RESUMEN

Adagrasib (MRTX849), an approved and promising KRAS G12C inhibitor, has shown the promising results for treating patients with advanced non-small cell lung cancer (NSCLC) or colorectal cancer (CRC) harboring KRAS-activating mutations. However, emergence of the acquired resistance limits its long-term efficacy and clinical application. Further understanding of the mechanism of the acquired resistance is crucial for developing more new effective therapeutic strategies. Herein, we firstly found a new connection between the acquired resistance to MRTX849 and nuclear factor erythroid 2-related factor 2 (Nrf2). The expression levels of Nrf2 and GLS1 proteins were substantially elevated in different CRC cell lines with the acquired resistance to MRTX849 in comparison with their corresponding parental cell lines. Next, we discovered that RA-V, one of natural cyclopeptides isolated from the roots of Rubia yunnanensis, could restore the response of resistant CRC cells to MRTX849. The results of molecular mechanisms showed that RA-V suppressed Nrf2 protein through the ubiquitin-proteasome-dependent degradation, leading to the induction of oxidative and ER stress, and DNA damage in CRC cell lines. Consequently, RA-V reverses the resistance to MRTX849 by inhibiting the Nrf2/GLS1 axis, which shows the potential for further developing into one of novel adjuvant therapies of MRTX849.

Asunto(s)

Neoplasias Colorrectales , Factor 2 Relacionado con NF-E2 , Péptidos Cíclicos , Factor 2 Relacionado con NF-E2/metabolismo , Factor 2 Relacionado con NF-E2/genética , Humanos , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/genética , Línea Celular Tumoral , Péptidos Cíclicos/farmacología , Péptidos Cíclicos/uso terapéutico , Resistencia a Antineoplásicos/efectos de los fármacos , Animales , Antineoplásicos Fitogénicos/farmacología , Ratones Desnudos

RESUMEN

AIMS: Intestinal ischemia reperfusion (II/R) is a common clinical emergency. Ferroptosis is reported to play a role in II/R injury. Our previous studies revealed that corilagin significantly attenuates intestinal ischemia/reperfusion injuries. However, the underlying molecular mechanism is unclear and requires further study. MATERIALS AND METHODS: DAO, GSSG/T-GSH, MDA, and Fe2+ were measured by assay kits, 4-HNE was assessed by IHC, and 15-LOX was measured by ELISA. Mitochondrial damage was observed by TEM and cellular oxidation levels were detected by C11-BODIPY 581/591 and DHE probes. LC3, p62, Beclin1, ACSL4, GPX4, NCOA4, and ferritin expression were examined by WB in vivo and in vitro. IF, co-IF, q-PCR, and constructed NCOA4-knock-down IEC-6 cells were used to evaluate the role of NCOA4 in the effect of corilagin against II/R injury. Temporal and nucleoplasmic variations with or without corilagin were observed by WB. Co-IP and molecular docking were used to investigate the NCOA4-ferritin interaction. KEY FINDINGS: Corilagin attenuated II/R-induced ferroptosis both in vitro and in vivo. Further study revealed that the anti-ferroptosis bioactivity of corilagin might be due to the modulation of iron homeostasis via inhibition of ferritinophagy in an NCOA4-dependent manner. SIGNIFICANCE: Corilagin might be a potential therapeutic agent for II/R-induced tissue injury.

Asunto(s)

Ferroptosis , Isquemia Mesentérica , Daño por Reperfusión , Animales , Ratones , Simulación del Acoplamiento Molecular , Daño por Reperfusión/tratamiento farmacológico , Ferritinas , Isquemia

RESUMEN

Ubiquitin proteasome activity is suppressed in enzalutamide resistant prostate cancer cells, and the heat shock protein 70/STIP1 homology and U-box-containing protein 1 (HSP70/STUB1) machinery are involved in androgen receptor (AR) and AR variant protein stabilization. Targeting HSP70 could be a viable strategy to overcome resistance to androgen receptor signaling inhibitor (ARSI) in advanced prostate cancer. Here, we showed that a novel HSP70 allosteric inhibitor, JG98, significantly suppressed drug-resistant C4-2B MDVR and CWR22Rv1 cell growth, and enhanced enzalutamide treatment. JG98 also suppressed cell growth in conditional reprogramed cell cultures (CRCs) and organoids derived from advanced prostate cancer patient samples. Mechanistically, JG98 degraded AR/AR-V7 expression in resistant cells and promoted STUB1 nuclear translocation to bind AR-V7. Knockdown of the E3 ligase STUB1 significantly diminished the anticancer effects and partially restored AR-V7 inhibitory effects of JG98. JG231, a more potent analog developed from JG98, effectively suppressed the growth of the drug-resistant prostate cancer cells, CRCs, and organoids. Notably, the combination of JG231 and enzalutamide synergistically inhibited AR/AR-V7 expression and suppressed CWR22Rv1 xenograft tumor growth. Inhibition of HSP70 using novel small-molecule inhibitors coordinates with STUB1 to regulate AR/AR-V7 protein stabilization and ARSI resistance.

Asunto(s)

Neoplasias de la Próstata Resistentes a la Castración , Receptores Androgénicos , Masculino , Humanos , Receptores Androgénicos/metabolismo , Antagonistas de Andrógenos , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Línea Celular Tumoral , Nitrilos/farmacología , Antagonistas de Receptores Androgénicos , Andrógenos/farmacología , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP70 de Choque Térmico/farmacología , Resistencia a Antineoplásicos , Ubiquitina-Proteína Ligasas

RESUMEN

The proteases of the mitochondrial inner membrane are challenging yet highly desirable drug targets for complex, multifactorial diseases prevalent mainly in the elderly. Among them, OMA1 with its substrates OPA1 and DELE1 safeguards mitochondrial homeostasis at the intersection of energy metabolism and apoptosis, which may have relevance for neurodegeneration, malignancy and heart failure, among other diseases. Little is known about OMA1. Its structure has not been solved and we are just beginning to understand the enzyme's context-dependent regulation. OMA1 appears dormant under physiological conditions as judged by OPA1's processing pattern. The protease is rapidly activated, however, when cells experience stress or undergo apoptosis. Intriguingly, genetic OMA1 ablation can delay or even prevent apoptosis in animal models for diseases that can be broadly categorized as ischemia-reperfusion related disorders. Three groups have reported their efforts implementing OMA1 drug screens. This article reviews some of the technical challenges encountered in these assays and highlights what can be learned for future screening campaigns, and about the OMA1 protease more broadly. OMA1 does not exists in a vacuum and potent OMA1 inhibitors are needed to tease apart OMA1's intricate interactions with the other mitochondrial proteases and enzymes. Furthermore, OMA1 inhibitors hold the promise of becoming a new class of cytoprotective medicines for disorders influenced by dysfunctional mitochondria, such as heart failure or Alzheimer's Disease.

Asunto(s)

Metaloendopeptidasas/antagonistas & inhibidores , Animales , Diseño de Fármacos , Ensayos Analíticos de Alto Rendimiento , Humanos , Metaloendopeptidasas/metabolismo

RESUMEN

Drug resistance in small cell lung cancer (SCLC) significantly affects the efficacy of chemotherapy treatment. However, due to the lack of tumor tissue samples, especially serial tumor samples during chemotherapy, the mechanism of chemotherapy resistance has not been fully studied. Circulating tumor DNA, which can be obtained in a noninvasive manner, can complement tumor sampling approaches for research in this field. We identified an SCLC patient with acquired drug resistance from 52 SCLC patients for whom follow-up data were available. By comparing somatic mutations in circulating tumor DNA before and after chemotherapy, for the first time, we found that the somatic mutation eIF3A R803K may be related to acquired chemotherapy resistance. Then, the association between the eIF3A R803K mutation and chemotherapy resistance was confirmed by samples from 254 lung cancer patients receiving chemotherapy. We found that the eIF3a R803K mutation weakened the proliferation ability of tumor cells but increased their resistance to chemotherapy. Further studies revealed that the eIF3A R803K mutation promotes cellular senescence. In addition, fisetin showed a synergistic effect with chemotherapy in eIF3A R803K mutant cells. These results suggest that the eIF3A R803K somatic mutation has the potential to predict chemotherapy resistance in SCLC. Moreover, the eIF3A R803K mutation promotes chemotherapy resistance by inducing senescence. Furthermore, a senolytic drug, fisetin, can reverse chemotherapy resistance mediated by the eIF3A R803K mutation.

Asunto(s)

Senescencia Celular/genética , Resistencia a Antineoplásicos/genética , Factor 3 de Iniciación Eucariótica/genética , Neoplasias Pulmonares/genética , Carcinoma Pulmonar de Células Pequeñas/genética , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular , Movimiento Celular , Supervivencia Celular , Femenino , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/mortalidad , Masculino , Persona de Mediana Edad , Mutación , Inhibidores de la Síntesis de la Proteína/farmacología , Inhibidores de la Síntesis de la Proteína/uso terapéutico , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico , Carcinoma Pulmonar de Células Pequeñas/mortalidad

RESUMEN

The ubiquitin-proteasome system constitutes a major pathway for protein degradation in the cell. Therefore the crosstalk of this pathway with mitochondria is a major topic with direct relevance to many mitochondrial diseases. Proteasome dysfunction triggers not only protein toxicity, but also mitochondrial dysfunction. The involvement of proteasomes in the regulation of protein transport into mitochondria contributes to an increase in mitochondrial function defects. On the other hand, mitochondrial impairment stimulates reactive oxygen species production, which increases protein damage, and protein misfolding and aggregation leading to proteasome overload. Concurrently, mitochondrial dysfunction compromises cellular ATP production leading to reduced protein ubiquitination and proteasome activity. In this review we discuss the complex relationship and interdependence of the ubiquitin-proteasome system and mitochondria. Furthermore, we describe pharmacological inhibition of proteasome activity as a novel strategy to treat a group of mitochondrial diseases.

Asunto(s)

Mitocondrias/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina/metabolismo , Animales , Humanos , Enfermedades Mitocondriales/tratamiento farmacológico , Enfermedades Mitocondriales/metabolismo , Péptidos/metabolismo

RESUMEN

MEF2D-fusion (M-fusion) genes are newly discovered recurrent gene abnormalities that are detected in approximately 5 % of acute lymphoblastic leukemia (ALL) cases. Their introduction to cells has been reported to transform cell lines or increase the colony formation of bone marrow cells, suggesting their survival-supporting ability, which prompted us to examine M-fusion-targeting drugs. To identify compounds that reduce the protein expression level of MEF2D, we developed a high-throughput screening system using 293T cells stably expressing a fusion protein of MEF2D and luciferase, in which the protein expression level of MEF2D was easily measured by a luciferase assay. We screened 3766 compounds with known pharmaceutical activities using this system and selected staurosporine as a potential inducer of the proteolysis of MEF2D. Staurosporine induced the proteolysis of M-fusion proteins in M-fusion (+) ALL cell lines. Proteolysis was inhibited by caspase inhibitors, not proteasome inhibitors, suggesting caspase dependency. Consistent with this result, the growth inhibitory effects of staurosporine were stronger in M-fusion (+) ALL cell lines than in negative cell lines, and caspase inhibitors blocked apoptosis induced by staurosporine. We identified the cleavage site of MEF2D-HNRNPUL1 by caspases and confirmed that its caspase cleavage-resistant mutant was resistant to staurosporine-induced proteolysis. Based on these results, we investigated another Food and Drug Administration-approved caspase activator, venetoclax, and found that it exerted similar effects to staurosporine, namely, the proteolysis of M-fusion proteins and strong growth inhibitory effects in M-fusion (+) ALL cell lines. The present study provides novel insights into drug screening strategies and the clinical indications of venetoclax.

Asunto(s)

Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Caspasas/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Estaurosporina/farmacología , Sulfonamidas/farmacología , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Fusión Génica , Células HEK293 , Humanos , Factores de Transcripción MEF2/genética , Factores de Transcripción MEF2/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Proteolisis , Transducción de Señal

RESUMEN

Cardiac injury is followed by fibrosis, characterized by myofibroblast activation. Excessive deposition of extracellular matrix (ECM) impairs the plasticity of myocardium and results in myocardial systolic and diastolic dysfunction. Mangiferin is a xanthonoid derivative rich in plants mangoes and iris unguicularis, exhibiting the ability to ameliorate metabolic disorders. This study aims to investigate whether mangiferin attenuates cardiac fibrosis via redox regulation. The transverse aortic constriction (TAC) in mice induced cardiac fibrosis with impaired heart function. Oral administration of mangiferin (50 mg/kg, 4 weeks) inhibited myofibroblast activation with reduced formation of ECM. The impaired left ventricular contractive function was also improved by mangiferin. TGF-ß1 stimulation increased glutaminolysis to fuel intracellular glutamate pool for the increased demands of nutrients to support cardiac myofibroblast activation. Mangiferin degraded Keap1 to promote Nrf2 protein accumulation by improving its stability, leading to Nrf2 activation. Nrf2 transcriptionally promotes the synthesis of antioxidant proteins. By activating Nrf2, mangiferin promoted the synthesis of glutathione (GSH) in cardiac fibroblasts, likely due to the consumption of glutaminolysis-derived glutamate as a source. Meanwhile, mangiferin promoted the exchange of intracellular glutamate for the import of extracellular cystine to support GSH generation. As a result of redistribution, the reduced glutamate availability failed to support myofibroblast activation. In support of this, the addition of extracellular glutamate or α-ketoglutarate diminished the inhibitory effects of mangiferin on cardiac myofibroblast proliferation and activation. Moreover, cardiac knockdown of Nrf2 attenuated the cardioprotective effects of mangiferin in mice subjected to TAC. In conclusion, we demonstrated that activated myofibroblasts were sensitive to glutamate availability. Mangiferin activated Nrf2 and redistributed intracellular glutamate for the synthesis of GSH, consequently impairing cardiac myofibroblast activation due to decreased glutamate availability. These results address that pharmacological activation of Nrf2 could restrain cardiac fibrosis via metabolic regulation.

Asunto(s)

Cardiomiopatías/prevención & control , Ácido Glutámico/metabolismo , Glutatión/metabolismo , Miocardio/metabolismo , Miofibroblastos/efectos de los fármacos , Factor 2 Relacionado con NF-E2/agonistas , Xantonas/farmacología , Animales , Cardiomiopatías/genética , Cardiomiopatías/metabolismo , Cardiomiopatías/patología , Modelos Animales de Enfermedad , Fibrosis , Masculino , Ratones , Ratones Endogámicos C57BL , Miocardio/patología , Miofibroblastos/metabolismo , Miofibroblastos/patología , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Células 3T3 NIH , Ratas Sprague-Dawley , Transducción de Señal

RESUMEN

Salinomycin, a polyether antibiotic, acts as a highly selective potassium ionophore and has anticancer activity on various cancer cell lines. Cisplatin has been proved as chemotherapy drug for advanced human non-small cell lung cancer (NSCLC). Thymidylate synthase (TS) is a key enzyme in the pyrimidine salvage pathway, and increased expression of TS is thought to be associated with resistance to cisplatin. In this study, we showed that salinomycin (0.5-2µg/mL) treatment down-regulating of TS expression in an AKT inactivation manner in two NSCLC cell lines, human lung adenocarcinoma A549 and squamous cell carcinoma H1703 cells. Knockdown of TS using small interfering RNA (siRNA) or inhibiting AKT activity with PI3K inhibitor LY294002 enhanced the cytotoxicity and cell growth inhibition of salinomycin. A combination of cisplatin and salinomycin resulted in synergistic enhancement of cytotoxicity and cell growth inhibition in NSCLC cells, accompanied with reduced activation of phospho-AKT, and TS expression. Overexpression of a constitutive active AKT (AKT-CA) expression vector reversed the salinomycin and cisplatin-induced synergistic cytotoxicity. In contrast, pretreatment with LY294002 further decreased the cell viability in salinomycin and cisplatin cotreated cells. Our findings suggested that the down-regulation of AKT-mediated TS expression by salinomycin enhanced the cisplatin-induced cytotoxicity in NSCLC cells. These results may provide a rationale to combine salinomycin with cisplatin for lung cancer treatment.

Asunto(s)

Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Cisplatino/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Piranos/farmacología , Timidilato Sintasa/metabolismo , Antibacterianos/farmacología , Antineoplásicos/farmacología , Línea Celular Tumoral , Regulación hacia Abajo , Quimioterapia Combinada , Humanos , Proteínas Proto-Oncogénicas c-akt/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Timidilato Sintasa/genética

RESUMEN

The activation of autophagy has been demonstrated to exert protective roles during hypoxia-reoxygenation (H/R)-induced brain injuries. This study aimed to investigate whether and how preconditioning with a proteasome inhibitor (MG-132), a proteasome promoter (Adriamycin, ADM), an autophagy inhibitor (3-methyladenine, 3-MA) and an autophagy promoter (Rapamycin, Rap) affected endoplasmic reticulum stress (ERS), the ubiquitin-proteasome system (UPS), autophagy, inflammation and apoptosis. Ubiquitin protein and 26S proteasome activity levels were decreased by MG-132 pretreatment but increased by ADM pretreatment at 2h, 4h and 6h following H/R treatment. MG-132 pretreatment led to the increased expression of autophagy-related genes, ER stress-associated genes and IκB but decreased the expression levels of NF-κB and caspase-3. ADM pretreatment led to the decreased expression of autophagy-related genes, ERS-associated genes and IκB but increased the expression of NF-κB and caspase-3. Pretreatment with 3-MA reduced the expression of autophagy-related genes, autophagy and UPS co-related genes, as well as apoptosis-related although the latter was increased by Rap pretreatment at 2h, 4h and 6h following H/R treatment. In vivo, pretreatment of rats with ADM, MG-132, 3-MA or Rap followed by ischemia-reperfusion (I/R) treatment resulted in similar changes. Proteasome inhibition preconditioning strengthened autophagy and ER stress but decreased apoptosis and inflammation. Autophagy promotion preconditioning exhibited similar changes. The combination of a proteasome inhibitor and an autophagy promoter might represent a new possible therapy to treat H/R or I/R injury-related diseases.

Asunto(s)

Autofagia , Estrés del Retículo Endoplásmico , Oxígeno/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Ubiquitina/metabolismo , Adenina/análogos & derivados , Adenina/farmacología , Animales , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Hipoxia de la Célula/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Doxorrubicina/farmacología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Histona Desacetilasa 6 , Histona Desacetilasas/metabolismo , Leupeptinas/farmacología , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Pulmón/patología , Masculino , FN-kappa B/metabolismo , Ratas , Sirolimus/farmacología

RESUMEN

Parkin is a Parkinson's disease (PD)-linked gene that plays an important role in the ubiquitin-proteasome system (UPS). This study explored whether carnosic acid (CA) from rosemary protects against 6-hydroxydopamine (6-OHDA)-induced neurotoxicity via upregulation of parkin in vivo and in vitro. We found that the reduction in proteasomal activity by 6-OHDA was attenuated in SH-SY5Y cells pretreated with 1 µM CA. Immunoblots showed that CA reversed the induction of ubiquitinated protein and the reduction of PTEN-induced putative kinase 1 (PINK1) and parkin protein in 6-OHDA-treated SH-SY5Y cells and rats. Moreover, in a transgenic OW13 Caenorhabditis elegans model of PD that expresses human α-synuclein in muscle cells, CA reduced α-synuclein accumulation in a dose-dependent manner. In cells pretreated with the proteasome inhibitor MG132, CA no longer reversed the 6-OHDA-mediated induction of cleavage of caspase 3 and poly(ADP)-ribose polymerase and no longer reversed the suppression of proteasome activity. When parkin expression was silenced by use of small interfering RNA, the ability of CA to inhibit apoptosis and induce proteasomal activity was significantly reduced. The reduction in 6-OHDA-induced neurotoxicity by CA was associated with the induction of parkin, which in turn upregulated the UPS and then decreased cell death.

Asunto(s)

Abietanos/farmacología , Citoprotección/fisiología , Oxidopamina/toxicidad , Ubiquitina-Proteína Ligasas/biosíntesis , Regulación hacia Arriba/fisiología , Animales , Animales Modificados Genéticamente , Antioxidantes/farmacología , Caenorhabditis elegans , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Línea Celular Tumoral , Citoprotección/efectos de los fármacos , Humanos , Masculino , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Regulación hacia Arriba/efectos de los fármacos , alfa-Sinucleína/biosíntesis

RESUMEN

Astaxanthin has been demonstrated to exhibit a wide range of beneficial effects, including anti-inflammatory and anti-cancer properties. However, the molecular mechanism of astaxanthin-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells has not been identified. Rad51 plays a central role in homologous recombination, and studies show that chemo-resistant carcinomas exhibit high levels of Rad51 expression. In this study, astaxanthin treatment inhibited cell viability and proliferation of two NSCLC cells, A549 and H1703. Astaxanthin treatment (2.5-20 µM) decreased Rad51 expression and phospho-AKT(Ser473) protein level in a time and dose-dependent manner. Furthermore, expression of constitutively active AKT (AKT-CA) vector rescued the decreased Rad51 mRNA and protein levels in astaxanthin-treated NSCLC cells. Combined treatment with phosphatidylinositol 3-kinase (PI3K) inhibitors (LY294002 or wortmannin) further decreased the Rad51 expression in astaxanthin-exposed A549 and H1703 cells. Knockdown of Rad51 expression by transfection with si-Rad51 RNA or cotreatment with LY294002 further enhanced the cytotoxicity and cell growth inhibition of astaxanthin. Additionally, mitomycin C (MMC) as an anti-tumor antibiotic is widely used in clinical NSCLC chemotherapy. Combination of MMC and astaxanthin synergistically resulted in cytotoxicity and cell growth inhibition in NSCLC cells, accompanied with reduced phospho-AKT(Ser473) level and Rad51 expression. Overexpression of AKT-CA or Flag-tagged Rad51 reversed the astaxanthin and MMC-induced synergistic cytotoxicity. In contrast, pretreatment with LY294002 further decreased the cell viability in astaxanthin and MMC co-treated cells. In conclusion, astaxanthin enhances MMC-induced cytotoxicity by decreasing Rad51 expression and AKT activation. These findings may provide rationale to combine astaxanthin with MMC for the treatment of NSCLC.

Asunto(s)

Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Neoplasias Pulmonares/metabolismo , Mitomicina/toxicidad , Proteína Oncogénica v-akt/metabolismo , Recombinasa Rad51/biosíntesis , Antibióticos Antineoplásicos/toxicidad , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Mitomicina/uso terapéutico , Proteína Oncogénica v-akt/antagonistas & inhibidores , Recombinasa Rad51/antagonistas & inhibidores , Xantófilas/toxicidad

RESUMEN

The activator of G protein signalling AGS2 (Tctex-1) forms protein complexes with Gßγ, and controls cell proliferation by regulating cell cycle progression. A direct interaction of Tctex-1 with various G protein-coupled receptors has been reported. Since the carboxyl terminal portion of CB2 carries a putative Tctex-1 binding motif, we investigated the potential interplay of CB2 and Tctex-1 in the absence and presence of Gßγ. The supposed interaction of cannabinoid receptor CB2 with Tctex-1 and the influence of CB2 on the formation of Tctex-1-Gßγ-complexes were studied by co- and/or immunoprecipitation experiments in transiently transfected HEK293 cells. The analysis on Tctex-1 protein was performed in the absence and presence of the ligands JWH 133, 2-AG, and AM 630, the protein biosynthesis inhibitor cycloheximide or the protein degradation blockers MG132, NH4Cl/leupeptin or bafilomycin. Our results show that CB2 neither directly nor indirectly via Gßγ interacts with Tctex-1, but competes with Tctex-1 in binding to Gßγ. The Tctex-1-Gßγ protein interaction was disrupted by CB2 receptor expression resulting in a release of Tctex-1 from the complex, and its degradation by the proteasome and partly by lysosomes. The decrease in Tctex-1 protein levels is induced by CB2 expression "dose-dependently" and is independent of stimulation by agonist or blocking by an inverse agonist treatment. The results suggest that CB2 receptor expression independent of its activation by agonists is sufficient to competitively disrupt Gßγ-Tctex-1 complexes, and to initiate Tctex-1 degradation. These findings implicate that CB2 receptor expression modifies the stability of intracellular protein complexes by a non-canonical pathway.

Asunto(s)

Dineínas/metabolismo , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Receptor Cannabinoide CB2/biosíntesis , Agonismo Inverso de Drogas , Regulación de la Expresión Génica , Células HEK293 , Humanos , Indoles/farmacología , Unión Proteica/fisiología , Receptor Cannabinoide CB2/antagonistas & inhibidores

RESUMEN

During tumor progression, hypoxia-inducible factor 1 (HIF-1) plays a critical role in tumor angiogenesis and tumor growth by regulating the transcription of several genes in response to a hypoxic environment and changes in growth factors. This study was designed to investigate the effects of cinnamic aldehyde (CA) on tumor growth and angiogenesis and the mechanisms underlying CA's anti-angiogenic activities. We found that CA administration inhibits tumor growth and blocks tumor angiogenesis in BALB/c mice. In addition, CA treatment decreased HIF-1α protein expression and vascular endothelial growth factor (VEGF) expression in mouse tumors and Renca cells exposed to hypoxia in vitro. Interestingly, CA treatment did not affect the stability of von Hippel-Lindau protein (pVHL)-associated HIF-1α and CA attenuated the activation of mammalian target of rapamycin (mTOR) pathway. Collectively, these findings strongly indicate that the anti-angiogenic activity of CA is, at least in part, regulated by the mTOR pathway-mediated suppression of HIF-1α protein expression and these findings suggest that CA may be a potential drug for human cancer therapy.

Asunto(s)

Acroleína/análogos & derivados , Antineoplásicos Fitogénicos/farmacología , Subunidad alfa del Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Hipoxia/metabolismo , Neoplasias Experimentales/irrigación sanguínea , Neovascularización Patológica/prevención & control , Acroleína/farmacología , Animales , Línea Celular Tumoral , Movimiento Celular , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Hipoxia/complicaciones , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Neoplasias Experimentales/tratamiento farmacológico , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo

RESUMEN

Minocycline is a semisynthetic tetracycline derivative; it has anti-inflammatory and anti-cancer effects distinct from its antimicrobial function. However, the molecular mechanism of minocycline-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells has not been identified. Rad51 plays a central role in homologous recombination and high levels of Rad51 expression are observed in chemo- or radioresistant carcinomas. Our previous studies have shown that the MKK1/2-ERK1/2 signal pathway maintains the expression of Rad51 in NSCLC cells. In this study, minocycline treatment inhibited cell viability and proliferation of two NSCLC cells, A549 and H1975. Treatment with minocycline decreased Rad51 mRNA and protein levels through MKK1/2-ERK1/2 inactivation. Furthermore, expression of constitutively active MKK1 (MKK1-CA) vectors significantly rescued the decreased Rad51 protein and mRNA levels in minocycline-treated NSCLC cells. However, combined treatment with MKK1/2 inhibitor U0126 and minocycline further decreased the Rad51 expression and cell viability of NSCLC cells. Knocking down Rad51 expression by transfection with small interfering RNA of Rad51 enhanced the cytotoxicity and cell growth inhibition of minocycline. Mitomycin C (MMC) is typically used as a first or second line regimen to treat NSCLC. Compared to a single agent alone, MMC combined with minocycline resulted in cytotoxicity and cell growth inhibition synergistically in NSCLC cells, accompanied with reduced activation of phospho-ERK1/2, and reduced Rad51 protein levels. Overexpression of MKK1-CA or Flag-tagged Rad51 could reverse the minocycline and MMC-induced synergistic cytotoxicity. These findings may have implications for the rational design of future drug regimens incorporating minocycline and MMC for the treatment of NSCLC.

Asunto(s)

Antibióticos Antineoplásicos/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Minociclina/farmacología , Mitomicina/farmacología , Recombinasa Rad51/genética , Antibióticos Antineoplásicos/administración & dosificación , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Regulación hacia Abajo , Sinergismo Farmacológico , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Minociclina/administración & dosificación , Mitomicina/administración & dosificación

RESUMEN

Transforming growth factor-ß (TGF-ß) has both tumor suppressive and oncogenic activities. Autocrine TGF-ß signaling supports tumor survival and growth in certain types of cancer, and the TGF-ß signaling pathway is a potential therapeutic target for these types of cancer. TGF-ß induces p21 expression, and p21 is considered as an oncogene as well as a tumor suppressor, due to its anti-apoptotic activity. Thus, we hypothesized that autocrine TGF-ß signaling maintains the expression of p21 at levels that can support cell growth. To verify this hypothesis, we sought to examine p21 expression and cell growth in various cancer cells following the inhibition of autocrine TGF-ß signaling using siRNAs targeting TGF-ß signaling components and SB431542, a TGF-ß receptor inhibitor. Results from the present study show that p21 expression and cell growth were reduced by knockdown of TGF-ß signaling components using siRNA in MDA-MB231 and A549 cells. Cell growth was also reduced in p21 siRNA-transfected cells. Downregulation of p21 expression induced cellular senescence in MDA-MB231 cells but did not induce apoptosis in both cells. These data suggest that autocrine TGF-ß signaling is required to sustain p21 levels for positive regulation of cell cycle. On the other hand, treatment with SB431542 up-regulated p21 expression while inhibiting cell growth. The TGF-ß signaling pathway was not associated with the SB431542-mediated induction of p21 expression. Specificity protein 1 (Sp1) was downregulated by treatment with SB431542, and p21 expression was increased by Sp1 knockdown. These findings suggest that downregulation of Sp1 expression is responsible for SB43154-induced p21 expression.

Asunto(s)

Benzamidas/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Dioxoles/farmacología , Proteínas Serina-Treonina Quinasas/efectos de los fármacos , Receptores de Factores de Crecimiento Transformadores beta/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta/farmacología , Animales , Comunicación Autocrina/efectos de los fármacos , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Células COS , Células CACO-2 , Proliferación Celular/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Chlorocebus aethiops , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Células HCT116 , Humanos , Células MCF-7 , Masculino , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Interferencia de ARN , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/genética , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Factor de Transcripción Sp1/genética , Factor de Transcripción Sp1/metabolismo , Factores de Tiempo , Transfección

RESUMEN

High-Voltage-Activated (HVA) Ca(2+) channels are known regulators of synapse formation and transmission and play fundamental roles in neuronal pathophysiology. Small GTPases of Rho and RGK families, via their action on both cytoskeleton and Ca(2+) channels are key molecules for these processes. While the effects of RGK GTPases on neuronal HVA Ca(2+) channels have been widely studied, the effects of RhoA on the HVA channels remains however elusive. Using heterologous expression in Xenopus laevis oocytes, we show that RhoA activity reduces Ba(2+) currents through CaV2.1, CaV2.2 and CaV2.3 Ca(2+) channels independently of CaVß subunit. This inhibition occurs independently of RGKs activity and without modification of biophysical properties and global level of expression of the channel subunit. Instead, we observed a marked decrease in the number of active channels at the plasma membrane. Pharmacological and expression studies suggest that channel expression at the plasma membrane is impaired via a ROCK-sensitive pathway. Expression of constitutively active RhoA in primary culture of spinal motoneurons also drastically reduced HVA Ca(2+) current amplitude. Altogether our data revealed that HVA Ca(2+) channels regulation by RhoA might govern synaptic transmission during development and potentially contribute to pathophysiological processes when axon regeneration and growth cone kinetics are impaired.

Asunto(s)

Canales de Calcio Tipo N/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Animales , Bario/metabolismo , Calcio/metabolismo , Canales de Calcio Tipo N/genética , Cationes/metabolismo , Membrana Celular/fisiología , Células Cultivadas , Electroporación , Potenciales de la Membrana/fisiología , Ratones Transgénicos , Neuronas Motoras/fisiología , Oocitos , Técnicas de Placa-Clamp , Médula Espinal/fisiología , Xenopus laevis , Proteínas de Unión al GTP rho/genética , Quinasas Asociadas a rho/metabolismo

RESUMEN

Inhibition of hypoxia inducible factor-prolyl hydroxylase-2 (HPH), leading to activation of hypoxia inducible factor (HIF)-1 is a potential therapeutic strategy for the treatment of colitis. Rosmarinic acid (RA), an ester of caffeic acid and 3,4-dihydroxyphenyllactic acid is a naturally occurring polyphenolic compound with two catechols, a or inhibition of HPH. To improve accessibility of highly hydrophilic RA to HPH, an intracellular target, RA was chemically modified to decrease hydrophilicity. Of the less-hydrophilic derivatives, rosmarinic acid methyl ester (RAME) most potently inhibited HPH. Accordingly, RAME prevented hydroxylation of HIF-1α and consequently stabilized HIF-1α protein in cells. RAME inhibition of HPH and induction of HIF-1α were diminished by elevated doses of the required factors of HPH, 2-ketoglutarate and ascorbate. RAME induction of HIF-1α led to activation of an ulcer healing pathway, HIF-1-vascular endothelial growth factor (VEGF), in human colon carcinoma cells. RAME administered rectally ameliorated TNBS-induced rat colitis and substantially decreased the levels of pro-inflammatory mediators in the inflamed colonic tissue. In parallel with the cellular effects of RAME, RAME up-regulated HIF-1α and VEGF in the inflamed colonic tissue. Thus, lipophilic modification of RA improves its ability to inhibit HPH, leading to activation of the HIF-1-VEGF pathway. RAME, a lipophilic RA derivative, may exert anti-colitic effects via activation of the ulcer healing pathway.

Asunto(s)

Cinamatos/química , Cinamatos/farmacología , Colitis/tratamiento farmacológico , Depsidos/química , Depsidos/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Interacciones Hidrofóbicas e Hidrofílicas , Prolina Dioxigenasas del Factor Inducible por Hipoxia/antagonistas & inhibidores , Animales , Ácido Ascórbico/farmacología , Ácidos Carboxílicos/química , Línea Celular Tumoral , Cinamatos/uso terapéutico , Coenzimas/fisiología , Colitis/inducido químicamente , Colitis/metabolismo , Colitis/patología , Depsidos/uso terapéutico , Inhibidores Enzimáticos/uso terapéutico , Ésteres , Humanos , Hidroxilación/efectos de los fármacos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Prolina Dioxigenasas del Factor Inducible por Hipoxia/metabolismo , Ácidos Cetoglutáricos/farmacología , Estabilidad Proteica/efectos de los fármacos , Ratas , Transducción de Señal/efectos de los fármacos , Relación Estructura-Actividad , Ácido Trinitrobencenosulfónico/efectos adversos , Factor A de Crecimiento Endotelial Vascular/metabolismo , Ácido Rosmarínico

RESUMEN

The pathogenic processes involving in the development of diabetes range from autoimmune destruction of pancreatic ß-cells with consequent insulin deficiency to abnormalities that result in resistance to insulin action. The major contributing factor for excessive ß-cell death includes oxidative stress-mediated mitochondrial damage, which creates an imbalance in redox homeostasis. Yet, ß-cells have evolved adaptive mechanisms to endure a wide range of stress conditions to safeguard its potential functions. These include 'Nrf2/Keap1' pathway, a key cellular defense mechanism, to combat oxidative stress by regulating phase II detoxifying and antioxidant genes. During diabetes, redox imbalance provokes defective Nrf2-dependent signaling and compromise antioxidant capacity of the pancreas which turnout ß-cells to become highly vulnerable against various insults. Hence, identification of small molecule activators of Nrf2/Keap1 pathway remains significant to enhance cellular defense to overcome the burden of oxidative stress related disturbances. This review summarizes the molecular mechanism behind Nrf2 activation and the impact of Nrf2 activators in diabetes and its complications.

Asunto(s)

Diabetes Mellitus/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Animales , Humanos , Proteína 1 Asociada A ECH Tipo Kelch , Oxidación-Reducción , Transducción de Señal , Estrés Fisiológico

RESUMEN

Calcium sensing receptor (CaSR) mutations or altered expression cause disorders of calcium handling. Recent studies suggest that reduced targeting to the plasma membrane is a feature common to many CaSR loss-of-function mutations. Allosteric agonists (calcimimetics) can rescue signaling of a subset of CaSR mutants. This review evaluates our current understanding of the subcellular site(s) for allosteric modulator rescue of CaSR mutants. Studies to date make a strong case for calcimimetic potentiation of signaling not only at plasma membrane-localized CaSR, but at the endoplasmic reticulum, acting as pharmacoperones to assist in navigation of multiple quality control checkpoints. The possible role of endogenous pharmacoperones, calcium and glutathione, in folding and stabilization of the CaSR extracellular and transmembrane domains are considered. Finally, the possibility that dihydropyridines act as unintended pharmacoperones of CaSR is proposed. While our understanding of pharmacoperone rescue of CaSR requires refinement, promising results to date argue that this may be a fruitful avenue for drug discovery.

Asunto(s)

Descubrimiento de Drogas , Receptores Sensibles al Calcio/metabolismo , Regulación Alostérica/efectos de los fármacos , Animales , Calcio/metabolismo , Dihidropiridinas/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Glutatión/metabolismo , Humanos , Mutación , Biosíntesis de Proteínas/efectos de los fármacos , Pliegue de Proteína/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Receptores Sensibles al Calcio/análisis , Receptores Sensibles al Calcio/genética