become new and attractive biomaterials applicable to in situ regenerative therapy.

The Combination of ATM and Chk1 Inhibitors Induces Synthetic Lethality in Colorectal Cancer Cells.

Genetic abnormalities induce the DNA damage response (DDR), which enables DNA repair at cell cycle checkpoints. Although the DDR is thought to function in preventing the onset and progression of cancer, DDR-related proteins are also thought to contribute to tumorigenesis, tumor progression, and drug resistance by preventing irreparable genomic abnormalities from inducing cell death. In the present study, the combination of ataxia telangiectasia-mutated serine/threonine kinase (ATM) and checkpoint kinase 1 (Chk1) inhibition exhibited synergistic antitumor effects and induced synergistic lethality in colorectal cancer cells at a low dose. The ATM and Chk1 inhibitors synergistically promoted the activation of cyclin-dependent kinase 1 by decreasing the phosphorylation levels of T14 and Y15. Furthermore, the combined treatment increased the number of sub-G1-stage cells, phospho-histone H2A.X-positive cells, and TdT-mediated dUTP nick-end labeling-positive cells among colon cancer cells, suggesting that the therapy induces apoptosis. Finally, the combined treatment exhibited a robust antitumor activity in syngeneic tumor model mice. These findings should contribute to the development of new treatments for colorectal cancer that directly exploit the genomic instability of cancer cells.

The DNMT3B Inhibitor Nanaomycin A as a Neuroblastoma Therapeutic Agent.

BACKGROUND: Neuroblastoma is one of the most common childhood solid tumors. Because tumor suppressor genes are often hypermethylated in cancers, DNA methylation has emerged as a target for cancer therapeutics. Nanaomycin A, an inhibitor of DNA methyltransferase 3B, which mediates de novo DNA methylation, reportedly induces death in several types of human cancer cells. OBJECTIVE: To study the antitumor activity of nanaomycin A against neuroblastoma cell lines and its mechanism. METHODS: The anti-tumor effect of nanaomycin A on neuroblastoma cell lines was evaluated based on cell viability, DNA methylation levels, apoptosis-related protein expression, and neuronal-associated mRNA expression. RESULTS: Nanaomycin A decreased genomic DNA methylation levels and induced apoptosis in human neuroblastoma cells. Nanaomycin A also upregulated the expression of mRNAs for several genes related to neuronal maturation. CONCLUSIONS: Nanaomycin A is an effective therapeutic candidate for treating neuroblastoma. Our findings also suggest that the inhibition of DNA methylation is a promising anti-tumor therapy strategy for neuroblastoma.