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CD73 is a cell-surface ectoenzyme that hydrolyzes the conversion of extracellular adenosine monophosphate to adenosine, which in turn can promote resistance to immune checkpoint blockade therapy. Immune response may therefore be improved by targeting tumor CD73, and this possibility underlines the need to non-invasively assess tumor CD73 level. In this study, we developed a cysteine site-specific 89Zr-labeled anti-CD73 (89Zr-CD73) IgG immuno-PET technique that can image tumor CD73 expression in living bodies. Anti-CD73 IgG was reduced with tris(2-carboxyethyl)phosphine, underwent sulfohydryl moiety-specific conjugation with deferoxamine-maleimide, and was radiolabeled with 89Zr. CT26 mouse colon cancer cells, CT26/CD73 cells engineered to constitutively overexpress CD73, and 4T1.2 mouse breast cancer cells underwent cell binding assays and western blotting. Balb/c nude mice bearing tumors underwent 89Zr-CD73 IgG PET imaging and biodistribution studies. 89Zr-CD73 IgG showed 20-fold higher binding to overexpressing CT26/CD73 cells compared to low-expressing CT26 cells, and moderate expressing 4T1.2 cells showed uptake that was 38.9 ± 1.51% of CT26/CD73 cells. Uptake was dramatically suppressed by excess unlabeled antibody. CD73 content proportionately increased in CT26 and CT26/CD73 cell mixtures was associated with linear increases in 89Zr-CD73 IgG uptake. 89Zr-CD73 IgG PET/CT displayed clear accumulation in CT26/CD73 tumors with greater uptake compared to CT26 tumors (3.13 ± 1.70%ID/g vs. 1.27 ± 0.31%ID/g at 8 days; P = 0.04). Specificity was further supported by low CT26/CD73 tumor-to-blood ratio of 89Zr-isotype-IgG compared to 89Zr-CD73 IgG (0.48 ± 0.08 vs. 2.68 ± 0.52 at 4 days and 0.53 ± 0.07 vs. 4.81 ± 1.02 at 8 days; both P < 0.001). Immunoblotting and immunohistochemistry confirmed strong CD73 expression in CT26/CD73 tumors and low expression in CT26 tumors. 4T1.2 tumor mice also showed clear 89Zr-CD73 IgG accumulation at 8 days (3.75 ± 0.70%ID/g) with high tumor-to-blood ratio compared to 89Zr-isotype-IgG (4.91 ± 1.74 vs. 1.20 ± 0.28; P < 0.005). 89Zr-CD73 IgG specifically targeted CD73 on high expressing cancer cells in vitro and tumors in vivo. Thus, 89Zr-CD73 IgG immuno-PET may be useful for the non-invasive monitoring of CD73 expression in tumors of living subjects.
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5'-Nucleotidasa , Neoplasias del Colon , Cisteína , Tomografía de Emisión de Positrones , Circonio , Animales , 5'-Nucleotidasa/metabolismo , Circonio/química , Neoplasias del Colon/diagnóstico por imagen , Neoplasias del Colon/metabolismo , Neoplasias del Colon/inmunología , Neoplasias del Colon/patología , Ratones , Línea Celular Tumoral , Tomografía de Emisión de Positrones/métodos , Cisteína/metabolismo , Humanos , Radioisótopos , Femenino , Ratones Endogámicos BALB C , Distribución Tisular , Ratones Desnudos , Proteínas Ligadas a GPI/metabolismo , Proteínas Ligadas a GPI/inmunología , Inmunoglobulina G/inmunología , Inmunoglobulina G/metabolismoRESUMEN
Bone homeostasis is maintained by tightly coordinated activities of bone-forming osteoblasts and bone-resorbing osteoclasts. In the present report, the role of Mer tyrosine kinase (MerTK) in bone metabolism was investigated. The expression of MerTK decreased upon BMP2 stimulation of osteoblast precursors. The femurs of Mertk-deficient mice showed significantly increased bone volume with concomitant increase of bone formation and reduction in bone resorption. These bone phenotypes were attributed to the increased osteoblast differentiation and mineralization accounted by the enhanced ß-catenin and Smad signaling in the absence of MerTK in osteoblast precursors. Although the Mertk-deficient bone marrow macrophages were predisposed to enhanced osteoclast differentiation via augmented Ca2+-NFATc1 signaling, the dramatic increase of Tnfsf11b/Tnfsf11 (Opg/Rankl) ratio in Mertk knockout bones and osteoblast precursors corroborated the reduction of osteoclastogenesis in Mertk deficiency. In ligature-induced periodontitis and ovariectomy models, the bone resorption was significantly attenuated in Mertk-deficient mice compared with wild-type control. Taken together, these data indicate novel role of MerTK in bone metabolism and suggest a potential strategy targeting MerTK in treating bone-lytic diseases including periodontitis and osteoporosis.
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Glioblastoma stem cells (GSCs) play a pivotal role in the initiation, progression, resistance to treatment, and relapse of glioblastoma multiforme (GBM). Thus, identifying potential therapeutic targets and drugs that interfere with the growth of GSCs may contribute to improved treatment outcomes for GBM. In this study, we first demonstrated the functional role of protein arginine methyltransferase 1 (PRMT1) in GSC growth. Furamidine, a PRMT1 inhibitor, effectively inhibited the proliferation and tumorsphere formation of U87MG-derived GSCs by inducing cell cycle arrest at the G0/G1 phase and promoting the intrinsic apoptotic pathway. Moreover, furamidine potently suppressed the in vivo tumor growth of U87MG GSCs in a chick embryo chorioallantoic membrane model. In particular, the inhibitory effect of furamidine on U87MG GSC growth was associated with the downregulation of signal transducer and activator of transcription 3 (STAT3) and key GSC markers, including CD133, Sox2, Oct4, Nanog, aldehyde dehydrogenase 1, and integrin α6. Our results also showed that the knockdown of PRMT1 by small interfering RNA significantly inhibited the proliferation of U87MG GSCs in vitro and in vivo through a molecular mechanism similar to furamidine. In addition, combined treatment with furamidine and berbamine, a calcium/calmodulin-dependent protein kinase II gamma (CaMKIIγ) inhibitor, inhibited the growth of U87MG GSCs more strongly than single-compound treatment. The increased antiproliferative effect of combining the two compounds resulted from a stronger downregulation of STAT3-mediated downstream GBM stemness regulators through dual PRMT1 and CaMKIIγ function blockade. In conclusion, these findings suggest that PRMT1 and its inhibitor, furamidine, are potential novel therapeutic targets and drug candidates for effectively suppressing GSC growth.
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Benzamidinas , Neoplasias Encefálicas , Glioblastoma , Embrión de Pollo , Animales , Humanos , Glioblastoma/metabolismo , Factor de Transcripción STAT3/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Células Madre Neoplásicas/metabolismo , Línea Celular Tumoral , Recurrencia Local de Neoplasia/patología , Proliferación Celular , Transducción de Señal , Neoplasias Encefálicas/metabolismo , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteínas Represoras/metabolismoRESUMEN
Very late antigen-4 (VLA4; CD49d) is a promising immune therapy target in treatment-resistant leukemia and multiple myeloma, and there is growing interest in repurposing the humanized monoclonal antibody (Ab), natalizumab, for this purpose. Positron emission tomography with radiolabeled Abs (immuno-PET) could facilitate this effort by providing information on natalizumab's in vivo pharmacokinetic and target delivery properties. In this study, we labeled natalizumab with 89Zr specifically on sulfhydryl moieties via maleimide-deferoxamine conjugation. High VLA4-expressing MOLT4 human T cell acute lymphoblastic leukemia cells showed specific 89Zr-natalizumab binding that was markedly blocked by excess Ab. In nude mice bearing MOLT4 tumors, 89Zr-natalizumab PET showed high-contrast tumor uptake at 7 days postinjection. Biodistribution studies confirmed that uptake was the highest in MOLT4 tumors (2.22 ± 0.41%ID/g) and the liver (2.33 ± 0.76%ID/g), followed by the spleen (1.51 ± 0.42%ID/g), while blood activity was lower at 1.12 ± 0.21%ID/g. VLA4-specific targeting in vivo was confirmed by a 58.1% suppression of tumor uptake (0.93 ± 0.15%ID/g) when excess Ab was injected 1 h earlier. In cultured MOLT4 cells, short-term 3 day exposure to the proteasome inhibitor bortezomib (BTZ) did not affect the α4 integrin level, but BTZ-resistant cells that survived the treatment showed increased α4 integrin expression. When the effects of BTZ treatment were tested in mice, there was no change of the α4 integrin level or 89Zr-natalizumab uptake in MOLT4 leukemia tumors, which underscores the complexity of tumor VLA4 regulation in vivo. In conclusion, 89Zr-natalizumab PET may be useful for noninvasive monitoring of tumor VLA4 and may assist in a more rational application of Ab-based therapies for hematologic malignancies.
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Integrina alfa4beta1 , Leucemia , Humanos , Animales , Ratones , Natalizumab/uso terapéutico , Cisteína , Integrina alfa4 , Ratones Desnudos , Distribución Tisular , Línea Celular Tumoral , Tomografía de Emisión de Positrones/métodos , Circonio/químicaRESUMEN
Triple-negative breast cancer (TNBC) is a highly aggressive type of breast cancer and has a poor prognosis. As standardized TNBC treatment regimens cause drug resistance and tumor recurrence, the development of new TNBC treatment strategies is urgently required. Bufotalin is a bufadienolide isolated from the skin and parotid venom glands of the toad Bufo gargarizan, and has several pharmacological properties, including antiviral, anti-inflammatory, and anticancer activities. However, the anticancer effect and underlying molecular mechanisms of action of bufotalin in TNBC have not been fully studied. In the current study, we investigated the effects of bufotalin on the growth and metastasis of MDA-MB-231 and HCC1937 TNBC cells. Bufotalin potently inhibited the proliferation of both TNBC cell lines by promoting cell cycle arrest and caspase-mediated apoptosis. Furthermore, bufotalin effectively suppressed the migration and invasion of both TNBC cell lines by regulating the expression of key epithelial-mesenchymal transition (EMT) biomarkers, matrix metalloproteinases (MMPs), and integrin α6. Notably, the anticancer effect of bufotalin in TNBC cells was associated with the downregulation of the signal transducer and activator of the transcription 3 (STAT3) signaling pathway. Collectively, our results suggest that the natural compound bufotalin may exert antiproliferative and antimetastatic activities in TNBC cells by modulating the apoptotic pathway and the STAT3/EMT axis.
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Bufanólidos , Neoplasias de la Mama Triple Negativas , Humanos , Neoplasias de la Mama Triple Negativas/metabolismo , Transición Epitelial-Mesenquimal , Recurrencia Local de Neoplasia , Apoptosis , Bufanólidos/farmacología , Proliferación Celular , Línea Celular Tumoral , Movimiento Celular , Factor de Transcripción STAT3/metabolismoRESUMEN
Gram-negative bacteria derived extracellular vesicles (EVs), also known as outer membrane vesicles, have attracted significant attention due to their pathogenic roles in various inflammatory diseases. We recently demonstrated that EVs secreted by the periodontopathogen Aggregatibacter actinomycetemcomitans (Aa) can cross the blood-brain barrier (BBB) and that their extracellular RNA cargo can promote the secretion of proinflammatory cytokines, such as IL-6 and TNF-α, in the brain. To gain more insight into the relationship between periodontal disease (PD) and neuroinflammatory diseases, we investigated the effect of Aa EVs in a mouse model of ligature-induced PD. When EVs were administered through intragingival injection or EV-soaked gel, proinflammatory cytokines were strongly induced in the brains of PD mice. The use of TLR (Toll-like receptor)-reporter cell lines and MyD88 knockout mice confirmed that the increased release of cytokines was triggered by Aa EVs via TLR4 and TLR8 signaling pathways and their downstream MyD88 pathway. Furthermore, the injection of EVs through the epidermis and gingiva resulted in the direct retrograde transfer of Aa EVs from axon terminals to the cell bodies of trigeminal ganglion (TG) neurons and the subsequent activation of TG neurons. We also found that the Aa EVs changed the action potential of TG neurons. These findings suggest that EVs derived from periodontopathogens such as Aa might be involved in pathogenic pathways for neuroinflammatory diseases, neuropathic pain, and other systemic inflammatory symptoms as a comorbidity of periodontitis.
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Vesículas Extracelulares , Enfermedades Periodontales , Periodontitis , Ratones , Animales , Enfermedades Neuroinflamatorias , Ganglio del Trigémino , Factor 88 de Diferenciación Mieloide/metabolismo , Periodontitis/metabolismo , Enfermedades Periodontales/metabolismo , Barrera Hematoencefálica/metabolismo , Citocinas/metabolismo , Ratones Noqueados , Vesículas Extracelulares/metabolismoRESUMEN
Non-small cell lung cancer (NSCLC) is a fatal malignant tumor with a high mortality rate. Cancer stem cells (CSCs) play pivotal roles in tumor initiation and progression, treatment resistance, and NSCLC recurrence. Therefore, the development of novel therapeutic targets and anticancer drugs that effectively block CSC growth may improve treatment outcomes in patients with NSCLC. In this study, we evaluated, for the first time, the effects of natural cyclophilin A (CypA) inhibitors, including 23-demethyl 8,13-deoxynargenicin (C9) and cyclosporin A (CsA), on the growth of NSCLC CSCs. C9 and CsA more sensitively inhibited the proliferation of epidermal growth factor receptor (EGFR)-mutant NSCLC CSCs than EGFR wild-type NSCLC CSCs. Both compounds suppressed the self-renewal ability of NSCLC CSCs and NSCLC-CSC-derived tumor growth in vivo. Furthermore, C9 and CsA inhibited NSCLC CSC growth by activating the intrinsic apoptotic pathway. Notably, C9 and CsA reduced the expression levels of major CSC markers, including integrin α6, CD133, CD44, ALDH1A1, Nanog, Oct4, and Sox2, through dual downregulation of the CypA/CD147 axis and EGFR activity in NSCLC CSCs. Our results also show that the EGFR tyrosine kinase inhibitor afatinib inactivated EGFR and decreased the expression levels of CypA and CD147 in NSCLC CSCs, suggesting close crosstalk between the CypA/CD147 and EGFR pathways in regulating NSCLC CSC growth. In addition, combined treatment with afatinib and C9 or CsA more potently inhibited the growth of EGFR-mutant NSCLC CSCs than single-compound treatments. These findings suggest that the natural CypA inhibitors C9 and CsA are potential anticancer agents that suppress the growth of EGFR-mutant NSCLC CSCs, either as monotherapy or in combination with afatinib, by interfering with the crosstalk between CypA/CD147 and EGFR.
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Antineoplásicos , Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Carcinoma de Pulmón de Células no Pequeñas/patología , Ciclofilina A/genética , Ciclofilina A/metabolismo , Afatinib/farmacología , Neoplasias Pulmonares/metabolismo , Línea Celular Tumoral , Receptores ErbB/metabolismo , Antineoplásicos/farmacología , Células Madre Neoplásicas/metabolismoRESUMEN
Gastric cancer stem cells (GCSCs) are a subgroup of gastric cancer (GC) cells with high self-renewal and multi-lineage differentiation abilities that lead to tumor initiation, metastasis, drug resistance, and tumor relapse. Therefore, the eradication of GCSCs can contribute to the effective treatment of advanced or metastatic GC. In our previous study, compound 9 (C9), a novel derivative of nargenicin A1, was identified as a potential natural anticancer agent that specifically targeted cyclophilin A (CypA). However, its therapeutic effect and molecular mechanisms of action on GCSC growth have not been assessed. In this study, we investigated the effects of natural CypA inhibitors, including C9 and cyclosporin A (CsA), on the growth of MKN45-derived GCSCs. Compound 9 and CsA effectively suppressed cell proliferation by inducing cell cycle arrest at the G0/G1 phase and promoted apoptosis by activating the caspase cascade in MKN45 GCSCs. In addition, C9 and CsA potently inhibited tumor growth in the MKN45 GCSC-grafted chick embryo chorioallantoic membrane (CAM) model. Furthermore, the two compounds significantly decreased the protein expression of key GCSC markers including CD133, CD44, integrin α6, Sox2, Oct4, and Nanog. Notably, the anticancer activities of C9 and CsA in MKN45 GCSCs were associated with the regulation of CypA/CD147-mediated AKT and mitogen-activated protein kinase (MAPK) signaling pathways. Collectively, our findings suggest that the natural CypA inhibitors C9 and CsA could be novel anticancer agents used to combat GCSCs by targeting the CypA/CD147 axis.
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Antineoplásicos , Basigina , Ciclofilina A , Células Madre Neoplásicas , Neoplasias Gástricas , Animales , Embrión de Pollo , Humanos , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Ciclofilina A/antagonistas & inhibidores , Ciclofilina A/metabolismo , Recurrencia Local de Neoplasia/tratamiento farmacológico , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Transducción de Señal , Neoplasias Gástricas/tratamiento farmacológico , Antineoplásicos/farmacología , Basigina/metabolismoRESUMEN
Based on the natural product terpestacin, seventeen derivatives (1-17) with various l-amino acid side chains were designed and synthesized. Their anticancer activities against U87MG-derived glioblastoma stem cells (GSCs) were evaluated, and compounds 5, 11, 13 and 15 showed strong abilities to inhibit the proliferation (IC50 = 2.8-6.9 µM) and tumorsphere formation of GSCs. Besides, compounds 13 and 15 could effectively induce apoptosis and significantly inhibit the invasion of GSCs (95 and 97 % inhibition, respectively, at 2.5 µM). The levels of CD133 marker in GSCs also decreased in dose-dependent manners after the treatment of these active compounds. Compared to terpestacin and the positive control A1938, our derivatives showed stronger activities and compounds 13 and 15 are promising candidates for further development as anticancer agents by targeting GSCs.
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Antineoplásicos , Glioblastoma , Humanos , Glioblastoma/tratamiento farmacológico , Aminoácidos/farmacología , Línea Celular Tumoral , Células Madre Neoplásicas , Antineoplásicos/farmacología , Antineoplásicos/uso terapéuticoRESUMEN
Liver cancer stem cells (LCSCs) contribute to the initiation, metastasis, treatment resistance, and recurrence of hepatocellular carcinoma (HCC). Therefore, exploring potential anticancer agents targeting LCSCs may offer new therapeutic options to overcome HCC treatment failure. Hovenia dulcis Thunberg (HDT), a tree from the buckthorn family found in Asia, exhibits various biological activities, including antifatigue, antidiabetic, neuroprotective, hepatoprotective, and antitumor activities. However, the therapeutic effect of HDT in eliminating LCSCs remains to be confirmed. In this study, we evaluated the inhibitory activity of ethanol, chloroform, and ethyl acetate extracts from HDT branches on the growth of Huh7-derived LCSCs. The ethyl acetate extract of HDT (EAHDT) exhibited the most potent inhibitory activity against the growth of Huh7 LCSCs among the three HDT extracts. EAHDT suppressed the in vitro self-renewal ability of Huh7 LCSCs and reduced tumor growth in vivo using the Huh7 LCSC-transplanted chick embryo chorioallantoic membrane model. Furthermore, EAHDT not only arrested the cell cycle in the G0/G1 phase but also induced receptor-interacting protein kinase 3/mixed-lineage kinase domain-like protein-mediated necroptosis and caspase-dependent apoptosis in Huh7 LCSCs in a concentration-dependent manner. Furthermore, the growth inhibitory effect of EAHDT on Huh7 LCSCs was associated with the downregulation of c-MET-mediated downstream signaling pathways and key cancer stemness markers. Based on these findings, we propose that EAHDT can be used as a new natural drug candidate to prevent and treat HCC by eradicating LCSCs.
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Acetatos , Carcinoma Hepatocelular , Neoplasias Hepáticas , Embrión de Pollo , Animales , Necroptosis , Neoplasias Hepáticas/tratamiento farmacológico , Células Madre Neoplásicas , ApoptosisRESUMEN
Glioblastoma multiforme (GBM) is the most aggressive form of brain tumor. Relapse is frequent and rapid due to glioblastoma stem-like cells (GSCs) that induce tumor initiation, drug resistance, high cancer invasion, immune evasion, and recurrence. Therefore, suppression of GSCs is a powerful therapeutic approach for GBM treatment. Natural compounds berbamine and arcyriaflavin A (ArcA) are known to possess anticancer activity by targeting calcium/calmodulin-dependent protein kinase II gamma (CaMKIIγ) and cyclin-dependent kinase 4 (CDK4), respectively. In this study, we evaluated the effects of concurrent treatment with both compounds on GSCs. Combined treatment with berbamine and ArcA synergistically inhibited cell viability and tumorsphere formation in U87MG- and C6-drived GSCs. Furthermore, simultaneous administration of both compounds potently inhibited tumor growth in a U87MG GSC-grafted chick embryo chorioallantoic membrane (CAM) model. Notably, the synergistic anticancer effect of berbamine and ArcA on GSC growth is associated with the promotion of reactive oxygen species (ROS)- and calcium-dependent apoptosis via strong activation of the p53-mediated caspase cascade. Moreover, co-treatment with both compounds significantly reduced the expression levels of key GSC markers, including CD133, integrin α6, aldehyde dehydrogenase 1A1 (ALDH1A1), Nanog, Sox2, and Oct4. The combined effect of berbamine and ArcA on GSC growth also resulted in downregulation of cell cycle regulatory proteins, such as cyclins and CDKs, by potent inactivation of the CaMKIIγ-mediated STAT3/AKT/ERK1/2 signaling pathway. In addition, a genetic knockdown study using small interfering RNAs (siRNAs) targeting either CaMKIIγ or CDK4 demonstrated that the synergistic anticancer effect of the two compounds on GSCs resulted from dual inhibition of CaMKIIγ and CDK4. Collectively, our findings suggest that a novel combination therapy involving berbamine and ArcA could effectively eradicate GSCs.
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Glioblastoma , Embrión de Pollo , Animales , Glioblastoma/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Calcio/metabolismo , Células Madre Neoplásicas , Proliferación CelularRESUMEN
Cyclophilin A (CypA), which has peptidyl-prolyl cis-trans isomerase (PPIase) activity, regulates multiple functions of cells by binding to its extracellular receptor CD147. The CypA/CD147 interaction plays a crucial role in the progression of several diseases, including inflammatory diseases, coronavirus infection, and cancer, by activating CD147-mediated intracellular downstream signaling pathways. Many studies have identified CypA and CD147 as potential therapeutic targets for cancer. Their overexpression promotes growth, metastasis, therapeutic resistance, and the stem-like properties of cancer cells and is related to the poor prognosis of patients with cancer. This review aims to understand the biology and interaction of CypA and CD147 and to review the roles of the CypA/CD147 interaction in cancer pathology and the therapeutic potential of targeting the CypA/CD147 axis. To validate the clinical significance of the CypA/CD147 interaction, we analyzed the expression levels of PPIA and BSG genes encoding CypA and CD147, respectively, in a wide range of tumor types using The Cancer Genome Atlas (TCGA) database. We observed a significant association between PPIA/BSG overexpression and poor prognosis, such as a low survival rate and high cancer stage, in several tumor types. Furthermore, the expression of PPIA and BSG was positively correlated in many cancers. Therefore, this review supports the hypothesis that targeting the CypA/CD147 interaction may improve treatment outcomes for patients with cancer.
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Ciclofilina A , Neoplasias , Basigina/genética , Basigina/metabolismo , Ciclofilina A/metabolismo , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Transducción de SeñalRESUMEN
Gastric cancer stem cells (GCSCs) are a major cause of radioresistance and chemoresistance in gastric cancer (GC). Therefore, targeting GCSCs is regarded as a powerful strategy for the effective treatment of GC. Atorvastatin is a widely prescribed cholesterol-lowering drug that inhibits 3-hydroxy-3-methylglutaryl-coenzyme A reductase, a rate-limiting enzyme in the mevalonate pathway. The anticancer activity of atorvastatin, a repurposed drug, is being investigated; however, its therapeutic effect and molecular mechanism of action against GCSCs remain unknown. In this study, we evaluated the anticancer effects of atorvastatin on MKN45-derived GCSCs. Atorvastatin significantly inhibited the proliferative and tumorsphere-forming abilities of MKN45 GCSCs in a mevalonate pathway-independent manner. Atorvastatin induced cell cycle arrest at the G0/G1 phase and promoted apoptosis by activating the caspase cascade. Furthermore, atorvastatin exerted an antiproliferative effect against MKN45 GCSCs by inhibiting the expression of cancer stemness markers, such as CD133, CD44, integrin α6, aldehyde dehydrogenase 1A1, Oct4, Sox2, and Nanog, through the downregulation of ß-catenin, signal transducer and activator of transcription 3, and protein kinase B activities. Additionally, the combined treatment of atorvastatin and sorafenib, a multi-kinase targeted anticancer drug, synergistically suppressed not only the proliferation and tumorsphere formation of MKN45 GCSCs but also the in vivo tumor growth in a chick chorioallantoic membrane model implanted with MKN45 GCSCs. These findings suggest that atorvastatin can therapeutically eliminate GCSCs.
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Methyltransferases transfer a methyl group to a diverse group of natural products, thus providing structural diversity, stability, and altered pharmacological properties to the molecules. A limited number of regiospecific sugar-O-methyltransferases are functionally characterized. Thus, discovery of such an enzyme could solve the difficulties of biological production of methoxy derivatives of glycosylated molecules. In the current study, a regiospecific sugar-O-methyltransferase, ThnM1, belonging to the biosynthetic gene cluster (BGC) of 1-(α-L-(2-O-methyl)-6-deoxymannopyranosyloxy)-3,6,8-trimethoxynaphthalene produced by Nocardia sp. strain CS682, was analyzed and functionally characterized. ThnM1 demonstrated promiscuity to diverse chemical structures such as rhamnose-containing anthraquinones and flavonoids with regiospecific methylation at the 2'-hydroxyl group of the sugar moiety. Compared with other compounds, anthraquinone rhamnosides were found to be the preferred substrates for methylation. Thus, the enzyme was further employed for whole-cell biotransformation using engineered Escherichia coli to produce a methoxy-rhamnosyl derivative of quinizarin, an anthraquinone derivative. The structure of the newly generated derivative from Escherichia coli fermentation was elucidated by liquid chromatography-mass spectrometry and nuclear magnetic resonance spectroscopic analyses and identified as quinizarin-4-O-α-l-2-O-methylrhamnoside (QRM). Further, the biological impact of methylation was studied by comparing the cytotoxicity of QRM with that of quinizarin against the U87MG, SNU-1, and A375SM cancer cell lines. IMPORTANCE ThnM1 is a putative sugar-O-methyltransferase produced by the Nocardia sp. strain CS682 and is encoded by a gene belonging to the biosynthetic gene cluster (BGC) of 1-(α-l-(2-O-methyl)-6-deoxymannopyranosyloxy)-3,6,8-trimethoxynaphthalene. We demonstrated that ThnM1 is a promiscuous enzyme with regiospecific activity at the 2'-OH of rhamnose. As regiospecific methylation of sugars by chemical synthesis is a challenging step, ThnM1 may fill the gap in the potential diversification of natural products by methylating the rhamnose moiety attached to them.
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Productos Biológicos , Nocardia , Productos Biológicos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Metiltransferasas/metabolismo , Nocardia/genética , Nocardia/metabolismo , Ramnosa/metabolismo , Azúcares/metabolismoRESUMEN
Glioblastoma stem-like cells (GSCs) drive tumor initiation, cancer invasion, immune evasion, and therapeutic resistance and are thus a key therapeutic target for improving treatment for glioblastoma multiforme (GBM). We previously identified calcium/calmodulin-dependent protein kinase II (CaMKII) as an emerging molecular target for eliminating GSCs. In this study, we aim to explore a new CaMKII-targeted synthetic lethal therapy for GSCs. Through high-throughput drug combination screening using CaMKII inhibitors and a bioactive compound library in GSCs, neurokinin 1 receptor (NK1R) inhibitors such as SR 140333 and aprepitant are found to be potential anticancer agents that exhibit chemical synthetic lethal interactions with CaMKII inhibitors, including hydrazinobenzoylcurcumin (HBC), berbamine, and KN93. Combined treatment with NK1R and CaMKII inhibitors markedly suppresses the viability and neurosphere formation of U87MG- and U373MG-derived GSCs. In addition, the combination of HBC and NK1R inhibitors significantly inhibits U87MG GSC tumor growth in a chick embryo chorioallantoic membrane (CAM) model. Furthermore, the synthetic lethal interaction is validated using RNA interference of CaMKIIγ and NK1R. Notably, the synthetic lethal effects in GSCs are associated with the activation of caspase-mediated apoptosis by inducing p53 expression and reactive oxygen species generation, as well as the suppression of stemness marker expression by reducing nuclear factor-kappa B (NF-κB) activity. This follows the downregulation of phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling and a decrease in intracellular calcium concentration. Moreover, NK1R affects CaMKIIγ activation. These findings demonstrate that NK1R is a potential synthetic lethal partner of CaMKII that is involved in eradicating GSCs, and they suggest a new CaMKII-targeted combination therapy for treating GBM.
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The differentiation and activity of bone-resorbing osteoclasts are tightly regulated to maintain the homeostasis of healthy bones. In this study, the role of protein tyrosine phosphatase 1B (PTP1B) during osteoclastogenesis was studied in myeloid-specific Ptpn1-deficient (conditional knockout [cKO]) mice. The mRNA and protein expression of PTP1B increased during the formation of mature osteoclasts from mouse bone macrophages on stimulation with macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor κB ligand (RANKL). The Ptpn1 cKO mice exhibited increased femoral trabecular bone volume with a decreased number and activity of osteoclasts compared with control mice. The in vitro culture of osteoclast precursors corroborated the inhibition of osteoclastogenesis in cKO cells compared with control, with concomitantly decreased RANKL-dependent proliferation, lower osteoclast marker gene expression, reduced nuclear expression of nuclear factor of activated T cells cytoplasmic 1 (NFATc1), diminished intracellular Ca2+ oscillations, and increased phosphorylation of proto-oncogene tyrosine-protein kinase Src on inhibitory tyrosine residue. In a ligature-induced periodontitis model, Ptpn1 cKO mice exhibited attenuated osteoclastogenesis and alveolar bone loss following the induction of inflammation. The Ptpn1-deficient mice were similarly protected from ovariectomy-induced bone loss compared with control mice. These results provide a novel regulatory role of PTP1B in osteoclastogenesis and suggest a potential as a therapeutic target for bone-lytic diseases. © 2021 American Society for Bone and Mineral Research (ASBMR).
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Resorción Ósea , Osteogénesis , Proteína Tirosina Fosfatasa no Receptora Tipo 1/metabolismo , Animales , Resorción Ósea/metabolismo , Diferenciación Celular , Femenino , Inflamación/metabolismo , Ratones , Ratones Endogámicos C57BL , Factores de Transcripción NFATC/metabolismo , Osteoclastos/metabolismo , Ovariectomía , Monoéster Fosfórico Hidrolasas/metabolismo , Ligando RANK/metabolismo , Tirosina/metabolismoRESUMEN
Hepatocellular carcinoma (HCC) is a malignant type of primary liver cancer with high incidence and mortality, worldwide. A major challenge in the treatment of HCC is chemotherapeutic resistance. It is therefore necessary to develop novel anticancer drugs for suppressing the growth of HCC cells and overcoming drug resistance for improving the treatment of HCC. Violacein is a deep violet-colored indole derivative that is produced by several bacterial strains, including Chromobacterium violaceum, and it possesses numerous pharmacological properties, including antitumor activity. However, the therapeutic effects of violacein and the mechanism underlying its antitumor effect against HCC remain to be elucidated. This study is the first to demonstrate that violacein inhibits the proliferation and stemness of Huh7 and Hep3B HCC cells. The antiproliferative effect of violacein was attributed to cell cycle arrest at the sub-G1 phase and the induction of apoptotic cell death. Violacein induced nuclear condensation, dissipated mitochondrial membrane potential (MMP), increased generation of reactive oxygen species (ROS), activated the caspase cascade, and upregulated p53 and p21. The anticancer effect of violacein on HCC cells was also associated with the downregulation of protein kinase B (AKT) and extracellular signal-regulated kinase (ERK)1/2 signaling. Violacein not only suppressed the proliferation and formation of tumorspheres of Huh7 and Hep3B cancer stem-like cells but also reduced the expression of key markers of cancer stemness, including CD133, Sox2, Oct4, and Nanog, by inhibiting the signal transducer and activator of transcription 3 (STAT3)/AKT/ERK pathways. These results suggest the therapeutic potential of violacein in effectively suppressing HCC by targeting the proliferation and stemness of HCC cells.
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Antineoplásicos/farmacología , Productos Biológicos/farmacología , Autorrenovación de las Células/efectos de los fármacos , Indoles/farmacología , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Productos Biológicos/química , Carcinoma Hepatocelular , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Indoles/química , Neoplasias Hepáticas , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factor de Transcripción STAT3/metabolismoRESUMEN
Glioblastoma (GBM) is one of the most malignant brain tumors and requires the formation of new blood vessels, called angiogenesis, for its growth and metastasis. Several proangiogenic factors, including vascular endothelial growth factor (VEGF) and brain-derived neurotrophic factor (BDNF), stimulate GBM angiogenesis. Accordingly, blocking the angiogenesis induced by angiogenic factors represents a promising modality for the treatment of GBM. In this study, we evaluated the inhibitory effects of berbamine, a plant-derived compound, on the angiogenesis induced by VEGF and BDNF in human umbilical vein endothelial cells (HUVECs). Berbamine effectively inhibited the angiogenic features stimulated by VEGF (such as proliferation, adhesion, invasion, tube formation, and reactive oxygen species (ROS) generation in HUVECs) as well as those by BDNF, at concentrations that do not affect endothelial cell viability. The antiangiogenic effects of berbamine were associated with the downregulation of VEGF/VEGF receptor 2 (VEGFR2)/Ca2+/calmodulin-dependent protein kinase IIγ (CaMKIIγ) and BDNF/tropomyosin receptor kinase B (TrkB)/CaMKIIγ signaling pathways. In addition, berbamine suppressed the expression of a key regulator of tumor angiogenesis, hypoxia-inducible factor-1α (HIF-1α), and its transcriptional target, VEGF, in U87MG GBM cells. Furthermore, berbamine significantly inhibited in vivo neovascularization as well as U87MG tumor growth in a chick embryo chorioallantoic membrane (CAM) model. All these findings suggest that berbamine may be utilized as a new antiangiogenic agent for the treatment of malignant brain tumors.
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Inhibidores de la Angiogénesis/farmacología , Antineoplásicos/farmacología , Bencilisoquinolinas/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/antagonistas & inhibidores , Glioblastoma/tratamiento farmacológico , Animales , Neoplasias Encefálicas/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Línea Celular Tumoral , Embrión de Pollo , Glioblastoma/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
Leukemia is a type of blood cancer caused by the rapid proliferation of abnormal white blood cells. Currently, several treatment options, including chemotherapy, radiation therapy, and bone marrow transplantation, are used to treat leukemia, but the morbidity and mortality rates of patients with leukemia are still high. Therefore, there is still a need to develop more selective and less toxic drugs for the effective treatment of leukemia. Ampelopsin, also known as dihydromyricetin, is a plant-derived flavonoid that possesses multiple pharmacological functions, including antibacterial, anti-inflammatory, antioxidative, antiangiogenic, and anticancer activities. However, the anticancer effect and mechanism of action of ampelopsin in leukemia remain unclear. In this study, we evaluated the antileukemic effect of ampelopsin against acute promyelocytic HL60 and chronic myelogenous K562 leukemia cells. Ampelopsin significantly inhibited the proliferation of both leukemia cell lines at concentrations that did not affect normal cell viability. Ampelopsin induced cell cycle arrest at the sub-G1 phase in HL60 cells but the S phase in K562 cells. In addition, ampelopsin regulated the expression of cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors differently in each leukemia cell. Ampelopsin also induced apoptosis in both leukemia cell lines through nuclear condensation, loss of mitochondrial membrane potential, increase in reactive oxygen species (ROS) generation, activation of caspase-9, caspase-3, and poly ADP-ribose polymerase (PARP), and regulation of Bcl-2 family members. Furthermore, the antileukemic effect of ampelopsin was associated with the downregulation of AKT and NF-κB signaling pathways. Moreover, ampelopsin suppressed the expression levels of leukemia stemness markers, such as Oct4, Sox2, CD44, and CD133. Taken together, our findings suggest that ampelopsin may be an attractive chemotherapeutic agent against leukemia.
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Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Flavonoides/farmacología , FN-kappa B/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Supervivencia Celular/efectos de los fármacos , Células HL-60 , Humanos , Células K562 , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
We recently discovered a novel nargenicin A1 analog, 23-demethyl 8,13-deoxynargenicin (compound 9), with potential anti-cancer and anti-angiogenic activities against human gastric adenocarcinoma (AGS) cells. To identify the key molecular targets of compound 9, that are responsible for its biological activities, the changes in proteome expression in AGS cells following compound 9 treatment were analyzed using two-dimensional gel electrophoresis (2-DE), followed by MALDI/TOF/MS. Analyses using chemical proteomics and western blotting revealed that compound 9 treatment significantly suppressed the expression of cyclophilin A (CypA), a member of the immunophilin family. Furthermore, compound 9 downregulated CD147-mediated mitogen-activated protein kinase (MAPK) signaling pathway, including c-Jun N-terminal kinase (JNK) and extracellular signal-regulated protein kinase 1/2 (ERK1/2) by inhibiting the expression of CD147, the cellular receptor of CypA. Notably, the responses of AGS cells to CypA knockdown were significantly correlated with the anticancer and antiangiogenic effects of compound 9. CypA siRNAs reduced the expression of CD147 and phosphorylation of JNK and ERK1/2. In addition, the suppressive effects of CypA siRNAs on proliferation, migration, invasion, and angiogenesis induction of AGS cells were associated with G2/M cell cycle arrest, caspase-mediated apoptosis, inhibition of MMP-9 and MMP-2 expression, inactivation of PI3K/AKT/mTOR pathway, and inhibition of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) expression. The specific interaction between compound 9 and CypA was also confirmed using the drug affinity responsive target stability (DARTS) and cellular thermal shift assay (CETSA) approaches. Moreover, in silico docking analysis revealed that the structure of compound 9 was a good fit for the cyclosporin A binding cavity of CypA. Collectively, these findings provide a novel molecular basis for compound 9-mediated suppression of gastric cancer progression through the targeting of CypA.