RESUMO
A new, simple and rapid method for the quantitative determination of the antimicrobial preservative 2-phenoxyethanol, based on reverse phase ultra-high-performance liquid chromatography has been developed. The validation was performed according the ICH Q2 guideline "Validation of Analytical Procedures". The desired chromatographic separation was achieved on a Waters Symmetry C18 (150 × 4.6 mm, 5 µm) column using an isocratic elution, with detection at 270 nm wavelength. The mobile phase consisted of acetonitrile/water (55:45, v/v), pumped at a flow rate of 1 mL/min. The calibration curve and the analytical procedure are linear (r2 = 0.999) from the concentration of 0.07 mg/mL to 1.1 mg/mL. The percent relative standard deviation for intra- and inter-day precision was <1%. The recovery of 2-phenoxyethanol in vaccines ranged between 96.5 and 100.60%. The limits of detection and quantitation were 1.3 × 10-4 and 2.7 × 10-4 mg/mL, respectively. The method was found to be robust by changing the column working temperature, the percentage of acetonitrile of the mobile phase and the flow rate. The validated method can be successfully and reliably used to quantify as well as to exclude presence of 2-phenoxyethanol preservative in marketed vaccines.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Etilenoglicóis , Conservantes Farmacêuticos , Vacinas , Acetonitrilas , Etilenoglicóis/química , Humanos , Conservantes Farmacêuticos/química , Vacinas/químicaRESUMO
Exosomes are nanovesicles released by normal and tumor cells, which are detectable in cell culture supernatant and human biological fluids, such as plasma. Functions of exosomes released by "normal" cells are not well understood. In fact, several studies have been carried out on exosomes derived from hematopoietic cells, but very little is known about NK cell exosomes, despite the importance of these cells in innate and adaptive immunity. In this paper, we report that resting and activated NK cells, freshly isolated from blood of healthy donors, release exosomes expressing typical protein markers of NK cells and containing killer proteins (i.e., Fas ligand and perforin molecules). These nanovesicles display cytotoxic activity against several tumor cell lines and activated, but not resting, immune cells. We also show that NK-derived exosomes undergo uptake by tumor target cells but not by resting PBMC. Exosomes purified from plasma of healthy donors express NK cell markers, including CD56+ and perforin, and exert cytotoxic activity against different human tumor target cells and activated immune cells as well. The results of this study propose an important role of NK cell-derived exosomes in immune surveillance and homeostasis. Moreover, this study supports the use of exosomes as an almost perfect example of biomimetic nanovesicles possibly useful in future therapeutic approaches against various diseases, including tumors.
Assuntos
Exossomos/imunologia , Exossomos/metabolismo , Células Matadoras Ativadas por Linfocina/imunologia , Células Matadoras Ativadas por Linfocina/metabolismo , Monitorização Imunológica , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Subpopulações de Linfócitos B/patologia , Linfoma de Burkitt/imunologia , Linfoma de Burkitt/patologia , Transformação Celular Neoplásica/imunologia , Transformação Celular Neoplásica/patologia , Técnicas de Cocultura , Exossomos/ultraestrutura , Proteína Ligante Fas/biossíntese , Humanos , Imunofenotipagem , Células Jurkat , Células K562 , Células Matadoras Ativadas por Linfocina/ultraestrutura , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Monitorização Imunológica/métodos , Perforina/biossínteseRESUMO
Exposure to traumatic events during childhood is common, and the consequences for physical and mental health can be severe. Adverse childhood experiences (ACEs) such as physical abuse, sexual abuse, emotional abuse, and neglect appear to contribute to the onset and severity of a variety of somatic inflictions, including obesity, diabetes, cancer, and heart disease. The aim of this scoping review was to try to gain insight into how this might occur. Given the evidence of indirect (i.e., through unhealthy behaviours such as excessive drinking or poor eating habits) and direct (i.e., through its impact on the endocrine, immune, and cardiovascular systems as well as on the brain) effects of attachment on health, we examined the possibility that insecure attachment might contribute to the development of somatic symptoms in adult survivors of childhood trauma. Eleven studies met our inclusion criteria. Findings from this review suggest that insecure and disorganized attachment orientations are related to DNA damage, metabolic syndrome and obesity, physical pain, functional neurological disorder, and somatization in adults exposed to childhood trauma. We discuss the implications of this for the conceptualization and treatment of trauma and stress disorders.
RESUMO
Therapeutic mAbs show a specific "charge fingerprint" that may affect safety and efficacy, and, as such, it is often identified as a critical quality attribute (CQA). Capillary iso-electric focusing (cIEF), commonly used for the evaluation of such CQA, provides an analytical tool to investigate mAb purity and identity across the product lifecycle. Here, we discuss the results of an analysis of a panel of antibody products by conventional and whole-column imaging cIEF systems performed as part of European Pharmacopoeia activities related to development of "horizontal standards" for the quality control of monoclonal antibodies (mAbs). The study aimed at designing and verifying an independent and transversal cIEF procedure for the reliable analysis of mAbs charge variants. Despite the use of comparable experimental conditions, discrepancies in the charge profile and measured isoelectric points emerged between the two cIEF systems. These data suggest that the results are method-dependent rather than absolute, an aspect known to experts in the field and pharmaceutical industry, but not suitably documented in the literature. Critical implications from analytical and regulatory perspectives, are herein thoughtfully discussed, with a special focus on the context of market surveillance and identification of falsified medicines.
Assuntos
Anticorpos Monoclonais , Eletroforese Capilar , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais/análise , Focalização Isoelétrica/métodos , Eletroforese Capilar/métodos , Ponto Isoelétrico , Controle de QualidadeRESUMO
Assessment of the viral load in hepatitis C virus (HCV) genotype 1-infected patients is critical before, during, and after antiviral therapy. In patients achieving a rapid virological response at week 4 of treatment, the viral load at the baseline is considered a predictive criterion of a sustained virological response 24 weeks after the discontinuation of treatment. A >or=2-log(10) drop in the viral load at week 12 of treatment (early virological response) triggers the continuation of therapy. We organized a multicenter study (MS) for diagnostic laboratories involved in the quantification of HCV RNA. Commercial assays, including two based on real-time reverse transcription-PCR (TaqMan system), and in-house methods, were used by the 61 participants. The overall reproducibility of the commercial quantitative nucleic acid amplification techniques (qNAT) was acceptable. As the intermethod variability among commercial qNAT for HCV RNA was still present, the manufacturers of these test kits should join efforts to harmonize the means of quantification of HCV RNA. This study also shows that caution should be exercised when the baseline viral load is evaluated and when the 2-log(10) reduction after 12 weeks of therapy is interpreted. Finally, this MS confirms the higher sensitivity of the commercial qNAT based on the TaqMan system, making them the elective assays for the monitoring of therapy.
Assuntos
Hepacivirus/isolamento & purificação , Hepatite C/virologia , RNA Viral/sangue , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Carga Viral/métodos , Genótipo , Hepacivirus/genética , Humanos , Técnicas de Diagnóstico Molecular/métodos , RNA Viral/genética , Kit de Reagentes para Diagnóstico , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
The phenomenon of cell cannibalism, which generally refers to the engulfment of cells within other cells, was described in malignant tumors, but its biological significance is still largely unknown. In the present study, we investigated the occurrence, the in vivo relevance, and the underlying mechanisms of cannibalism in human melanoma. As first evidence, we observed that tumor cannibalism was clearly detectable in vivo in metastatic lesions of melanoma and often involved T cells, which could be found in a degraded state within tumor cells. Then, in vitro experiments confirmed that cannibalism of T cells was a property of metastatic melanoma cells but not of primary melanoma cells. In particular, morphologic analyses, including time-lapse cinematography and electron microscopy, revealed a sequence of events, in which metastatic melanoma cells were able to engulf and digest live autologous melanoma-specific CD8(+) T cells. Importantly, this cannibalistic activity significantly increased metastatic melanoma cell survival, particularly under starvation condition, supporting the evidence that tumor cells may use the eating of live lymphocytes as a way to "feed" in condition of low nutrient supply. The mechanism underlying cannibalism involved a complex framework, including lysosomal protease cathepsin B activity, caveolae formation, and ezrin cytoskeleton integrity and function. In conclusion, our study shows that human metastatic melanoma cells may eat live T cells, which are instead programmed to kill them, suggesting a novel mechanism of tumor immune escape. Moreover, our data suggest that cannibalism may represent a sort of "feeding" activity aimed at sustaining survival and progression of malignant tumor cells in an unfavorable microenvironment.
Assuntos
Linfócitos/patologia , Melanoma/patologia , Melanoma/secundário , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Vesículas Citoplasmáticas/metabolismo , Vesículas Citoplasmáticas/fisiologia , Humanos , Concentração de Íons de Hidrogênio , Imuno-Histoquímica , Linfócitos/imunologia , Melanoma/imunologia , Melanoma/metabolismo , Fagocitose , Linfócitos T/imunologia , Linfócitos T/patologiaRESUMO
We organised a collaborative study to calibrate three new ISS reference preparations (ISS: Istituto Superiore di Sanità), one for HCV RNA, one for HIV RNA and one for HBV DNA, to be used for nucleic acid amplification techniques (NAT) in blood testing. Serial dilution of the ISS reference preparations and the respective international standards were tested in different days by each participating laboratory using two commercial NAT assays. Data were collected by the ISS for statistical analysis. Based on the mean potency of the HCV RNA and HIV RNA preparations, calculated from the results provided by the 12 participating laboratories, a definitive concentrations of 5700 IU/mL and 4000 IU/mL, respectively, were assigned to the reference materials. On the contrary, it was not possible to obtain a consensus titre for the HBV DNA reference material. These new Italian reference preparations (HCV RNA ISS/1005 and HIV RNA ISS/1005) calibrated against the respective international standards are available free of charge to any laboratory upon request.
Assuntos
DNA Viral/normas , HIV/genética , Hepacivirus/genética , Vírus da Hepatite B/genética , Técnicas de Amplificação de Ácido Nucleico/normas , RNA Viral/normas , Virologia/normas , Calibragem , Internacionalidade , Itália , Padrões de ReferênciaRESUMO
Natural killer (NK) cells were first identified for their ability to kill tumor cells of different origin in vitro. Similarly, gammadelta T lymphocytes display strong cytotoxic activity against various tumor cell lines. However, the ability of both the NK and gammadelta cells to mediate natural immune response against human malignant tumors in vivo is still poorly defined. Severe combined immunodeficient (SCID) mice have been successfully engrafted with human tumors. In this study, the antitumor effect of local as well as of systemic treatments based on NK cells or Vdelta1 or Vdelta2 gamma/delta T lymphocytes against autologous melanoma cells was investigated in vivo. The results show that all three of the populations were effective in preventing growth of autologous human melanomas when both tumor and lymphoid cells were s.c. inoculated at the same site. However, when lymphoid cells were infused i.v., only NK cells and Vdelta1 gamma/delta T lymphocytes could either prevent or inhibit the s.c. growth of autologous melanoma. Accordingly, both NK cells and Vdelta1 gammadelta T lymphocytes could be detected at the s.c. tumor site. In contrast, Vdelta2 gammadelta T lymphocytes were only detectable in the spleen of the SCID mice. Moreover, NK cells maintained their inhibitory effect on tumor growth even after discontinuation of the treatment. Indeed they were present at the tumor site for a longer period. These data support the possibility to exploit NK cells and Vdelta1 gammadelta T lymphocytes in tumor immunotherapy. Moreover, our study emphasizes the usefulness of human tumor/SCID mouse models for preclinical evaluation of immunotherapy protocols against human tumors.
Assuntos
Células Matadoras Naturais/imunologia , Melanoma/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Linfócitos T/imunologia , Animais , Antígenos CD/análise , Antígenos CD/genética , Divisão Celular , DNA/análise , DNA/genética , Feminino , Humanos , Camundongos , Camundongos SCID , Reação em Cadeia da Polimerase , Transplante Heterólogo/métodosRESUMO
Quality by design (QbD) is an innovative approach to drug development that has started to be implemented into the regulatory framework, but currently mainly for chemical drugs. The recent marketing authorization of the first monoclonal antibody developed using extensive QbD concepts in the European Union paves the way for future further regulatory approvals of complex products employing this cutting-edge technological concept. In this paper, we report and comment on insights and lessons learnt from the non-public discussions in the European Medicines Agency's Biologicals Working Party and Committee for Medicinal Products for Human Use on the key issues during evaluation related to the implementation of an extensive QbD approach for biotechnology-derived medicinal products. Sharing these insights could prove useful for future developments in QbD for biotech products in general and monoclonal antibodies in particular.
Assuntos
Anticorpos Monoclonais , Biotecnologia/normas , Biotecnologia/métodos , União Europeia , Humanos , Controle de QualidadeRESUMO
BACKGROUND: A second Italian external quality assessment programme was run in 2011 to assess the performance of blood transfusion centres in detecting West Nile virus RNA in plasma. MATERIALS AND METHODS: Each participant received two panels containing negative samples and samples positive for West Nile virus lineages 1 and 2, some of which with a viral concentration close to or below the 95% limit of detection of the respective commercial nucleic acid amplification test assay: the PROCLEIX WNV assay or the Cobas TaqScreen West Nile virus test. RESULTS: Eleven laboratories took part in the external quality assessment programme. All of them correctly identified the positive samples with a viral concentration above the 95% limit of detection. No false positive results or pre-/post-analytical errors were observed. DISCUSSION: The External quality assessment programme run in 2011 allowed participants to assess the performance of the nucleic acid amplification test methods applied in their seasonal routine screening of blood donations. The results confirm the 95% limit of detection reported by the test kits' manufacturers for both West Nile virus lineages.
Assuntos
Seleção do Doador/métodos , Técnicas de Amplificação de Ácido Nucleico , Garantia da Qualidade dos Cuidados de Saúde , RNA Viral/sangue , Febre do Nilo Ocidental/sangue , Vírus do Nilo Ocidental , Reações Falso-Positivas , Feminino , Humanos , Masculino , RNA Viral/genética , Febre do Nilo Ocidental/genéticaRESUMO
Cytoskeleton plays a crucial role in natural killer cell function. In this study the expression and subcellular distribution of ezrin, radixin and moesin, a family of proteins that connect actin filaments to many membrane structures, were evaluated in human NK cells. The results showed that NK cells expressed all these proteins, while NK cell-deprived peripheral blood leukocytes and purified T lymphocytes did not express radixin. Only ezrin changed its distribution following IL-2 activation and all three ezrin, moesin and radixin were polarized on uropods of adherent natural killer cells. Ezrin and radixin co-localized with the perforin granules at the intimate sites of contact between NK and the target cells, while moesin remains uniformly distributed on the membrane of NK cells. Ezrin, radixin and perforin co-localization was undetected in non-lytic conjugates and inhibited by treatment with actin depolymerizing agents. These results suggest that ezrin and radixin may exert a role in NK activity, particularly in the trafficking of perforin granules to the NK/target cells contact site. Moreover, our data suggest that radixin may represent an additional biological marker of human NK cells and that this protein may hold a specific role in NK cell function.
Assuntos
Proteínas Sanguíneas/análise , Proteínas do Citoesqueleto/análise , Células Matadoras Naturais/química , Proteínas de Membrana/análise , Proteínas dos Microfilamentos/análise , Fosfoproteínas/análise , Proteínas Sanguíneas/genética , Western Blotting , Proteínas do Citoesqueleto/genética , Humanos , Ativação Linfocitária , Glicoproteínas de Membrana/análise , Proteínas de Membrana/genética , Proteínas dos Microfilamentos/genética , Microscopia Confocal , Perforina , Fosfoproteínas/genética , Proteínas Citotóxicas Formadoras de Poros , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Features of phagocytosis have been observed in human tumors, but the phagocytic apparatus of tumor cells and the mechanism(s) underlying this phenomenon have yet to be defined. To address the phenomenon of phagocytosis, its underlying mechanism(s), and its possible role in tumor biology, we used human melanoma cells as a prototypic model. Our results showed that a process of phagocytosis of apoptotic cells occurs in vivo in human melanoma. This finding was consistent with evidence that human melanoma cells in vitro express all of the known lysosomal and phagocytic markers on their cytoplasmic vesicles and that a process of phagocytosis occurs in these vesicles. However, exclusively human melanoma cells deriving from metastatic lesions possess an efficient phagocytic machinery responsible for a macrophage-like activity against latex beads, yeast, and apoptotic cells of different origins, which was comparable to that of human primary macrophages. Moreover, the actin-binding protein ezrin was expressed on phagocytic vacuoles of melanoma cells and of cells deriving from a human adenocarcinoma; both treatment with cytochalasin B and specific inhibition of ezrin synthesis strongly affected the phagocytic activity of melanoma cells. This suggests that the association with the actin cytoskeleton is a crucial requirement for the development of this phenomenon. Hence our data provide evidence for a potent phagocytic activity exerted by metastatic melanoma cells possibly involved in determining the level of aggressiveness of human melanoma. This suggests that the assessment of phagocytic activity may be exploited as a new tool to evaluate the malignancy of human melanoma. Moreover, our data suggest that gene therapy or drug treatments aimed at inhibiting actin assembly to the phagosomal membranes may be proposed as a new strategy for the control of tumor aggressiveness.
Assuntos
Melanoma/metabolismo , Neurofibromina 2/metabolismo , Fagocitose/fisiologia , Neoplasias Cutâneas/metabolismo , Apoptose/fisiologia , Biomarcadores/análise , Linhagem Celular Tumoral , Citoesqueleto/fisiologia , Citometria de Fluxo , Humanos , Técnicas Imunoenzimáticas , Marcação In Situ das Extremidades Cortadas , Melanoma/secundário , Neoplasias Cutâneas/patologiaRESUMO
The CD95 (Fas/APO-1) linkage to the actin cytoskeleton through ezrin is an essential requirement for susceptibility to the CD95-mediated apoptosis in CD4+ T cells. We have previously shown that moesin was not involved in the binding to CD95. Here we further support the specificity of the ezrin/CD95 binding, showing that radixin did not bind CD95. The ezrin region specifically and directly involved in the binding to CD95 was located in the middle lobe of the ezrin FERM domain, between amino acids 149 and 168. In this region, ezrin, radixin, and moesin show 60-65% identity, as compared with the 86% identity in the whole FERM domain. Transfection of two different human cell lines with a green fluorescent protein-tagged ezrin mutated in the CD95-binding epitope, induced a marked inhibition of CD95-mediated apoptosis. In these cells, the mutated ezrin did not co-localize or co-immunoprecipitate with CD95. Further analysis showed that the mutated ezrin, while unable to bind CD95, was fully able to bind actin, thus preventing the actin linkage to CD95. Altogether, our results support the specificity of ezrin in the association to CD95 and the importance of the ezrin-to-CD95 linkage in CD95-mediated apoptosis. Moreover, this study suggests that a major role of ezrin is to connect CD95 to actin, thus allowing the CD95 polarization on the cells and the occurrence of the following multiple cascades of the CD95 pathway.
Assuntos
Fosfoproteínas/análise , Receptor fas/metabolismo , Actinas/metabolismo , Apoptose/fisiologia , Sítios de Ligação , Proteínas do Citoesqueleto , Células HeLa , Humanos , Mutação , Fosfoproteínas/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Transdução de Sinais , Receptor fas/genéticaRESUMO
P-glycoprotein is a 170-kd glycosylated transmembrane protein, expressed in a variety of human cells and belonging to the adenosine triphosphate-binding cassette transporter family, whose membrane expression is functionally associated with the multidrug resistance phenotype. However, the mechanisms underlying the regulation of P-glycoprotein functions remain unclear. On the basis of some evidence suggesting P-glycoprotein-actin cytoskeleton interaction, this study investigated the association of P-glycoprotein with ezrin, radixin, and moesin, a class of proteins that cross-link actin filaments with plasma membrane in a human cell line of lymphoid origin and that have been shown to link other ion-pump-related proteins. To this purpose, a multidrug-resistant variant of CCRF-CEM cells (CEM-VBL100) was used as a model to investigate the following: (1) the cellular localizations of P-glycoprotein and ezrin, radixin, and moesin and their molecular associations; and (2) the effects of ezrin, radixin, and moesin antisense oligonucleotides on multidrug resistance and P-glycoprotein function. The results showed that: (1) P-glycoprotein colocalized and coimmunoprecipitated with ezrin, radixin, and moesin; and (2) treatment with antisense oligonucleotides for ezrin, radixin, and moesin restored drug susceptibility consistently with inhibition of both drug efflux and actin-P-glycoprotein association and induction of cellular redistribution of P-glycoprotein. These data suggest that P-glycoprotein association with the actin cytoskeleton through ezrin, radixin, and moesin is key in conferring to human lymphoid cells a multidrug resistance phenotype. Strategies aimed at inhibiting P-glycoprotein-actin association may be helpful in increasing the efficiency of both antitumor and antiviral therapies.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Actinas/metabolismo , Proteínas Sanguíneas/metabolismo , Proteínas do Citoesqueleto/metabolismo , Linfócitos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Proteínas Sanguíneas/genética , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Proteínas do Citoesqueleto/genética , Citoesqueleto/metabolismo , Resistência a Múltiplos Medicamentos , Humanos , Linfoma de Células T/patologia , Substâncias Macromoleculares , Proteínas de Membrana/genética , Proteínas dos Microfilamentos/genética , Microscopia Confocal , Família Multigênica , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Fosfoproteínas/genética , Testes de Precipitina , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Tionucleotídeos/farmacologia , Células Tumorais CultivadasRESUMO
The identification of appropriate mouse models could be useful in carefully evaluating the actual role of the in vivo development of antigen-loss variants during antigen-specific vaccine therapy of human tumors. In this study we investigated the level of efficacy of a MART-1/Melan-A-specific CD8+ T cell clone against its autologous melanoma in a severe combined immunodeficiency (SCID) mouse model, in which the tumor cells expressed in vivo heterogeneous and suboptimal levels of MART-1. The subcutaneous co-injection of the MART-1/Melan-A-reactive T cell clone A42 with MART-1/Melan-A+ autologous human melanoma cells into SCID mice caused a total inhibition of tumor growth. However, the systemic treatment with A42 clone lymphocytes resulted in only 50-60% inhibition of tumor growth, although the T cell clone targeted the tumors and the MART-1+ cells virtually disappeared from the tumors. This study suggests that an immunotherapy based on the expansion of an antigen-specific T cell clone generated in vitro is highly efficient in abolishing tumor growth when the target antigen is fully expressed, but leads to in vivo immunoselection of antigen-loss variants in the presence of suboptimal levels of antigen expression. Furthermore, this work shows that human tumors/SCID mouse models may be useful in evaluating the in vivo efficacy of adoptive immunotherapies.
Assuntos
Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/imunologia , Epitopos/imunologia , Imunoterapia Adotiva , Melanoma/imunologia , Proteínas de Neoplasias/imunologia , Evasão Tumoral , Animais , Antígenos de Neoplasias/análise , Antígenos de Neoplasias/genética , Linfócitos T CD8-Positivos/transplante , Células Clonais/imunologia , Células Clonais/transplante , Epitopos/análise , Feminino , Humanos , Injeções Intravenosas , Ativação Linfocitária , Melanoma/terapia , Camundongos , Camundongos SCID , Proteínas de Neoplasias/análise , Seleção Genética , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/transplante , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Resistance to antitumor agents is a major cause of treatment failure in patients with cancer. Some mechanisms of tumor resistance to cytotoxic drugs may involve increased acidification of extracellular compartments. We investigated whether proton pump inhibitors (PPIs), currently used in the anti-acid treatment of peptic disease, could inhibit the acidification of the tumor microenvironment and increase the sensitivity of tumor cells to cytotoxic agents. METHODS: We pretreated cell lines derived from human melanomas, adenocarcinomas, and lymphomas with the PPIs omeprazole, esomeprazole, or pantoprazole and tested their response to cytotoxic drugs in cell death assays. We also evaluated extracellular and intracellular pH and vacuolar-H+-ATPase (V-H+-ATPase) expression, distribution, and activity in PPI-pretreated cells by using western blot analyses, immunocytochemistry, laser scanning confocal analysis, and bioluminescence assays. Finally, we evaluated human melanoma growth and cisplatin sensitivity with or without omeprazole pretreatment in xenografted SCID/SCID mice. RESULTS: PPI pretreatment sensitized tumor cell lines to the effects of cisplatin, 5-fluorouracil, and vinblastine, with an IC50 value reduction up to 2 logs. PPI pretreatment was associated with the inhibition of V-H+-ATPase activity and increases in both extracellular pH and the pH of lysosomal organelles. PPI pretreatment induced a marked increase in the cytoplasmic retention of the cytotoxic drugs, with clear targeting to the nucleus in the case of doxorubicin. In in vivo experiments, oral pretreatment with omeprazole was able to induce sensitivity of human solid tumors to cisplatin. CONCLUSION: Our results open new possibilities for the treatment of drug-resistant tumors through combination strategies based on the use of well-tolerated pH modulators such as PPIs.