RESUMEN
BACKGROUND: Hyperthermic intraperitoneal chemotherapy (HIPEC) is advised as a treatment option for epithelial ovarian cancer (EOC) with peritoneal carcinomatosis. This study was designed to define the pharmacokinetics of cisplatin (CDDP) and paclitaxel (PTX) administered together during HIPEC. METHODS: Thirteen women with EOC underwent cytoreductive surgery (CRS) and HIPEC, with CDDP and PTX. Blood, peritoneal perfusate and tissue samples were harvested to determine drug exposure by high-performance liquid chromatography and matrix-assisted laser desorption ionization imaging mass spectrometry (IMS). RESULTS: The mean maximum concentrations of CDDP and PTX in perfusate were, respectively, 24.8±10.4 µg ml(-1) and 69.8±14.3 µg ml(-1); in plasma were 1.87±0.4 µg ml(-1) and 0.055±0.009 µg ml(-1). The mean concentrations of CDDP and PTX in peritoneum at the end of HIPEC were 23.3±8.0 µg g(-1) and 30.1±18.3 µg(-1)g(-1), respectively. The penetration of PTX into the peritoneal wall, determined by IMS, was about 0.5 mm. Grade 3-4 surgical complications were recorded in four patients, five patients presented grade 3 and two patients presented grade 4 hematological complications. CONCLUSIONS: HIPEC with CDDP and PTX after CRS is feasible with acceptable morbidity and has a favorable pharmacokinetic profile: high drug concentrations are achieved in peritoneal tissue with low systemic exposure. Larger studies are needed to demonstrate its efficacy in patients with microscopic postsurgical residual tumours in the peritoneal cavity.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Carcinoma/tratamiento farmacológico , Neoplasias Glandulares y Epiteliales/tratamiento farmacológico , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Peritoneales/tratamiento farmacológico , Adulto , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Área Bajo la Curva , Carcinoma/secundario , Cisplatino/administración & dosificación , Femenino , Humanos , Hipertermia Inducida , Infusiones Parenterales , Masculino , Persona de Mediana Edad , Neoplasias Glandulares y Epiteliales/secundario , Neoplasias Ováricas/patología , Paclitaxel/administración & dosificación , Neoplasias Peritoneales/secundario , Peritoneo/metabolismoRESUMEN
Mass Spectrometry Imaging (MSI) is a widespread technique used to qualitatively describe in two dimensions the distribution of endogenous or exogenous compounds within tissue sections. Absolute quantification of drugs using MSI is a recent challenge that just in the last years has started to be addressed. Starting from a two dimensional MSI protocol, we developed a three-dimensional pipeline to study drug penetration in tumors and to develop a new drug quantification method by MALDI MSI. Paclitaxel distribution and concentration in different tumors were measured in a 3D model of Malignant Pleural Mesothelioma (MPM), which is known to be a very heterogeneous neoplasm, highly resistant to different drugs. The 3D computational reconstruction allows an accurate description of tumor PTX penetration, adding information about the heterogeneity of tumor drug distribution due to the complex microenvironment. The use of an internal standard, homogenously sprayed on tissue slices, ensures quantitative results that are similar to those obtained using HPLC. The 3D model gives important information about the drug concentration in different tumor sub-volumes and shows that the great part of each tumor is not reached by the drug, suggesting the concept of pseudo-resistance as a further explanation for ineffective therapies and tumors relapse.
Asunto(s)
Antineoplásicos Fitogénicos/análisis , Neoplasias/diagnóstico por imagen , Paclitaxel/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Animales , Antineoplásicos Fitogénicos/metabolismo , Antineoplásicos Fitogénicos/uso terapéutico , Línea Celular Tumoral , Cromatografía Líquida de Alta Presión , Femenino , Humanos , Imagenología Tridimensional , Mesotelioma/química , Mesotelioma/diagnóstico por imagen , Mesotelioma/tratamiento farmacológico , Mesotelioma/patología , Nanopartículas del Metal/química , Ratones , Ratones Desnudos , Neoplasias/química , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Paclitaxel/metabolismo , Paclitaxel/uso terapéutico , Titanio/química , Trasplante HeterólogoRESUMEN
We provide an overview of the available information on the distribution of chemotherapeutics in human tumors, highlighting the progress made to assess the heterogeneity of drug concentrations in relation to the complex neoplastic tissue using novel analytical methods, e.g., mass spectrometry imaging. The increase in interstitial fluid pressure due to abnormal vascularization and stiffness of tumor stroma explains the variable and heterogeneous drug concentrations. Therapeutic strategies to enhance tumor drug distribution, thus possibly increasing efficacy, are discussed.
Asunto(s)
Antineoplásicos/metabolismo , Antineoplásicos/uso terapéutico , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Animales , Antineoplásicos/farmacología , Diagnóstico por Imagen/métodos , Humanos , Neoplasias/diagnóstico , Distribución Tisular/efectos de los fármacos , Distribución Tisular/fisiologíaRESUMEN
Urinary exosomes (UE) are nanovesicles released by every epithelial cell facing the urinary space and they are considered a promising source of molecular markers for renal dysfunction and structural injury. Exosomal proteomics has emerged as a powerful tool for understanding the molecular composition of exosomes and has potential to accelerate biomarker discovery. We employed this strategy in the study of diabetic nephropathy (DN) and the consequent end stage renal disease, which represent the dramatic evolution of diabetes, often leading the patients to dialysis or kidney transplantation. The identification of DN biomarkers is likely to help monitoring the disease onset and progression. A label free LC-MS/MS approach was applied to investigate the alteration of the proteome of urinary exosomes isolated from the Zucker diabetic fatty rats (ZDF), as a model of type 2 DN. We collected 24 hour urine samples from 7 ZDF and from 7 control rats at different ages (6, 12 and 20 weeks old) to monitor the development of DN. Exosomes were isolated by ultracentrifugation and their purity assessed by immunoblotting for known exosomal markers. Exosomal proteins from urine samples of 20 week old rats were pooled and analyzed by nLC-ESI-UHR-QToF-MS/MS after pre-filtration and tryptic digestion, leading to the identification and label free quantification of 286 proteins. Subcellular localization and molecular functions were assigned to each protein by UniprotKB, showing that the majority of identified proteins were membrane-associated or cytoplasmic and involved in transport, signalling and cellular adhesion, typical functions of exosomal proteins. We further validated label free mass spectrometry results by immunoblotting, as exemplified by: Xaa-Pro dipeptidase, Major Urinary Protein 1 and Neprilysin, which resulted increased, decreased and not different, respectively, in exosomes isolated from diabetic urine samples compared to controls, by both techniques. In conclusion we show the potential of exosome proteomics for DN biomarker discovery.
Asunto(s)
Diabetes Mellitus Tipo 2/metabolismo , Nefropatías Diabéticas/metabolismo , Exosomas/química , Proteínas/análisis , Proteómica , Sistema Urinario/metabolismo , Animales , Biomarcadores/metabolismo , Biomarcadores/orina , Diabetes Mellitus Tipo 2/patología , Nefropatías Diabéticas/genética , Nefropatías Diabéticas/patología , Exosomas/genética , Masculino , Espectrometría de Masas , Proteoma/análisis , Proteoma/metabolismo , Ratas , Ratas Zucker , Sistema Urinario/patologíaRESUMEN
Renal cell carcinoma (RCC) accounts for about 3% of all human malignancies and its incidence is increasing. There are no standard biomarkers currently used in the clinical management of patients with renal cell carcinoma. A promising strategy for new biomarker detection is comparative proteomics of urinary exosomes (UE), nanovesicles released by every epithelial cell facing the urinary space, enriched in renal proteins and excluding high-abundance plasmatic proteins, such as albumin. Aim of the work is to establish the protein profile of exosomes isolated from urines of RCC patient compared with control subjects. We enrolled 29 clear cell RCC patients and 23 control healthy subjects (CTRL), age and sex-matched, for urine collection and vesicle isolation by differential centrifugation. Such vesicles were morphologically and biochemically characterized and proved to share exosome properties. Proteomic analysis, performed on 9 urinary exosome (UE) pooled samples by gel based digestion followed by LC-MS/MS, led to the identification of 261 proteins from CTRL subject UE and 186 from RCC patient UE, and demonstrated that most of the identified proteins are membrane associated or cytoplasmic. Moreover, about a half of identified proteins are not shared between RCC and control UE. Starting from these observations, and from the literature, we selected a panel of 10 proteins, whose UE differential content was subjected to immunoblotting validation. Results show for the first time that RCC UE protein content is substantially and reproducibly different from control UE, and that these differences may provide clues for new RCC biomarker discovery.
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Carcinoma de Células Renales/metabolismo , Exosomas/metabolismo , Neoplasias Renales/metabolismo , Proteoma/análisis , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/patología , Femenino , Humanos , Riñón/metabolismo , Neoplasias Renales/genética , Neoplasias Renales/patología , Masculino , Persona de Mediana Edad , Análisis por Matrices de Proteínas , Proteínas/análisis , ProteómicaRESUMEN
Renal cell carcinoma (RCC) is representing about 3% of all adult cancers. A promising strategy for cancer biomarker discovery is subcellular comparative proteomics, allowing enriching specific cell compartments and assessing differences in protein expression patterns. We investigated the proteomic profile of a peculiar RCC subcellular compartment, plasma membrane microdomains (MD), involved in cell signalling, transport, proliferation and in many human diseases, such as cancer. Subcellular fractions were prepared by differential centrifugation from surgical samples of RCC and adjacent normal kidney (ANK). MD were isolated from plasma-membrane-enriched fractions after Triton X-100 treatment and sucrose density gradient ultracentrifugation. MD derived from RCC and ANK tissues were analyzed after SDS-PAGE separation by LC-ESI-MS/MS. We identified 93 proteins from MD isolated from RCC tissue, and 98 proteins from ANK MD. About 70% of the identified proteins are membrane-associated and about half of these are known as microdomain-associated. GRAVY scores assignment shows that most identified proteins (about 70%) are in the hydrophobic range. We chose a panel of proteins to validate their differential expression by WB. In conclusion, our work shows that RCC microdomain proteome is reproducibly different from ANK, and suggests that mining into such differences may support new biomarker discovery.
Asunto(s)
Carcinoma de Células Renales/genética , Neoplasias Renales/genética , Riñón/citología , Proteínas de Neoplasias/genética , Análisis por Matrices de Proteínas/métodos , Adulto , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/patología , Electroforesis en Gel de Poliacrilamida , Femenino , Perfilación de la Expresión Génica , Humanos , Riñón/metabolismo , Riñón/patología , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Masculino , Microdominios de Membrana/genética , Microdominios de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Octoxinol , Proteoma/genética , Proteómica/métodos , Espectrometría de Masas en TándemRESUMEN
Some chemical compounds used in intensive agriculture have been found to induce estrogenic effects; therefore a histological analysis of the testes and an evaluation of plasma levels of sex steroid, thyroid hormones, and vitellogenin were carried out in adult male water frogs of two coexisting taxa (Rana lessonae and the hemiclonal hybrid Rana esculenta) sampled in agricultural and pristine areas. Differences in seasonal profiles of hormones were found in water frogs living in the agricultural area where the presence of endocrine disrupting compounds was suspected on the basis of a previous study. In R. esculenta, sampled in the pristine area, high androgen levels were found in May; the opposite trend was found for R. esculenta sampled in agricultural areas in which the highest androgen levels were found in September, significantly lower compared with those found in R. esculenta sampled in the pristine area. Low androgen levels were also recorded in R. lessonae males sampled both in pristine and agricultural areas, while the highest levels were found in September. Regarding the trend of estradiol-17beta, an increase of this hormone was found in July both in esculenta and lessonae sampled in the agricultural area, and in the same month an estradiol-17beta peak, even though lower, was also found both in esculenta and lessonae males captured in the pristine area; detectable vitellogenin was found neither in males captured in the agricultural area, nor in those sampled in the pristine one. Moreover, while no significant changes of thyroid hormones were found either in the esculenta or lessonae males sampled in the pristine area, increased T3 and T4 titers were found in July in both esculenta and lessonae captured in the agricultural area. Morphological differences of the testes in males of parental species captured in the agricultural area were also observed. These findings indicate alterations in endocrine and reproductive function in frogs in the agricultural area, that could suggest the presence of endocrine disrupting compounds.