RESUMO
A series of degrasyn-like symmetrical compounds have been designed, synthesized, and screened against B cell malignancy (multiple myeloma, mantle cell lymphoma) cell lines. The lead compounds T5165804 and CP2005 showed higher nanomolar potency against these tumor cells in comparison to degrasyn and inhibited Usp9x activity in vitro and in intact cells. These observations suggest that this new class of compounds holds promise as cancer therapeutic agents.
Assuntos
Antineoplásicos/química , Nitrilas/química , Piridinas/química , Antineoplásicos/uso terapêutico , Antineoplásicos/toxicidade , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cianoacrilatos , Dimerização , Humanos , Modelos Moleculares , Mieloma Múltiplo/tratamento farmacológico , Nitrilas/farmacologia , Nitrilas/uso terapêutico , Piridinas/farmacologia , Piridinas/uso terapêutico , Ubiquitina Tiolesterase/antagonistas & inibidores , Ubiquitina Tiolesterase/metabolismoRESUMO
INTRODUCTION: Previous studies demonstrated that the lactose-binding protein (hepatocellular carcinoma-intestine-pancreas and pancreatitis-associated proteins (HIP/PAP)) is upregulated >130 times in peritumoral pancreatic tissue as compared to normal pancreatic tissue. Therefore, we developed a new radiolabeled ligand of HIP/PAP, the ethyl-ß-D-galactopyranosyl-(1,4')-2'-deoxy-2'-[¹8F]fluoro-ß-D-glucopyranoside (Et-[¹8F]FDL) for noninvasive imaging of pancreatic carcinoma using positron emission tomography and computerized tomography (PET/CT). METHODS: The novel precursor and radiolabeling methods for synthesis of Et-[¹8F]FDL produced no isomers; the average decay-corrected radiochemical yield was 68%, radiochemical purity >99%, and specific activity >74 GBq/µmol. The radioligand properties of Et-[¹8F]FDL were evaluated using an ex vivo autoradiography and immunohistochemistry in pancreatic tissue sections obtained from mice-bearing orthotopic pancreatic tumor xenografts. RESULTS AND DISCUSSION: Et-[¹8F]FDL binding to peritumoral pancreatic tissue sections strongly correlated with HIP/PAP expression (r = 0.81) and could be completely blocked by treatment with 1 mM lactose. CONCLUSION: These results suggest that Et-[¹8F]FDL is a promising agent which should be evaluated for detection of early pancreatic carcinomas by PET/CT imaging.
Assuntos
Autorradiografia/métodos , Dissacarídeos/síntese química , Detecção Precoce de Câncer , Glucosídeos/síntese química , Lactose/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Pancreáticas/diagnóstico por imagem , Tomografia por Emissão de Pósitrons , Animais , Antígenos de Neoplasias/metabolismo , Biomarcadores Tumorais/metabolismo , Cromatografia Líquida de Alta Pressão , Dissacarídeos/química , Dissacarídeos/isolamento & purificação , Glucosídeos/química , Glucosídeos/isolamento & purificação , Halogenação , Imuno-Histoquímica , Lectinas Tipo C/metabolismo , Ligantes , Camundongos , Proteínas Associadas a Pancreatite , Compostos Radiofarmacêuticos/síntese química , Compostos Radiofarmacêuticos/químicaRESUMO
BACKGROUND: Early diagnosis of pancreatic carcinoma with highly sensitive diagnostic imaging methods could save lives of many thousands of patients, because early detection increases resectability and survival rates. Current non-invasive diagnostic imaging techniques have inadequate resolution and sensitivity for detection of small size ( approximately 2-3 mm) early pancreatic carcinoma lesions. Therefore, we have assessed the efficacy of positron emission tomography and computer tomography (PET/CT) imaging with beta-O-D-galactopyranosyl-(1,4')-2'-deoxy-2'-[(18)F]fluoroethyl-D-glucopyranose ([(18)F]FEDL) for detection of less than 3 mm orthotopic xenografts of L3.6pl pancreatic carcinomas in mice. [(18)F]FEDL is a novel radioligand of hepatocarcinoma-intestine-pancreas/pancreatitis-associated protein (HIP/PAP), which is overexpressed in peritumoral pancreatic acinar cells. METHODOLOGY/PRINCIPAL FINDINGS: Dynamic PET/CT imaging demonstrated rapid accumulation of [(18)F]FEDL in peritumoral pancreatic tissue (4.04+/-2.06%ID/g), bi-exponential blood clearance with half-lives of 1.65+/-0.50 min and 14.14+/-3.60 min, and rapid elimination from other organs and tissues, predominantly by renal clearance. Using model-independent graphical analysis of dynamic PET data, the average distribution volume ratio (DVR) for [(18)F]FEDL in peritumoral pancreatic tissue was estimated as 3.57+/-0.60 and 0.94+/-0.72 in sham-operated control pancreas. Comparative analysis of quantitative autoradiographic images and densitometry of immunohistochemically stained and co-registered adjacent tissue sections demonstrated a strong linear correlation between the magnitude of [(18)F]FEDL binding and HIP/PAP expression in corresponding regions (r = 0.88). The in situ analysis demonstrated that at least a 2-4 fold apparent lesion size amplification was achieved for submillimeter tumors and to nearly half a murine pancreas for tumors larger than 3 mm. CONCLUSION/SIGNIFICANCE: We have demonstrated the feasibility of detection of early pancreatic tumors by non-invasive imaging with [(18)F]FEDL PET/CT of tumor biomarker HIP/PAP over-expressed in peritumoral pancreatic tissue. Non-invasive non-invasive detection of early pancreatic carcinomas with [(18)F]FEDL PET/CT imaging should aid the guidance of biopsies and additional imaging procedures, facilitate the resectability and improve the overall prognosis.
Assuntos
Antígenos de Neoplasias/metabolismo , Biomarcadores Tumorais/metabolismo , Carcinoma/metabolismo , Radioisótopos de Flúor/farmacologia , Lactose/farmacologia , Lectinas Tipo C/metabolismo , Pâncreas/metabolismo , Neoplasias Pancreáticas/diagnóstico , Tomografia por Emissão de Pósitrons/métodos , Compostos Radiofarmacêuticos/farmacologia , Tomografia Computadorizada de Emissão de Fóton Único/métodos , Tomografia Computadorizada por Raios X/métodos , Animais , Carcinoma de Células Acinares/metabolismo , Ligantes , Camundongos , Modelos Biológicos , Transplante de Neoplasias , Proteínas Associadas a PancreatiteRESUMO
BACKGROUND: Imatinib is a tyrosine kinase inhibitor that is used to treat chronic myelogenous leukemia (CML). BCR-ABL mutations are associated with failure of imatinib treatment in many CML patients. LYN kinase regulates survival and responsiveness of CML cells to inhibition of BCR-ABL kinase, and differences in LYN regulation have been found between imatinib-sensitive and -resistant CML cell lines. METHODS: We evaluated cells from 12 imatinib-resistant CML patients with mutation-negative BCR-ABL and from six imatinib-sensitive patients who discontinued therapy because of imatinib intolerance. Phosphorylation of BCR-ABL and LYN was assessed in patient cells and cell lines by immunoblotting with activation state-specific antibodies, co-immunoprecipitation studies, and mass spectroscopy analysis of phosphopeptides. Cell viability, caspase activation, and apoptosis were also measured. Mutations were analyzed by sequencing. The effect of silencing LYN with short interfering RNAs (siRNAs) or reducing activation by treatment with tyrosine kinase inhibitors was evaluated in cell lines and patient cells. RESULTS: Imatinib treatment suppressed LYN phosphorylation in cells from imatinib-sensitive CML patients and imatinib-sensitive cell lines. Imatinib treatment blocked BCR-ABL signaling but did not suppress LYN phosphorylation in cells from imatinib-resistant patients, and persistent activation of LYN kinase was not associated with mutations in LYN kinase or its carboxyl-terminal regulatory domains. Unique LYN phosphorylation sites (tyrosine-193 and tyrosine-459) and associated proteins (c-Cbl and p80) were identified in cells from imatinib-resistant patients. Reducing LYN expression (siRNA) or activation (dasatinib) was associated with loss of cell survival and cytogenetic or complete hematologic responses in imatinib-resistant disease. CONCLUSIONS: LYN activation was independent of BCR-ABL in cells from imatinib-resistant patients. Thus, LYN kinase may be involved in imatinib resistance in CML patients with mutation-negative BCR-ABL and its direct inhibition is consistent with clinical responses in these patients.
Assuntos
Antineoplásicos/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Leucemia Mieloide Crônica Atípica BCR-ABL Negativa/enzimologia , Piperazinas/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirimidinas/uso terapêutico , Quinases da Família src/metabolismo , Adulto , Idoso , Animais , Antineoplásicos/efeitos adversos , Apoptose/efeitos dos fármacos , Benzamidas , Caspases/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Feminino , Proteínas de Fusão bcr-abl , Humanos , Mesilato de Imatinib , Immunoblotting , Imunoprecipitação , Leucemia Mieloide Crônica Atípica BCR-ABL Negativa/tratamento farmacológico , Leucemia Mieloide Crônica Atípica BCR-ABL Negativa/genética , Masculino , Espectrometria de Massas , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Fosforilação , Piperazinas/efeitos adversos , Inibidores de Proteínas Quinases/efeitos adversos , Proteínas Tirosina Quinases/metabolismo , Pirimidinas/efeitos adversosRESUMO
Methyl-2-cyano-3,12 dioxoolean-1,9 diene-28-oate (CDDO-Me) is a synthetic oleanolic acid derivative that displays antitumorigenic and anti-inflammatory activities, and we have previously reported that this agent potently activates the intrinsic apoptotic pathway in leukemia cells. In this study, we demonstrate that mitochondrial dysfunction induced by CDDO-Me is mediated by direct permeabilization of the inner mitochondrial membrane, which results in the rapid depletion of mitochondrial glutathione (GSXm), loss of cardiolipin, and inhibition of mitochondrial respiration. More importantly, we demonstrate that in addition to activating the intrinsic apoptotic pathway, the mitochondrial effects of CDDO-Me may mediate its anti-inflammatory activity by modulating the generation of superoxide anion (O2*). It is noteworthy that CDDO-Me did not increase the generation of O2* and pretreatment of leukemia cells with CDDO-Me prevented the increase of this reactive oxygen species elicited by inhibition of complex I or III in the absence of de novo protein synthesis. CDDO-Me, but not other inhibitors of respiration, induced a time- and dose-dependent, cyclosporin A-independent permeability transition (PT) of isolated mitochondria that was sensitive to sulfhydryl antioxidants but not to EDTA. PT induced by CDDO-Me and Ca2+ was accompanied by loss of GSXm, suggesting that the increased permeability of the inner mitochondrial membrane facilitates the loss of this antioxidant. Finally, transmission electron microscopy revealed that CDDO-Me rapidly induced caspase-independent mitochondrial swelling and loss of inner membrane structure before the release of cytochrome c. Taken together, our results indicate that CDDO-Me is a novel mitochondriotoxic agent that induces apoptosis and inhibits mitochondrial electron transport via perturbations in inner mitochondrial membrane integrity.