RESUMEN
Oncogenic multidrug resistance is commonly intrinsic to renal cancer based on the physiological expression of detoxification transporters, particularly ABCB1, thus hampering chemotherapy. ABCB1 activity is directly dependent on its lipid microenvironment, localizing to cholesterol- and sphingomyelin (SM)-rich domains. As ceramides are the sole source for SMs, we hypothesized that ceramide synthase (CerS)-derived ceramides regulate ABCB1 activity. Using data from RNA-Seq databases, we found that patient kidney tumors exhibited increased CerS2 mRNA, which was inversely correlated with CerS6 mRNA in ABCB1+ clear cell carcinomas. Endogenous elevated CerS2 and lower CerS5/6 mRNA and protein resulted in disproportionately higher CerS2 to CerS5/6 activities (approximately twofold) in chemoresistant ABCB1high (A498, Caki-1) compared with chemosensitive ABCB1low (ACHN, normal human proximal convoluted tubule cell) cells. In addition, lipidomics analyses by HPLC-MS/MS showed bias toward CerS2-associated C20:0/C20:1-ceramides compared with CerS5/6-associated C14:0/C16:0-ceramides (2:1). SMs were similarly altered. We demonstrated that chemoresistance to doxorubicin in ABCB1high cells was partially reversed by inhibitors of de novo ceramide synthesis (l-cycloserine) and CerS (fumonisin B1) in cell viability assays. Downregulation of CerS2/6, but not CerS5, attenuated ABCB1 mRNA, protein, plasma membrane localization, rhodamine 123+ efflux transport activity, and doxorubicin resistance. Similar findings were observed with catalytically inactive CerS6-H212A. Furthermore, CerS6-targeting siRNA shifted ceramide and SM composition to ultra long-chain species (C22-C26). Inhibitors of endoplasmic reticulum-associated degradation (eeyarestatin I) and the proteasome (MG132, bortezomib) prevented ABCB1 loss induced by CerS2/6 downregulation. We conclude that a critical balance in ceramide/SM species is prerequisite to ABCB1 expression and functionalization, which could be targeted to reverse multidrug resistance in renal cancers.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP , Neoplasias Renales , Proteínas de la Membrana , Esfingolípidos , Esfingosina N-Aciltransferasa , Proteínas Supresoras de Tumor , Subfamilia B de Transportador de Casetes de Unión a ATP/biosíntesis , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Ceramidas/metabolismo , Doxorrubicina/farmacología , Resistencia a Antineoplásicos , Degradación Asociada con el Retículo Endoplásmico , Femenino , Humanos , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/genética , Neoplasias Renales/metabolismo , Masculino , Proteínas de la Membrana/metabolismo , ARN Mensajero/genética , Esfingolípidos/metabolismo , Esfingosina N-Aciltransferasa/genética , Esfingosina N-Aciltransferasa/metabolismo , Espectrometría de Masas en Tándem , Microambiente TumoralRESUMEN
The overexpression of the human ATP-binding cassette (ABC) drug transporter ABCB1 (P-glycoprotein, P-gp) or ABCG2 (breast cancer resistance protein, BCRP) in cancer cells often contributes significantly to the development of multidrug resistance (MDR) in cancer patients. Previous reports have demonstrated that some epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) could modulate the activity of ABCB1 and/or ABCG2 in human cancer cells, whereas some EGFR TKIs are transport substrates of these transporters. Almonertinib (HS-10296) is a promising, orally available third-generation EGFR TKI for the treatment of EGFR T790M mutation-positive non-small cell lung cancer (NSCLC) in patients who have progressed on or after other EGFR TKI therapies. Additional clinical trials are currently in progress to study almonertinib as monotherapy and in combination with other agents in patients with NSCLC. In the present work, we found that neither ABCB1 nor ABCG2 confers significant resistance to almonertinib. More importantly, we discovered that almonertinib was able to reverse MDR mediated by ABCB1, but not ABCG2, in multidrug-resistant cancer cells at submicromolar concentrations by inhibiting the drug transport activity of ABCB1 without affecting its expression level. These findings are further supported by in silico docking of almonertinib in the drug-binding pocket of ABCB1. In summary, our study revealed an additional activity of almonertinib to re-sensitize ABCB1-overexpressing multidrug-resistant cancer cells to conventional chemotherapeutic drugs, which may be beneficial for cancer patients and warrant further investigation.
Asunto(s)
Acrilamidas/farmacología , Antineoplásicos/farmacología , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Indoles/farmacología , Pirimidinas/farmacología , Subfamilia B de Transportador de Casetes de Unión a ATP/antagonistas & inhibidores , Subfamilia B de Transportador de Casetes de Unión a ATP/biosíntesis , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos/fisiología , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/biosíntesis , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Estructura Secundaria de ProteínaRESUMEN
The development of multidrug resistance is the major obstacle to successful lung cancer chemotherapy. Cancer cells gain resistance through increased levels of Pglycoprotein (Pgp), which is encoded by the multidrug resistanceassociated protein 1 (MDR1) gene. Leucinerich PPR motifcontaining protein (LRPPRC), a member of the PPR family, has been verified to regulate the transcription of MDR1. This regulation is influenced by the methylation status of the GC 100 box in the MDR1 promoter. The present study aimed to investigate the effect of LRPPRC on cisplatin (DDP) resistance in lung cancer cells and explore the underlying mechanism. DDPresistant nonsmall cell lung cancer cell lines (A549/DDP, H1299/DDP) were generated. The expression levels of LRPPRC and Pgp/MDR1, investigated by qPCR and western blot analysis, were increased in the A549/DDP and H1299/DDP cells compared with that in the parental cells. LRPPRC silencing with shRNA increased DDP sensitivity in vitro and in vivo. LRPPRC silencing inhibited the level of LRPPRC binding with the MDR1 promoter, investigated by chromatin immunoprecipitationqPCR, and the corresponding MDR1 expression. Demethylation treatment rescued the decrease in the level of LRPPRC binding with MDR1 and the corresponding expression of MDR1 and the increase in DDP sensitivity due to LRPPRC silencing. Our study suggests that LRPPRC contributes to DDP resistance in lung cancer cells by regulating MDR1 transcription. Thus, LRPPRC may serve as a potential molecular target for chemoresistance reversal in lung cancer.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Cisplatino/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Proteínas de Neoplasias/metabolismo , Células A549 , Subfamilia B de Transportador de Casetes de Unión a ATP/biosíntesis , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Animales , Antineoplásicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/genética , 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 , Metilación de ADN , Resistencia a Antineoplásicos , Xenoinjertos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Mensajero/metabolismoAsunto(s)
Resistencia a Múltiples Medicamentos/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Mieloma Múltiple , Proteínas de Neoplasias/biosíntesis , Oligopéptidos/uso terapéutico , Regulación hacia Arriba/efectos de los fármacos , Subfamilia B de Transportador de Casetes de Unión a ATP/biosíntesis , Humanos , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patologíaRESUMEN
BACKGROUND/AIMS: Obstructive sleep apnea (OSA) is characterized by repeated episodes of complete or partial obstruction of the upper airways, leading to chronic intermittent hypoxia (IH). OSA patients are considered at high cerebrovascular risk and may also present cognitive impairment. One hypothesis explored is that disturbances may be linked to blood-brain barrier (BBB) dysfunction. The BBB is a protective barrier separating the brain from blood flow. The BBB limits the paracellular pathway through tight and adherens junctions, and the transcellular passage by efflux pumps (ABC transporters). The aims of this study were to evaluate the impact of IH and sustained hypoxia (SH) on a validated in vitro BBB model and to investigate the factors expressed under both conditions. METHODS: Exposure of endothelial cells (HBEC-5i) in our in vitro model of BBB to hypoxia was performed using IH cycles: 1% O2-35 min/18% O2-25 min for 6 cycles or 6 h of SH at 1% O2. After exposure, we studied the cytotoxicity and the level of ROS in our cells. We measured the apparent BBB permeability using sodium fluorescein, FITC-dextran and TEER measurement. Whole cell ELISA were performed to evaluate the expression of tight junctions, ABC transporters, HIF-1α and Nrf2. The functionality of ABC transporters was evaluated with accumulation studies. Immunofluorescence assays were also conducted to illustrate the whole cell ELISAs. RESULTS: Our study showed that 6 h of IH or SH induced a BBB disruption marked by a significant decrease in junction protein expressions (claudin-5, VE-cadherin, ZO-1) and an increase in permeability. We also observed an upregulation in P-gp protein expression and functionality and a downregulation in BCRP. Hypoxia induced production of ROS, Nrf2 and HIF-1α. They were expressed in both sustained and intermittent conditions, but the expression and the activity of P-gp and BCRP were different. CONCLUSION: Understanding these mechanisms seems essential in order to propose new therapeutic strategies for patients with OSA.
Asunto(s)
Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/biosíntesis , Barrera Hematoencefálica/metabolismo , Regulación de la Expresión Génica , Hipoxia Encefálica/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Modelos Cardiovasculares , Factor 2 Relacionado con NF-E2/metabolismo , Proteínas de Neoplasias/biosíntesis , Subfamilia B de Transportador de Casetes de Unión a ATP/biosíntesis , Barrera Hematoencefálica/patología , Línea Celular , Humanos , Hipoxia Encefálica/patologíaRESUMEN
Treatment of acute lymphoblastic leukemia (ALL) requires the combination of multiple drugs to integrate a complete remission. The different prognostic factors (age, leukocytes, risk, cytogenetic alterations) allow identifying those patients with a high risk of relapse, but there are few described factors that impact the induction response. The objective was to identify the utility of different risk factors (overexpression of the ABCB1 drug resistance gene, favorable response to steroids (FRS) and early response at day + 8 of treatment) on the percentage of complete remissions and overall survival. This is a prospective, observational study in adult patients with B-ALL without specific cytogenetic alterations, who started induction treatment based on a pretreatment with prednisone and subsequently vincristine (1.6 mg/m2 subcutaneous) plus daunorubicin (45 mg/m2 subcutaneously) on days + 1, + 8, + 15. The ABCB1 resistance gene was evaluated at diagnosis, the FRS at the end of the pretreatment and the early response during day + 8. A total of 53 adult patients diagnosed with ALL Philadelphia negative chromosome (Ph-), with immunophenotype B, with a normal karyotype, were studied. Cases with genetic abnormalities with a poor prognosis were excluded in order to reduce bias. The mean age was 48 years (range 17-68 years). 62.3% of patients were at high risk of relapse. When analyzing the risk factors, 30.2% showed high levels of the ABCB1 resistance gene, without showing an impact on the induction response (OR: 1.218, p = 0.743), but its overexpression was associated with a poor response to steroids as in the absence of early response. Individually, both the FRS (OR: 5.7, p = 0.004) and the absence of early response to day + 8 (OR: 6.42, p = 0.002) showed significance. By combining the different factors, having more than 2 was directly related to a failure (OR: 9.514, p = 0.000). The identification of factors such as FRS such as the persistence of blasts at the end of the first week of treatment is useful to identify patients at risk of failure in induction.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Quimioterapia de Inducción , Proteínas de Neoplasias/biosíntesis , Leucemia-Linfoma Linfoblástico de Células Precursoras , Subfamilia B de Transportador de Casetes de Unión a ATP/biosíntesis , Adolescente , Adulto , Anciano , Daunorrubicina/administración & dosificación , Supervivencia sin Enfermedad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidad , Prednisolona/administración & dosificación , Estudios Retrospectivos , Tasa de Supervivencia , Factores de Tiempo , Vincristina/administración & dosificaciónRESUMEN
Malaria in pregnancy (MiP) induces intrauterine growth restriction (IUGR) and preterm labour (PTL). However, its effects on yolk sac morphology and function are largely unexplored. We hypothesized that MiP modifies yolk sac morphology and efflux transport potential by modulating ABC efflux transporters. C57BL/6 mice injected with Plasmodium berghei ANKA (5 × 105 infected erythrocytes) at gestational day (GD) 13.5 were subjected to yolk sac membrane harvesting at GD 18.5 for histology, qPCR and immunohistochemistry. MiP did not alter the volumetric proportion of the yolk sac's histological components. However, it increased levels of Abcb1a mRNA (encoding P-glycoprotein) and macrophage migration inhibitory factor (Mif chemokine), while decreasing Abcg1 (P < 0.05); without altering Abca1, Abcb1b, Abcg2, Snat1, Snat2, interleukin (Il)-1ß and C-C Motif chemokine ligand 2 (Ccl2). Transcripts of Il-6, chemokine (C-X-C motif) ligand 1 (Cxcl1), Glut1 and Snat4 were not detectible. ABCA1, ABCG1, breast cancer resistance protein (BCRP) and P-gp were primarily immunolocalized to the cell membranes and cytoplasm of endodermic epithelium but also in the mesothelium and in the endothelium of mesodermic blood vessels. Intensity of P-gp labelling was stronger in both endodermic epithelium and mesothelium, whereas ABCA1 labelling increased in the endothelium of the mesodermic blood vessels. The presence of ABC transporters in the yolk sac wall suggests that this fetal membrane acts as an important protective gestational barrier. Changes in ABCA1 and P-gp in MiP may alter the biodistribution of toxic substances, xenobiotics, nutrients and immunological factors within the fetal compartment and participate in the pathogenesis of malaria-induced IUGR and PTL.
Asunto(s)
Transportador 1 de Casete de Unión a ATP/biosíntesis , Subfamilia B de Transportador de Casetes de Unión a ATP/biosíntesis , Regulación de la Expresión Génica , Malaria/metabolismo , Complicaciones Infecciosas del Embarazo/metabolismo , Saco Vitelino/metabolismo , Transportador 1 de Casete de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Animales , Transporte Biológico , Citocinas/biosíntesis , Citocinas/genética , Femenino , Retardo del Crecimiento Fetal/etiología , Inflamación , Malaria/complicaciones , Malaria/genética , Proteínas de Transporte de Membrana/biosíntesis , Proteínas de Transporte de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Tamaño de los Órganos , Plasmodium berghei , Embarazo , Complicaciones Infecciosas del Embarazo/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Saco Vitelino/ultraestructuraRESUMEN
The ATP Binding Cassette transporter B1 (ABCB1) induces chemoresistance in osteosarcoma, because it effluxes doxorubicin, reducing the intracellular accumulation, toxicity, and immunogenic cell death induced by the drug. The ATP Binding Cassette transporter A1 (ABCA1) effluxes isopentenyl pyrophosphate (IPP), a strong activator of anti-tumor Vγ9Vδ2 T-cells. Recruiting this population may represent an alternative strategy to rescue doxorubicin efficacy in ABCB1-expressing osteosarcoma. In this work, we analyzed how ABCA1 and ABCB1 are regulated in osteosarcoma, and if increasing the ABCA1-dependent activation of Vγ9Vδ2 T-cells could be an effective strategy against ABCB1-expressing osteosarcoma. We used 2D-cultured doxorubicin-sensitive human U-2OS and Saos-2 cells, their doxorubicin-resistant sublines (U-2OS/DX580 and Saos-2/DX580), and 3D cultures of U-2OS and Saos-2 cells. DX580-sublines and 3D cultures had higher levels of ABCB1 and higher resistance to doxorubicin than parental cells. Surprisingly, they had reduced ABCA1 levels, IPP efflux, and Vγ9Vδ2 T-cell-induced killing. In these chemo-immune-resistant cells, the Ras/Akt/mTOR axis inhibits the ABCA1-transcription induced by Liver X Receptor α (LXRα); Ras/ERK1/2/HIF-1α axis up-regulates ABCB1. Targeting the farnesylation of Ras with self-assembling nanoparticles encapsulating zoledronic acid (NZ) simultaneously inhibited both axes. In humanized mice, NZ reduced the growth of chemo-immune-resistant osteosarcomas, increased intratumor necro-apoptosis, and ABCA1/ABCB1 ratio and Vγ9Vδ2 T-cell infiltration. We suggest that the ABCB1highABCA1low phenotype is indicative of chemo-immune-resistance. We propose aminobisphosphonates as new chemo-immune-sensitizing tools against drug-resistant osteosarcomas.
Asunto(s)
Transportador 1 de Casete de Unión a ATP/metabolismo , Neoplasias Óseas/metabolismo , Osteosarcoma/metabolismo , Transportador 1 de Casete de Unión a ATP/biosíntesis , Transportador 1 de Casete de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/biosíntesis , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Animales , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/genética , Neoplasias Óseas/inmunología , Línea Celular Tumoral , Doxorrubicina/farmacología , Resistencia a Antineoplásicos , Femenino , Xenoinjertos , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Osteosarcoma/tratamiento farmacológico , Osteosarcoma/genética , Osteosarcoma/inmunología , Linfocitos T/inmunología , TransfecciónRESUMEN
BACKGROUND: Docetaxel is the preferred chemotherapeutic agent for hormone-refractory prostate cancer (PC) patients. However, patients eventually develop docetaxel resistance, and no effective treatment options are available for them. OBJECTIVE: We aimed to establish docetaxel resistance in castration-resistant prostate cancer (CRPC) cell lines (DU145/TXR, PC-3/TXR, and CWR22/TXR) and characterized transcriptional changes upon acquiring resistance to the docetaxel. METHODS: Human PC cells (DU145, PC-3, CWR22) and all docetaxel-resistant cells were maintained in Roswell Park Memorial Institute Medium (RPMI) 1640 media supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. ABCB1 was detected by using both parental and docetaxel-resistant CRPCs prepared for flow cytometry. For the evaluation of tumor-suppressive effects under each chemotherapeutic agent, subcutaneous xenografts of DU145 or DU145/TXR were implanted at the mouse flank. RESULTS: The P-glycoprotein-encoding gene ABCB1 was distinctively upregulated in the resistant cells, and its overexpression played an essential role in docetaxel resistance in CRPC. When tested for the cytotoxicity of gemcitabine, another option for chemotherapy, the docetaxel-resistant cells were shown to become sensitive to the drug, implying additional phenotypic transformation in the docetaxel-resistant cells. Studies using xenograft animal models demonstrated that the growth of tumors composed of both docetaxel-sensitive and docetaxel-resistant cells was deterred most profoundly when docetaxel and gemcitabine were administered together. CONCLUSION: This study suggests that when a drug develops therapeutic resistance, sensitivity tests could be another option, ultimately providing insight into a novel alternative clinical strategy.
Asunto(s)
Desoxicitidina/análogos & derivados , Docetaxel/farmacología , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/biosíntesis , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Animales , Ciclo Celular/efectos de los fármacos , Procesos de Crecimiento Celular/efectos de los fármacos , Línea Celular Tumoral , Desoxicitidina/farmacología , Resistencia a Antineoplásicos/genética , Femenino , Humanos , Masculino , Ratones , Ratones Desnudos , Células PC-3 , Neoplasias de la Próstata Resistentes a la Castración/genética , Neoplasias de la Próstata Resistentes a la Castración/patología , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/genética , Transcriptoma , Transfección , Regulación hacia Arriba , Ensayos Antitumor por Modelo de Xenoinjerto , GemcitabinaRESUMEN
The drug efflux pump ABCB1 is a key driver of chemoresistance, and high expression predicts treatment failure in acute myeloid leukemia (AML). In this study, we identified and functionally validated the network of enhancers that controls expression of ABCB1. We show that exposure of leukemia cells to daunorubicin activated an integrated stress response-like transcriptional program to induce ABCB1 through remodeling and activation of an ATF4-bound, stress-responsive enhancer. Protracted stress primed enhancers for rapid increases in activity following re-exposure of cells to daunorubicin, providing an epigenetic memory of prior drug treatment. In primary human AML, exposure of fresh blast cells to daunorubicin activated the stress-responsive enhancer and led to dose-dependent induction of ABCB1. Dynamic induction of ABCB1 by diverse stressors, including chemotherapy, facilitated escape of leukemia cells from targeted third-generation ABCB1 inhibition, providing an explanation for the failure of ABCB1 inhibitors in clinical trials. Stress-induced upregulation of ABCB1 was mitigated by combined use of the pharmacologic inhibitors U0126 and ISRIB, which inhibit stress signaling and have potential for use as adjuvants to enhance the activity of ABCB1 inhibitors.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Elementos de Facilitación Genéticos , Leucemia Mieloide Aguda , Subfamilia B de Transportador de Casetes de Unión a ATP/biosíntesis , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Acetamidas/farmacología , Factor de Transcripción Activador 4/genética , Factor de Transcripción Activador 4/metabolismo , Butadienos/farmacología , Ciclohexilaminas/farmacología , Daunorrubicina/farmacología , Resistencia a Antineoplásicos/genética , Epigénesis Genética/efectos de los fármacos , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Humanos , Células K562 , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Nitrilos/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
OBJECTIVE: The aim of this study was to explore the effect of circ MTHFD2 on resistance to pemetrexed (MTA) in gastric cancer by regulating the expression of micro ribonucleic acid (miR)-124. MATERIALS AND METHODS: Human gastric cancer MGC-803 cells were induced by MTA at an increasing concentration. MGC-803/MTA resistant cell model was successfully established after 5 months. The half-maximal inhibitory concentration (IC50) of MTA on the two kinds of cells was detected via cell counting kit-8 (CCK-8) assay. Differentially expressed circular RNAs (circRNAs) were screened using circRNA microarray analysis. Meanwhile, the target miRNAs of circRNAs were predicted using bioinformatics tool and verified via luciferase reporter assay, respectively. In MGC-803 cells, the effects of overexpression and knockdown of circ MTHFD2 on the expression of miR-124 were detected via quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Finally, the effects of circ MTHFD2 on the protein expressions of FDZ5 and multidrug resistance gene-1 (MDR-1) were detected via Western blotting. RESULTS: MGC-803/MTA resistant cell lines were successfully constructed via persistent drug exposure to MTA at an increasing concentration for 5 months. Compared with parental cells, MGC-803/MTA resistant cells showed significantly enhanced drug resistance. Subsequently, differentially expressed circRNAs were screened using circRNA microarray analysis. A total of 673 circRNAs were screened out based on Fold Change >3 and adjusted p-value. The results of qRT-PCR showed that the expression levels of all differentially expressed circRNAs were significantly changed when compared with MGC-803/MTA resistant cells. Compared with MGC-803/MTA resistant cells, the drug resistance of cells increased significantly in circ MTHFD2 overexpression group. However, it markedly decreased in circ MTHFD2 knockdown group. According to the results of bioinformatics and luciferase reporter assay, circ MTHFD2 could target bind to miR-124. In MGC-803/MTA cells, miR-124 could remarkably increase the resistance of MGC-803/MTA cells to MTA. Western blotting results revealed that overexpression of circ MTHFD2 significantly increased the protein expressions of FDZ5 and MDR-1. However, miR-124 mimics reversed the inhibitory effect of circ MTHFD2 on FDZ5 and MDR-1. CONCLUSIONS: Circ MTHFD2 directly binds to miR-124 through the molecular sponge effect. This may induce increased protein expression of MDR-1, ultimately enhancing the drug resistance of MGC-803/MTA cells.
Asunto(s)
Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/fisiología , MicroARNs/fisiología , Pemetrexed/farmacología , ARN Circular/biosíntesis , Neoplasias Gástricas/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/biosíntesis , Línea Celular Tumoral , Receptores Frizzled/biosíntesis , Técnicas de Silenciamiento del Gen , Humanos , Concentración 50 Inhibidora , MicroARNs/biosíntesis , MicroARNs/metabolismo , Análisis por Micromatrices , Imitación Molecular , Motivos de Unión al ARNRESUMEN
In amyotrophic lateral sclerosis (ALS), upregulation in expression and activity of the ABC transporter P-glycoprotein (P-gp) driven by disease advancement progressively reduces CNS penetration and efficacy of the ALS drug, riluzole. Post-mortem spinal cord tissues from ALS patients revealed elevated P-gp expression levels in endothelial cells of the blood-spinal cord barrier compared to levels measured in control, non-diseased individuals. We recently found that astrocytes expressing familial ALS-linked SOD1 mutations regulate expression levels of P-gp in endothelial cells, which also exhibit a concomitant, significant increase in reactive oxygen species production and NFκB nuclear translocation when exposed to mutant SOD1 astrocyte conditioned media. In this study, we found that glutamate, which is abnormally secreted by mutant SOD1 and sporadic ALS astrocytes, drives upregulation of P-gp expression and activity levels in endothelial cells via activation of N-Methyl-D-Aspartic acid (NMDA) receptors. Surprisingly, astrocyte-secreted glutamate regulation of endothelial P-gp levels is not a mechanism shared by all forms of ALS. C9orf72-ALS astrocytes had no effect on endothelial cell P-gp expression and did not display increased glutamate secretion. Utilizing an optimized in vitro human BBB model consisting of patient-derived induced pluripotent stem cells, we showed that co-culture of endothelial cells with patient-derived astrocytes increased P-gp expression levels and transport activity, which was significantly reduced when endothelial cells were incubated with the NMDAR antagonist, MK801. Overall, our findings unraveled a complex molecular interplay between astrocytes of different ALS genotypes and endothelial cells potentially occurring in disease that could differentially impact ALS prognosis and efficacy of pharmacotherapies.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/biosíntesis , Esclerosis Amiotrófica Lateral/metabolismo , Astrocitos/metabolismo , Células Endoteliales/metabolismo , Ácido Glutámico/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Animales , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/patología , Capilares/metabolismo , Células Cultivadas , Medios de Cultivo Condicionados , Maleato de Dizocilpina/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Humanos , Mutación/genética , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/biosíntesis , Receptores de N-Metil-D-Aspartato/genética , Superóxido Dismutasa-1/genética , Regulación hacia ArribaRESUMEN
Gastric cancer (GC) is one of the most malignant tumors, accounting for 10% of deaths caused by all cancers. Chemotherapy is often necessary for treatment of GC; the FOLFOX regimen is extensively applied. However, multidrug resistance (MDR) of GC cells prevents wider application of this treatment. Ubenimex, an inhibitor of CD13, is used as an immune adjuvant to treat hematological malignancies. Here, we demonstrate that CD13 expression positively correlates with MDR development in GC cells. Moreover, Ubenimex reverses the MDR of SGC7901/X and MKN45/X cells and enhances their sensitivity to FOLFOX, in part by decreasing CD13 expression, which is accompanied by downregulation of Bcl-xl, Bcl-2, and survivin expression; increased expression of Bax; and activation of the caspase-3-mediated apoptotic cascade. In addition, Ubenimex downregulates expression of membrane transport proteins, such as P-gp and MRP1, by inhibiting phosphorylation in the PI3K/AKT/mTOR pathway to increase intracellular accumulations of 5-fluorouracil and oxaliplatin, a process for which downregulation of CD13 expression is essential. Therefore, the present results reveal a previously uncharacterized function of CD13 in promoting MDR development in GC cells and suggest that Ubenimex is a candidate for reversing the MDR of GC cells.
Asunto(s)
Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Leucina/análogos & derivados , Proteínas de Neoplasias/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/biosíntesis , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Adulto , Anciano , Apoptosis/genética , Antígenos CD13/biosíntesis , Antígenos CD13/genética , Línea Celular Tumoral , Resistencia a Múltiples Medicamentos/genética , Resistencia a Antineoplásicos/genética , Femenino , Humanos , Leucina/farmacología , Masculino , Persona de Mediana Edad , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/biosíntesis , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas de Neoplasias/genéticaRESUMEN
There is conflicting evidence that MDR1, MRP2 and LRP expression is responsible for chemotherapy resistance. We conducted this study to explore their role in AML therapy outcomes. Bone marrow and peripheral blood samples of 90 AML patients, receiving chemotherapy, were analyzed by real time PCR. Gene expression was calculated by the 2-ΔΔCt method. The patients who had a persistent remission were labelled 'Good Responder' (GRes) whereas, those with relapse or drug resistance were labelled 'Poor Responders' (PRes). Higher LRP expression in bone marrow, but not in peripheral blood, was positively associated with persistent remission (p = 0.001), GRes (p = 0.002), 1-year overall as well as disease-free survival (p = 0.02 and p = 0.007, respectively). Marrow and blood MDR1 and MRP2 expression did not differ significantly between the above groups. Logistic regression analysis showed that only a diagnosis of acute promyelocytic leukemia (APL; M3) or high marrow LRP expression significantly predicted a favorable therapeutic outcome. This is the first report showing that high bone marrow LRP expression predicts significant favorable therapeutic outcome. Peripheral blood LRP expression as well as marrow and blood MDR1 and MRP2 expression have no predictive value in AML patients treated with standard dose cytarabine and daunorubicin 3+7 regimen.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Médula Ósea/metabolismo , Resistencia a Antineoplásicos , Leucemia Promielocítica Aguda , Proteínas de Neoplasias/biosíntesis , Partículas Ribonucleoproteicas en Bóveda/biosíntesis , Subfamilia B de Transportador de Casetes de Unión a ATP/biosíntesis , Adolescente , Adulto , Médula Ósea/patología , Citarabina/administración & dosificación , Daunorrubicina/administración & dosificación , Femenino , Humanos , Leucemia Promielocítica Aguda/tratamiento farmacológico , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/mortalidad , Masculino , Persona de Mediana Edad , Miembro 4 de la Subfamilia B de Casete de Unión a ATPRESUMEN
Overexpression of ATP-binding cassette (ABC) transporters is one of the most important mechanisms responsible for the development of multidrug resistance (MDR). Selonsertib, a serine/threonine kinase inhibitor, targets apoptosis signal-regulating kinase 1 (ASK1) and is now in phase III clinical trial for the treatment of non-alcoholic steatohepatitis (NASH). In this study, we investigated whether selonsertib could reverse MDR-mediated by ABC transporters, including ABCB1, ABCG2, ABCC1 and ABCC10. The results showed that selonsertib significantly reversed ABCB1- and ABCG2-mediated MDR, but not MDR-mediated by ABCC1 or ABCC10. Mechanism studies indicated that the reversal effect of selonsertib was related to the attenuation of the efflux activity of ABCB1 and ABCG2 transporters, without the protein level decrease or change in the subcellular localization of ABCB1 or ABCG2. Selonsertib stimulated the ATPase activity of ABCB1 and ABCG2 in a concentration-dependent manner, and in silico docking study showed selonsertib could interact with the substrate-binding sites of both ABCB1 and ABCG2. This study provides a clue into a novel treatment strategy, which includes a combination of selonsertib with antineoplastic drugs to attenuate MDR-mediated by ABCB1 or ABCG2 in cancer cells overexpressing these transporters.
Asunto(s)
Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/antagonistas & inhibidores , Anticuerpos Monoclonales Humanizados/farmacología , Proteínas de Neoplasias/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Subfamilia B de Transportador de Casetes de Unión a ATP/antagonistas & inhibidores , Subfamilia B de Transportador de Casetes de Unión a ATP/biosíntesis , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/biosíntesis , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Adenosina Trifosfatasas/metabolismo , Antineoplásicos/farmacocinética , Antineoplásicos Fitogénicos/farmacocinética , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/metabolismo , Línea Celular Tumoral , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/metabolismo , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Células HEK293 , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , MAP Quinasa Quinasa Quinasa 5/antagonistas & inhibidores , MAP Quinasa Quinasa Quinasa 5/metabolismo , Mitoxantrona/farmacocinética , Modelos Moleculares , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Paclitaxel/farmacocinética , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
PURPOSE: To investigate the expressions of class III ß-tubulin (TUBB3), nucleotide excision repair cross-complementary gene 1 (ERCC1) and P-glycoprotein (P-gp) in ovarian cancer tissues and adjacent normal tissues and their clinical significance. METHODS: Ovarian cancer patients undergoing surgical resection at the Department of Oncology of the Affiliated Hospital of Shandong Medical College from March 2012 to May 2016 were enrolled in this study, from which 166 cases of pathologically confirmed cancer tissues and 50 cases of adjacent normal tissues were collected. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the messenger RNA (mRNA) expression levels of TUBB3, ERCC1 and P-gp in ovarian cancer tissues and adjacent normal tissues, and their relationships with ovarian cancer clinical stage and grade of pathological differentiation were analyzed. RESULTS: The expression levels of TUBB3, ERCC1 and P-gp in ovarian cancer tissues were significantly higher than those in adjacent normal tissues (p<0.05). The later the clinical stage of ovarian cancer was, the higher the expression levels of TUBB3, ERCC1 and P-gp were (p<0.05). The lower the pathological differentiation grade of ovarian cancer was, the higher the expression levels of TUBB3, ERCC1 and P-gp were (p<0.05). TUBB3, ERCC1 and P-gb were positively correlated with clinical stage and pathological differentiation grade. CONCLUSION: TUBB3, ERCC1 and P-gp are involved in the occurrence and development of ovarian cancer and can be used as important indexes judging the severity of ovarian cancer, providing a reference for the occurrence and development of the disease in ovarian cancer patients in clinical practice.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/biosíntesis , Proteínas de Unión al ADN/biosíntesis , Endonucleasas/biosíntesis , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Tubulina (Proteína)/biosíntesis , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endonucleasas/genética , Endonucleasas/metabolismo , Femenino , Humanos , Neoplasias Ováricas/patología , Tubulina (Proteína)/metabolismoRESUMEN
BACKGROUND: Vascular endothelial cells (EC) are constantly exposed to endo- and exogenous compounds, which may disturb EC function. One of the protecting mechanisms against chemicals consists of drug metabolizing enzymes and transporter proteins regulated by nuclear receptors and transcription factors. Therefore, the aim of the current study was to assess the regulation of nuclear receptors and their coordinated genes in Human Umbilical Vein Endothelial Cells (HUVEC). METHODS: HUVEC were exposed to TCDD (10nM), oltipraz (100µM) and simvastatin (1µM) for 24h. Gene expressions were evaluated using quantitative real-time PCR. The protein expression levels were determined by Western blotting. Enzymatic activity of CYP1A1/CYP1B1 was assessed by luciferin-labelled CYPs substrate. RESULTS: Our study confirmed that nuclear receptor AhR and nuclear factor Nrf2 are highly expressed in HUVECs. Treatment of HUVECs with TCDD (AhR inducer) resulted in a significant induction of AHR target genes CYP1A1, CYP1B1 and NQO1. Oltipraz (Nrf2 inducer) also markedly increased expression of NQO1 but did not affect Nrf2 mRNA nor protein levels. Under simvastatin stimulation PXR and NRF2 target transcripts were not altered, however AHR-regulated genes: CYP1A1, CYP1B1 and MDR1 were significantly induced. Western blot analysis confirmed CYP1B1 induction in TCDD-treated HUVECs, but not in the simvastatin group. Moreover, HUVEC exposure to TCDD resulted in induction of CYP1A1/CYP1B1 enzymatic activity. CONCLUSIONS: This study revealed functional expression of AhR and Nrf2 in HUVECs. Moreover, it was defined that simvastatin induced AhR and its related genes.
Asunto(s)
Citocromo P-450 CYP1A1/biosíntesis , Citocromo P-450 CYP1B1/biosíntesis , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , NAD(P)H Deshidrogenasa (Quinona)/biosíntesis , Factor 2 Relacionado con NF-E2/biosíntesis , Receptores de Hidrocarburo de Aril/biosíntesis , Simvastatina/farmacología , Subfamilia B de Transportador de Casetes de Unión a ATP/biosíntesis , Células Cultivadas , Regulación de la Expresión Génica/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/enzimología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Receptor de la Señal 1 de Direccionamiento al Peroxisoma/biosíntesis , Dibenzodioxinas Policloradas/farmacología , Pirazinas/farmacología , Tionas , TiofenosRESUMEN
Essential oils (EOs) are extensively used in food industry, gastronomy and alternative medicine. They are multicomponent mixtures of bioactive compounds; hence, their potential for food-drug interactions is substantial. In this study, we investigated the effects of 31 EOs of culinary herbs and spices on the transcriptional activity of pregnane X receptor (PXR) and expression of cytochrome P450 3A4 (CYP3A4), using human intestinal and hepatic in vitro models. All tested EOs activated PXR in intestinal LS180 cells transiently transfected with PXR, as revealed by a reporter gene assay. Consistently, all EOs induced CYP3A4 mRNA expression in PXR-transfected LS180 cells, primary human hepatocytes and wild-type hepatic progenitor HepaRG cells. EO-mediated induction of CYP3A4 mRNA expression was nullified in PXR-knock out HepaRG cells, suggesting the involvement of PXR in these effects. Collectively, we showed that EOs of culinary herbs and spices might be common activators of PXR and inducers of CYP3A4 at doses present in foods, thereby, they might have a potential for food-drug interactions. Follow-up studies are warranted to identify the bioactive constituents in the tested EOs.
Asunto(s)
Citocromo P-450 CYP3A/biosíntesis , Mucosa Intestinal/metabolismo , Hígado/metabolismo , Aceites Volátiles/farmacología , Receptores de Esteroides/metabolismo , Especias/análisis , Subfamilia B de Transportador de Casetes de Unión a ATP/biosíntesis , Línea Celular , Citocromo P-450 CYP2B6/biosíntesis , Citocromo P-450 CYP2B6/genética , Inducción Enzimática/efectos de los fármacos , Genes Reporteros , Hepatocitos/efectos de los fármacos , Humanos , Intestinos/efectos de los fármacos , Hígado/efectos de los fármacos , Receptor X de Pregnano , Cultivo Primario de Células , Receptores de Esteroides/efectos de los fármacos , Activación Transcripcional/efectos de los fármacosRESUMEN
Ricolinostat is the first orally available, selective inhibitor of histone deacetylase 6 (HDAC6), currently under evaluation in clinical trials in patients with various malignancies. It is likely that the inevitable emergence of resistance to ricolinostat is likely to reduce its clinical effectiveness in cancer patients. In this study, we investigated the potential impact of multidrug resistance-linked ATP-binding cassette (ABC) transporters ABCB1 and ABCG2 on the efficacy of ricolinostat, which may present a major hurdle to its development as an anticancer drug in the future. We demonstrated that the overexpression of ABCB1 or ABCG2 reduces the intracellular accumulation of ricolinostat, resulting in reduced efficacy of ricolinostat to inhibit the activity of HDAC6 in cancer cells. Moreover, the efficacy of ricolinostat can be fully restored by inhibiting the drug efflux function of ABCB1 and ABCG2 in drug-resistant cancer cells. In conclusion, our results provide some insights into the basis for the development of resistance to ricolinostat and suggest that co-administration of ricolinostat with a modulator of ABCB1 or ABCG2 could overcome ricolinostat resistance in human cancer cells, which may be relevant to its use in the clinic.
Asunto(s)
Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/biosíntesis , Resistencia a Antineoplásicos/efectos de los fármacos , Histona Desacetilasa 6/antagonistas & inhibidores , Inhibidores de Histona Desacetilasas/farmacología , Ácidos Hidroxámicos/farmacología , Proteínas de Neoplasias/biosíntesis , Pirimidinas/farmacología , Subfamilia B de Transportador de Casetes de Unión a ATP/biosíntesis , Antineoplásicos/farmacología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos/fisiología , Células HEK293 , Histona Desacetilasa 6/metabolismo , Humanos , Células MCF-7RESUMEN
The acceleration of drug efflux activity realized by plasma membrane transporters in neoplastic cells, particularly by P-glycoprotein (P-gp, ABCB1 member of the ABC transporter family), represents a frequently observed molecular cause of multidrug resistance (MDR). This multiple resistance represents a real obstacle in the effective chemotherapy of neoplastic diseases. Therefore, identifying cytotoxic substances that are also effective in P-gp overexpressing cells may be useful for the rational design of substances for the treatment of malignancies with developed MDR. Here, we showed that triorganotin derivativestributyltin-chloride (TBT-Cl), tributyltin-bromide (TBT-Br), tributyltin-iodide (TBT-I) and tributyltin-isothiocyanate (TBT-NCS) or triphenyltin-chloride (TPT-Cl) and triphenyltin-isothiocyanate (TPT-NCS)could induce the death of L1210 mice leukemia cells at a submicromolar concentration independently of P-gp overexpression. The median lethal concentration obtained for triorganotin derivatives did not exceed 0.5 µM in the induction of cell death of either P-gp negative or P-gp positive L1210 cells. Apoptosis related to regulatory pathway of Bcl-2 family proteins seems to be the predominant mode of cell death in either P-gp negative or P-gp positive L1210 cells. TBT-Cl and TBT-Br were more efficient with L1210 cells overexpressing P-gp than with their counterpart P-gp negative cells. In contrast, TBT-I and TPT-NCS induced a more pronounced cell death effect on P-gp negative cells than on P-gp positive cells. Triorganotin derivatives did not affect P-gp efflux in native cells measured by calcein retention within the cells. Taken together, we assumed that triorganotin derivatives represent substances suitable for suppressing the viability of P-gp positive malignant cells.