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Background: Cetuximab is one of the most widely used monoclonal antibodies to treat patients with RAS/BRAF wild-type metastatic colorectal cancer (mCRC). Unfortunately, cetuximab resistance often occurs during targeted therapy. However, the underlying epigenetic mechanisms remain unclear. Our previous study demonstrated that the exosomal transfer of urothelial carcinoma-associated 1 (UCA1) confers cetuximab resistance to CRC cells. The goal of this study was to elucidate the detailed role of UCA1 in cetuximab resistance in CRC and the underlying molecular mechanism. Methods: In vitro and in vivo functional studies were performed to assess the role of UCA1 in cetuximab resistance in CRC cell lines and xenograft models. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to examine UCA1 localization and expression. Bioinformatics analysis was performed to predict the potential mechanism of UCA1, which was further validated by the dual-luciferase reporter assay and the RNA immunoprecipitation (RIP) assay. Cells treated with indicators were subjected to Cell Counting Kit-8 (CCK-8) and western blotting to investigate the role of hepatocyte growth factor (HGF)/c-mesenchymal-epithelial transition (c-MET) signalling in UCA1-mediated cetuximab resistance. Results: We showed that UCA1 decreased CRC cell sensitivity to cetuximab by suppressing apoptosis. Mechanistic studies revealed that UCA1 promoted cetuximab resistance by competitively binding miR-495 to facilitate HGF and c-MET expression in CRC cells. Moreover, HGF was shown to attenuate the cetuximab-induced inhibition of cell proliferation by activating the HGF/c-MET pathway in CRC cells. Conclusion: We provide the first evidence of a UCA1-miR-495-HGF/c-MET regulatory network involved in cetuximab resistance in CRC. Therefore, UCA1 has potential as a predictor and therapeutic target for cetuximab resistance.
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BACKGROUND: Primary or acquired resistance to cetuximab often occurs during targeted therapy in metastatic colorectal cancer (mCRC) patients. In many cancers, the key role of the long noncoding RNA (lncRNA) urothelial carcinoma-associated 1 (UCA1) in anticancer drug resistance has been confirmed. Emerging evidence has shown that specific exosomal lncRNAs may serve as meaningful biomarkers. In this study, we hypothesize that exosomal UCA1 might predict the response to cetuximab in CRC patients. METHODS: First, acquired cetuximab-resistant cell lines were generated, and UCA1 expressions in these cells and their exosomes were compared. We also systematically evaluate the stability of exosomal UCA1. Thereafter, the predictive value of exosomal UCA1 in CRC patients treated with cetuximab was evaluated. Finally, through cell apoptosis assays and immunofluorescence staining, we analyzed the role of UCA1-containing exosomes in conferring cetuximab resistance. RESULTS: UCA1 expression was markedly higher in cetuximab-resistant cancer cells and their exosomes. Exosomal UCA1 was shown to be detectable and stable in serum from CRC patients. In addition, circulating UCA1-containing exosomes could predict the clinical outcome of cetuximab therapy in CRC patients, and UCA1 expression was considerably higher in the progressive disease/stable disease patients than in the partial response/complete response patients. Furthermore, exosomes derived from cetuximab-resistant cells could alter UCA1 expression and transmit cetuximab resistance to sensitive cells. CONCLUSIONS: We discovered a novel role of UCA1-containing exosomes, showed their capability to transmit drug resistance and investigated their potential clinical use in predicting cetuximab resistance.
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The human EGFR family consists of four type-1 transmembrane tyrosine kinase receptors: HER1 (EGFR, ErbB1), HER2 (Neu, ErbB2), HER3 (ErbB3), and HER4 (ErbB4). HER3 can dimerize with EGFR, HER2 and even c-Met and likely plays a central role in the response to EGFR-targeted therapy. Because HER3 lacks significant kinase activity and cannot be inhibited by tyrosine kinase inhibitors, neutralizing antibodies and alternative inhibitors of HER3 have been sought as cancer therapeutics. Here, we describe the stable suppression of HER3 mRNA and protein using siRNA. The inhibition of HER3 expression decreased cell proliferation, suppressed cell cycle progression, induced apoptosis and inhibited cell motility, migration, invasiveness, and soft agar growth. In addition, we found that gefitinib treatment increased the HER3 and HER2 mRNA levels. The administration of various concentrations of gefitinib to HER3-knockdown cells enhanced antitumour activity and sensitivity due to the downregulation of protein phosphorylation via PI3K/AKT and ERK signalling. Our results support the use of combined treatments targeting multiple EGFR receptors, particularly the use of HER3 inhibitors combined with EGFR inhibitors, such as gefitinib.
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Sonodynamic therapy (SDT), a promising modality for cancer treatment, involves the synergistic interaction of ultrasound and some chemical compounds termed sonosensitizers. However, its effect on pancreatic cancer cells remains unclear. In our study, we sought to identify the cytotoxic effects of ultrasound-activated 5-aminolevulinic acid on human pancreatic cancer Capan-1 cells. Cell viability was determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) analysis; mitochondrial membrane potential was assessed using the fluorescent probe jc-1; apoptosis was evaluated by flow cytometry; cell morphology was investigated by scanning electron microscopy; apoptosis-related protein expression was analyzed by Western blot assay. We found that SDT significantly decreased the survival rate of cells, and this effect increased with 5-aminolevulinic acid concentration and ultrasound exposure time. The mechanism underlying the effect of SDT involves, in part, the induction of a conspicuous loss in mitochondrial membrane potential and, in part, the induction of apoptosis through upregulation of Bax expression, downregulation of Bcl-2 and increased activation of procaspase-3. These results indicate that the ultrasonically induced cell killing effect could be enhanced by 5-ALA and that the mitochondrial pathway might be involved in the cell damage process. We conclude that SDT is a promising new methodology for pancreatic cancer treatment.
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Ácido Aminolevulínico/uso terapéutico , Neoplasias Pancreáticas/terapia , Terapia por Ultrasonido/métodos , Apoptosis/efectos de los fármacos , Western Blotting/métodos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Citometría de Flujo/métodos , Humanos , Fármacos Fotosensibilizantes/uso terapéuticoRESUMEN
Colorectal cancer (CRC) is one of the most common cancers worldwide. Long non-coding RNAs (lncRNAs) have been shown to play important regulatory roles in cancer biology, and functional lncRNAs can be used for cancer diagnosis and prognosis. One lncRNA that has attracted significant attention is urothelial carcinoma-associated 1 (UCA1), which is significantly up-regulated in most tumour tissues and cancer cells. However, the contributions of UCA1 to CRC remain largely unknown. Thus, the aim of the current study was to investigate the clinical significance and biological function of UCA1 in CRC.First, we evaluated whether UCA1 is detectable or altered in CRC tissues or cell lines compared to adjacent normal tissues or normal cell lines by quantitative real-time polymerase chain reaction. The potential relationship between UCA1 levels in tumour tissues and the clinicopathological features of CRC was then investigated. Finally, we assessed whether UCA1 influences cell proliferation, apoptosis, cell cycle distribution and migration in vitro.Our results demonstrated that UCA1 levels were markedly increased in CRC tissues and cells compared to controls, and this high level of UCA1 expression was significantly correlated with larger tumour size, less differentiated histology and greater tumour depth. In addition, patients with high UCA1 expression had a significantly poorer prognosis than those with low UCA1 expression. Moreover, UCA1 was found to influence the proliferation, apoptosis and cell cycle progression of CRC cells. These data suggest an important role for UCA1 in the molecular aetiology of CRC and suggest a potential application for UCA1 in CRC diagnosis, progression and therapy.
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Apoptosis/genética , Ciclo Celular/genética , Proliferación Celular/genética , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , ARN Largo no Codificante/biosíntesis , Anciano , Movimiento Celular/genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia ArribaRESUMEN
BACKGROUND: Photodynamic therapy (PDT) is a new treatment for esophageal cancer which has been shown to be effective in the elimination of tumor. However, PDT could induce the activation of nuclear factor-kappa B (NF-κB) in many photosensitizers based PDT, which plays a negative role in PDT. In addition, our previous results have shown that dihydroartemisinin (DHA), which was the most potent one of artemisinin derivatives, has anticancer activity in esophageal cancer cells. METHODS: Cell viability was determined by MTT analysis, and apoptosis was evaluated by flow cytometry. Nuclear extract was obtained for determining NF-κB DNA-binding activity, while total protein extract obtained for downstream gene expression by western blot. RESULTS: We demonstrated DHA enhanced PDT-induced growth inhibition and apoptosis in both human esophageal cancer cell lines Eca109 and Ec9706 in vitro. The mechanism was at least partially due to DHA deactivated PDT-induced NF-κB activation, so as to decrease tremendously the expression of its target gene Bcl-2. CONCLUSION: Our results demonstrate that DHA augments PDT-induced growth inhibition and apoptosis in esophageal cancer cells, and that inactivation of NF-κB activity is a potential mechanism by which DHA sensitizes esophageal cancer cells to PDT-induced growth inhibition and apoptosis.
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Antineoplásicos/farmacología , Artemisininas/farmacología , Neoplasias Esofágicas/tratamiento farmacológico , FN-kappa B/antagonistas & inhibidores , Fotoquimioterapia , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Artemisininas/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patología , Humanos , FN-kappa B/metabolismo , Relación Estructura-ActividadRESUMEN
Estrogen-related genes and the fat mass and obesity-associated (FTO) gene play a critical role in estrogen metabolism, and those polymorphisms are associated with a poor prognosis in breast cancer. However, little is known about the association between these polymorphisms and the efficacy of anastrozole. The aim was to investigate the impact of the genetic polymorphisms, CYP19A1, 17-ß-HSD-1 and FTO, on the response to anastrozole in metastatic breast carcinoma (MBC) and to evaluate the impact of those polymorphisms on various clinicopathologic features. Two-hundred seventy-two women with hormone receptor-positive MBC treated with anastrozole were identified retrospectively. DNA was extracted from peripheral blood and genotyped for five variants in three candidate genes. Time to progression was improved in patients carrying the variant alleles of rs4646 when compared to patients with the wild-type allele (16.40 months versus 13.52 months; p = 0.049). The rs4646 variant alleles were significantly associated with longer overall survival (37.3 months versus 31.6 months; p = 0.007). This relationship was not observed with the rs10046, rs2830, rs9926298 and rs9939609 polymorphisms. The findings of this study indicate that rs4646 polymorphism in the CYP19A1 gene may serve as a prognostic maker of the response to anastrozole in patients with MBC who are treated with anastrozole.
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Inhibidores de la Aromatasa/uso terapéutico , Aromatasa/genética , Neoplasias de la Mama/genética , Nitrilos/uso terapéutico , Triazoles/uso terapéutico , 17-Hidroxiesteroide Deshidrogenasas/genética , Regiones no Traducidas 3' , Adulto , Anciano , Anciano de 80 o más Años , Alelos , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato , Anastrozol , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Femenino , Frecuencia de los Genes , Genotipo , Humanos , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Proteínas/genética , Estudios Retrospectivos , Análisis de SupervivenciaRESUMEN
Dihydroartemisinin (DHA) has recently been shown anti-tumor activity in various cancer cells. However, its effect on esophageal cancer remains unclear. In this study, for the first time, we demonstrated that DHA reduced viability of esophageal cancer cells in a dose-dependent manner. The mechanism was at least partially due to DHA induced apoptosis by upregulating the expression of Bax, downregulating Bcl-2, Bcl-xL and Procaspase-3, and increasing caspase-9 activation, induced cell cycle arrest by downregulating cyclin E, CDK2 and CDK4. Furthermore, we firstly found that DHA induced autophagy in cancer cells. We concluded DHA might be a novel agent against esophageal cancer.
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Apoptosis/efectos de los fármacos , Artemisininas/farmacología , Neoplasias Esofágicas/tratamiento farmacológico , Antineoplásicos/farmacología , Artemisininas/administración & dosificación , Autofagia/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/efectos de los fármacos , Neoplasias Esofágicas/patología , Humanos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
AIMS: The epidermal growth factor receptor (EGFR) is abnormally activated in many tumours. Two different categories of compounds targeting EGFR, monoclonal antibodies (mAbs) and low molecular weight tyrosine kinase inhibitors (TKIs), which target extracellular and intracellular domains of the receptor, respectively, have shown antitumour activity. We decided to explore whether the combined administration of cetuximab, a mAb, and gefitinib, a TKI, had superior antitumour activity than either agent given alone. METHODS: We studied the effects of cetuximab alone, gefitinib alone and the combination of cetuximab and gefitinib in two colon cancer cell lines, HT-29 and LoVo. The effects of these two agents on cell proliferation and induction of apoptosis were evaluated. RESULTS: Dose-dependent activity of cetuximab alone or gefitinib alone or the combination was observed for both colon cancer cell lines. In addition, the combined treatment with cetuximab and gefitinib resulted in a synergistic and more pronounced growth effect on cell proliferation and induction of apoptosis than either single-agent treatment. CONCLUSIONS: Our findings suggest that combined treatment with distinct EGFR inhibitory agents can augment the antitumour response over that realised with a single EGFR inhibitor. New and tempting treatment strategies on the EGFR target consisting of a double hit with a mAb and a TKI may improve the therapeutic ratio for colorectal cancer in future clinical trials.