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1.
Adv Exp Med Biol ; 1188: 227-237, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31820391

RESUMO

Understanding cellular heterogeneity is a challenge for cell biology, particularly in the fields of stem cell and cancer biology. We recently established a reverse phase protein array (RPPA)-modified method, colony lysate array (CoLA), which enables proteomic profiling of individual subpopulations (i.e., colonies) derived from various cell proliferative conditions. Here we describe two independent CoLA assays for a functional subpopulation, drug-tolerant persisters (DTPs), which are considered to mimic the origin of chemotherapeutic cancer relapse. In the first study, we analyzed individual DTPs derived from different cell types grown in the presence of different drugs. A hierarchical clustering analysis of DTPs using CoLA revealed two types of clonal populations, including those that expressed stem cell-associated proteins as well as epithelium-associated proteins. Subsequent principal component analyses demonstrated that the DTPs clustered on the basis of their proteomic profiles, which could change in response to drugs and doses. Another study was designed to identify signaling pathways that may be associated with drug resistance using a pair of 5-fluorouracil-tolerant and parental gastric cancer cell lines. Although a hierarchical clustering analysis did not show any specific clusters for the tolerant line, a drug dose-response analysis revealed that phosphorylation of PI3K and AKT increased and decreased, respectively, specifically in the tolerant cell line. These frameworks that readily profile small subpopulations isolated from an external stresses may be applicable to a wide variety of heterogeneous cellular samples.

2.
Sci Rep ; 9(1): 17332, 2019 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-31757997

RESUMO

Genome-wide association studies (GWAS) have successfully identified about 70 genomic loci associated with breast cancer. Owing to the complexity of linkage disequilibrium and environmental exposures in different populations, it is essential to perform regional GWAS for better risk prediction. This study aimed to investigate the genetic architecture and to assess common genetic risk model of breast cancer with 6,669 breast cancer patients and 21,930 female controls in the Japanese population. This GWAS identified 11 genomic loci that surpass genome-wide significance threshold of P < 5.0 × 10-8 with nine previously reported loci and two novel loci that include rs9862599 on 3q13.11 (ALCAM) and rs75286142 on 21q22.12 (CLIC6-RUNX1). Validation study was carried out with 981 breast cancer cases and 1,394 controls from the Aichi Cancer Center. Pathway analyses of GWAS signals identified association of dopamine receptor medicated signaling and protein amino acid deacetylation with breast cancer. Weighted genetic risk score showed that individuals who were categorized in the highest risk group are approximately 3.7 times more likely to develop breast cancer compared to individuals in the lowest risk group. This well-powered GWAS is a representative study to identify SNPs that are associated with breast cancer in the Japanese population.

3.
J Surg Oncol ; 120(7): 1154-1161, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31578743

RESUMO

BACKGROUND: Postoperative adjuvant chemotherapy is not indicated for T1N1M0/T2N0M0/T3N0M0 gastric cancer. However, approximately 10% to 30% of these patients experience recurrence and metastasis. METHODS: Among 658 patients with gastric cancer who received gastrectomy with curative intent, 130 T1N1M0/T2N0M0 and 73 T3N0M0 patients were enrolled. Overall survival (OS) and relapse-free survival (RFS) were analyzed based on TP53 codon 72 polymorphisms Arg/Arg, Arg/Pro, and Pro/Pro. The hazard ratio (HR) for each subgroup was compared by TP53 codon 72 polymorphisms. RESULTS: Of the 189 patients for whom polymorphism analysis results were available, the 5- and 10-year OS was 84.9% and 65.1%, respectively. The 5- and 10-year RFS was 81.8% and 65.4%, respectively. When the study cohort was divided into two groups according to polymorphism status (ie, "Arg/Arg and Arg/Pro" vs Pro/Pro), both the OS (HR, 2.799; 95% confidence interval [CI], 1.071-7.315; P = .036) and RFS (HR, 2.639; 95% CI, 1.025-6.794; P = .044) of the Pro/Pro group were significantly lower than those for the Arg/Arg and Arg/Pro groups across the entire observation period. CONCLUSIONS: The TP53 codon 72 Pro/Pro polymorphism may isolate a relatively high-risk patient group in T1N1M0/T2N0M0/T3N0M0 gastric cancer.


Assuntos
Adenocarcinoma/cirurgia , Gastrectomia/efeitos adversos , Recidiva Local de Neoplasia/diagnóstico , Polimorfismo de Nucleotídeo Único , Medição de Risco/métodos , Neoplasias Gástricas/cirurgia , Proteína Supressora de Tumor p53/genética , Adenocarcinoma/genética , Adenocarcinoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Códon , Feminino , Seguimentos , Predisposição Genética para Doença , Genótipo , Humanos , Incidência , Japão/epidemiologia , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/epidemiologia , Recidiva Local de Neoplasia/genética , Estadiamento de Neoplasias , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Taxa de Sobrevida
4.
Methods Mol Biol ; 1908: 229-241, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30649732

RESUMO

Circulating tumor DNA (ctDNA) is emerging as a promising biomarker for cancer diagnosis. However, the system to detect gene mutations with very low frequencies from plasma remains to be established in terms of technical aspects of sequencing technologies and cost for universal use. One strategy is to employ a cancer sequencing panel to detect mutations in a primary tumor in a time- and cost-effective manner, and subsequently assess these mutations with a digital PCR technology from plasma ctDNA. This strategy enables the accurate detection of low frequency mutations (i.e., less than 1% allele frequency) from ctDNA, since both comprehensive coverage of genes and quantitative mutation detection with very low frequencies are required for cancer diagnosis from plasma samples. Here, we present a pipeline can be used to detect mutations from plasma ctDNA with very low allele frequencies using a next-generation sequencing technology for comprehensive coverage of primary tumors and droplet digital PCR for sensitive detection from plasma ctDNA.


Assuntos
DNA Tumoral Circulante/sangue , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mutação , Neoplasias/diagnóstico , Análise de Sequência de DNA/métodos , Biomarcadores Tumorais , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Humanos , Neoplasias/genética , Neoplasias/metabolismo
5.
Adv Exp Med Biol ; 1103: 219-241, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30484232

RESUMO

Cellular compensation from extrahepatic resources is expected to improve the prognosis of liver diseases. Currently, liver dysfunction is treated by a variety of modalities including drugs, cytokines, vascular interventions, energy devices, surgery, and liver transplantation; however, in recent years there have been few significant advancements in treatment efficacy. A next-generation therapeutic strategy for liver disease, cellular compensatory therapy (i.e., cell therapy), is now being considered for clinical practice. Liver dysfunction is attributed to a lack of sufficient functional cells. However, processes involved in recovery of liver function are not fully elucidated, which has complicated the interpretation of treatment effects at the cellular level. Our genotyping study of living donor liver transplantation revealed that a variety of graft liver tissues contained the donor genotype, indicating that extrahepatic cells had differentiated into liver component cells during liver regeneration. Multilineage-differentiating stress-enduring (Muse) cells appear to be a strong candidate for extrahepatic resources that can contribute to liver regeneration. Muse cells are defined as stage-specific embryonic antigen 3-expressing cells that contribute to tissue regeneration and have the potential to differentiate into three germ layers. The significant advantage of Muse cells over other "pluripotent cells" is that Muse cells are present in bone marrow/blood as well as a variety of connective tissues, which provides safety and ethical advantages for clinical applications. Here, we review current therapeutic topics in liver diseases and discuss the potential for cell therapy using Muse cells based on our recent studies of Muse cell administration in a mouse model of physical partial hepatectomy.


Assuntos
Regeneração Hepática , Células-Tronco Pluripotentes/citologia , Transplante de Células-Tronco , Animais , Diferenciação Celular , Humanos , Fígado , Camundongos
6.
J Surg Oncol ; 117(5): 947-956, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29355977

RESUMO

BACKGROUND AND OBJECTIVES: Limited information exists regarding beneficial effects of Helicobacter pylori. To examine the effect in advanced gastric cancer, we compared survival for patients treated with surgery-only or adjuvant chemotherapy on the basis of H. pylori infection status. METHODS: A cohort of 491 patients who underwent R0 resection for locally advanced gastric cancer between 2000 and 2009 at 12 institutions in northern Japan was included. H. pylori infection status, was assessed from paraffin-embedded formalin-fixed samples. Overall survival (OS) and disease-free survival (DFS) in surgery-only (Surgery) and adjuvant chemotherapy (S-1) groups were analyzed. A propensity score matching was employed to correct for confounding factors by indication. RESULTS: H. pylori infection was positive in 175 patients and negative in 316 patients. H. pylori-positive patients showed significantly better survival than H. pylori-negative patients in both OS (hazard ratio [HR] 0.593, 95% confidence interval [CI] 0.417-0.843; P = 0.003]) and DFS (HR 0.679, 95%CI 0.492-0.937; P = 0.018). Propensity score matching further confirmed that S-1 was virtually only effective when tumors were H. pylori-positive. CONCLUSIONS: The favorable outcome of H. pylori-positive patients implies that the host immune system is modulated by H. pylori enhancing the chemotherapeutic efficacy.


Assuntos
Antimetabólitos Antineoplásicos/uso terapêutico , Infecções por Helicobacter/complicações , Helicobacter pylori/isolamento & purificação , Ácido Oxônico/uso terapêutico , Neoplasias Gástricas/mortalidade , Tegafur/uso terapêutico , Idoso , Quimioterapia Adjuvante , Estudos de Coortes , Combinação de Medicamentos , Feminino , Seguimentos , Infecções por Helicobacter/virologia , Humanos , Masculino , Prognóstico , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/patologia , Neoplasias Gástricas/virologia , Taxa de Sobrevida
7.
Anal Chem ; 89(17): 8626-8631, 2017 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-28753272

RESUMO

Functional heterogeneity of cancer cells is one of the key properties to understanding relapse after drug treatment. Hence, clarification is needed with regard to which types of subgroups of cancer cells dominantly contribute to the initiation of relapse. Recently, we established the colony lysate array (CoLA), which is a method that allows comparison of individual colonies at the protein level to assess the initiation of anticancer drug-tolerant persisters (DTPs) based on the reverse-phase protein array (RPPA) system. DTPs grow in various drug concentrations and types showing 2-dimensional growth (∼1 mm) on a flat surface. The size of DTPs are larger than spheroids (∼0.3 mm) in agarose gel, which makes them easy to handle for a number of assays. DTPs provide functional information during the process of their formation, initiating from the origin of a drug-tolerant single cell. Using >2000 DTPs generated from various drugs and doses profiled on the basis of 44 proteins, we demonstrate that the DTPs are clustered on the basis of their proteomic profiles changing in response to drugs and doses. Of interest, nine transcription factors in the DTPs, such as STAT3 and OCT4A, were identified as having decreased or increased levels of proteins in response to gefitinib. Importantly, these results can be obtained only by individual proteomic colony profiling, which may identify alternative therapeutic targets and biomarkers for DTPs that may harbor critical mechanisms for cancer relapse.


Assuntos
Bioensaio/métodos , Resistencia a Medicamentos Antineoplásicos/fisiologia , Neoplasias/metabolismo , Proteômica/métodos , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos
8.
Sci Rep ; 7(1): 2262, 2017 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-28536445

RESUMO

Drug-tolerant cancer cell subpopulations are responsible for relapse after chemotherapy. By continuously exposing the gastric cancer cell line MKN45 to 5-FU for >100 passages, we established a 5-fluorouracil (5-FU)-tolerant line, MKN45/5FU. Orthotopic xenografts of MKN45/5FU cells in the stomach of nude mice revealed that these cells had a high potential to metastasize to sites such as the liver. Levels of phosphorylated phosphatidylinositide 3-kinase (PI3K) increased both in 5-FU-tolerant subpopulations according to the 5-FU dose, and in gastric submucosal orthotopic xenografts of MKN45/5FU cells. Sequential administration of 5-FU and a PI3K inhibitor, GDC-0941, targeted the downstream ribosomal S6 kinase phosphorylation to significantly suppress 5-FU-tolerant subpopulations and tumor propagation of orthotopic MKN45/5FU xenografts. These results suggest that administration of 5-FU followed by GDC-0941 may suppress disease relapse after 5-FU-based gastric cancer chemotherapy.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Fluoruracila/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Classe I de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Classe I de Fosfatidilinositol 3-Quinases/genética , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Códon , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/genética , Variação Genética , Xenoenxertos , Humanos , Camundongos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Fenótipo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteoma , Proteômica/métodos , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Transdução de Sinais
9.
J Surg Res ; 212: 195-204, 2017 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-28550907

RESUMO

BACKGROUND: Although surgery and chemotherapy have extended advanced gastric cancer patient survival, some patients still experience relapse and metastasis. We postulated that PI3K pathway proteins could be prognostic biomarkers for the advanced gastric cancer patients. METHODS: A retrospective cohort of 160 advanced gastric cancer patients receiving potentially curative surgery with/without chemotherapy was investigated for PIK3CA mutation and PI3K pathway protein level in the context of overall survival and relapse-free survival. RESULTS: Thirteen patients (13 of 111, 11.7%) had PIK3CA mutations in codon 545, whereas one patient (1 of 94, 1.1%) had a mutation in PIK3CA codon 1047. PI3K pathway protein immunohistochemistry demonstrated that phosphorylated AKT positive [p-AKT (+)] patients in the surgery-only group had a good prognosis in terms of overall survival and relapse-free survival. No significant association between PIK3CA mutations and PI3K pathway protein level was seen. CONCLUSIONS: This study revealed that (1) PIK3CA hotspot mutations occurred with low frequency in gastric cancer; (2) PIK3CA hotspot mutations were not directly associated with PI3K pathway activation; and (3) p-AKT (+) may be a biomarker for better outcomes for gastric cancer patients undergoing gastrectomy regardless of the PIK3CA mutation status.


Assuntos
Biomarcadores Tumorais/genética , Mutação , Fosfatidilinositol 3-Quinases/metabolismo , Neoplasias Gástricas/genética , Adulto , Idoso , Biomarcadores Tumorais/metabolismo , Quimioterapia Adjuvante , Classe I de Fosfatidilinositol 3-Quinases , Feminino , Gastrectomia , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Fosfatidilinositol 3-Quinases/genética , Prognóstico , Reação em Cadeia da Polimerase em Tempo Real , Estudos Retrospectivos , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/mortalidade , Neoplasias Gástricas/terapia , Análise de Sobrevida
10.
R Soc Open Sci ; 3(9): 160267, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27703689

RESUMO

Elucidation of tumour suppression mechanisms is a major challenge in cancer biology. Therefore, Peto's paradox, or low cancer incidence in large animals, has attracted focus. According to the gene-abundance hypothesis, which considers the increase/decrease in cancer-related genes with body size, researchers evaluated the associations between gene abundance and body size. However, previous studies only focused on a few specific gene functions and have ignored the alternative hypothesis (metabolic rate hypothesis): in this hypothesis, the cellular metabolic rate and subsequent oxidative stress decreases with increasing body size. In this study, we have elected to explore the gene-abundance hypothesis taking into account the metabolic rate hypothesis. Thus, we comprehensively investigated the correlation between the number of genes in various functional categories and body size while at the same time correcting for the mass-specific metabolic rate (Bc). A number of gene functions that correlated with body size were initially identified, but they were found to be artefactual due to the decrease in Bc with increasing body size. By contrast, immune system-related genes were found to increase with increasing body size when the correlation included this correction for Bc. These findings support the gene-abundance hypothesis and emphasize the importance of also taking into account the metabolic rate when evaluating gene abundance-body size relationships. This finding may be useful for understanding cancer evolution and tumour suppression mechanisms as well as for determining cancer-related genes and functions.

11.
Lab Invest ; 96(11): 1211-1222, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27617400

RESUMO

Partial hepatectomy models in mice have been widely used for liver regeneration studies. A typical procedure removes ~2/3 of the liver by lobular ligation without tissue dissection. However, hepatectomy in humans involves physical damage (ie, physical partial hepatectomy, PPHx). Therefore, the liver regeneration process after PPHx should involve reactions to acute local injury followed by systematic remodeling. To clarify the liver regeneration process after PPHx, we used a murine liver injury model that mimics the actual human surgical procedure. A 20-30% PPHx was performed by transection of the left lobe of the liver using an ultrasonically activated scalpel in mice. Gene expression and morphological characteristics were analyzed during the liver regeneration process. Liver weight continuously increased by hypertrophic reaction of hepatocytes, whereas Ki67 staining showed hepatocyte proliferation. At the transected border, emergence of ductular reactions, a representative process of hepatic tissue remodeling that contain liver stem/progenitor cells, were observed. Gene expression of the transected border and non-damaged lobes revealed that inflammatory cytokine- and extracellular matrix-associated genes were significantly upregulated at the transected border. Our PPHx model triggered local extracellular matrix remodeling that resulted in ductular reactions. These processes occurred during the tissue repair process in local inflammatory responses as well as compensatory hepatocyte hypertrophy of the entire liver. These findings may provide insight for elucidating the mechanism of tissue repair and regeneration of the liver after PPHx.


Assuntos
Hepatectomia , Regeneração Hepática , Fígado/patologia , Actinas/metabolismo , Animais , Proliferação de Células , Matriz Extracelular/fisiologia , Feminino , Perfilação da Expressão Gênica , Células Estreladas do Fígado/metabolismo , Hepatócitos/fisiologia , Hipertrofia , Quinase I-kappa B/metabolismo , Fígado/enzimologia , Fígado/imunologia , Metaloproteinases da Matriz/metabolismo , Camundongos Endogâmicos C57BL , Modelos Animais , NF-kappa B/metabolismo , Células-Tronco/fisiologia , Fator de Necrose Tumoral alfa/metabolismo
12.
Sci Rep ; 6: 25895, 2016 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-27181033

RESUMO

Cancer relapse occurs with substantial frequency even after treatment with curative intent. Here we studied drug-tolerant colonies (DTCs), which are subpopulations of cancer cells that survive in the presence of drugs. Proteomic characterization of DTCs identified stemness- and epithelial-dominant subpopulations, but functional screening suggested that DTC formation was regulated at the transcriptional level independent from protein expression patterns. We consistently found that α-amanitin, an RNA polymerase II (RNAPII) inhibitor, effectively inhibited DTCs by suppressing TAF15 expression, which binds to RNA to modulate transcription and RNA processing. Sequential administration of α-amanitin and cisplatin extended overall survival in a cancer-relapse mouse model, namely peritonitis carcinomatosa. Therefore, post-treatment cancer relapse may occur through non-distinct subpopulations and may be effectively prevented by α-amanitin to disrupt transcriptional machinery, including TAF15.


Assuntos
Alfa-Amanitina/administração & dosagem , Resistência a Medicamentos/efeitos dos fármacos , Inibidores Enzimáticos/administração & dosagem , Neoplasias Peritoneais/tratamento farmacológico , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Alfa-Amanitina/farmacologia , Animais , Linhagem Celular Tumoral , Cisplatino/administração & dosagem , Cisplatino/uso terapêutico , Regulação para Baixo , Inibidores Enzimáticos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HCT116 , Células HT29 , Células HeLa , Humanos , Células MCF-7 , Camundongos , Neoplasias Peritoneais/genética , Neoplasias Peritoneais/metabolismo , Proteômica/métodos , Prevenção Secundária , Transcrição Genética/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
13.
PLoS One ; 11(1): e0146275, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26727500

RESUMO

BACKGROUND: Circulating tumor DNA (ctDNA) carries information on tumor burden. However, the mutation spectrum is different among tumors. This study was designed to examine the utility of ctDNA for monitoring tumor burden based on an individual mutation profile. METHODOLOGY: DNA was extracted from a total of 176 samples, including pre- and post-operational plasma, primary tumors, and peripheral blood mononuclear cells (PBMC), from 44 individuals with colorectal tumor who underwent curative resection of colorectal tumors, as well as nine healthy individuals. Using a panel of 50 cancer-associated genes, tumor-unique mutations were identified by comparing the single nucleotide variants (SNVs) from tumors and PBMCs with an Ion PGM sequencer. A group of the tumor-unique mutations from individual tumors were designated as individual marker mutations (MMs) to trace tumor burden by ctDNA using droplet digital PCR (ddPCR). From these experiments, three major objectives were assessed: (a) Tumor-unique mutations; (b) mutation spectrum of a tumor; and (c) changes in allele frequency of the MMs in ctDNA after curative resection of the tumor. RESULTS: A total of 128 gene point mutations were identified in 27 colorectal tumors. Twenty-six genes were mutated in at least 1 sample, while 14 genes were found to be mutated in only 1 sample, respectively. An average of 2.7 genes were mutated per tumor. Subsequently, 24 MMs were selected from SNVs for tumor burden monitoring. Among the MMs found by ddPCR with > 0.1% variant allele frequency in plasma DNA, 100% (8 out of 8) exhibited a decrease in post-operation ctDNA, whereas none of the 16 MMs found by ddPCR with < 0.1% variant allele frequency in plasma DNA showed a decrease. CONCLUSIONS: This panel of 50 cancer-associated genes appeared to be sufficient to identify individual, tumor-unique, mutated ctDNA markers in cancer patients. The MMs showed the clinical utility in monitoring curatively-treated colorectal tumor burden if the allele frequency of MMs in plasma DNA is above 0.1%.


Assuntos
Adenocarcinoma/sangue , Neoplasias Colorretais/sangue , Análise Mutacional de DNA , DNA de Neoplasias/sangue , Genes Neoplásicos , Mutação Puntual , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA/métodos , Carga Tumoral , Adenocarcinoma/genética , Adenocarcinoma/patologia , Adenocarcinoma/cirurgia , Adulto , Idoso , Alelos , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Neoplasias Colorretais/cirurgia , Primers do DNA , Feminino , Humanos , Leucócitos Mononucleares/química , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Multiplex , Análise de Sequência de DNA/instrumentação
14.
Drug Metab Pharmacokinet ; 31(1): 35-45, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26822993

RESUMO

The ultimate goal of cancer biomarker discovery is to use individual patient-derived biochemical information to improve patients' outcomes. A recent increase in the spectrum of therapeutic options for cancer requires improved approaches to identify patients likely to benefit from them. The ability to elucidate biological characteristics of tumors and to understand both inter and intra tumoral heterogeneity using large-scale data sets generated from both model systems and patient samples provides a unique opportunity to identify useful cancer biomarkers. Since the reverse-phase protein array (RPPA) was introduced in the early 2000s, it has played a central role in a wide range of uses including cancer biology, biomarker discovery, and therapeutic target evaluation. The quantitative output from RPPAs is readily combined with that from other "omics" platforms using a variety of information-intensive approaches at the molecular and population levels. A vast and rapidly growing body of related proteomic is now publicly available, and leading researchers in the field organized the Global RPPA Society in 2011 to facilitate the sharing of resources and data. The RPPA community is embracing the new paradigm of data sharing, which is designed to accelerate the discovery of cancer biomarkers and implementation of them in cancer patient management.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias/metabolismo , Análise Serial de Proteínas , Pesquisa Biomédica/métodos , Humanos , Proteômica/métodos
15.
J Proteome Res ; 15(1): 205-15, 2016 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-26625007

RESUMO

In this study we monitored protein dynamics in response to cisplatin, 5-fluorouracil, and irinotecan with different concentrations and administration modes using "reverse-phase" protein arrays (RPPAs) in order to gain comprehensive insight into the protein dynamics induced by genotoxic drugs. Among 666 protein time-courses, 38% exhibited an increasing trend, 32% exhibited a steady decrease, and 30% fluctuated within 24 h after drug exposure. We analyzed almost 12,000 time-course pairs of protein levels based on the geometrical similarity by correlation distance (dCor). Twenty-two percent of the pairs showed dCor > 0.8, which indicates that each protein of the pair had similar dynamics. These trends were disrupted by a proteasome inhibitor, MG132, suggesting that the protein degradation system was activated in response to the drugs. Among the pairs with high dCor, the average dCor of pairs with apoptosis-related protein was significantly higher than those without, indicating that regulation of protein levels was induced by the drugs. These results suggest that the levels of numerous functionally distinct proteins may be regulated by common degradation machinery induced by genotoxic drugs.


Assuntos
Camptotecina/análogos & derivados , Cisplatino/toxicidade , Fluoruracila/toxicidade , Mutagênicos/toxicidade , Proteólise/efeitos dos fármacos , Apoptose , Camptotecina/toxicidade , Dano ao DNA , Células HCT116 , Humanos , Irinotecano , Leupeptinas/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Proteoma/metabolismo
16.
Mol Cell Proteomics ; 13(7): 1625-43, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24777629

RESUMO

Reverse phase protein array (RPPA) technology introduced a miniaturized "antigen-down" or "dot-blot" immunoassay suitable for quantifying the relative, semi-quantitative or quantitative (if a well-accepted reference standard exists) abundance of total protein levels and post-translational modifications across a variety of biological samples including cultured cells, tissues, and body fluids. The recent evolution of RPPA combined with more sophisticated sample handling, optical detection, quality control, and better quality affinity reagents provides exquisite sensitivity and high sample throughput at a reasonable cost per sample. This facilitates large-scale multiplex analysis of multiple post-translational markers across samples from in vitro, preclinical, or clinical samples. The technical power of RPPA is stimulating the application and widespread adoption of RPPA methods within academic, clinical, and industrial research laboratories. Advances in RPPA technology now offer scientists the opportunity to quantify protein analytes with high precision, sensitivity, throughput, and robustness. As a result, adopters of RPPA technology have recognized critical success factors for useful and maximum exploitation of RPPA technologies, including the following: preservation and optimization of pre-analytical sample quality, application of validated high-affinity and specific antibody (or other protein affinity) detection reagents, dedicated informatics solutions to ensure accurate and robust quantification of protein analytes, and quality-assured procedures and data analysis workflows compatible with application within regulated clinical environments. In 2011, 2012, and 2013, the first three Global RPPA workshops were held in the United States, Europe, and Japan, respectively. These workshops provided an opportunity for RPPA laboratories, vendors, and users to share and discuss results, the latest technology platforms, best practices, and future challenges and opportunities. The outcomes of the workshops included a number of key opportunities to advance the RPPA field and provide added benefit to existing and future participants in the RPPA research community. The purpose of this report is to share and disseminate, as a community, current knowledge and future directions of the RPPA technology.


Assuntos
Imunoensaio/métodos , Análise Serial de Proteínas/métodos , Pesquisa Médica Translacional/métodos , Educação , Humanos , Processamento de Proteína Pós-Traducional
17.
PLoS One ; 9(2): e90155, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24587255

RESUMO

Despite of remarkable improvement of postoperative 5-FU-based adjuvant chemotherapy, the relapse rate of gastric cancer patients who undergo curative resection followed by the adjuvant chemotherapy remains substantial. Therefore, it is important to identify prediction markers for the chemotherapeutic efficacy of 5-FU. We recently identified NF-κB as a candidate relapse prediction biomarker in gastric cancer. To evaluate the biological significance of NF-κB in the context of 5-FU-based chemotherapy, we analyzed the NF-κB-dependent biological response upon 5-FU treatment in gastric cancer cell lines. Seven genes induced by 5-FU treatment in an NF-κB-dependent manner were identified, five of which are known p53 targets. Knockdown of RELA, which encodes the p65 subunit of NF-κB, decreased both p53 and p53 target protein levels. In contrast, NF-κB was not affected by TP53 knockdown. We also demonstrated that cell lines bearing Pro/Pro homozygosity in codon72 of p53 exon4, which is important for NF-κB binding to p53, are more resistant to 5-FU than those with Arg/Arg homozygosity. We conclude that NF-κB plays an important role in the response to 5-FU treatment in gastric cancer cell lines, with a possible compensatory function of p53. These results suggest that NF-κB is a potential 5-FU-chemosensitivity prediction marker that may reflect 5-FU-induced stress-response pathways, including p53.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Fluoruracila/farmacologia , NF-kappa B/metabolismo , Neoplasias Gástricas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Antimetabólitos Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Códon , Resistencia a Medicamentos Antineoplásicos/genética , Fluoruracila/uso terapêutico , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Ligação Proteica , Transporte Proteico , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/genética , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo , Proteína Supressora de Tumor p53/genética
18.
World J Surg Oncol ; 11: 11, 2013 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-23339659

RESUMO

BACKGROUND: The use of standard chemotherapy regimens has changed the application of chemosensitivity tests from all chemotherapy-eligible patients to those who have failed standard chemotherapy, which includes patients with highly advanced, relapsed, or chemoresistant tumors. METHODS: We evaluated a total of 43 advanced primary and relapsed gastric cancers for chemosensitivity based on drug dose response curves to improve the objectivity and quality of quantitative measurements. The dose response curves were classified based on seven expected patterns. Instead of a binary chemosensitivity evaluation, we ranked drug sensitivity according to curve shapes and comparison with the peak plasma concentration (ppc) of each drug. RESULTS: A total of 193 dose response curves were obtained. The overall informative rate was 67.4%, and 85.3% for cases that had a sufficient number of cells. Paclitaxel (PXL)and docetaxel tended to show a higher rank, while cisplatin (CIS) and 5-fluorouracil (5-FU) tended to show resistance, particularly among the 20 cases (46.5%) that had recurrent disease after receiving chemotherapy with CIS and S-1 (5-FU). As such, we speculate that the resistant pattern of the chemosensitivity test suggests that cells with acquired drug resistance were selected by chemotherapy. Indeed, we observed a change in the chemosensitivity pattern of a sample before and after chemotherapy in terms of PXL sensitivity, which was used after primary chemotherapy. CONCLUSIONS: These results suggest that: (i) the dose-response pattern provides objective information for predicting chemosensitivity; and (ii) chemotherapy may select resistant cancer cell populations as a result of the therapy.


Assuntos
Antineoplásicos/farmacologia , Ascite/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Recidiva Local de Neoplasia/tratamento farmacológico , Neoplasias Gástricas/classificação , Neoplasias Gástricas/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Ascite/patologia , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/patologia , Estadiamento de Neoplasias , Neoplasias Gástricas/patologia , Células Tumorais Cultivadas , Adulto Jovem
19.
PLoS One ; 7(8): e43236, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22905237

RESUMO

To confirm the clinical significance of NF-κB and JNK protein expression from experimentally identified candidates for predicting prognosis for patients with 5-FU treatment, we evaluated the protein expression of surgically removed specimens. A total of 79 specimens were obtained from 30 gastric and 49 colorectal cancer patients who underwent R0 resection followed by postoperative 5-FU based adjuvant chemotherapy. Immunohistochemical examinations of NF-κB and JNK on tissue microarrays (TMAs) revealed that significantly shorter time-to-relapse (TTR) in both NF-κB(+) and JNK(-) subgroups in both gastric (NF-κB(+), p = 0.0002, HR11.7. 95%CI3 3.2-43.4; JNK(-), p = 0.0302, HR4.4, 95%CI 1.2-16.6) and colon (NF-κB(+), p = 0.0038, HR36.9, 95%CI 3.2-426.0; JNK(-), p = 0.0098, HR3.2, 95%CI 1.3-7.7) cancers. These protein expression patterns also show strong discriminately power in gastric cancer patients for overall survival rate, suggesting a potential utility as prognostic or chemosensitivity markers. Baseline expression of these proteins using gastric cancer cell lines demonstrated the reciprocal patterns between NF-κB and JNK, while 5-FU exposure of these cell lines only induced NF-κB, suggesting that NF-κB plays a dominant role in the response to 5-FU. Subsequent siRNA experiments confirmed that gene knockdown of NF-κB increased 5-FU-specific sensitivity, whereas that of JNK did not affect the chemosensitivity. These results suggest that the expression of these proteins may aid in the decisions involved with adjuvant chemotherapy for gastrointestinal tract cancers.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Biomarcadores Tumorais/metabolismo , Quimioterapia Adjuvante/métodos , Neoplasias Colorretais/tratamento farmacológico , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica , Neoplasias Gástricas/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Análise por Conglomerados , Neoplasias Colorretais/metabolismo , Humanos , Imuno-Histoquímica/métodos , MAP Quinase Quinase 4/metabolismo , Pessoa de Meia-Idade , Prognóstico , RNA Interferente Pequeno/metabolismo , Recidiva , Neoplasias Gástricas/metabolismo
20.
Methods Mol Biol ; 785: 65-77, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21901594

RESUMO

Theoretical models describing complex biological phenomena have been accumulating. However, most of these models have been created with hypothetical parameter determination without seeing actual cell reactions. The parameter determination requires high-dimensional data monitoring, particularly at the protein level. It has been a difficult task to develop the standard model system because of the lack of an appropriate validation technique. Reverse-phase protein lysate microarray (RPA) is one of the most potent technologies for high-dimensional proteomic monitoring. Therefore, proteomic monitoring by RPA may contribute substantially to develop theoretical protein network models based on experimental validation.


Assuntos
Modelos Biológicos , Análise Serial de Proteínas/métodos , Mapas de Interação de Proteínas/genética , Proteômica/métodos , Transdução de Sinais/genética , Western Blotting , Linhagem Celular Tumoral , Humanos , Técnicas In Vitro
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