RESUMEN
Owing to intrinsic and acquired chemoresistance, the response of gastric adenocarcinoma (GAC) to chemotherapy is very poor. Here we have investigated the role of transportome in reducing the intracellular content of anticancer drugs and conferring multidrug resistance (MDR) phenotype. Tumors specimens and paired adjacent tissue were analyzed to determine the MDR signature by TaqMan Low-Density Arrays and single-gene qPCR. Strategies of sensitization were evaluated in vitro using the GAC-derived cell line AGS and in vivo using a subcutaneous xenograft model in immunodeficient nude mice. Several transporters involved in drug uptake and export, which are present in healthy stomach, were highly expressed in GAC. In contrast, the cancer-type OATP1B3 was almost exclusively expressed in tumor tissue. The transportome profile varied depending on tumor anatomical location, differentiation, and stage. Immunofluorescence analysis revealed high MRP1 and MRP4 expression at the plasma membrane of tumor cells as well as AGS cells in culture, in which MRP inhibition resulted in selective sensitization to cytotoxic MRP substrates, such as sorafenib, docetaxel, etoposide, and doxorubicin. In mice with subcutaneous tumors formed by AGS cells, sorafenib alone failed to prevent tumor growth. In contrast, this drug induced a marked inhibitory effect when it was co-administered with diclofenac. In conclusion, MRP1 and MRP4 play an important role in the lack of response of GAC to drugs that are transported by these export pumps. Moreover, agents, such as sorafenib, considered at present useless to treat GAC, may become active antitumor drugs when co-administered with non-toxic MRP inhibitors, such as diclofenac.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Adenocarcinoma/tratamiento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Gástricas/tratamiento farmacológico , Transportadoras de Casetes de Unión a ATP/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Anciano , Anciano de 80 o más Años , Animales , Línea Celular Tumoral , Diclofenaco/administración & dosificación , Docetaxel/administración & dosificación , Doxorrubicina/administración & dosificación , Resistencia a Múltiples Medicamentos/genética , Resistencia a Antineoplásicos/genética , Femenino , Humanos , Masculino , Ratones Desnudos , Persona de Mediana Edad , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Sorafenib/administración & dosificación , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Although the multi-tyrosine kinase inhibitor sorafenib is useful in the treatment of several cancers, cholangiocarcinoma (CCA) is refractory to this drug. Among other mechanisms of chemoresistance, impaired uptake through human organic cation transporter type 1 (hOCT1) (gene SLC22A1) has been suggested. Here we have investigated the events accounting for this phenotypic characteristic and have evaluated the interest of selective gene therapy strategies to overcome this limitation. Gene expression and DNA methylation of SLC22A1 were analyzed using intrahepatic (iCCA) and extrahepatic (eCCA) biopsies (Copenhagen and Salamanca cohorts; n = 132) and The Cancer Genome Atlas (TCGA)-CHOL (n = 36). Decreased hOCT1 mRNA correlated with hypermethylation status of the SLC22A1 promoter. Treatment of CCA cells with decitabine (demethylating agent) or butyrate (histone deacetylase inhibitor) restored hOCT1 expression and increased sorafenib uptake. MicroRNAs able to induce hOCT1 mRNA decay were analyzed in paired samples of TCGA-CHOL (n = 9) and Copenhagen (n = 57) cohorts. Consistent up-regulation in tumor tissue was found for miR-141 and miR-330. High proportion of aberrant hOCT1 mRNA splicing in CCA was also seen. Lentiviral-mediated transduction of eCCA (EGI-1 and TFK-1) and iCCA (HuCCT1) cells with hOCT1 enhanced sorafenib uptake and cytotoxic effects. In chemically induced CCA in rats, reduced rOct1 expression was accompanied by impaired sorafenib uptake. In xenograft models of eCCA cells implanted in mouse liver, poor response to sorafenib was observed. However, tumor growth was markedly reduced by cotreatment with sorafenib and adenoviral vectors encoding hOCT1 under the control of the BIRC5 promoter, a gene highly up-regulated in CCA. Conclusion: The reason for impaired hOCT1-mediated sorafenib uptake by CCA is multifactorial. Gene therapy capable of selectively inducing hOCT1 in tumor cells can be considered a potentially useful chemosensitization strategy to improve the response of CCA to sorafenib.
Asunto(s)
Neoplasias de los Conductos Biliares/tratamiento farmacológico , Colangiocarcinoma/tratamiento farmacológico , Regulación hacia Abajo/genética , Factor 1 de Transcripción de Unión a Octámeros/genética , Inhibidores de Proteínas Quinasas/farmacología , Sorafenib/farmacología , Animales , Neoplasias de los Conductos Biliares/genética , Neoplasias de los Conductos Biliares/patología , Línea Celular Tumoral/efectos de los fármacos , Colangiocarcinoma/genética , Colangiocarcinoma/patología , Metilación de ADN/genética , Modelos Animales de Enfermedad , Resistencia a Medicamentos/genética , Terapia Genética/métodos , Humanos , Immunoblotting , Masculino , ARN Mensajero/genética , Distribución Aleatoria , Ratas , Ratas Wistar , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Estadísticas no ParamétricasRESUMEN
BACKGROUND: Sorafenib is the drug of choice in the treatment of advanced hepatocellular carcinoma (HCC). Beneficial effects are limited by mechanisms of chemoresistance, which include downregulation and/or impaired function of plasma membrane transporters accounting for drug uptake. The organic cation transporter 1 (OCT1) plays a major role in sorafenib uptake and decreased expression in HCC has been associated with poorer response. METHODS: The multicenter retrospective TRANSFER study involved tumor biopsies from 39 patients with advanced HCC and sorafenib therapy for ≥4 wk. Endpoint was the relationship between clinicopathological features and immunohistological result. Immunostaining was performed using specific primary anti-OCT1-head and anti-OCT1-tail antibodies. Tumors were classified according to a simplified staining score as absent, weak, moderate or strong, taking into account the localization of the staining at the plasma membrane as positive or negative. RESULTS: Results confirmed OCT1 downregulation in half of the cases investigated (10% absent, 38% weak). However, only one third of tumors expressing OCT1 displayed plasma membrane location (15% vs. 36% cytosolic expression). When comparing HCC with and without OCT1 expression, no different sorafenib response was found. When tumors expressing OCT1 at the plasma membrane were considered separately, a marked longer survival was found (Log Rank p<0.001). No association between OCT1 expression at the plasma membrane with tumor stage, previous treatment with TACE or radiological response was seen.In conclusion, these results indicate that the presence of OCT1 at the plasma membrane, rather than its expression levels, is related to better outcome of HCC patients treated with sorafenib.
Asunto(s)
Carcinoma Hepatocelular/tratamiento farmacológico , Neoplasias Hepáticas/tratamiento farmacológico , Niacinamida/análogos & derivados , Factor 1 de Transcripción de Unión a Octámeros/metabolismo , Compuestos de Fenilurea/uso terapéutico , Inhibidores de Proteínas Quinasas/uso terapéutico , Anciano , Anciano de 80 o más Años , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Membrana Celular/metabolismo , Femenino , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Masculino , Persona de Mediana Edad , Niacinamida/farmacocinética , Niacinamida/uso terapéutico , Factor 1 de Transcripción de Unión a Octámeros/deficiencia , Compuestos de Fenilurea/farmacocinética , Inhibidores de Proteínas Quinasas/farmacocinética , Estudios Retrospectivos , SorafenibRESUMEN
UNLABELLED: Reduced drug uptake is an important mechanism of chemoresistance. Down-regulation of SLC22A1 encoding the organic cation transporter-1 (OCT1) may affect the response of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CGC) to sorafenib, a cationic drug. Here we investigated whether SLC22A1 variants may contribute to sorafenib chemoresistance. Complete sequencing and selective variant identification were carried out to detect single nucleotide polymorphisms (SNPs) in SLC22A1 complementary DNA (cDNA). In HCC and CGC biopsies, in addition to previously described variants, two novel alternative spliced variants and three SNPs were identified. To study their functional consequences, these variants were mimicked by directed mutagenesis and expressed in HCC (Alexander and SK-Hep-1) and CGC (TFK1) cells. The two novel described variants, R61S fs*10 and C88A fs*16, encoded truncated proteins unable to reach the plasma membrane. Both variants abolished OCT1-mediated uptake of tetraethylammonium, a typical OCT1 substrate, and were not able to induce sorafenib sensitivity. In cells expressing functional OCT1 variants, OCT1 inhibition with quinine prevented sorafenib-induced toxicity. Expression of OCT1 variants in Xenopus laevis oocytes and determination of quinine-sensitive sorafenib uptake by high-performance liquid chromatography-dual mass spectrometry confirmed that OCT1 is able to transport sorafenib and that R61S fs*10 and C88A fs*16 abolish this ability. Screening of these SNPs in 23 HCC and 15 CGC biopsies revealed that R61S fs*10 was present in both HCC (17%) and CGC (13%), whereas C88A fs*16 was only found in HCC (17%). Considering all SLC22A1 variants, at least one inactivating SNP was found in 48% HCC and 40% CGC. CONCLUSION: Development of HCC and CGC is accompanied by the appearance of aberrant OCT1 variants that, together with decreased OCT1 expression, may dramatically affect the ability of sorafenib to reach active intracellular concentrations in these tumors.
Asunto(s)
Neoplasias de los Conductos Biliares/tratamiento farmacológico , Conductos Biliares Intrahepáticos , Carcinoma Hepatocelular/tratamiento farmacológico , Colangiocarcinoma/tratamiento farmacológico , Neoplasias Hepáticas/tratamiento farmacológico , Niacinamida/análogos & derivados , Transportador 1 de Catión Orgánico/genética , Compuestos de Fenilurea/uso terapéutico , Polimorfismo de Nucleótido Simple/genética , Secuencia de Aminoácidos , Animales , Antineoplásicos/uso terapéutico , Neoplasias de los Conductos Biliares/metabolismo , Neoplasias de los Conductos Biliares/patología , Biopsia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Células Cultivadas , Colangiocarcinoma/metabolismo , Colangiocarcinoma/patología , Femenino , Humanos , Técnicas In Vitro , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Datos de Secuencia Molecular , Niacinamida/uso terapéutico , Oocitos/citología , Oocitos/metabolismo , Transportador 1 de Catión Orgánico/química , Transportador 1 de Catión Orgánico/metabolismo , Farmacogenética , Sorafenib , Resultado del Tratamiento , Xenopus laevisRESUMEN
The antiviral effect against hepatitis B virus (HBV) of artemisinin, its derivative artesunate and other compounds highly purified from traditional Chinese medicine remedies, were investigated. HBV production by permanently transfected HepG2 2.2.15 cells was determined by measuring the release of surface protein (HBsAg) and HBV-DNA after drug exposure (0.01-100 microM) for 21 days. The forms of HBV-DNA released were investigated by Southern-blotting. Neutral Red retention test was used to evaluate drug-induced toxicity on host cells. The compounds were classified according to their potential interest as follows: (i) none: they had no effect on viral production (daidzein, daidzin, isonardosinon, nardofuran, nardosinon, tetrahydronardosinon and quercetin); (ii) low: they were able to markedly reduce viral production, but also induced toxicity on host cells (berberine and tannic acid) or they had no toxic effect on host cells but only had a moderate ability to reduce viral production (curcumin, baicalein, baicalin, bufalin, diallyl disulphide, glycyrrhizic acid and puerarin); (iii) high: they induced strong inhibition of viral production at concentrations at which host cell viability was not affected (artemisinin and artesunate). Moreover, artesunate in conjunction with lamivudine had synergic anti-HBV effects, which warrants further evaluation of artemisinin/artesunate as antiviral agents against HBV infection.