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1.
Photodiagnosis Photodyn Ther ; 35: 102420, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34242818

RESUMO

BACKGROUND: Accurate diagnosis of peritoneal metastasis in gastric cancer (GC) is important to determine the appropriate treatment. This study aimed to examine whether matrix metalloprotease-14 (MMP-14) was a candidate enzyme in fluorescence imaging for the diagnosis of peritoneal metastasis in GC. METHODS: GC and normal peritoneal (NP) tissues from 96 and 20 patients, respectively were evaluated for MMP-14 expression. Live cell imaging of GC cell lines (NUGC4, MKN45, MKN74, HGC-27, and Kato-III) was performed using the MMP-14-activatable fluorescence probe; BODIPY-MMP. Furthermore, the overall survival (OS) was calculated in all patients (n = 96). RESULTS: MMP-14 expression was significantly higher in GC tissues (median: 3.57 ng/mg protein; range:0.64-24.4 ng/mg protein) than in NP tissues (median: 1.34 ng/mg protein; median: 0.53-3.09 ng/mg protein) (P < 0.01). Receiver operating characteristic curves showed that the area under the curve, sensitivity, and specificity were 0.907, 84.4%, and 90.0%, respectively. In live cell imaging using the BODIPY-MMP, fluorescence was observed in five GC cell lines. In the analysis of OS, the high expression of the MMP-14 group had a significantly poorer OS rate than the low expression of the MMP-14 group (P = 0.02). In the multivariate analyses, MMP-14 expression was an independent risk factor for OS (hazard ratio: 2.33; 95 % confidence interval: 1.05-5.45; P = 0.04). CONCLUSION: MMP-14 is a promising enzyme in intraoperative fluorescence imaging for peritoneal metastasis in GC, especially in patients with poor prognosis.


Assuntos
Neoplasias Peritoneais , Fotoquimioterapia , Neoplasias Gástricas , Biomarcadores Tumorais , Humanos , Metaloproteinase 14 da Matriz , Neoplasias Peritoneais/diagnóstico por imagem , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes , Prognóstico , Neoplasias Gástricas/diagnóstico por imagem
2.
Clin Cancer Res ; 27(14): 3936-3947, 2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-34031057

RESUMO

PURPOSE: Five-aminolevulinic acid (5-ALA) is widely used as an intraoperative fluorescent probe for radical resection of high-grade glioma, and thus aids in extending progression-free survival of patients. However, there exist some cases where 5-ALA fails to fluoresce. In some other cases, it may undergo fluorescence quenching but cannot be orally readministered during surgery. This study aimed to develop a novel hydroxymethyl rhodamine green (HMRG)-based fluorescence labeling system that can be repeatedly administered as a topical spray during surgery for the detection of glioblastoma. EXPERIMENTAL DESIGN: We performed a three-stage probe screening using tumor lysates and fresh tumor tissues with our probe library consisting of a variety of HMRG probes with different dipeptides. We then performed proteome and transcript expression analyses to detect candidate enzymes responsible for cleaving the probe. Moreover, in vitro and ex vivo studies using U87 glioblastoma cell line were conducted to validate the findings. RESULTS: The probe screening identified proline-arginine-HMRG (PR-HMRG) as the optimal probe that distinguished tumors from peritumoral tissues. Proteome analysis identified calpain-1 (CAPN1) to be responsible for cleaving the probe. CAPN1 was highly expressed in tumor tissues which reacted to the PR-HMRG probe. Knockdown of this enzyme suppressed fluorescence intensity in U87 glioblastoma cells. In situ assay using a mouse U87 xenograft model demonstrated marked contrast of fluorescence with the probe between the tumor and peritumoral tissues. CONCLUSIONS: The novel fluorescent probe PR-HMRG is effective in detecting glioblastoma when applied topically. Further investigations are warranted to assess the efficacy and safety of its clinical use.

3.
World J Surg ; 44(12): 4245-4253, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32909125

RESUMO

BACKGROUND: Bile leakage is the most common postoperative complication associated with hepatobiliary and pancreatic surgery. Until now, however, a rapid, accurate diagnostic method for monitoring intraoperative and postoperative bile leakage had not been established. METHOD: Bilirubin levels in drained abdominal fluids collected from 23 patients who had undergone hepatectomy (n = 22) or liver transplantation (n = 1) were measured using a microplate reader with excitation/emission wavelengths of 497/527 nm after applying 5 µM of UnaG to the samples. UnaG was also sprayed directly on hepatic raw surfaces in swine hepatectomy models to identify bile leaks by fluorescence imaging. RESULTS: The bilirubin levels measured by UnaG fluorescence imaging showed favorable correlations with the results of the conventional light-absorptiometric methods (indirect bilirubin: rs = 0.939, p < 0.001; direct bilirubin: rs = 0.929, p < 0.001). Approximate time required for bilirubin measurements with UnaG was 15 min, whereas it took about 40 min with the conventional method at a hospital laboratory. Following administration of UnaG on hepatic surfaces, the fluorescence imaging identified bile leaks not only on the resected specimens but also in the abdominal cavity of the swine hepatectomy models. CONCLUSION: Fluorescence imaging techniques using UnaG may enable real-time identification of bile leaks during hepatectomy and on-site rapid diagnosis of bile leaks after surgery.


Assuntos
Bile , Bilirrubina , Animais , Drenagem , Hepatectomia/efeitos adversos , Humanos , Fígado , Complicações Pós-Operatórias/diagnóstico , Suínos
4.
BMC Cancer ; 20(1): 64, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31992267

RESUMO

BACKGROUND: It is still difficult to detect and diagnose early adenocarcinoma of the esophagogastric junction (EGJ) using conventional endoscopy or image-enhanced endoscopy. A glutamylprolyl hydroxymethyl rhodamine green (EP-HMRG) fluorescent probe that can be enzymatically activated to become fluorescent after the cleavage of a dipeptidyl peptidase (DPP)-IV-specific sequence has been developed and is reported to be useful for the detection of squamous cell carcinoma of the head and neck, and esophagus; however, there is a lack of studies that focuses on detecting EGJ adenocarcinoma by fluorescence molecular imaging. Therefore, we investigated the visualization of early EGJ adenocarcinoma by applying EP-HMRG and using clinical samples resected by endoscopic submucosal dissection (ESD). METHODS: Fluorescence imaging with EP-HMRG was performed in 21 clinical samples resected by ESD, and the fluorescence intensity of the tumor and non-tumor regions of interest was prospectively measured. Immunohistochemistry was also performed to determine the expression of DPP-IV. RESULTS: Fluorescence imaging of the clinical samples showed that the tumor lesions were visualized within a few minutes after the application of EP-HMRG, with a sensitivity, specificity, and accuracy of 85.7, 85.7, and 85.7%, respectively. However, tumors with a background of intestinal metaplasia did not have a sufficient contrast-to-background ratio since complete intestinal metaplasia also expresses DPP-IV. Immunohistochemistry measurements revealed that all fluorescent tumor lesions expressed DPP-IV. CONCLUSIONS: Fluorescence imaging with EP-HMRG could be useful for the detection of early EGJ adenocarcinoma lesions that do not have a background of intestinal metaplasia.


Assuntos
Adenocarcinoma/diagnóstico , Dipeptidil Peptidase 4/metabolismo , Neoplasias Esofágicas/diagnóstico , Junção Esofagogástrica/patologia , Corantes Fluorescentes/farmacologia , Rodaminas/farmacologia , Adenocarcinoma/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Detecção Precoce de Câncer , Neoplasias Esofágicas/metabolismo , Junção Esofagogástrica/metabolismo , Feminino , Corantes Fluorescentes/química , Humanos , Masculino , Pessoa de Meia-Idade , Imagem Molecular , Imagem Óptica , Estudos Prospectivos , Rodaminas/química , Sensibilidade e Especificidade
6.
Bioconjug Chem ; 30(4): 1055-1060, 2019 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-30920803

RESUMO

We have developed an activatable red fluorescence probe for dipeptidylpeptidase-IV (DPP-IV) by precisely controlling the photoinduced electron transfer (PeT) process of a red fluorescent scaffold, SiR600. The developed probe exhibited an extremely low background signal and showed significant fluorescence activation upon reaction with DPP-IV, enabling sensitive detection of esophageal cancer in clinical specimens from cancer patients.


Assuntos
Dipeptidil Peptidase 4/metabolismo , Neoplasias Esofágicas/diagnóstico , Corantes Fluorescentes/química , Dipeptidil Peptidase 4/química , Neoplasias Esofágicas/enzimologia , Humanos , Sensibilidade e Especificidade , Espectrometria de Fluorescência
7.
Sci Rep ; 8(1): 17781, 2018 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-30542087

RESUMO

Rapid diagnosis of metastatic lymph nodes (mLNs) of colorectal cancer (CRC) is desirable either intraoperatively or in resected fresh specimens. We have developed a series of activatable fluorescence probes for peptidase activities that are specifically upregulated in various tumors. We aimed to discover a target enzyme for detecting mLNs of CRC. Among our probes, we found that gGlu-HMRG, a gamma-glutamyl transpeptidase (GGT)-activatable fluorescence probe, could detect mLNs. This was unexpected, because we have previously reported that gGlu-HMRG could not detect primary CRC. We confirmed that the GGT activity of mLNs was high, whereas that of non-metastatic lymph nodes and CRC cell lines was low. We investigated the reason why GGT activity was upregulated in mLNs, and found that GGT was induced under conditions of hypoxia or low nutritional status. We utilized this feature to achieve rapid detection of mLNs with gGlu-HMRG. GGT appears to be a promising candidate enzyme for fluorescence imaging of mLNs.


Assuntos
Neoplasias Colorretais/metabolismo , Corantes Fluorescentes/metabolismo , Linfonodos/metabolismo , Metástase Linfática/patologia , gama-Glutamiltransferase/metabolismo , Animais , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Feminino , Fluorescência , Células HCT116 , Células HT29 , Humanos , Linfonodos/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Regulação para Cima/fisiologia
8.
J Gastric Cancer ; 18(2): 134-141, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29984063

RESUMO

Purpose: Postoperative pancreatic fistula is a serious and fatal complication of gastrectomy for gastric cancer. Blunt trauma to the parenchyma of the pancreas can result from an assistant's forceps compressing and retracting the pancreas, which in turn may result in pancreatic juice leakage. However, no published studies have focused on blunt trauma to the pancreas during laparoscopic surgery. Our aim was to investigate the relationship between compression of the pancreas and pancreatic juice leakage in a swine model. Materials and Methods: Three female pigs were used in this study. The pancreas was gently compressed dorsally for 15 minutes laparoscopically with gauze grasped with forceps. Pancreatic juice leakage was visualized by fluorescence imaging after topical administration of chymotrypsin-activatable fluorophore in real time. Amylase concentrations in ascites collected at specified times was measured. In addition, pancreatic tissue was fixed with formalin, and the histology of the compressed sites was evaluated. Results: Fluorescence imaging enabled visualization of pancreatic juice leaking into ascites around the pancreas. Median concentrations of pancreatic amylase in ascites increased from 46 U/L preoperatively to 12,509 U/L 4 hours after compression. Histological examination of tissues obtained 4 hours after compression revealed necrotic pancreatic acinar cells extending from the surface to deep within the pancreas and infiltration of inflammatory cells. Conclusions: Pancreatic compression by the assistant's forceps can contribute to pancreatic juice leakage. These findings will help to improve the procedure for lymph node dissection around the pancreas during laparoscopic gastrectomy.

9.
Chem Commun (Camb) ; 54(50): 6939-6942, 2018 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-29862387

RESUMO

Si-Rhodamines are bright fluorophores with red to near-infrared (NIR) emission, and are widely used for fluorescence imaging of biological phenomena. Here, in order to extend the scope of Si-rhodamine fluorophores, we established a versatile synthesis of unsymmetrical Si-rhodamines. To illustrate its value, we used one of these new fluorophores to synthesize a far-red to NIR fluorescence probe for hypoxia, and showed that it can visualize hepatic ischemia in mice in vivo.

10.
Head Neck ; 40(7): 1466-1475, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29509281

RESUMO

BACKGROUND: A fluorescent probe glutamylprolyl hydroxymethyl rhodamine green (EP-HMRG), which becomes fluorescent after cleavage by dipeptidyl peptidase-IV (DPP-IV), has been reported to be useful for the detection of esophageal cancer. Thus, we investigated whether head and neck squamous cell carcinoma (HNSCC) can be detected by spraying EP-HMRG. METHODS: Fluorescence imaging of 17 cases of HNSCCs resected using endoscopic or surgical resection was performed ex vivo after spraying EP-HMRG, and then the fluorescence intensity of the tumors and normal mucosa were measured. RESULTS: Iodine-voiding lesions became fluorescent within a few minutes after the application of EP-HMRG in 12 resected tumors without a history of radiotherapy but this was not observed in the normal mucosa. Fluorescence intensity in tumor lesions was significantly higher than normal lesions. However, 5 other tumors that developed after radiotherapy did not have sufficient contrast against normal mucosa. CONCLUSION: Fluorescence imaging with EP-HMRG would be useful for rapid detection of superficial HNSCC without a history of radiotherapy.


Assuntos
Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/enzimologia , Dipeptidil Peptidase 4/metabolismo , Corantes Fluorescentes , Neoplasias de Cabeça e Pescoço/diagnóstico , Neoplasias de Cabeça e Pescoço/enzimologia , Microscopia de Fluorescência , Administração Tópica , Idoso , Carcinoma de Células Escamosas/cirurgia , Linhagem Celular Tumoral , Detecção Precoce de Câncer/métodos , Feminino , Neoplasias de Cabeça e Pescoço/cirurgia , Humanos , Masculino , Rodaminas
11.
J Am Chem Soc ; 140(5): 1767-1773, 2018 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-29368925

RESUMO

Carboxypeptidases (CPs) are a family of hydrolases that cleave one or more amino acids from the C-terminal of peptides or proteins. However, methodology to monitor the activities of CPs is poorly developed. Here, we present the first versatile design strategy to obtain activatable fluorescent probes for CPs by utilizing intramolecular spirocyclization of rhodamine to translate the "aliphatic carboxamide to aliphatic carboxylate" structural conversion catalyzed by CPs into dynamic fluorescence activation. Based on this novel strategy, we developed probes for carboxypeptidases A and B. One of these probes was able to detect pancreatic juice leakage in mice ex vivo, suggesting that its suitability for intraoperative diagnosis of pancreatic fistula. This design strategy should be broadly applicable to CPs, as well as other previously untargetable enzymes, enabling development of fluorescent probes to study various pathological and biological processes.

12.
Ann Surg ; 2017 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-29135497

RESUMO

OBJECTIVE: To assess the predictive value of chymotrypsin activity in pancreatic juice on clinically relevant postoperative pancreatic fistula (CR-POPF) after pancreatic resection, since pancreatic peptidases rather than glycolytic enzymes play a pivotal role in causing tissue damage due to pancreatic leakage. BACKGROUND: The risk of CR-POPF has been estimated based on amylase level in abdominal drainage fluid. METHODS: Eighty-one consecutive patients underwent pancreatoduodenectomy, and postoperative pancreatic juice and drainage fluids were collected for 14 days. The chymotrypsin activity and fluid amylase level in these fluids were measured, and their susceptibility to the elapsed postoperative time and circadian rhythm were evaluated. The predictive value for the development of CR-POPF was compared between assessment of pancreatic chymotrypsin activity versus fluid amylase level. RESULTS: No significant differences in the daily pancreatic chymotrypsin activity were observed, whereas the amylase level in pancreatic juice was susceptible to the postoperative interval and circadian rhythm. CR-POPF developed in 19 patients (23%). Assessment of pancreatic chymotrypsin activity on the first postoperative day predicted CR-POPF with a sensitivity/specificity of 84/87% (area under the curve, 0.855; cut-off value, 0.5 arbitrary units), which was better than measurement of fluid amylase level. Independent predictors of CR-POPF were the day-1 pancreatic chymotrypsin activity (≥0.5 arbitrary units, P < 0.001) and the main pancreatic duct index (<0.25, P = 0.039). CONCLUSIONS: Assessment of pancreatic chymotrypsin activity may allow for more rapid and accurate prediction of CR-POPF than use of conventional diagnostic criteria based on fluid amylase level, enabling individualized surgical procedures and postoperative drain management.

13.
J Am Chem Soc ; 139(9): 3465-3472, 2017 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-28191944

RESUMO

Cellular homeostasis is maintained by a complex network of reactions catalyzed by enormous numbers of enzymatic activities (the enzymome), which serve to determine the phenotypes of cells. Here, we focused on the enzymomics of proteases and peptidases because these enzymes are an important class of disease-related proteins. We describe a system that (A) simultaneously evaluates metabolic activities of peptides using a series of exogenous peptide substrates and (B) identifies the enzymes that metabolize the specified peptide substrate with high throughput. We confirmed that the developed system was able to discover cell-type-specific and disease-related exo- and endopeptidase activities and identify the responsible enzymes. For example, we found that the activity of the endopeptidase neurolysin is highly elevated in human colorectal tumor tissue samples. This simple but powerful enzymomics platform should be widely applicable to uncover cell-type-specific reactions and altered enzymatic functions with potential value as biomarkers or drug targets in various disease states and to investigate the mechanisms of the underlying pathologies.


Assuntos
Peptídeo Hidrolases/metabolismo , Peptídeos/metabolismo , Linhagem Celular Tumoral , Endopeptidases/química , Endopeptidases/metabolismo , Humanos , Conformação Molecular , Especificidade de Órgãos , Peptídeo Hidrolases/química , Peptídeos/química
14.
Sci Rep ; 6: 26399, 2016 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-27245876

RESUMO

Early detection of esophageal squamous cell carcinoma (ESCC) is an important prognosticator, but is difficult to achieve by conventional endoscopy. Conventional lugol chromoendoscopy and equipment-based image-enhanced endoscopy, such as narrow-band imaging (NBI), have various practical limitations. Since fluorescence-based visualization is considered a promising approach, we aimed to develop an activatable fluorescence probe to visualize ESCCs. First, based on the fact that various aminopeptidase activities are elevated in cancer, we screened freshly resected specimens from patients with a series of aminopeptidase-activatable fluorescence probes. The results indicated that dipeptidylpeptidase IV (DPP-IV) is specifically activated in ESCCs, and would be a suitable molecular target for detection of esophageal cancer. Therefore, we designed, synthesized and characterized a series of DPP-IV-activatable fluorescence probes. When the selected probe was topically sprayed onto endoscopic submucosal dissection (ESD) or surgical specimens, tumors were visualized within 5 min, and when the probe was sprayed on biopsy samples, the sensitivity, specificity and accuracy reached 96.9%, 85.7% and 90.5%. We believe that DPP-IV-targeted activatable fluorescence probes are practically translatable as convenient tools for clinical application to enable rapid and accurate diagnosis of early esophageal cancer during endoscopic or surgical procedures.


Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/diagnóstico por imagem , Dipeptidil Peptidase 4/metabolismo , Neoplasias Esofágicas/diagnóstico por imagem , Carcinoma de Células Escamosas/enzimologia , Linhagem Celular Tumoral , Detecção Precoce de Câncer , Neoplasias Esofágicas/enzimologia , Corantes Fluorescentes/química , Humanos , Microscopia de Fluorescência , Rodaminas/química , Sensibilidade e Especificidade
15.
J Am Chem Soc ; 137(38): 12187-90, 2015 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-26360463

RESUMO

Tissue-restricted bioreactions can be utilized to design chemical-biological tools and prodrugs. We have developed a fluorescent-substrate-library-based enzyme discovery approach to screen tissue extracts for enzymatic activities of interest. Assay-positive candidate proteins were identified by diced electrophoresis gel assay followed by peptide mass fingerprinting. We discovered that pyruvyl anilide is specifically hydrolyzed by carboxylesterase 2 (CES2), which is predominantly localized in the liver and kidney. We show that the pyruvyl targeting group/CES2 enzyme pair can be used to deliver the 7-amino-4-methylcoumarin fluorophore specifically to the liver and kidney in vivo. Our screening approach should be useful to find other masking group/enzyme pairs suitable for development of fluorescent substrates and prodrugs.


Assuntos
Anilidas/metabolismo , Carboxilesterase/metabolismo , Cumarínicos/metabolismo , Fluorescência , Anilidas/química , Cumarínicos/química , Ativação Enzimática , Humanos , Rim/enzimologia , Rim/metabolismo , Fígado/enzimologia , Fígado/metabolismo , Especificidade por Substrato
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