RESUMO
Patient-derived xenograft (PDX) assay has been widely used in preclinical research in patients with multidrug-resistant lung cancer. One hundred patients with non-small cell lung cancer (NSCLC) were divided into MiniPDX group and conventional group, with 50 cases in each group. The MiniPDX assay was established by enriching high-purity tumor cells using microfluidic technology to detect the drug sensitivity of NSCLC cells. All patients underwent conventional computed tomography (CT) scans of lung and mediastinum at baseline and during follow-up. Kaplan-Meier method was used to compare the overall survival and progression-free survival of two groups. The sensitivity of the same drug in different tumor xenograft varied greatly. The overall survival, progression-free survival, and clinical benefit rate of patients in the MiniPDX-guided chemotherapy group were significantly longer than those in the conventional chemotherapy group. MiniPDX assay may be an effective tool for screening chemotherapy regimens in NSCLC patients.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Técnicas Analíticas Microfluídicas/métodos , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) are malignant clonal diseases of the hematopoietic system with an unsatisfactory overall prognosis. The main obstacle is the increased resistance of AML and ALL cells to chemotherapy. The development and validation of new therapeutic strategies for acute leukemia require preclinical models that accurately recapitulate the genetic, pathological, and clinical features of acute leukemia. A patient-derived orthotopic xenograft (PDOX) model is established using surgical orthotopic implantation. They closely resemble human tumor progression and microenvironment and are more reliable translational research tools than subcutaneous-transplant models. In this study, we established PDOX models by direct intrafemoral injection of bone marrow and peripheral blood cells from AML and ALL patients, characterized their pathology, cytology, and genetics, and compared the model's characteristics and drug responsiveness with those of the corresponding patients.
Assuntos
Leucemia Mieloide Aguda , Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Medula Óssea/patologia , Xenoenxertos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Prognóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Microambiente TumoralRESUMO
As one of the leading causes of death from disease, cancer continues to pose a serious threat to human health globally. Despite the development of novel therapeutic regimens and drugs, the long-term survival of cancer patients is still very low, especially for those whose diagnosis is not caught early enough. Meanwhile, our understanding of tumorigenesis is still limited. Suitable research models are essential tools for exploring cancer mechanisms and treatments. Herein we review and compare several widely used in vitro and in vivo murine cancer models, including syngeneic tumor models, genetically engineered mouse models (GEMM), cell line-derived xenografts (CDX), patient-derived xenografts (PDX), conditionally reprogrammed (CR) cells, organoids, and MiniPDX. We will summarize the methodology and feasibility of various models in terms of their advantages and limitations in the application prospects for drug discovery and development and precision medicine.
Assuntos
Neoplasias , Humanos , Camundongos , Animais , Modelos Animais de Doenças , Organoides , Medicina de Precisão/métodos , XenoenxertosRESUMO
Current liquid biopsy assays lack sufficient sensitivity to detect copy number loss, which limits the interrogation of critical tumor suppressor gene deletions during cancer progression and treatment. Here we describe a liquid biopsy assay with improved sensitivity for detection of copy number loss in blood samples with low levels of circulating tumor DNA, and demonstrate its utility by profiling PTEN, RB1, and TP53 genetic loss in metastatic prostate cancer patients.
RESUMO
BACKGROUND: Treatment guidelines for a variety of cancers have been increasingly used in clinical practice, and have resulted in major improvement in patient outcomes. However, recommended regimens (even first-line treatments) are clearly not ideal for every patients. In the present study, we used mini patient-derived xenograft (mini-PDX) and next-generation sequencing to develop personalized treatment in a patient with metastatic duodenal adenocarcinoma. METHODS: Resected metachronous metastatic tumor tissues were implanted into SCID mice to determine the sensitivity to a variety of drug regimens. Mutation profiles were assessed using both DNA whole-exome sequencing (DNA-WES) and RNA sequencing. The results of the analyses were used to select optimal treatment for the patient with metastatic duodenal adenocarcinoma. RESULTS: Assessment with mini-PDX models took only 7 days. The results showed high sensitivity to S-1 plus cisplatin, gemcitabine plus cisplatin and everolimus alone. The patient received gemcitabine plus cisplatin initially, but the treatment was terminated due to toxicity. The patient was then switched to treatment with S-1 alone. The overall disease-free survival was 34 months. DNA-WES and RNA sequencing identified KRAS mutation (A146T), TP53 (C229Yfs*10) and RICTOR amplification in the metastatic duodenal adenocarcinoma. These findings provided further support to the results of the mini-PDX, and suggest mTOR inhibitors should be used if and when relapse eventually occurs in this patient. CONCLUSIONS: Mini-PDX model combined with WES/RNA sequencing can rapidly assess drug sensitivity in cancer patients and reveal key genetic alterations. Further research on this technology for personalized therapy in patients with refractory malignant tumors is warranted.
Assuntos
Adenocarcinoma/tratamento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Duodenais/tratamento farmacológico , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Neoplasias Duodenais/genética , Neoplasias Duodenais/patologia , Everolimo/uso terapêutico , Humanos , Imunossupressores/uso terapêutico , Masculino , Camundongos SCID , Pessoa de Meia-Idade , Mutação , Metástase Neoplásica , Medicina de Precisão/métodosRESUMO
BACKGROUND: Non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutations or anaplastic lymphoma kinase (ALK) fusions show dramatic responses to specific tyrosine kinase inhibitors (TKIs); however, after 10-12 months, secondary mutations arise that confer resistance. We generated a murine xenograft model using patient-derived NSCLC cells isolated from the pleural fluid of two patients with NSCLC to investigate the mechanisms of resistance against the ALK- and EGFR-targeted TKIs crizotinib and osimertinib, respectively. METHODS: Genotypes of patient biopsies and xenograft tumors were determined by whole exome sequencing (WES), and patients and xenograft-bearing mice received targeted treatment (crizotinib or osimertinib) accordingly. Xenograft mice were also treated for prolonged periods to identify whether the development of drug resistance and/or treatment responses were associated with tumor size. Finally, the pathology of patients biopsies and xenograft tumors were compared histologically. RESULTS: The histological characteristics and chemotherapy responses of xenograft tumors were similar to the actual patients. WES showed that the genotypes of the xenograft and patient tumors were similar (an echinoderm microtubule-associated protein-like 4-ALK (EML4-ALK) gene fusion (patient/xenograft: CTC15035EML4-ALK) and EGFR L858R and T790M mutations (patient/xenograft: CTC15063EGFR L858R, T790M)). After continuous crizotinib or osimertinib treatment, WES data suggested that acquired ALK E1210K mutation conferred crizotinib resistance in the CTC15035EML4-ALK xenograft, while decreased frequencies of EGFR L858R and T790M mutations plus the appearance of v-RAF murine sarcoma viral oncogene homolog B (BRAF) G7V mutations and phosphatidylinositol-4-phosphate 3-kinase catalytic subunit type 2 alpha (PIK3C2A) A86fs frame shift mutations led to osimertinib resistance in the CTC15063EGFR L858R, T790M xenografts. CONCLUSIONS: We successfully developed a new method of generating drug resistance xenograft models from liquid biopsies using microfluidic technology, which might be a useful tool to investigate the mechanisms of drug resistance in NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Acrilamidas , Quinase do Linfoma Anaplásico/genética , Compostos de Anilina , Animais , Carcinoma Pulmonar de Células não Pequenas/complicações , Carcinoma Pulmonar de Células não Pequenas/genética , Crizotinibe/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/genética , Feminino , Humanos , Neoplasias Pulmonares/complicações , Neoplasias Pulmonares/genética , Camundongos SCID , Mutação , Fosfatidilinositol 3-Quinases/genética , Piperazinas/farmacologia , Derrame Pleural Maligno/complicações , Proteínas Proto-Oncogênicas B-raf/genéticaRESUMO
BACKGROUND: Patient-derived organoids and xenografts (PDXs) have emerged as powerful models in functional diagnostics with high predictive power for anticancer drug response. However, limitations such as engraftment failure and time-consuming for establishing and expanding PDX models followed by testing drug efficacy, and inability to subject to systemic drug administration for ex vivo organoid culture hinder realistic and fast decision-making in selecting the right therapeutics in the clinic. The present study aimed to develop an advanced PDX model, namely MiniPDX, for rapidly testing drug efficacy to strengthen its value in personalized cancer treatment. METHODS: We developed a rapid in vivo drug sensitivity assay, OncoVee® MiniPDX, for screening clinically relevant regimens for cancer. In this model, patient-derived tumor cells were arrayed within hollow fiber capsules, implanted subcutaneously into mice and cultured for 7 days. The cellular activity morphology and pharmacokinetics were systematically evaluated. MiniPDX performance (sensitivity, specificity, positive and negative predictive values) was examined using PDX as the reference. Drug responses were examined by tumor cell growth inhibition rate and tumor growth inhibition rate in PDX models and MiniPDX assays respectively. The results from MiniPDX were also used to evaluate its predictive power for clinical outcomes. RESULTS: Morphological and histopathological features of tumor cells within the MiniPDX capsules matched those both in PDX models and in original tumors. Drug responses in the PDX tumor graft assays correlated well with those in the corresponding MiniPDX assays using 26 PDX models generated from patients, including 14 gastric cancer, 10 lung cancer and 2 pancreatic cancer. The positive predictive value of MiniPDX was 92%, and the negative predictive value was 81% with a sensitivity of 80% and a specificity of 93%. Through expanding to clinical tumor samples, MiniPDX assay showed potential of wide clinical application. CONCLUSIONS: Fast in vivo MiniPDX assay based on capsule implantation was developed-to assess drug responses of both PDX tumor grafts and clinical cancer specimens. The high correlation between drug responses of paired MiniPDX and PDX tumor graft assay, as well as translational data suggest that MiniPDX assay is an advanced tool for personalized cancer treatment.
Assuntos
Biomarcadores Farmacológicos/química , Animais , Modelos Animais de Doenças , Camundongos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Constitutive activation of the NF-kappaB pathway is required for survival of the activated B cell-like (ABC) subgroup of diffuse large B-cell lymphoma (DLBCL). Here we show that a small molecule IkappaB kinase (IKK) inhibitor, PS-1145, and related compounds are toxic for ABC DLBCL cell lines but not for cell lines derived from the other prevalent form of DLBCL, germinal center B cell-like DLBCL. Treatment of ABC lines with these inhibitors rapidly induced a series of gene expression changes that were attributable to cessation of constitutive IKK activity, similar to changes induced by acute expression of genetic inhibitors of NF-kappaB, confirming the effectiveness and specificity of this compound. Before cell death, inhibition of IKK also induced features of apoptosis and an arrest in the G1 phase of the cell cycle. To test further the specificity of this toxicity, an inducible form of NF-kappaB was created by fusing the p65 NF-kappaB subunit with the ligand-binding domain of the estrogen receptor (p65-ERD). In the presence of tamoxifen, p65-ERD reversed the toxicity of IKK inhibition and restored expression of many NF-kappaB target genes. Another subgroup of DLBCL, primary mediastinal B-cell lymphoma (PMBL), also expresses NF-kappaB target genes, and treatment of a PMBL cell line with an IKK inhibitor was toxic and induced gene expression changes of a distinct group of NF-kappaB target genes. These studies validate the NF-kappaB pathway as a promising therapeutic target in ABC DLBCL, PMBL, and other lymphomas that depend on the activity of NF-kappaB for survival and proliferation.
Assuntos
Regulação Neoplásica da Expressão Gênica , Linfoma de Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Antineoplásicos/farmacologia , Apoptose , Carbolinas/farmacologia , Ciclo Celular , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Relação Dose-Resposta a Droga , Regulação para Baixo , Inibidores Enzimáticos/farmacologia , Genes Dominantes , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Quinase I-kappa B , Concentração Inibidora 50 , L-Lactato Desidrogenase/metabolismo , Leucócitos Mononucleares/citologia , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Piridinas/farmacologia , Receptores de Estrogênio/metabolismo , Tamoxifeno/farmacologia , Fatores de Tempo , Regulação para CimaRESUMO
Hepatocellular carcinoma (HCC) is a common cancer with poor prognosis worldwide and the molecular mechanism is not well understood. This study aimed to establish a collection of human HCC cell lines from patient-derived xenograft (PDX) models. From the 20 surgical HCC sample collections, 7 tumors were successfully developed in immunodeficient mice and further established 7 novel HCC cell lines (LIXC002, LIXC003, LIXC004, LIXC006, LIXC011, LIXC012 and CPL0903) by primary culture. The characterization of cell lines was defined by morphology, growth kinetics, cell cycle, chromosome analysis, short tandem repeat (STR) analysis, molecular profile, and tumorigenicity. Additionally, response to clinical chemotherapeutics was validated both in vitro and in vivo. STR analysis indicated that all cell lines were unique cells different from known cell lines and free of contamination by bacteria or mycoplasma. The other findings were quite heterogeneous between individual lines. Chromosome aberration could be found in all cell lines. Alpha-fetoprotein was overexpressed only in 3 out of 7 cell lines. 4 cell lines expressed high level of vimentin. Ki67 was strongly stained in all cell lines. mRNA level of retinoic acid induced protein 3 (RAI3) was decreased in all cell lines. The 7 novel cell lines showed variable sensitivity to 8 tested compounds. LIXC011 and CPL0903 possessed multiple drug resistance property. Sorafenib inhibited xenograft tumor growth of LIXC006, but not of LIXC012. Our results indicated that the 7 novel cell lines with low passage maintaining their clinical and pathological characters could be good tools for further exploring the molecular mechanism of HCC and anti-cancer drug screening.
Assuntos
Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral/patologia , Aberrações Cromossômicas , Efeito Fundador , Neoplasias Hepáticas/patologia , Proteínas de Neoplasias/genética , Animais , Antineoplásicos/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/metabolismo , Feminino , Expressão Gênica , Xenoenxertos , Humanos , Cariotipagem , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos , Camundongos SCID , Proteínas de Neoplasias/metabolismo , Especificidade de Órgãos , Cultura Primária de Células , Sequências de Repetição em Tandem , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cancer stem cells (CSCs) have received considerable attention from the research community since they were first reported in human acute myeloid leukemia 15 years ago. Accumulating evidence suggests that CSCs are responsible for tumor initiation and progression, drug resistance, and metastasis in both liquid and solid tumors. These findings lead to the development of novel compounds targeting CSC populations that is becoming increasingly important for eradicating CSCs in heterogeneous tumor masses and to cure the cancer. Since 2003, we have participated in CSC studies and encountered crucial early events in the field. This article reviews the history of CSC biology, clarifies the term and its definition, and further addresses the issue of how to utilize CSCs in therapeutic target discovery and drug development based on our substantial experience.
Assuntos
Antineoplásicos/uso terapêutico , Descoberta de Drogas , Neoplasias/tratamento farmacológico , Células-Tronco Neoplásicas , Animais , Antineoplásicos/história , Biomarcadores Tumorais , Descoberta de Drogas/história , História do Século XX , História do Século XXI , Humanos , Neoplasias/história , Neoplasias/patologia , Células Tumorais CultivadasRESUMO
There are reports of early evidence that suggest the involvement of chronic low-grade inflammation in the pathogenesis of Type 2 diabetes. Thus, substances that have effects in reducing inflammation could be potential drugs for Type 2 diabetes. Leonurine (4-guanidino-n-butyl syringate; SCM-198) is an alkaloid in HL (Herba leonuri), which was reported to possess anti-inflammatory properties. We hypothesize that SCM-198 may have beneficial effects on Type 2 diabetes. In the present study, we attempted to test this hypothesis by evaluating the anti-diabetic effect of SCM-198 and the possible underlying mechanisms of its effects in db/db mice. SCM-198 (50, 100 and 200 mg/kg of body weight), pioglitazone (50 mg/kg of body weight, as a positive control) or 1% CMC-Na (sodium carboxymethylcellulose) were administered to the db/db or db/m mice once daily for 3 weeks. After 3 weeks, SCM-198 (200 mg/kg of body weight) treatment significantly reduced the fasting blood glucose level and increased the plasma insulin concentration in the db/db mice, meanwhile it significantly lowered the plasma TAG (triacylglycerol) concentration and increased the HDL (high-density lipoprotein)-cholesterol concentration. Moreover, the dysregulated transcription of the hepatic glucose metabolic enzymes, including GK (glucokinase), G6Pase (glucose-6-phosphatase) and PEPCK (phosphoenolpyruvate carboxykinase), was recovered by an Akt-dependent pathway. The pro-inflammatory mediators {such as TNFα (tumour necrosis factor α), IL (interleukin)-6, IL-1ß, degradation of IκB [inhibitor of NF-κB (nuclear factor-κB)] α and thereafter activation of NF-κB} were reversed by SCM-198 treatment in the db/db mice. The present study provides first evidence that SCM-198 exhibits anti-inflammatory activity and has an ameliorating effect on diabetic symptoms via inhibiting of NF-κB/IKK (IκB kinase) pathway. Consequently, we suggest that SCM-198 may be a prospective agent for prevention and/or moderation of the progress of Type 2 diabetes.
Assuntos
Anti-Inflamatórios/farmacologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Ácido Gálico/análogos & derivados , Hipoglicemiantes/farmacologia , Leonurus/química , Fígado/efeitos dos fármacos , NF-kappa B/antagonistas & inibidores , Tecido Adiposo Branco/efeitos dos fármacos , Animais , Glicemia/metabolismo , Peso Corporal/efeitos dos fármacos , Diabetes Mellitus Tipo 2/imunologia , Diabetes Mellitus Tipo 2/metabolismo , Ácido Gálico/farmacologia , Mediadores da Inflamação/imunologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/imunologiaRESUMO
The limited therapeutic effect of gemcitabine on pancreatic cancer is largely attributed to pre-existing or acquired resistance of the tumor cells. This study was aimed at screening for candidate resistance-related gene(s) and elucidating the underlying mechanisms. NME5 was found to be highly expressed in an innate gemcitabine-resistant human pancreatic cancer sample and the cell line PAXC002 derived from the sample. Downregulation of NME5 significantly reversed gemcitabine resistance in PAXC002 cells, whereas NME5 overexpression induced gemcitabine resistance in the pancreatic cancer cell line BxPC-3. NME5 attenuated the induction of apoptosis and cell cycle arrest induced by gemcitabine, probably accounting for the blunted sensitivity to gemcitabine. Furthermore, NME5 was demonstrated to play its role in a nuclear factor kappaB (NF-κB)-dependent manner. NME5 was capable of directly binding NF-κB, and possibly regulated its expression level in PAXC002 cells. Our results also suggest that NF-κB is a key executor of NME5 in regulating apoptosis and cell cycle. All of these data suggest that NME5 is a promising target for relieving innate gemcitabine resistance in pancreatic cancer cells.
Assuntos
Antimetabólitos Antineoplásicos/uso terapêutico , Desoxicitidina/análogos & derivados , Resistencia a Medicamentos Antineoplásicos/fisiologia , Nucleosídeo NM23 Difosfato Quinases/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/fisiopatologia , Animais , Apoptose/fisiologia , Ciclo Celular/fisiologia , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Desoxicitidina/uso terapêutico , Regulação para Baixo , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Camundongos SCID , NF-kappa B/genética , NF-kappa B/metabolismo , Nucleosídeo NM23 Difosfato Quinases/genética , Transplante de Neoplasias , GencitabinaRESUMO
Pancreatic ductal adenocarcinoma (PDA) remains one of the most lethal malignancies in the world, often diagnosed at an advanced stage, resistant to conventional chemotherapy and having high invasive and metastatic potential. The mechanism of drug resistance of PDA is still not clear. In the present study, we established two novel pancreatic cancer cell lines PAXC-002 and PAXC-003 from human primary xenograft models. The cell lines were characterized by morphology, karyotype, pancreatic cancer marker and short tandem repeat (STR) analysis, and growth kinetics and tumorigenicity. The in vitro anti-proliferation test revealed that PAXC-002 cell was intrinsically resistant to the standard of care chemotherapy-gemcitabine, compared with that of PAXC-003 and other widely used pancreatic cancer cell lines. Interestingly, the gemcitabine resistant PAXC-002 cell line was more potent in forming colonies in 3-Dimensional matrigel culture conditions and had a higher percentage of CD133 positive cells, which is recognized as a cancer stem cell marker, compared to the gemcitabine-sensitive PAXC-003 cell line. In this study, we present two novel pancreatic cancer cell lines which could be used for gemcitabine resistance investigation, mechanism identification of pancreatic cancer and anticancer drug screening. The preliminary data indicate that the drug resistance of pancreatic carcinoma cells is associated with a cancer stem cell-like phenotype.
Assuntos
Antineoplásicos/farmacologia , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/patologia , Desoxicitidina/análogos & derivados , Células-Tronco Neoplásicas/efeitos dos fármacos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Animais , Antimetabólitos Antineoplásicos/farmacologia , Biomarcadores Tumorais/metabolismo , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Desoxicitidina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Cariótipo , Camundongos , Camundongos SCID , Repetições de Microssatélites , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Transplante Heterólogo , GencitabinaRESUMO
Non-metastatic cells 5 (NME5), a recently found gene belonging to the NDPK-like molecules gene family, is highly expressed in testis and some types of human cancer. Current studies have revealed diverse potential functions of NME5 and we have reported that NME5 is associated with innate resistance to gemcitabine in human pancreatic cancer cells in previous study. However, the mechanism underlying the transcriptional regulation of NME5 has not been elucidated yet. In this study, we analyzed the 5'-flanking region of the human NME5 gene and revealed its transcription start site (TSS) at -35 bp relative to its translation start codon ATG. Using 5' unidirectional deletion analysis, we demonstrated that the proximal promoter of NME5 is located within -1051 bp to +35 bp. Two functional GC-boxes (-300 bp and -323 bp) were identified within the promoter region. Mutation of either GC-box led to significant reduction in NME5 promoter activity, whereas overexpression of Sp1 activated NME5 promoter activity in MIA PaCa-2 and 293T cells. In silico analysis predicted that transcription factor Sp1 binds to both GC-boxes, which were confirmed by EMSA and ChIP. In addition, we found that compared with MIA PaCa-2, Sp1 was highly expressed in PAXC002, a well characterized human pancreatic cancer cell line with innate gemcitabine resistance where NME5 was reported to be highly expressed, indicating that Sp1 induces NEM5 expression in PAXC002 cells. In conclusion, our study characterized for the first time the human NME5 promoter which is controlled by Sp1 transcription factor in pancreatic cancer.
Assuntos
Nucleosídeo NM23 Difosfato Quinases/genética , Neoplasias Pancreáticas/genética , Regiões Promotoras Genéticas , Fator de Transcrição Sp1/metabolismo , Ativação Transcricional , Sequência de Bases , Linhagem Celular Tumoral , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Sequências Reguladoras de Ácido Nucleico , Análise de Sequência de DNA , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp3/biossíntese , Fator de Transcrição Sp3/genética , Fator de Transcrição Sp3/metabolismo , Sítio de Iniciação de Transcrição , Transcrição Gênica , GencitabinaRESUMO
The transcription factor NF-kappaB plays a central role in regulating inflammation and apoptosis, making it a compelling target for drug development. We identified a small molecule inhibitor (ML120B) that specifically inhibits IKKbeta, an Ikappa-B kinase that regulates NF-kappaB. IKKbeta and NF-kappaB are required in vivo for prevention of TNFalpha-mediated apoptosis. ML120B sensitized mouse bone marrow progenitors and granulocytes, but not mature B cells to TNFalpha killing in vitro, and induced apoptosis in vivo in the bone marrow and spleen within 6 hours of a single oral dose. In vivo inhibition of IKKbeta with ML120B resulted in depletion of thymocytes and B cells in all stages of development in the bone marrow but did not deplete granulocytes. TNF receptor-deficient mouse thymocytes and B cells were resistant to ML120B-induced depletion in vivo. Surprisingly, surviving bone marrow granulocytes expressed TNFR1 and TNFR2 after dosing in vivo with ML120B. Our results show that inhibition of IKKbeta with a small molecule in vivo leads to rapid TNF-dependent depletion of T and B cells. This observation has several implications for potential use of IKKbeta inhibitors for the treatment of inflammatory disease and cancer.
Assuntos
Quinase I-kappa B/antagonistas & inibidores , Depleção Linfocítica/métodos , Receptores Tipo I de Fatores de Necrose Tumoral/fisiologia , Animais , Apoptose/efeitos dos fármacos , Linfócitos B/efeitos dos fármacos , Células da Medula Óssea/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Granulócitos/efeitos dos fármacos , Granulócitos/metabolismo , Células-Tronco Hematopoéticas/efeitos dos fármacos , Camundongos , Camundongos Knockout , Receptores do Fator de Necrose Tumoral , Receptores Tipo I de Fatores de Necrose Tumoral/deficiência , Receptores Tipo II do Fator de Necrose Tumoral/deficiência , Receptores Tipo II do Fator de Necrose Tumoral/fisiologia , Receptores Chamariz do Fator de Necrose Tumoral , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
IkappaB kinase (IKK) beta is essential for inflammatory cytokine-induced activation of nuclear factor kappaB (NF-kappaB). NF-kappaB plays a pivotal role in the function of major cell types that contribute to the pathophysiological process of rheumatoid arthritis (RA). Here, we report the mechanism and the effect of the IKKbeta inhibitor N-(6-chloro-7-methoxy-9H-beta-carbolin-8-yl)-2-methylnicotinamide (ML120B), a beta-carboline derivative, on NF-kappaB signaling and gene activation in RA-relevant cell systems. ML120B is a potent, selective, reversible, and ATP-competitive inhibitor of IKKbeta with an IC50 of 60 nM when evaluated in an IkappaBalpha kinase complex assay. ML120B does not inhibit other IKK isoforms or a panel of other kinases. ML120B concentration-dependently inhibits tumor necrosis factor alpha (TNFalpha)-stimulated NF-kappaB signaling via inhibition of IkappaBalpha phosphorylation, degradation, and NF-kappaB translocation into the nucleus. For the first time, we have demonstrated that in human fibroblast-like synoviocytes, TNFalpha- or interleukin (IL)-1beta-induced monocyte chemoattractant protein-1 regulated on activation, normal T cell expressed and secreted and production is IKKbeta-dependent. In addition, for the first time, we have demonstrated that lipopolysaccharide- or peptidoglycan-induced cytokine production in human cord blood-derived mast cells is IKKbeta-dependent. In addition, in human chondrocytes, ML120B inhibited IL-1beta-induced matrix metalloproteinase production with an IC50 of approximately 1 microM. ML120B also blocked IL-1beta-induced prostaglandin E2 production. In summary, ML120B blocked numerous NF-kappaB-regulated cell responses that are involved in inflammation and destructive processes in the RA joint. Our findings support the evaluation of IKKbeta inhibitors as anti-inflammatory agents for the treatment of RA.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Células do Tecido Conjuntivo , Inibidores Enzimáticos/farmacologia , Quinase I-kappa B/antagonistas & inibidores , NF-kappa B/antagonistas & inibidores , Anti-Inflamatórios não Esteroides/química , Condrócitos/efeitos dos fármacos , Condrócitos/enzimologia , Condrócitos/imunologia , Células do Tecido Conjuntivo/efeitos dos fármacos , Células do Tecido Conjuntivo/enzimologia , Células do Tecido Conjuntivo/imunologia , Citocinas/imunologia , Dinoprostona/imunologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Fibroblastos/imunologia , Células HeLa , Humanos , Mastócitos/efeitos dos fármacos , Mastócitos/enzimologia , Mastócitos/imunologia , Estrutura Molecular , NF-kappa B/imunologia , Transdução de Sinais/efeitos dos fármacos , Membrana Sinovial/citologiaRESUMO
OBJECTIVE: The IKK complex regulates NF-kappaB activation, an important pathway implicated in the rheumatoid arthritis (RA) disease process. This study was undertaken to assess the efficacy of N-(6-chloro-7-methoxy-9H-beta-carbolin-8-yl)-2-methylnicotinamide (ML120B), a potent and selective small molecule inhibitor of IKKbeta. METHODS: Polyarthritis was induced in rats by injection of Freund's complete adjuvant into the hind footpad. ML120B was administered orally twice daily, either prophylactically or therapeutically. Paw volumes and body weights were measured every 2-3 days throughout the study. We assessed bone erosions by several methods: histologic evaluation, quantitative micro-computed tomography (micro-CT) imaging analysis, and measurement of type I collagen fragments in the serum. Quantitative polymerase chain reaction was used to evaluate expression of messenger RNA for genes related to inflammation and to bone and cartilage integrity. RESULTS: Oral administration of ML120B inhibited paw swelling in a dose-dependent manner (median effective dosage 12 mg/kg twice daily) and offered significant protection against arthritis-induced weight loss as well as cartilage and bone erosion. We were able to directly demonstrate that NF-kappaB activity in arthritic joints was reduced after ML120B administration. Also, we observed that down-regulation of the NF-kappaB pathway via IKKbeta inhibition dampened the chronic inflammatory process associated with rat adjuvant-induced arthritis. CONCLUSION: The results of the present study suggest that IKKbeta inhibition is an effective therapeutic approach to treat both the inflammation and the bone/cartilage destruction observed in RA. Methods for the determination of serum markers for bone and cartilage destruction, as well as micro-CT analysis, may aid in predicting and evaluating the therapeutic efficacy of IKKbeta inhibition therapy in humans.