RESUMEN
Gemcitabine is a nucleoside analog widely used as an anticancer agent against several types of cancer. Although gemcitabine sometimes shows excellent effectiveness, cancer cells are often poorly responsive to or resistant to the drug. Recently, specific strains or dysbiosis of the human microbiome were correlated with drug reactivity and resistance acquisition. Therefore, we aimed to identify antibiotic compounds that can modulate the microbiome to enhance the responsiveness to gemcitabine. To achieve this, we confirmed the gemcitabine responsiveness based on public data and conducted drug screening on a set of 250 antibiotics compounds. Subsequently, we performed experiments to investigate whether the selected compounds could enhance the responsiveness to gemcitabine. First, we grouped a total of seven tumor cell lines into resistant and sensitive group based on the IC50 value (1 µM) of gemcitabine obtained from the public data. Second, we performed high-throughput screening with compound treatments, identifying seven compounds from the resistant group and five from the sensitive group based on dose dependency. Finally, the combination of the selected compound, puromycin dihydrochloride, with gemcitabine in gemcitabine-resistant cell lines resulted in extensive cell death and a significant increase in cytotoxic efficacy. Additionally, mRNA levels associated with cell viability and stemness were reduced. Through this study, we screened antibiotics to further improve the efficacy of existing anticancer drugs and overcome resistance. By combining existing anticancer agents and antibiotic substances, we hope to establish various drug combination therapies and ultimately improve cancer treatment efficacy.
Asunto(s)
Antibacterianos , Desoxicitidina , Resistencia a Antineoplásicos , Ensayos de Selección de Medicamentos Antitumorales , Gemcitabina , Ensayos Analíticos de Alto Rendimiento , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Humanos , Ensayos Analíticos de Alto Rendimiento/métodos , Resistencia a Antineoplásicos/efectos de los fármacos , Línea Celular Tumoral , Antibacterianos/farmacología , Ensayos de Selección de Medicamentos Antitumorales/métodos , Puromicina/farmacología , Antimetabolitos Antineoplásicos/farmacología , Sinergismo Farmacológico , Antineoplásicos/farmacología , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
Acute myeloid leukemia (AML) is the most common type of hematological disease in adults, and has a very poor outcome [1]. Based on its wide range of efficacy in AML models, a small-molecule inhibitor of the anti-apoptotic protein BCL-2, venetoclax (ABT-199/GDC-0199), was developed for clinical trials. However, venetoclax showed limited monotherapy activity [2]. The overexpression of myeloid cell leukemia sequence-1 protein (Mcl-1)-due to mutations in Fms-like tyrosine kinase 3 internal tandem duplication (FLT-3 ITD)-was considered to be the main reason for low efficacy of venetoclax in clinical trials [3-5]. To achieve venetoclax sensitization in AML, targeting CDK-9 with venetoclax is a promising therapeutic strategy. In this study, we developed A09-003 as a potent inhibitor of CDK-9, with an IC50 value of 16 nM. A09-003 inhibited cell proliferation in various leukemia cell lines. In particular, the proliferation inhibitory effect of A09-003 was most potent in MV4-11 and Molm-14 cells, harboring the FLT-3 ITD mutation with a high expression profile of Mcl-1. Marker analysis revealed that A09-003 reduced CDK-9 phosphorylation and reduced RNA polymerase II activity with decreased Mcl-1 expression. Finally, combining A09-003 with venetoclax induced apoptotic cell death in a synergistic manner. In summary, this study shows the potential of A09-003 in AML therapy.
Asunto(s)
Apoptosis , Leucemia Mieloide Aguda , Adulto , Humanos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Línea Celular Tumoral , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Leucemia Mieloide Aguda/tratamiento farmacológico , Quinasas Ciclina-Dependientes , Células Mieloides/metabolismoRESUMEN
Hepatocellular carcinoma (HCC) is an extremely aggressive malignancy that ranks as the sixth-leading cause of cancer-associated death worldwide. Recently, various epigenetic mechanisms including gene methylation were reported to be potential next era HCC therapeutics and biomarkers. Although inhibition of epigenetic enzymes including histone lysine demethylase 4 (KDM4) enhanced cell death in HCC cells, the detailed mechanism of cell death machinery is poorly understood. In this study, we found that ML324, a small molecule KDM4-specific inhibitor, induced the death of HCC cells in a general cell culture system and 3D spheroid culture with increased cleavage of caspase-3. Mechanistically, we identified that unfolded protein responses (UPR) were involved in ML324-induced HCC cell death. Incubation of HCC cells with ML324 upregulated death receptor 5 (DR5) expression through the activation transcription factor 3 (ATF3)-C/EBP homologous protein (CHOP)-dependent pathway. Moreover, we identified BIM protein as a mediator of ML324-induced apoptosis using CRISPR/Cas9 knockout analysis. We showed that the loss of Bim suppressed ML324-induced apoptosis by flow cytometry analysis, colony formation assay, and caspase-3 activation assay. Interestingly, BIM protein expression by ML324 was regulated by ATF3, CHOP, and DR5 which are factors involved in UPR. Specifically, we confirmed the regulating roles of KDM4E in Bim and CHOP expression using a chromatin immune precipitation (ChIP) assay. Physical binding of KDM4E to Bim and CHOP promoters decreased the response to ML324. Our findings suggest that KDM4 inhibition is a potent anti-tumor therapeutic strategy for human HCC, and further studies of UPR-induced apoptosis and the associated epigenetic functional mechanisms may lead to the discovery of novel target for future cancer therapy.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Proteína 11 Similar a Bcl2/metabolismo , Histona Demetilasas con Dominio de Jumonji/metabolismo , Oxiquinolina/farmacología , Respuesta de Proteína Desplegada/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Antineoplásicos/química , Proteína 11 Similar a Bcl2/genética , Benzamidas/química , Benzamidas/farmacología , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Histona Demetilasas con Dominio de Jumonji/antagonistas & inhibidores , Neoplasias Hepáticas/patología , Oxiquinolina/química , Unión Proteica , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/antagonistas & inhibidores , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/genética , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Factor de Transcripción CHOP/genética , Factor de Transcripción CHOP/metabolismoRESUMEN
The unfolded protein response (UPR) is an emerging target pathway for cancer treatment owing to its ability to induce cell death. In our previous analysis of UPR-modulating small molecules, we had reported that piperazine oxalate derivative compounds (AMC-01-04) are able to promote increased phosphorylation of eukaryotic translation initiation factor-2 alpha (eIF2α). In this study, we found that AMC-04 induces apoptotic cell death via the activation of UPR in human breast and liver cancer cells. AMC-04 upregulated the expression of activating transcription factor-4 (ATF4)-C/EBP homologous protein (CHOP) and death receptor 5 (DR5) in cancer cells, as revealed by microarray analysis, small-interference RNA assay, and western blotting. From a mechanistic perspective, cytotoxic UPR pathway activation by AMC-04 is mediated by reactive oxygen species (ROS) and p38 mitogen-activated protein kinase (p38 MAPK) signaling. A chemical informatics approach predicted that AMC-04 modulates histone methyltransferase activity. Based on biochemical analysis, the activity of histone methyltransferases, including SUV39H1, SUV39H2, SETDB1, and EHMT1, was inhibited by AMC-04. Furthermore, chemical inhibition of the identified target proteins induced UPR activation and apoptotic cell death, suggesting that inhibition of histone methyltransferases is a promising strategy for cancer therapy. Taken together, we showed that the small molecule AMC-04 modulates epigenetic enzyme activity and mediates the link between cytotoxic UPR and histone modifications.
Asunto(s)
Factor de Transcripción Activador 4/metabolismo , Apoptosis/efectos de los fármacos , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Transducción de Señal , Bibliotecas de Moléculas Pequeñas/farmacología , Factor de Transcripción CHOP/metabolismo , Línea Celular Tumoral , Estrés del Retículo Endoplásmico/efectos de los fármacos , Estrés del Retículo Endoplásmico/genética , Inhibidores Enzimáticos/farmacología , Histona Metiltransferasas/antagonistas & inhibidores , Histona Metiltransferasas/metabolismo , Humanos , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Respuesta de Proteína Desplegada/efectos de los fármacos , Respuesta de Proteína Desplegada/genética , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
The use of human mesenchymal stem cells (hMSCs) in clinical applications requires large-scale cell expansion prior to administration. However, the prolonged culture of hMSCs results in cellular senescence, impairing their proliferation and therapeutic potentials. To understand the role of microRNAs (miRNAs) in regulating cellular senescence in hMSCs, we globally depleted miRNAs by silencing the DiGeorge syndrome critical region 8 (DGCR8) gene, an essential component of miRNA biogenesis. DGCR8 knockdown hMSCs exhibited severe proliferation defects and senescence-associated alterations, including increased levels of reactive oxygen species (ROS). Transcriptomic analysis revealed that the antioxidant gene superoxide dismutase 2 (SOD2) was significantly downregulated in DGCR8 knockdown hMSCs. Moreover, we found that DGCR8 silencing in hMSCs resulted in hypermethylation in CpG islands upstream of SOD2. 5-aza-2'-deoxycytidine treatment restored SOD2 expression and ROS levels. We also found that these effects were dependent on the epigenetic regulator DNA methyltransferase 3 alpha (DNMT3A). Using computational and experimental approaches, we demonstrated that DNMT3A expression was regulated by miR-29a-3p and miR-30c-5p. Overexpression of miR-29a-3p and/or miR-30c-5p reduced ROS levels in DGCR8 knockdown hMSCs and rescued proliferation defects, mitochondrial dysfunction, and premature senescence. Our findings provide novel insights into hMSCs senescence regulation by the miR-29a-3p/miR-30c-5p/DNMT3A/SOD2 axis.
Asunto(s)
ADN (Citosina-5-)-Metiltransferasas/genética , Células Madre Mesenquimatosas , MicroARNs/genética , Mitocondrias , Estrés Oxidativo , Superóxido Dismutasa/metabolismo , ADN Metiltransferasa 3A , Epigénesis Genética , Técnicas de Silenciamiento del Gen , Humanos , Células Madre Mesenquimatosas/metabolismo , Proteínas de Unión al ARNRESUMEN
Dimethylation of the histone H3 protein at lysine residue 9 (H3K9) is mediated by euchromatin histone methyltransferase II (EHMT2) and results in transcriptional repression of target genes. Recently, chemical inhibition of EHMT2 was shown to induce various physiological outcomes, including endoplasmic reticulum stress-associated genes transcription in cancer cells. To identify genes that are transcriptionally repressed by EHMT2 during apoptosis, and cell stress responses, we screened genes that are upregulated by BIX-01294, a chemical inhibitor of EHMT2. RNA sequencing analyses revealed 77 genes that were upregulated by BIX-01294 in all four hepatic cell carcinoma (HCC) cell lines. These included genes that have been implicated in apoptosis, the unfolded protein response (UPR), and others. Among these genes, the one encoding the stress-response protein Ras-related GTPase C (RRAGC) was upregulated in all BIX-01294-treated HCC cell lines. We confirmed the regulatory roles of EHMT2 in RRAGC expression in HCC cell lines using proteomic analyses, chromatin immune precipitation (ChIP) assay, and small guide RNA-mediated loss-of-function experiments. Upregulation of RRAGC was limited by the reactive oxygen species (ROS) scavenger N-acetyl cysteine (NAC), suggesting that ROS are involved in EHMT2-mediated transcriptional regulation of stress-response genes in HCC cells. Finally, combined treatment of cells with BIX-01294 and 5- Aza-cytidine induced greater upregulation of RRAGC protein expression. These findings suggest that EHMT2 suppresses expression of the RRAGC gene in a ROS-dependent manner and imply that EHMT2 is a key regulator of stress-responsive gene expression in liver cancer cells. [BMB Reports 2020; 53(11): 576-581].
Asunto(s)
Antígenos de Histocompatibilidad/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Apoptosis/genética , Azepinas/farmacología , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Línea Celular Tumoral , Proliferación Celular/genética , Inmunoprecipitación de Cromatina/métodos , Eucromatina/genética , Expresión Génica/genética , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Antígenos de Histocompatibilidad/fisiología , Histona Metiltransferasas/genética , Histona Metiltransferasas/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/fisiología , Histonas/genética , Histonas/metabolismo , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Proteínas de Unión al GTP Monoméricas/fisiología , Proteómica , Quinazolinas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Estrés Fisiológico/genética , Transcriptoma/genéticaRESUMEN
Human mesenchymal stem cells (hMSCs) have self-renewal and differentiation capabilities but the regulatory mechanisms of MSC fate determination remain poorly understood. Here, we aimed to identify microRNAs enriched in hMSCs that modulate differentiation commitments. Microarray analysis revealed that miR-140-5p is commonly enriched in undifferentiated hMSCs from various tissue sources. Moreover, bioinformatic analysis and luciferase reporter assay validated that miR-140-5p directly represses bone morphogenic protein 2 (BMP2). Furthermore, blocking miR-140-5p in hMSCs increased the expression of BMP signaling components and critical regulators of osteogenic differentiation. We propose that miR-140-5p functionally inhibits osteogenic lineage commitment in undifferentiated hMSCs.
Asunto(s)
Proteína Morfogenética Ósea 2/genética , Diferenciación Celular , Células Madre Mesenquimatosas/citología , MicroARNs/genética , MicroARNs/metabolismo , Osteogénesis , Linaje de la Célula , Regulación de la Expresión Génica/genética , Humanos , Transducción de SeñalRESUMEN
A Gram-stain-positive, strictly aerobic, short-rod-shaped, non-motile strain (designated MJ32(T)) was isolated from a sludge sample of the Daejeon sewage disposal plant in South Korea. A polyphasic approach was applied to study the taxonomic position of strain MJ32(T). Strain MJ32(T) showed highest 16S rRNA gene sequence similarity to Gordonia hirsuta DSM 44140(T) (98.1%) and Gordonia hydrophobica DSM 44015(T) (97.0%); levels of sequence similarity to the type strains of other recognized Gordonia species were less than 97.0%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain MJ32(T) belonged to the clade formed by members of the genus Gordonia in the family Gordoniaceae. The G+C content of the genomic DNA of strain MJ32(T) was 69.2 mol%. Chemotaxonomically, strain MJ32(T) showed features typical of the genus Gordonia. The predominant respiratory quinone was MK-9(H(2)), the mycolic acids present had C(56)-C(60) carbon atoms, and the major fatty acids were C(16:0) (34.6%), tuberculostearic acid (21.8%), C(16:1)ω7c (19.5%) and C(18:1)ω9c (12.7%). The peptidoglycan type was based on meso-2,6-diaminopimelic acid as the diagnostic diamino acid with glycolated sugars. On the basis of phylogenetic inference, fatty acid profile and other phenotypic properties, strain MJ32(T) is considered to represent a novel species of the genus Gordonia, for which the name Gordonia caeni sp. nov. is proposed. The type strain is MJ32(T) (=KCTC 19771(T)=JCM 16923(T)).