RESUMEN
AIM: Cetuximab is an essential drug for the treatment of wild-type K-RAS colorectal cancer (CRC). It improves the overall survival of patients. However, acquired resistance prevents its clinical efficacy. Tumor heterogeneity may be a nonnegligible reason for cetuximab resistance. We attempted to explore the corresponding molecular mechanism. METHODS: Cetuximab-resistant CRC cell RKO and cetuximab-sensitive CRC cell Caco-2 were applied in this study. Cells were centrifuged to determine the concentration in the culture supernatant (CS). MTT, EdU, and colony formation assays were utilized to evaluate cell survival and proliferation. Chromatin immunoprecipitation (ChIP) and promoter-luciferase reporter assays were employed to confirm the direct binding of transcription factors. Western blot and reverse transcription-polymerase chain reaction (RT-PCR) assays were used to detect the expression of molecular markers in the pathway. RESULTS: Hepatocyte growth factor (HGF) was up-regulated in RKO cell culture supernatant and induced cetuximab resistance in Caco-2 cells. SRY-Box Transcription Factor 8 (SOX8) bound to the promoter sequence of HGF. HGF activated the HGF/MET bypass pathway and induced cetuximab resistance in Caco-2 cells. CONCLUSION: The SOX8/HGF/MET axis played a crucial role in the communication between cetuximab-resistant cells and cetuximab-sensitive cells, inducing treatment resistance.
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Neoplasias Colorrectales , Factor de Crecimiento de Hepatocito , Células CACO-2 , Línea Celular Tumoral , Proliferación Celular , Cetuximab/farmacología , Cetuximab/uso terapéutico , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Resistencia a Antineoplásicos , Factor de Crecimiento de Hepatocito/genética , Factor de Crecimiento de Hepatocito/farmacología , Factor de Crecimiento de Hepatocito/uso terapéutico , Humanos , Proteínas Proto-Oncogénicas c-met/genética , Proteínas Proto-Oncogénicas c-met/metabolismo , Factores de Transcripción SOXERESUMEN
BACKGROUND: Tamoxifen is an important choice in endocrine therapy for patients with oestrogen receptor-positive (ER+) breast cancer, and disease progression-associated resistance to tamoxifen therapy is still challenging. Flap endonuclease-1 (FEN1) is used as a prognostic biomarker and is considered to participate in proliferation, migration, and drug resistance in multiple cancers, especially breast cancer, but the prognostic function of FEN1 in ER+ breast cancer, and whether FEN1 is related to tamoxifen resistance or not, remain to be explored. METHODS: On-line database Kaplan-Meier (KM) plotter, GEO datasets, and immunohistochemistry were used to analyse the prognostic value of FEN1 in ER+ breast cancer from mRNA and protein levels. Cell viability assay and colony formation assays showed the response of tamoxifen in MCF-7 and T47D cells. Microarray data with FEN1 siRNA versus control group in MCF-7 cells were analysed by Gene Set Enrichment Analysis (GSEA). The protein levels downstream of FEN1 were detected by western blot assay. RESULTS: ER+ breast cancer patients who received tamoxifen for adjuvant endocrine therapy with poor prognosis showed a high expression of FEN1. MCF-7 and T47D appeared resistant to tamoxifen after FEN1 over-expression and increased sensitivity to tamoxifen after FEN1 knockdown. Importantly, FEN1 over-expression could activate tamoxifen resistance through the ERα/cyclin D1/Rb axis. CONCLUSIONS: As a biomarker of tamoxifen effectiveness, FEN1 participates in tamoxifen resistance through ERα/cyclin D1/Rb axis. In the future, reversing tamoxifen resistance by knocking-down FEN1 or by way of action as a small molecular inhibitor of FEN1 warrants further investigation.
RESUMEN
Flap endonuclease1 (FEN1), a structurespecific nuclease participating in DNA replication and repair processes, has been confirmed to promote the proliferation and drug resistance of tumor cells. However, the biological functions of FEN1 in cancer cell migration and invasion have not been defined. In the present study, using online database analysis and immunohistochemistry of the specimens, it was found that FEN1 expression was associated with a highly invasive triplenegative breast cancer (TNBC) subtype in both breast cancer samples from the Oncomine database and from patients recruited into the study. Furthermore, FEN1 was an important biomarker of lymph node metastasis and poor prognosis in patients with TNBC. FEN1 promoted migration of TNBC cell lines and FEN1 knockdown reduced the number of spontaneous lung metastasis in vivo. Ingenuity Pathway Analysis of FEN1related transcripts in 198 patients with TNBC demonstrated that the pololike kinase family may be the downstream target of FEN1. PLK4 was further identified as a critical target of FEN1 mediating TNBC cell migration, by regulating actin cytoskeleton rearrangement. The results of the present study validate FEN1 as a therapeutic target in patients with TNBC and revealed a new role for FEN1 in regulating TNBC invasion and metastasis.
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Biomarcadores de Tumor/metabolismo , Endonucleasas de ADN Solapado/metabolismo , Neoplasias Pulmonares/secundario , Recurrencia Local de Neoplasia/epidemiología , Neoplasias de la Mama Triple Negativas/patología , Animales , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/genética , Mama/patología , Mama/cirugía , Línea Celular Tumoral , Proliferación Celular , Supervivencia sin Enfermedad , Femenino , Endonucleasas de ADN Solapado/análisis , Endonucleasas de ADN Solapado/genética , Estudios de Seguimiento , Técnicas de Silenciamiento del Gen , Humanos , Metástasis Linfática/patología , Mastectomía , Ratones , Persona de Mediana Edad , Recurrencia Local de Neoplasia/patología , Estadificación de Neoplasias , Pronóstico , RNA-Seq , Análisis de Matrices Tisulares , Neoplasias de la Mama Triple Negativas/diagnóstico , Neoplasias de la Mama Triple Negativas/mortalidad , Neoplasias de la Mama Triple Negativas/cirugía , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: Several clinical trials have suggested that adjuvant chemotherapy improves the survival of patients with resected gastric cancer, but the optimal time at which to initiate post-operative adjuvant chemotherapy has not been studied. This study investigated the association between time to adjuvant chemotherapy and survival in gastric cancer. METHODS: We retrospectively identified 266 patients with stage IB-IIIC gastric cancer who received fluorouracil-based adjuvant chemotherapy after radical gastrectomy. Overall survival (OS) was compared between patients grouped according to time from surgery to adjuvant chemotherapy (<45 and ≥45 days). The Cox proportional hazards model was used to analyze the effects of time to initiation of chemotherapy and other clinical covariates on survival. RESULTS: Of 266 patients, 141 (53%) started adjuvant chemotherapy within 45 days after surgery and 125 (47%) started adjuvant chemotherapy more than 45 days after surgery. The 3-year OS rates were 81.2 and 65.8% for patients starting chemotherapy within 45 days and after 45 days, respectively (p=0.006). Multivariate analysis identified early initiation of adjuvant chemotherapy, completion of the planned chemotherapy, and early-stage disease as favorable prognostic factors in terms of OS (p<0.05). Subgroup analysis suggested that starting chemotherapy within 45 days after surgery was associated with significant OS benefit compared with initiation of chemotherapy after 45 days from surgery in most subgroups. CONCLUSIONS: This retrospective analysis suggests that delaying adjuvant chemotherapy for longer than 45 days after surgery may be associated with poorer survival in patients with resected gastric cancer.
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Antimetabolitos Antineoplásicos/administración & dosificación , Fluorouracilo/administración & dosificación , Gastrectomía , Neoplasias Gástricas/terapia , Adulto , Anciano , Antimetabolitos Antineoplásicos/efectos adversos , Quimioterapia Adyuvante , Distribución de Chi-Cuadrado , Esquema de Medicación , Femenino , Fluorouracilo/efectos adversos , Gastrectomía/efectos adversos , Gastrectomía/mortalidad , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Análisis Multivariante , Estadificación de Neoplasias , Modelos de Riesgos Proporcionales , Estudios Retrospectivos , Factores de Riesgo , Neoplasias Gástricas/mortalidad , Neoplasias Gástricas/patología , Factores de Tiempo , Resultado del TratamientoRESUMEN
Prognostic models are generally used to predict gastric cancer outcomes. However, no model combining patient-, tumor- and host-related factors has been established to predict outcomes after radical gastrectomy, especially outcomes of patients without nodal involvement. The aim of this study was to develop a prognostic model based on the systemic inflammatory response and clinicopathological factors of resectable gastric cancer and determine whether the model can improve prognostic accuracy in node-negative patients. We reviewed the clinical, laboratory, histopathological and survival data of 1397 patients who underwent radical gastrectomy between 2007 and 2013. Patients were split into development and validation sets of 1123 and 274 patients, respectively. Among all 1397 patients, 545 had node-negative gastric cancer; 440 were included in the development set, 105 were included in the validation set. A prognostic model was constructed from the development set. The scoring system was based on hazard ratios in a Cox proportional hazard model. In the multivariate analysis, age, tumor size, Lauren type, depth of invasion, lymph node metastasis, and the neutrophil--lymphocyte ratio were independent prognostic indicators of overall survival. A prognostic model was then established based on the significant factors. Patients were categorized into five groups according to their scores. The 3-year survival rates for the low- to high-risk groups were 98.9%, 92.8%, 82.4%, 58.4%, and 36.9%, respectively (P < 0.001). The prognostic model clearly discriminated patients with stage pT1-4N0M0 tumor into four risk groups with significant differences in the 3-year survival rates (P < 0.001). Compared with the pathological T stage, the model improved the predictive accuracy of the 3-year survival rate by 5% for node-negative patients. The prognostic scores also stratified the patients with stage pT4aN0M0 tumor into significantly different risk groups (P = 0.004). Furthermore, the predictive value of this model was validated in an independent set of 274 patients. This model, which included the systemic inflammatory markers and clinicopathological factors, is more effective in predicting the prognosis of node-negative gastric cancer than traditional staging systems. Patients in the high-risk group might be good candidates for adjuvant chemotherapy.
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Neoplasias Gástricas/epidemiología , Neoplasias Gástricas/patología , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores , Femenino , Estudios de Seguimiento , Gastrectomía , Humanos , Estimación de Kaplan-Meier , Metástasis Linfática , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Estadificación de Neoplasias , Evaluación de Resultado en la Atención de Salud , Pronóstico , Curva ROC , Reproducibilidad de los Resultados , Estudios Retrospectivos , Neoplasias Gástricas/mortalidad , Neoplasias Gástricas/cirugía , Síndrome de Respuesta Inflamatoria Sistémica/epidemiología , Síndrome de Respuesta Inflamatoria Sistémica/mortalidad , Carga TumoralRESUMEN
Interleukin- (IL-) 2 is the major growth factor for T-cell activation and proliferation. IL-2 has multiple functions in the regulation of immunological processes. Although most studies focus on T-cell immunomodulation, T-cell activation by IL-2 is the foundation of priming the feedback loop. Here, we investigated the effect of MAPK/ERK and PI3K/Akt signaling pathways on IL-2-induced cell activation and the regulatory mechanisms of upstream ubiquitin ligase Cbl-b and c-Cbl. Morphological analysis of Jurkat T cells was performed by cytospin preparations with Wright-Giemsa stain. CD25 expression on Jurkat T cells was determined by flow cytometry. Changes in cell activation proteins such as p-ERK, ERK, p-Akt, Akt, and ubiquitin ligase Casitas B-cell Lymphoma (Cbl) proteins were analyzed by western blot. Following IL-2-induced activation of Jurkat T cells, p-ERK expression was upregulated, while there was no change in p-Akt, ERK, or Akt expression. Thus, the MAPK/ERK signaling pathway, but not PI3K/Akt, was involved in IL-2-induced T-cell activation. Either using PD98059 (a specific inhibitor for p-ERK) or depletion of ERK with small interfering RNA (siRNA) reduced the expression of CD25. This study also showed that ubiquitin ligase proteins Cbl-b and c-Cbl might be involved in IL-2-induced Jurkat T-cell activation by negatively regulating the MAPK/ERK signaling pathway.
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Interleucina-2/inmunología , Activación de Linfocitos/inmunología , Proteínas Proto-Oncogénicas c-cbl , Apoptosis/efectos de los fármacos , Humanos , Interleucina-2/metabolismo , Células Jurkat , Activación de Linfocitos/genética , Sistema de Señalización de MAP Quinasas , Proteína Oncogénica v-akt/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-cbl/genética , Proteínas Proto-Oncogénicas c-cbl/inmunología , Proteínas Proto-Oncogénicas c-cbl/metabolismo , Transducción de Señal/efectos de los fármacos , Regulación hacia ArribaRESUMEN
BACKGROUND: Although several clinical trials have suggested that postoperative adjuvant chemotherapy can improve survival of patients with gastric cancer, the optimal treatment duration has not been studied. This retrospective analysis evaluated the outcomes of patients with gastric cancer treated with six cycles of fluorouracil-based treatment compared with a cohort treated with four or eight cycles. METHODS: We retrospectively identified 237 patients with stage IB-IIIC gastric cancer who received four, six, or eight cycles of fluorouracil-based adjuvant chemotherapy administered every 3 weeks after radical gastrectomy. The endpoint was overall survival (OS). Factors associated with prognosis were also analyzed. RESULTS: The estimated 3-year OS rates for the four-, six-, and eight-cycle cohorts were 54.4%, 76.1%, and 68.9%, respectively; and the estimated 5-year OS rates were 41.2%, 74.0%, and 65.8%, respectively. Patients who received six cycles were more likely to have a better OS than those who received four cycles (P = 0.002). Eight cycles failed to show an additional survival benefit (P = 0.454). In the multivariate analysis, the number of chemotherapy cycles was associated with OS independent of clinical covariates (P<0.05). Subgroup analysis suggested that among patients in all age groups examined, male patients, and subgroups of fluorouracil plus oxaliplatin combined chemotherapy, stage III, poor differentiation, and gastrectomy with D2 lymphadenectomy, six cycles of adjuvant chemotherapy were associated with a statistically significant benefit of OS compared with four cycles (P<0.05). CONCLUSIONS: Six cycles of adjuvant chemotherapy might lead to a favorable outcome for patients with gastric cancer, and two further cycles could not provide an additional clinical benefit.
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Fluorouracilo/uso terapéutico , Neoplasias Gástricas/tratamiento farmacológico , Adulto , Anciano , Quimioterapia Adyuvante/efectos adversos , Quimioterapia Adyuvante/métodos , Estudios de Cohortes , Femenino , Fluorouracilo/administración & dosificación , Fluorouracilo/efectos adversos , Humanos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Neoplasias Gástricas/cirugía , Análisis de Supervivencia , Factores de TiempoRESUMEN
BACKGROUND: Tamoxifen (TAM) is a nonsteroidal antiestrogen that has been widely used in the treatment of breast cancer through its anti-estrogen activity. Recent studies show that TAM is cytotoxic to both estrogen receptor (ER)-positive and ER-negative cells via the induction of apoptosis. However, the molecular mechanisms of this effect are not well understood. In the present study, we investigated the roles of c-Src, ERK, AKT and c-Cbl ubiquitin ligases during TAM-induced apoptosis of MCF-7 cells. MATERIAL AND METHODS: MCF-7 cell proliferation and apoptosis were measured by 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, and flow cytometry, respectively. c-Cbl expression, and the activity of c-Src, ERK, AKT were assayed by Western blotting. Overexpression of the wild and the dominant-negative type of c-Cbl (70Z/Cbl) were achieved by transient transfection of plasmids encoding c-Cbl and 70Z/Cbl, respectively, and were confirmed by Western blotting. Statistical analysis was performed using the t-test, and a p-value <0.05 was considered to be statistically significant. RESULTS: A high concentration of TAM (25 µM) induced a time-dependent apoptosis of MCF-7 cells. ERK1/2 and AKT were activated during TAM-induced apoptosis. The ERK1/2 inhibitor PD98059, the PI3K/Akt inhibitor LY294002, and the c-Src inhibitor PP2 all enhanced TAM action. Moreover, the ubiquitin ligase c-Cbl was up-regulated during this process. Over-expression of c-Cbl significantly enhanced the apoptosis-inducing effects of TAM, while 70Z/Cbl suppressed the apoptosis-inducing effects of TAM. Further investigation revealed that, overexpression of c-Cbl significantly downregulated the c-Src protein levels and TAM-induced AKT activity. But 70Z/Cbl significantly upregulated TAM-induced ERK and AKT activity. CONCLUSIONS: This study demonstrates that c-Src, ERK, and AKT played a protective role during TAM-induced apoptosis, and that c-Cbl sensitized MCF-7 cells to TAM by modulating the expression of c-Src, and TAM-induced ERK and AKT activity.
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Apoptosis/efectos de los fármacos , Neoplasias de la Mama/patología , Carcinoma/patología , Proteínas Proto-Oncogénicas c-cbl/fisiología , Tamoxifeno/farmacología , Apoptosis/genética , Neoplasias de la Mama/metabolismo , Carcinoma/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Antagonistas de Estrógenos/farmacología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Humanos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-cbl/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/fisiologíaRESUMEN
Background/Aims Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) as a newly identified biological agent has shown promising antitumor effects in a wide range of cancers. However, gastric cancer cells are less sensitive than other cancer cells to TRAIL-induced apoptosis.Here, we combined TRAIL with bortezomib, a proteasomal inhibitor to induce apoptosis in three gastric cancer cell lines.Methods After the cells were treated with TRAIL and/or bortezomib, the cell viability, apoptosis and cell cycle distribution were examined. The levels of death receptors and the mitochondrial membrane potential were also detected. The expression of apoptosis-associated proteins was determined by Western blot.Results Bortezomib at low concentration significantly(P<0.05) enhanced the cytotoxic effect of TRAIL by enhancing apoptosis as well as cell cycle arrest at G2/M phase. The enhancement of efficiency of TRAIL by bortezomib involved up-regulation of death receptor 4 and 5, as well as reduction of the mitochondrial membrane potential. Further study showed that combined treatment with TRAIL and bortezomib down-regulated anti-apoptotic protein cIAP-1, and over expression of cIAP-1 significantly(P\0.05) reduced the synergistic effect between TRAIL and bortezomib.Conclusions Bortezomib synergizes TRAIL-induced apoptosis in human gastric cancer cells. The synergistic effect between these two drugs is associated with up-regulation of death receptors and down-regulation of cIAP-1.The combination of TRAIL and bortezomib might be an effective regimen for the treatment of advanced gastric cancer.
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Antineoplásicos/farmacología , Ácidos Borónicos/farmacología , Pirazinas/farmacología , Neoplasias Gástricas/tratamiento farmacológico , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Apoptosis/efectos de los fármacos , Bortezomib , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Sinergismo Farmacológico , HumanosRESUMEN
BACKGROUND. Exosomes are nanometer-sized vesicles with immunomodulatory functions, which are released by a diverse range of living cells. Although recent studies have shown that tumor-derived exosomes can suppress the function of T cells, the molecular mechanisms are not well understood. In the present study, we investigated the role of the Casitas B lineage lymphoma (cbl) family of ubiquitin ligases in gastric cancer exosome-induced apoptosis of Jurkat T cells. MATERIALS AND METHODS. By serial centrifugation and sucrose gradient ultracentrifugation, we isolated and purified the exosomes from gastric cancer SGC7901 cells, and identified them by electron microscopy and Western blotting. Cell apoptosis was detected using propidium iodide staining. Western blotting and RT-PCR was exploited to evaluate the expression of proteins and mRNA, respectively. RESULTS. Gastric cancer exosomes induced Jurkat T cell apoptosis in a time- and dose-dependent manner and activated caspases 3, 8 and 9. The expression of Cbl-b and c-Cbl was up-regulated during exosome-induced apoptosis of cells. Meanwhile, exosomes induced ubiquitination of the p85 subunit of phosphoinositide 3-kinase (PI3K) and reduced downstream Akt activity. Inhibition of proteasome led to partial restoration of Akt activity and cell apoptosis. DISCUSSION AND CONCLUSIONS. The Cbl family of ubiquitin ligases might be involved in regulation of exosome-induced apoptosis of Jurkat T cells by increasing PI3K proteasome degradation, inactivation of PI3K/Akt signaling, thus mediating some effects of caspase activation.
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Apoptosis/fisiología , Caspasas/metabolismo , Exosomas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Proto-Oncogénicas c-cbl/fisiología , Neoplasias Gástricas/enzimología , Neoplasias Gástricas/inmunología , Linfocitos T/fisiología , Ubiquitina-Proteína Ligasas/fisiología , Adenocarcinoma , Western Blotting , Caspasa 3/metabolismo , Caspasa 8/metabolismo , Caspasa 9/metabolismo , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Humanos , Células Jurkat , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-cbl/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Neoplasias Gástricas/ultraestructura , Ubiquitina-Proteína Ligasas/metabolismo , Regulación hacia ArribaRESUMEN
The study was aimed to explore the mechanism of SYK and CBL family of ubiquitin ligases in Bufalin-induced HL-60 cells apoptosis. Cell viability was tested by trypan blue staining and apoptosis was detected by using flow cytometry. The expressions of CBL and CBL-b and the phosphorylation of SYK were detected by using immunoprecipitation and Western blot. The results showed that Bufalin inhibited HL-60 cell proliferation in time- and dose-dependent manners. IC(50) of suppressing cell viability at 24, 48 and 72 hours were about 26.3, 7.8 and 2.0 nmol/L respectively. The high dose of bufalin already induced apoptosis of HL-60 cells at 8 hours. SYK was quickly phosphorylated, and the expressions of CBL and CBL-b were down-regulated after treatment with Bufalin. It is concluded that SYK activation and CBL protein down-regulation may be involved in Bufalin-induced HL-60 cell apoptosis.