RESUMEN
Skp2 is overexpressed in multiple cancers and plays a critical role in tumor development through ubiquitin/proteasome-dependent degradation of its substrate proteins. Drugs targeting Skp2 have exhibited promising anticancer activity. Here, we identified a plant-derived Skp2 inhibitor, betulinic acid (BA), via high-throughput structure-based virtual screening of a phytochemical library. BA significantly inhibited the proliferation and migration of non-small cell lung cancer (NSCLC) through targeting Skp2-SCF E3 ligase both in vitro and in vivo. Mechanistically, BA binding to Skp2, especially forming H-bonds with residue Lys145, decreases its stability by disrupting Skp1-Skp2 interactions, thereby inhibiting the Skp2-SCF E3 ligase and promoting the accumulation of its substrates; that is, E-cadherin and p27. In both subcutaneous and orthotopic xenografts, BA significantly inhibited the proliferation and metastasis of NSCLC through targeting Skp2-SCF E3 ligase and upregulating p27 and E-cadherin protein levels. Taken together, BA can be considered a valuable therapeutic candidate to inhibit metastasis of NSCLC.
Asunto(s)
Antineoplásicos Fitogénicos/administración & dosificación , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Inhibidores Enzimáticos/administración & dosificación , Neoplasias Pulmonares/tratamiento farmacológico , Triterpenos Pentacíclicos/administración & dosificación , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Células A549 , Animales , Antineoplásicos Fitogénicos/farmacología , Sitios de Unión , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Detección Precoz del Cáncer , Inhibidores Enzimáticos/farmacología , Ensayos Analíticos de Alto Rendimiento , Humanos , Neoplasias Pulmonares/metabolismo , Masculino , Ratones , Triterpenos Pentacíclicos/farmacología , Unión Proteica/efectos de los fármacos , Proteínas Quinasas Asociadas a Fase-S/química , Ensayos Antitumor por Modelo de Xenoinjerto , Ácido BetulínicoRESUMEN
Neuroinflammation is one of the critical events in neurodegenerative diseases, whereas microglia play an important role in the pathogenesis of neuroinflammation. In this study, we investigated the effects of a natural sesquiterpene lactone, 6-O-angeloylplenolin (6-OAP), isolated from the traditional Chinese medicine Centipeda minima (L.) A.Br., on neuroinflammation and the underlying mechanisms. We showed that treatment with lipopolysaccharide (LPS) caused activation of BV2 and primary microglial cells and development of neuroinflammation in vitro, evidenced by increased production of inflammatory cytokines TNF-α and IL-1ß, the phosphorylation and nuclear translocation of NF-κB, and the transcriptional upregulation of COX-2 and iNOS, leading to increased production of proinflammatory factors NO and PGE2. Moreover, LPS treatment induced oxidative stress through increasing the expression levels of NOX2 and NOX4. Pretreatment with 6-OAP (0.5-4 µM) dose-dependently attenuated LPS-induced NF-κB activation and oxidative stress, thus suppressed neuroinflammation in the cells. In a mouse model of LPS-induced neuroinflammation, 6-OAP (5-20 mg·kg-1·d-1, ip, for 7 days before LPS injection) dose-dependently inhibited the production of inflammatory cytokines, the activation of the NF-κB signaling pathway, and the expression of inflammatory enzymes in brain tissues. 6-OAP pretreatment significantly ameliorated the activation of microglia and astrocytes in the brains. 6-OAP at a high dose caused a much stronger antineuroinflammatory effect than dexamethansone (DEX). Furthermore, we demonstrated that 6-OAP pretreatment could inhibit LPS-induced neurite and synaptic loss in vitro and in vivo. In conclusion, our results demonstrate that 6-OAP exerts antineuroinflammatory effects and can be considered a novel drug candidate for the treatment of neuroinflammatory diseases.
Asunto(s)
Inflamación/tratamiento farmacológico , Lactonas/farmacología , Lipopolisacáridos/antagonistas & inhibidores , Enfermedades Neurodegenerativas/tratamiento farmacológico , Fármacos Neuroprotectores/farmacología , Sesquiterpenos/farmacología , Animales , Asteraceae/química , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Relación Dosis-Respuesta a Droga , Inflamación/inducido químicamente , Inflamación/metabolismo , Lactonas/química , Lactonas/aislamiento & purificación , Lipopolisacáridos/farmacología , Masculino , Medicina Tradicional China , Ratones , Ratones Endogámicos C57BL , Conformación Molecular , Enfermedades Neurodegenerativas/inducido químicamente , Enfermedades Neurodegenerativas/metabolismo , Fármacos Neuroprotectores/química , Fármacos Neuroprotectores/aislamiento & purificación , Óxido Nítrico/antagonistas & inhibidores , Óxido Nítrico/biosíntesis , Oxidación-Reducción , Sesquiterpenos/química , Sesquiterpenos/aislamiento & purificaciónRESUMEN
Aberrant activation of the Hedgehog (Hh) pathway is implicated in the pathogenesis and development of multiple cancers, especially Hh-driven medulloblastoma (MB). Smoothened (SMO) is a promising therapeutic target of the Hh pathway in clinical cancer treatment. However, SMO mutations frequently occur, which leads to drug resistance and tumor relapse. Novel inhibitors that target both the wild-type and mutant SMO are in high demand. In this study, we identified a novel Hh pathway inhibitor, pseudolaric acid B (PAB), which significantly inhibited the expression of Gli1 and its transcriptional target genes, such as cyclin D1 and N-myc, thus inhibiting the proliferation of DAOY and Ptch1+/- primary MB cells. Mechanistically, PAB can potentially bind to the extracellular entrance of the heptahelical transmembrane domain (TMD) of SMO, based on molecular docking and the BODIPY-cyclopamine binding assay. Further, PAB also efficiently blocked ciliogenesis, demonstrating the inhibitory effects of PAB on the Hh pathway at multiple levels. Thus, PAB may overcome drug-resistance induced by SMO mutations, which frequently occurs in clinical setting. PAB markedly suppressed tumor growth in the subcutaneous allografts of Ptch1+/- MB cells. Together, our results identified PAB as a potent Hh pathway inhibitor to treat Hh-dependent MB, especially cases resistant to SMO antagonists.
Asunto(s)
Neoplasias Cerebelosas/tratamiento farmacológico , Diterpenos/farmacología , Medicamentos Herbarios Chinos/uso terapéutico , Proteínas Hedgehog/antagonistas & inhibidores , Meduloblastoma/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos , Células A549 , Animales , Neoplasias Cerebelosas/metabolismo , Neoplasias Cerebelosas/patología , Diterpenos/química , Diterpenos/uso terapéutico , Relación Dosis-Respuesta a Droga , Medicamentos Herbarios Chinos/química , Medicamentos Herbarios Chinos/farmacología , Células HEK293 , Células HeLa , Proteínas Hedgehog/química , Proteínas Hedgehog/metabolismo , Humanos , Masculino , Meduloblastoma/metabolismo , Meduloblastoma/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Desnudos , Ratones Transgénicos , Células 3T3 NIH , Estructura Secundaria de Proteína , Transducción de Señal/fisiología , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
BACKGROUND: Intrinsic and acquired chemoresistance remains a critical challenge in lung cancer chemotherapy. Fanconi anemia (FA) pathway plays an important role in antagonizing the cytotoxic effects of chemotherapeutics by repairing DNA damage. We recently demonstrated that the traditional Chinese medicinal herb, Centipeda minima (C. minima), possessed anti-inflammatory and antioxidant properties. However, the potential anticancer application of C. minima and the underlying mechanisms remain unclear. PURPOSE: We aimed to investigate the combined anticancer effects of the ethanol extract of C. minima (ECM) and DNA-crosslinking agents on non-small cell lung cancer (NSCLC) and elucidate the underlying mechanisms. METHODS: Cell viability and flow cytometry assay were performed to determine the synergistic cytotoxicity of ECM and DNA-crosslinking agents, cisplatin (CDDP) or mitomycin C (MMC), in NSCLC cells. Western blotting and immunofluorescence were conducted to examine the effects of ECM on protein expression in DNA damage repair pathway. Comet assay was applied to evaluate DNA damage levels. Subcutaneous xenografts of NSCLC were established to evaluate the combined anticancer effects of ECM and CDDP. RESULTS: Combined treatments with ECM and DNA-crosslinking agents exhibited synergistic cytotoxic effects against A549 and H1299 cells. FANCD2 was highly expressed in NSCLC that correlates with poor prognosis of NSCLC patients, based on the online database analysis. ECM significantly inhibited DNA damage-induced monoubiquitination and nuclear foci formation of FANCD2, thereby sensitizing NSCLC to CDDP- or MMC-induced DNA damage and apoptosis, as evidenced by increased expression of γ-H2AX, increased cleavage of caspases-3 and PARP, and enhanced Annexin V-FITC/PI staining. Further, ECM can also decrease the protein level of FANCD2 that contributes to the chemosensitizing effects. Moreover, ECM significantly attenuated CDDP-mediated S-phase arrest by antagonizing the activation of ATR/Chk1 pathway in NSCLC cells. Animal experiments further demonstrated that ECM and CDDP combination treatment synergistically inhibited tumor growth by decreasing FANCD2 protein level in tumor tissues. CONCLUSION: Our results demonstrated that ECM can inhibit DNA-crosslinking agents-induced activation of FA pathway by attenuating both the expression and monoubiquitination of FANCD2. ECM and CDDP combination therapy exhibited synergistic anticancer effects both in vitro and in vivo, indicating that ECM and its active components might serve as novel anticancer drugs in the combination chemotherapy.
Asunto(s)
Antineoplásicos Fitogénicos , Asteraceae/química , Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Extractos Vegetales , Animales , Antineoplásicos Fitogénicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Línea Celular Tumoral , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Extractos Vegetales/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
In this paper, we present an in-built N+ pocket electrically doped tunnel FET (ED-TFET) based on the polarity bias concept that enhances the DC and analog/RF performance. The proposed device begins with a MOSFET like structure (n-p-n) with a control gate (CG) and a polarity gate (PG). The PG is biased at -0.7 V to induce a P+ region at the source side, leaving an N+ pocket between the source and the channel. This technique yields an N+ pocket that is realized in the in-built architecture and removes the need for additional chemical doping. Calibrated 2-D simulations have demonstrated that the introduction of the N+ pocket yields a higher ION and a steeper average subthreshold swing when compared to conventional ED-TFET. Further, a local minimum on the conduction band edge (EC) curve at the tunneling junction is observed, leading to a dramatic reduction in the tunneling width. As a result, the in-built N+ pocket ED-TFET significantly improves the DC and analog/RF figure-of-merits and, hence, can serve as a better candidate for low-power applications.
RESUMEN
Alzheimer's disease (AD) is an age-related neurodegenerative disease. The main active component in Angelica sinensis, ligustilide, has been reported to have the protective effect on AD. Whether ligustilide could protect against age-induced dementia is still unknown. In this study, we used an aging model, SAMP8 mice to investigate the neuroprotective effect of ligustilide. The behavioral tests (Morris water maze, object recognition task, open field test and elevated plus maze) results showed that ligustilide could improve the memory deficit in SAMP8 mice. For mechanism study, we found that the protein level of P-Drp1 (fission) was decreased and the levels of Mfn1 and Mfn2 (fusion) were increased after ligustilide treatment in animals and cells. Ligustilide increased P-AMPK and ATP levels. Malondialdehyde and superoxide dismutase activity results indicated that ligustilide exerts antioxidant effects by reducing the level of oxidative stress markers. In addition, ligustilide improved neural function and alieved apoptosis and neuroinflammation. These findings have shown that ligustilide treatment improves mitochondrial function in SAMP8 mice, and improves memory loss.
Asunto(s)
4-Butirolactona/análogos & derivados , Envejecimiento/metabolismo , Inflamación/tratamiento farmacológico , Aprendizaje por Laberinto/efectos de los fármacos , Trastornos de la Memoria/tratamiento farmacológico , Mitocondrias/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , 4-Butirolactona/farmacología , 4-Butirolactona/uso terapéutico , Animales , Apoptosis/efectos de los fármacos , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Inflamación/metabolismo , Masculino , Trastornos de la Memoria/metabolismo , Ratones , Mitocondrias/metabolismo , Fármacos Neuroprotectores/uso terapéutico , Estrés Oxidativo/efectos de los fármacos , Memoria Espacial/efectos de los fármacosRESUMEN
Oxidative stress is implicated in the pathogenesis of neurodegeneration and other aging-related diseases. Previous studies have found that the whole herb of Centipeda minima has remarkable antioxidant activities. However, there have been no reports on the neuroprotective effects of C. minima, and the underlying mechanism of its antioxidant properties is unclear. Here, we examined the underlying mechanism of the antioxidant activities of the ethanol extract of C. minima (ECM) both in vivo and in vitro and found that ECM treatment attenuated glutamate and tert-butyl hydroperoxide (tBHP)-induced neuronal death, reactive oxygen species (ROS) production, and mitochondria dysfunction. tBHP-induced phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun N-terminal kinases (JNK) was reduced by ECM, and ECM sustained phosphorylation level of extracellular signal regulated kinase (ERK) in SH-SY5Y and PC12 cells. Moreover, ECM induced the activation of nuclear factor erythroid 2-related factor 2 (Nrf2) and the upregulation of phase II detoxification enzymes, including heme oxygenase-1 (HO-1), superoxide dismutase-2 (SOD2), and NAD(P)H quinone oxidoreductase-1 (NQO-1) in both two cell types. In a D-galactose (D-gal) and aluminum muriate (AlCl3)-induced neurodegenerative mouse model, administration of ECM improved the learning and memory of mice in the Morris water maze test and ameliorated the effects of neurodegenerative disorders. ECM sustained the expression level of postsynaptic density 95 (PSD95) and synaptophysin (SYN), activated the Nrf2 signaling pathway, and restored the levels of cellular antioxidants in the hippocampus of mice. In addition, four sesquiterpenoids were isolated from C. minima to identify the bioactive components responsible for the antioxidant activity of C. minima; 6-O-angeloylplenolin and arnicolide D were found to be the active compounds responsible for the activation of the Nrf2 signaling pathway and inhibition of ROS production. Our study examined the mechanism of C. minima and its active components in the amelioration of oxidative stress, which holds the promise for the treatment of neurodegenerative disease.