RESUMO
Design and efficacy of bioactive drugs is restricted by their (in)ability to traverse cellular membranes. Therapy resistance, a major cause of ineffective cancer treatment, is frequently due to suboptimal intracellular accumulation of the drug. We report a molecular mechanism that promotes trans-membrane movement of a stereotypical, widely used anti-cancer agent to counteract resistance. Well-defined lipid analogues adapt to the amphiphilic drug doxorubicin, when co-inserted into the cell membrane, and assemble a transient channel that rapidly facilitates the translocation of the drug onto the intracellular membrane leaflet. Molecular dynamic simulations unveiled the structure and dynamics of membrane channel assembly. We demonstrate that this principle successfully addresses multi-drug resistance of genetically engineered mouse breast cancer models. Our results illuminate the role of the plasma membrane in restricting the efficacy of established therapies and drug resistance - and provide a mechanism to overcome ineffectiveness of existing and candidate drugs.
Assuntos
Antineoplásicos/farmacocinética , Membrana Celular/metabolismo , Resistencia a Medicamentos Antineoplásicos , Glicoesfingolipídeos/metabolismo , Animais , Antineoplásicos/administração & dosagem , Transporte Biológico , Linhagem Celular , Membrana Celular/química , Proliferação de Células , Modelos Animais de Doenças , Doxorrubicina/administração & dosagem , Doxorrubicina/farmacocinética , Feminino , Glicerofosfolipídeos/química , Glicerofosfolipídeos/metabolismo , Glicoesfingolipídeos/química , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Lipossomos , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/mortalidade , Neoplasias Mamárias Experimentais/patologia , Camundongos , Modelos Biológicos , Carga TumoralRESUMO
PURPOSE: To improve nanoliposomal-doxorubicin (DoxNL) delivery in tumor cells using liposome membrane-incorporated short-chain sphingolipids (SCS) with selective membrane-permeabilizing properties. DoxNL bilayers contained synthetic short-chain derivatives of known membrane microdomain-forming sphingolipids; C8-glucosylceramide (C8-GluCer), C8-galactosylceramide (C8-GalCer) or C8-lactosylceramide (C8-LacCer). METHODS: DoxNL enriched with C8-GluCer or C8-GalCer were developed, optimized and characterized with regard to size, stability and drug retention. In vitro cytotoxic activity was studied in a panel of human tumor cell lines and normal cells. Intracellular Dox delivery was measured by flow cytometry and visualized by fluorescence microscopy. For a further understanding of the involved drug delivery mechanism confocal microscopy studies addressed the cellular fate of the nanoliposomes, the SCS and Dox in living cells. RESULTS: C8-LacCer-DoxNL aggregated upon Dox loading. In tumor cell lines SCS-DoxNL with C8-GluCer or C8-GalCer demonstrated strongly increased Dox delivery and cytotoxicity compared to standard DoxNL. Surprisingly, this effect was much less pronounced in normal cells. Nanoliposomes were not internalized, SCS however transfered from the nanoliposomal bilayer to the cell membrane and preceded cellular uptake and subsequent nuclear localization of Dox. CONCLUSION: C8-GluCer or C8-GalCer incorporated in DoxNL selectively improved intracellular drug delivery upon transfer to tumor cell membranes by local enhancement of cell membrane permeability.
Assuntos
Antibióticos Antineoplásicos/administração & dosagem , Membrana Celular/efeitos dos fármacos , Doxorrubicina/administração & dosagem , Lipossomos/metabolismo , Esfingolipídeos/metabolismo , Antibióticos Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular/efeitos dos fármacos , Doxorrubicina/farmacocinética , Galactosilceramidas/química , Galactosilceramidas/metabolismo , Glucosilceramidas/química , Glucosilceramidas/metabolismo , Humanos , Lactosilceramidas/química , Lactosilceramidas/metabolismo , Lipossomos/química , Neoplasias/tratamento farmacológico , Esfingolipídeos/químicaRESUMO
Synthetic alkylphospholipids (ALPs), such as edelfosine, miltefosine, perifosine, erucylphosphocholine and erufosine, represent a relatively new class of structurally related antitumor agents that act on cell membranes rather than on DNA. They selectively target proliferating (tumor) cells, inducing growth arrest and apoptosis, and are potent sensitizers of conventional chemo- and radiotherapy. ALPs easily insert in the outer leaflet of the plasma membrane and cross the membrane via an ATP-dependent CDC50a-containing 'flippase' complex (in carcinoma cells), or are internalized by lipid raft-dependent endocytosis (in lymphoma/leukemic cells). ALPs resist catabolic degradation, therefore accumulate in the cell and interfere with lipid-dependent survival signaling pathways, notably PI3K-Akt and Raf-Erk1/2, and de novo phospholipid biosynthesis. At the same time, stress pathways (e.g. stress-activated protein kinase/JNK) are activated to promote apoptosis. In many preclinical and clinical studies, perifosine was the most effective ALP, mainly because it inhibits Akt activity potently and consistently, also in vivo. This property is successfully exploited clinically in highly malignant tumors, such as multiple myeloma and neuroblastoma, in which a tyrosine kinase receptor/Akt pathway is amplified. In such cases, perifosine therapy is most effective in combination with conventional anticancer regimens or with rapamycin-type mTOR inhibitors, and may overcome resistance to these agents. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.
Assuntos
Antineoplásicos/farmacologia , Neoplasias/tratamento farmacológico , Organofosfatos/farmacologia , Éteres Fosfolipídicos/farmacologia , Fosforilcolina/análogos & derivados , Compostos de Amônio Quaternário/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Endocitose , Humanos , Neoplasias/metabolismo , Organofosfatos/uso terapêutico , Fosfatidilinositol 3-Quinase/genética , Fosfatidilinositol 3-Quinase/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Éteres Fosfolipídicos/uso terapêutico , Fosforilcolina/farmacologia , Fosforilcolina/uso terapêutico , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Compostos de Amônio Quaternário/uso terapêutico , Transdução de Sinais/efeitos dos fármacosRESUMO
Epidermal growth factor receptor (EGFR) activation is negatively regulated by protein kinase C (PKC) signaling. Stimulation of A431 cells with EGF, bradykinin or UTP increased EGFR phosphorylation at Thr654 in a PKC-dependent manner. Inhibition of PKC signaling enhanced EGFR activation, as assessed by increased phosphorylation of Tyr845 and Tyr1068 residues of the EGFR. Diacylglycerol is a physiological activator of PKC that can be removed by diacylglycerol kinase (DGK) activity. We found, in A431 and HEK293 cells, that the DGKθ isozyme translocated from the cytosol to the plasma membrane, where it co-localized with the EGFR and subsequently moved into EGFR-containing intracellular vesicles. This translocation was dependent on both activation of EGFR and PKC signaling. Furthermore, DGKθ physically interacted with the EGFR and became tyrosine-phosphorylated upon EGFR stimulation. Overexpression of DGKθ attenuated the bradykinin-stimulated, PKC-mediated EGFR phosphorylation at Thr654, and enhanced the phosphorylation at Tyr845 and Tyr1068. SiRNA-induced DGKθ downregulation enhanced this PKC-mediated Thr654 phosphorylation. Our data indicate that DGKθ translocation and activity is regulated by the concerted activity of EGFR and PKC and that DGKθ attenuates PKC-mediated Thr654 phosphorylation that is linked to desensitisation of EGFR signaling.
Assuntos
Diacilglicerol Quinase/metabolismo , Receptores ErbB/metabolismo , Proteína Quinase C/metabolismo , Bradicinina/farmacologia , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Ativação Enzimática/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Fosforilação/efeitos dos fármacos , Fosfotreonina/metabolismo , Fosfotirosina/metabolismo , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Uridina Trifosfato/farmacologiaRESUMO
Diacylglycerol kinases (DGKs) metabolize diacylglycerol to phosphatidic acid. In T lymphocytes, DGKα acts as a negative regulator of TCR signaling by decreasing diacylglycerol levels and inducing anergy. In this study, we show that upon costimulation of the TCR with CD28 or signaling lymphocyte activation molecule (SLAM), DGKα, but not DGKζ, exits from the nucleus and undergoes rapid negative regulation of its enzymatic activity. Inhibition of DGKα is dependent on the expression of SAP, an adaptor protein mutated in X-linked lymphoproliferative disease, which is essential for SLAM-mediated signaling and contributes to TCR/CD28-induced signaling and T cell activation. Accordingly, overexpression of SAP is sufficient to inhibit DGKα, whereas SAP mutants unable to bind either phospho-tyrosine residues or SH3 domain are ineffective. Moreover, phospholipase C activity and calcium, but not Src-family tyrosine kinases, are also required for negative regulation of DGKα. Finally, inhibition of DGKα in SAP-deficient cells partially rescues defective TCR/CD28 signaling, including Ras and ERK1/2 activation, protein kinase C membrane recruitment, induction of NF-AT transcriptional activity, and IL-2 production. Thus SAP-mediated inhibition of DGKα sustains diacylglycerol signaling, thereby regulating T cell activation, and it may represent a novel pharmacological strategy for X-linked lymphoproliferative disease treatment.
Assuntos
Diacilglicerol Quinase/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ativação Linfocitária/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/imunologia , Western Blotting , Diglicerídeos/metabolismo , Imunofluorescência , Humanos , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Células Jurkat , Transporte Proteico/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Proteína Associada à Molécula de Sinalização da Ativação Linfocitária , Linfócitos T/imunologia , Linfócitos T/metabolismo , TransfecçãoRESUMO
S49 mouse lymphoma cells undergo apoptosis in response to the ALP (alkyl-lysophospholipid) edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine), FasL (Fas ligand) and DNA damage. S49 cells made resistant to ALP (S49(AR)) are defective in sphingomyelin synthesis and ALP uptake, and also have acquired resistance to FasL and DNA damage. However, these cells can be re-sensitized following prolonged culturing in the absence of ALP. The resistant cells show sustained ERK (extracellular-signal-regulated kinase)/Akt activity, consistent with enhanced survival signalling. In search of a common mediator of the observed cross-resistance, we found that S49(AR) cells lacked the PtdIns(3,4,5)P(3) phosphatase SHIP-1 [SH2 (Src homology 2)-domain-containing inositol phosphatase 1], a known regulator of the Akt survival pathway. Re-sensitization of the S49(AR) cells restored SHIP-1 expression as well as phosphoinositide and sphingomyelin levels. Knockdown of SHIP-1 mimicked the S49(AR) phenotype in terms of apoptosis cross-resistance, sphingomyelin deficiency and altered phosphoinositide levels. Collectively, the results of the present study suggest that SHIP-1 collaborates with sphingomyelin synthase to regulate lymphoma cell death irrespective of the nature of the apoptotic stimulus.
Assuntos
Éteres Fosfolipídicos/farmacologia , Monoéster Fosfórico Hidrolases/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Dano ao DNA , Regulação para Baixo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteína Ligante Fas/metabolismo , Inositol Polifosfato 5-Fosfatases , Linfoma/patologia , Camundongos , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismoRESUMO
For amphiphilic anticancer drugs, such as the anthracyclin doxorubicin (Dox), uptake by tumor cells involves slow diffusion across the plasma membrane, a limiting factor in clinical oncology. Previously, we discovered that preinsertion of short-chain sphingolipids such as N-octanoyl-glucosylceramide (GC) in the tumor cell membrane enhances cellular Dox uptake. In the present study, we apply this strategy in vitro and in vivo by coadministering GC and Dox in a lipid nanovesicle (LNV). GC enrichment of Dox-LNVs strongly enhanced in vitro cytotoxicity toward B16 melanoma and A431 carcinoma, as evidenced by 6-fold decreased IC(50) values compared with Dox-LNVs. This correlated with enhanced cellular Dox uptake observed by confocal microscopy. Intravital optical imaging in window chamber-bearing mice with orthotopically implanted B16 melanoma demonstrated enhanced GC-mediated Dox delivery to tumor cells. Treatment of nude mice bearing human A431 xenografts with 6 mg/kg GC-Dox-LNVs almost doubled the tumor growth delay compared with Dox-LNVs. A second administration of 5 mg/kg after 3 d induced even 3-fold delay in tumor growth, while no systemic toxicity was found. GC-enriched Dox-LNVs displayed superior in vitro and in vivo antitumor activity, without systemic toxicity. This new drug delivery concept, aiming at increased membrane permeability for amphiphilic drugs, provides an opportunity to improve cancer chemotherapy.
Assuntos
Doxorrubicina/farmacologia , Glucosilceramidas/química , Nanoestruturas/química , Neoplasias/tratamento farmacológico , Animais , Antibióticos Antineoplásicos/administração & dosagem , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Doxorrubicina/administração & dosagem , Doxorrubicina/química , Sistemas de Liberação de Medicamentos/métodos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Microscopia Confocal , Neoplasias/patologia , Resultado do Tratamento , Lipossomas Unilamelares/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Gossypol, a naturally occurring polyphenolic compound has been identified as a small molecule inhibitor of anti-apoptotic Bcl-2 family proteins. It induces apoptosis in a wide range of tumor cell lines and enhances chemotherapy- and radiation-induced cytotoxicity both in vitro and in vivo. Bcl-2 and related proteins are important inhibitors of apoptosis and frequently overexpressed in human tumors. Increased levels of these proteins confer radio- and chemoresistance and may be associated with poor prognosis. Consequently, inhibition of the anti-apoptotic functions of Bcl-2 family members represents a promising strategy to overcome resistance to anticancer therapies. METHODS: We tested the effect of (-)-gossypol, also denominated as AT-101, radiation and the combination of both on apoptosis induction in human leukemic cells, Jurkat T and U937. Because activation of the SAPK/JNK pathway is important for apoptosis induction by many different stress stimuli, and Bcl-X(L) is known to inhibit activation of SAPK/JNK, we also investigated the role of this signaling cascade in AT-101-induced apoptosis using a pharmacologic and genetic approach. RESULTS: AT-101 induced apoptosis in a time- and dose-dependent fashion, with ED50 values of 1.9 and 2.4 microM in Jurkat T and U937 cells, respectively. Isobolographic analysis revealed a synergistic interaction between AT-101 and radiation, which also appeared to be sequence-dependent. Like radiation, AT-101 activated SAPK/JNK which was blocked by the kinase inhibitor SP600125. In cells overexpressing a dominant-negative mutant of c-Jun, AT-101-induced apoptosis was significantly reduced. CONCLUSION: Our data show that AT-101 strongly enhances radiation-induced apoptosis in human leukemic cells and indicate a requirement for the SAPK/JNK pathway in AT-101-induced apoptosis. This type of apoptosis modulation may overcome treatment resistance and lead to the development of new effective combination therapies.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Gossipol/análogos & derivados , Proteínas Quinases JNK Ativadas por Mitógeno/efeitos dos fármacos , Proteínas Quinases JNK Ativadas por Mitógeno/efeitos da radiação , Apoptose/efeitos da radiação , Western Blotting , Linhagem Celular Tumoral , Terapia Combinada , Relação Dose-Resposta à Radiação , Gossipol/farmacologia , Humanos , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Radiossensibilizantes/farmacologia , Radioterapia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiaçãoRESUMO
The ALP (alkyl-lysophospholipid) edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine) induces apoptosis in S49 mouse lymphoma cells. A variant cell line, S49AR, made resistant to ALP, was found previously to be impaired in ALP uptake via lipid-raft-mediated endocytosis. In the present paper, we report that these cells display cross-resistance to Fas/CD95 ligation [FasL (Fas ligand)], and can be gradually resensitized by prolonged culturing in the absence of ALP. Fas and ALP activate distinct apoptotic pathways, since ALP-induced apoptosis was not abrogated by dominant-negative FADD (Fas-associated protein with death domain), cFLIP(L) [cellular FLICE (FADD-like interleukin 1beta-converting enzyme)-inhibitory protein long form] or the caspase 8 inhibitor Z-IETD-FMK (benzyloxycarbonyl-Ile-Glu-Thr-Asp-fluoromethylketone). ALP-resistant cells showed decreased Fas expression, at both the mRNA and protein levels, in a proteasome-dependent fashion. The proteasome inhibitor MG132 partially restored Fas expression and resensitized the cells to FasL, but not to ALP. Resistant cells completely lacked SM (sphingomyelin) synthesis, which seems to be a unique feature of the S49 cell system, having very low SM levels in parental cells. Lack of SM synthesis did not affect cell growth in serum-containing medium, but retarded growth under serum-free (SM-free) conditions. SM deficiency determined in part the resistance to ALP and FasL. Exogenous short-chain (C12-) SM partially restored cell-surface expression of Fas in lipid rafts and FasL sensitivity, but did not affect Fas mRNA levels or ALP sensitivity. We conclude that the acquired resistance of S49 cells to ALP is associated with down-regulated SM synthesis and Fas gene transcription and that SM in lipid rafts stabilizes Fas expression at the cell surface.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Lisofosfolipídeos/farmacologia , Esfingomielinas/metabolismo , Receptor fas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo , Proteína Ligante Fas/farmacologia , Proteína de Domínio de Morte Associada a Fas/genética , Proteína de Domínio de Morte Associada a Fas/metabolismo , Citometria de Fluxo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Immunoblotting , Leupeptinas/farmacologia , Linfoma/genética , Linfoma/metabolismo , Linfoma/patologia , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/metabolismo , Camundongos , Microscopia Confocal , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esfingomielinas/deficiência , Transfecção , Receptor fas/genéticaRESUMO
Synthetic anticancer alkylphospholipids (APLs), such as edelfosine, miltefosine and perifosine, are a group of structurally related lipids that act on cellular membranes rather than the DNA. APLs have essentially one long hydrocarbon chain that allows easy partitioning into membrane lipid bilayers, but they resist catabolic degradation. APLs therefore accumulate in cell membranes and can interfere with normal lipid metabolism and lipid-dependent signal transduction. This action, often leading to apoptosis, is most effective in metabolically active, proliferating cells, such as cancer cells, but not in quiescent normal cells. This review describes the general mechanisms of APL cellular uptake and action. Most important for their biological effect are the inhibition of phosphatidylcholine synthesis, the inhibition of the MAP-kinase/ERK proliferative and phosphatidylinositol 3-kinase/ Akt survival pathways and the stimulation of the Stress-activated protein kinase/JNK pathway, which may lead to apoptosis in cancer cells. APLs are most promising in combination with conventional cancer therapies. For example, ALPs increase the cancer cell sensitivity to radiotherapy in vitro and in vivo. We highlight the clinical potential of perifosine, an orally available APL.
Assuntos
Antineoplásicos/farmacologia , Neoplasias/tratamento farmacológico , Éteres Fosfolipídicos/farmacologia , Animais , Antineoplásicos/farmacocinética , Apoptose/efeitos dos fármacos , Transporte Biológico , Ensaios Clínicos como Assunto , Terapia Combinada , Resistencia a Medicamentos Antineoplásicos , Humanos , Neoplasias/fisiopatologia , Neoplasias/radioterapia , Éteres Fosfolipídicos/farmacocinética , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacocinética , Fosforilcolina/farmacologia , Transdução de Sinais/efeitos dos fármacosAssuntos
Ciclo Celular/fisiologia , Diacilglicerol Quinase/fisiologia , Animais , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Fase G1/efeitos dos fármacos , Humanos , Camundongos , Osteossarcoma/metabolismo , Proteína do Retinoblastoma/metabolismo , Células Swiss 3T3RESUMO
Synthetic alkylphospholipids (APLs), such as edelfosine, miltefosine and perifosine, constitute a new class of antineoplastic compounds with various clinical applications. Here we have evaluated the antiangiogenic properties of APLs. The sensitivity of three types of vascular endothelial cells (ECs) (bovine aortic ECs, human umbilical vein ECs and human microvascular ECs) to APL-induced apoptosis was dependent on the proliferative status of these cells and correlated with the cellular drug incorporation. Although confluent, nondividing ECs failed to undergo apoptosis, proliferating ECs showed a 3-4-fold higher uptake and significant levels of apoptosis after APL treatment. These findings raised the question of whether APLs interfere with new blood vessel formation. To test the antiangiogenic properties in vitro, we studied the effect of APLs using two different experimental models. The first one tested the ability of human microvascular ECs to invade a three-dimensional human fibrin matrix and form capillary-like tubular networks. In the second model, bovine aortic ECs were grown in a collagen gel sandwich to allow tube formation. We found that all three APLs interfered with endothelial tube formation in a dose-dependent manner, with a more than 50% reduction at 25 micromol/l. Interference with the angiogenic process represents a novel mode of action of APLs and might significantly contribute to the antitumor effect of these compounds.
Assuntos
Inibidores da Angiogênese/farmacologia , Antineoplásicos/farmacologia , Endotélio Vascular/efeitos dos fármacos , Fosfolipídeos/farmacologia , Alquilação , Inibidores da Angiogênese/química , Inibidores da Angiogênese/metabolismo , Animais , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Capilares/efeitos dos fármacos , Capilares/crescimento & desenvolvimento , Bovinos , Linhagem Celular Tumoral , Células Cultivadas , Relação Dose-Resposta a Droga , Células Endoteliais/efeitos dos fármacos , Endotélio Vascular/citologia , Endotélio Vascular/crescimento & desenvolvimento , Humanos , Indicadores e Reagentes , Inibidores de Fosfodiesterase/farmacologia , Éteres Fosfolipídicos/farmacologia , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacologiaRESUMO
Diacylglycerol kinases (Dgk) phosphorylate diacylglycerol (DG) to phosphatidic acid (PA), thus turning off and on, respectively, DG-mediated and PA-mediated signaling pathways. We previously showed that hepatocyte growth factor (HGF), vascular endothelial growth factor, and anaplastic lymphoma kinase activate Dgkalpha in endothelial and leukemia cells through a Src-mediated mechanism and that activation of Dgkalpha is required for chemotactic, proliferative, and angiogenic signaling in vitro. Here, we investigate the downstream events and signaling pathways regulated by Dgkalpha, leading to cell scatter and migration upon HGF treatment and v-Src expression in epithelial cells. We report that specific inhibition of Dgkalpha, obtained either pharmacologically by R59949 treatment, or by expression of Dgkalpha dominant-negative mutant, or by small interfering RNA-mediated down-regulation of endogenous Dgkalpha, impairs 1) HGF- and v-Src-induced cell scatter and migration, without affecting the loss of intercellular adhesions; 2) HGF-induced cell spreading, lamellipodia formation, membrane ruffling, and focal adhesions remodeling; and 3) HGF-induced Rac activation and membrane targeting. In summary, we provide evidence that Dgkalpha, activated downstream of tyrosine kinase receptors and Src, regulates crucial steps directing Rac activation and Rac-dependent remodeling of actin cytoskeleton and focal contacts in migrating epithelial cells.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Diacilglicerol Quinase/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Fator de Crescimento de Hepatócito/farmacologia , Proteínas rac de Ligação ao GTP/metabolismo , Animais , Caderinas/metabolismo , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Diacilglicerol Quinase/genética , Regulação para Baixo/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Células Epiteliais/citologia , Humanos , Ligação ProteicaRESUMO
Perifosine is a member of the class of synthetic alkylphospholipids (APLs) and is being evaluated as anti-cancer agent in several clinical trials. These single-chain APLs accumulate in cellular membranes and disturb lipid-dependent signal transduction, ultimately causing apoptosis in a variety of tumor cells. The APL prototype edelfosine was previously found to be endocytosed by S49 mouse lymphoma cells via lipid rafts. An edelfosine-resistant cell variant, S49(AR), was found to be cross-resistant to other APLs, including perifosine. This resistance was due to defective synthesis of the raft constituent sphingomyelin, which abrogated APL cellular uptake. Sensitivity of S49 cells to edelfosine was higher than perifosine, which correlated with a relatively higher uptake. Human KB epidermal carcinoma cells were much more sensitive to APLs than S49 cells. Their much higher APL uptake was highly dependent on intracellular ATP and ambient temperature, and was blocked by chlorpromazine, independent of canonical endocytic pathways. We found no prominent role of lipid rafts for APL uptake in these KB cells; contrary to S49(AR) cells, perifosine-resistant KBr cells display normal sphingomyelin synthesis, whereas APL uptake by the responsive KB cells was insensitive to treatment with methyl-beta-cyclodextrin, a cholesterol-sequestrator and inhibitor of raft-mediated endocytosis. In conclusion, different mechanisms determine APL uptake and consequent apoptotic toxicity in lymphoma versus carcinoma cells. In the latter cells, APL uptake is mainly determined by a raft- and endocytosis-independent process, but metabolic energy-dependent process, possibly by a lipid transporter.
Assuntos
Antineoplásicos/farmacologia , Carcinoma de Células Escamosas/metabolismo , Resistencia a Medicamentos Antineoplásicos , Linfoma/metabolismo , Microdomínios da Membrana/metabolismo , Éteres Fosfolipídicos/farmacologia , Fosforilcolina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Animais , Apoptose , Linhagem Celular Tumoral , Humanos , Células KB , Camundongos , Fosfolipídeos/metabolismo , Fosforilcolina/farmacologiaRESUMO
Single-chain alkylphospholipids, unlike conventional chemotherapeutic drugs, act on cell membranes to induce apoptosis in tumor cells. We tested four different alkylphospholipids, i.e., edelfosine, perifosine, erucylphosphocholine, and compound D-21805, as inducers of apoptosis in the mouse lymphoma cell line S49. We compared their mechanism of cellular entry and their potency to induce apoptosis through inhibition of de novo biosynthesis of phosphatidylcholine at the endoplasmic reticulum. Alkylphospholipid potency closely correlated with the degree of phosphatidylcholine synthesis inhibition in the order edelfosine > D-21805 > erucylphosphocholine > perifosine. In all cases, exogenous lysophosphatidylcholine, an alternative source for cellular phosphatidylcholine production, could partly rescue cells from alkylphospholipid-induced apoptosis, suggesting that phosphatidylcholine biosynthesis is a direct target for apoptosis induction. Cellular uptake of each alkylphospholipid was dependent on lipid rafts because pretreatment of cells with the raft-disrupting agents, methyl-beta-cyclodextrin, filipin, or bacterial sphingomyelinase, reduced alkylphospholipid uptake and/or apoptosis induction and alleviated the inhibition of phosphatidylcholine synthesis. Uptake of all alkylphospholipids was inhibited by small interfering RNA (siRNA)-mediated blockage of sphingomyelin synthase (SMS1), which was previously shown to block raft-dependent endocytosis. Similar to edelfosine, perifosine accumulated in (isolated) lipid rafts independent on raft sphingomyelin content per se. However, perifosine was more susceptible than edelfosine to back-extraction by fatty acid-free serum albumin, suggesting a more peripheral location in the cell due to less effective internalization. Overall, our results suggest that lipid rafts are critical membrane portals for cellular entry of alkylphospholipids depending on SMS1 activity and, therefore, are potential targets for alkylphospholipid anticancer therapy.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linfoma/patologia , Microdomínios da Membrana/efeitos dos fármacos , Fosfolipídeos/farmacologia , Animais , Antineoplásicos/química , Linhagem Celular Tumoral , Endocitose/efeitos dos fármacos , Células HeLa , Humanos , Camundongos , Fosfatidilcolinas/biossíntese , Éteres Fosfolipídicos/química , Éteres Fosfolipídicos/farmacologia , Fosfolipídeos/química , Fosforilcolina/análogos & derivados , Fosforilcolina/química , Fosforilcolina/farmacologia , Esfingomielinas/biossíntese , Fatores de TempoRESUMO
Concurrent treatment with radiotherapy and chemotherapy has emerged as an effective strategy to improve clinical outcome of cancer. In addition to combining radiation with classical anticancer agents, several new biological response modifiers are under investigation in pre-clinical and clinical studies. Synthetic alkylphospholipids are anticancer agents that in contrast to most anticancer drugs, do not target DNA, but insert in the plasma membrane and subsequently induce a broad range of biological effects, ultimately leading to cell death. Alkylphospholipids kill tumor cells directly by induction of both apoptotic and non-apoptotic cell death, and indirectly by interference with critical signal transduction pathways involved in phospholipid metabolism and survival. Due to their distinct mode of action, these drugs are considered as attractive candidates to combine with radiotherapy. In this review, we will discuss several alkylphospholipids that reached clinical application. These include first-generation alkyl-lysophospholipids edelfosine and ilmofosine, second-generation alkylphosphocholine-prototype miltefosine and more recently developed analogues perifosine and erucylphosphocholine. We focus on mechanisms of action and the rationale to combine these agents with radiotherapy. The preclinical results on molecular targeting underlying this approach will be reviewed, concluded with first clinical data on combined treatment of radiotherapy with perifosine.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias/tratamento farmacológico , Neoplasias/radioterapia , Éteres Fosfolipídicos/uso terapêutico , Fosforilcolina/uso terapêutico , Terapia Combinada , Humanos , Fosforilcolina/análogos & derivadosRESUMO
We previously showed that the retinoblastoma protein (pRB), a key regulator of G1 to S-phase transition of the cell cycle, binds to and stimulates diacylglycerol kinase-zeta (DGKzeta) to phosphorylate the lipid second messenger diacylglycerol into phosphatidic acid. pRB binds to the MARCKS phosphorylation-site domain of DGKzeta that can be phosphorylated by protein kinase C (PKC). Here, we report that activation of PKC by phorbol ester inhibits DGKzeta binding to pRB. Ro 31-8220, a specific inhibitor of PKC, alleviated this inhibition of binding. Mimicking of PKC phosphorylation of serine residues (by S/D but not S/N mutations) within the DGKzeta-MARCKS phosphorylation-site domain also prevented DGKzeta binding to pRB, suggesting that PKC phosphorylation of these residues negatively regulates the interaction between DGKzeta and pRB. In PKC overexpression studies, it appeared that activation of particularly the (wild-type) PKCalpha isoform inhibits DGKzeta binding to pRB, whereas dominant-negative PKCalpha neutralized this inhibition. PKCalpha activation thus prevents DGKzeta regulation by pRB, which may have implications for nuclear diacylglycerol and phosphatidic acid levels during the cell cycle.
Assuntos
Diacilglicerol Quinase/metabolismo , Proteína Quinase C/metabolismo , Proteína do Retinoblastoma/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Chlorocebus aethiops , Diacilglicerol Quinase/química , Diacilglicerol Quinase/classificação , Diacilglicerol Quinase/genética , Ativação Enzimática , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Isoenzimas/classificação , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Mutação/genética , Substrato Quinase C Rico em Alanina Miristoilada , Fosfosserina/metabolismo , Ligação Proteica , Proteína Quinase C/genética , Proteína do Retinoblastoma/genéticaRESUMO
The ALP (alkyl-lysophospholipid) edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine; Et-18-OCH3) induces apoptosis in S49 mouse lymphoma cells. To this end, ALP is internalized by lipid raft-dependent endocytosis and inhibits phosphatidylcholine synthesis. A variant cell-line, S49AR, which is resistant to ALP, was shown previously to be unable to internalize ALP via this lipid raft pathway. The reason for this uptake failure is not understood. In the present study, we show that S49AR cells are unable to synthesize SM (sphingomyelin) due to down-regulated SMS1 (SM synthase 1) expression. In parental S49 cells, resistance to ALP could be mimicked by small interfering RNA-induced SMS1 suppression, resulting in SM deficiency and blockage of raft-dependent internalization of ALP and induction of apoptosis. Similar results were obtained by treatment of the cells with myriocin/ISP-1, an inhibitor of general sphingolipid synthesis, or with U18666A, a cholesterol homoeostasis perturbing agent. U18666A is known to inhibit Niemann-Pick C1 protein-dependent vesicular transport of cholesterol from endosomal compartments to the trans-Golgi network and the plasma membrane. U18666A reduced cholesterol partitioning in detergent-resistant lipid rafts and inhibited SM synthesis in S49 cells, causing ALP resistance similar to that observed in S49AR cells. The results are explained by the strong physical interaction between (newly synthesized) SM and available cholesterol at the Golgi, where they facilitate lipid raft formation. We propose that ALP internalization by lipid-raft-dependent endocytosis represents the retrograde route of a constitutive SMS1- and lipid-raft-dependent membrane vesicular recycling process.
Assuntos
Apoptose/efeitos dos fármacos , Microdomínios da Membrana/química , Éteres Fosfolipídicos/farmacologia , Transferases (Outros Grupos de Fosfato Substituídos)/biossíntese , Androstenos/farmacologia , Animais , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Colesterol/análise , Colesterol/metabolismo , Regulação para Baixo , Endocitose/efeitos dos fármacos , Ácidos Graxos Monoinsaturados/farmacologia , Expressão Gênica/efeitos dos fármacos , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/metabolismo , Camundongos , Norbornanos , Éteres Fosfolipídicos/metabolismo , RNA Interferente Pequeno/farmacologia , Esfingomielinas/análise , Esfingomielinas/metabolismo , Tiocarbamatos , Tionas/farmacologia , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Células Tumorais CultivadasRESUMO
PURPOSE: Combined modality treatment has improved outcome in various solid tumors. Besides classic anticancer drugs, a new generation of biological response modifiers has emerged that increases the efficacy of radiation. Here, we have investigated whether perifosine, an orally applicable, membrane-targeted alkylphospholipid, enhances the antitumor effect of radiation in vitro and in vivo. EXPERIMENTAL DESIGN: Several long-term and short-term in vitro assays (clonogenic survival, sulforhodamine B cytotoxicity, apoptosis, and cell cycle analysis) were used to assess the cytotoxic effect of perifosine in combination with radiation. In vivo, the response of human KB squamous cell carcinoma xenografts was measured after treatment with perifosine, irradiation, and the combination. Radiolabeled perifosine was used to determine drug disposition in tumor and normal tissues. At various intervals after treatment, tumor specimens were collected to document histopathologic changes. RESULTS: In vitro, perifosine reduced clonogenic survival, enhanced apoptosis, and increased cell cycle arrest after radiation. In vivo, radiation and perifosine alone induced a dose-dependent tumor growth delay. When combining multiple perifosine administrations with single or split doses of radiation, complete and sustained tumor regression was observed. Histopathologic analysis of tumor specimens revealed a prominent apoptotic response after combined treatment with radiation and perifosine. Radiation-enhanced tumor response was observed at clinically relevant plasma perifosine concentrations and accumulating drug disposition of >100 microg/g in tumor tissue. CONCLUSIONS: Perifosine enhances radiation-induced cytotoxicity, as evidenced by reduced clonogenic survival and increased apoptosis induction in vitro and by complete tumor regression in vivo. These data provide strong support for further development of this combination in clinical studies.
Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Fosforilcolina/análogos & derivados , Radiossensibilizantes/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/radioterapia , Terapia Combinada , Feminino , Fase G2/efeitos dos fármacos , Fase G2/efeitos da radiação , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias de Cabeça e Pescoço/radioterapia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fosforilcolina/farmacocinética , Fosforilcolina/uso terapêutico , Radiossensibilizantes/farmacocinética , Rodaminas/metabolismo , Transplante Heterólogo , Células Tumorais Cultivadas , Ensaio Tumoral de Célula-Tronco , Raios XRESUMO
The retinoblastoma protein (pRB) is a tumor suppressor and key regulator of the cell cycle. We have previously shown that pRB interacts with phosphatidylinositol-4-phosphate 5-kinases, lipid kinases that can regulate phosphatidylinositol 4,5-bisphosphate levels in the nucleus. Here, we investigated pRB binding to another lipid kinase in the phosphoinositide cycle, diacylglycerol kinase (DGK) that phosphorylates the second messenger diacylglycerol to yield phosphatidic acid. We found that DGKzeta, but not DGKalpha or DGK, interacts with pRB in vitro and in vivo. Binding of DGKzeta to pRB is dependent on the phosphorylation status of pRB, since only hypophosphorylated pRB interacts with DGKzeta. DGKzeta also binds to the pRB-related pocket proteins p107 and p130 in vitro and in cells. Although DGKzeta did not affect the ability of pRB to regulate E2F-mediated transcription, we found that pRB, p107, and p130 potently stimulate DGKzeta activity in vitro. Finally, overexpression of DGKzeta in pRB-null fibroblasts reconstitutes a cell cycle arrest induced by gamma-irradiation. These results suggest that DGKzeta may act in vivo as a downstream effector of pRB to regulate nuclear levels of diacylglycerol and phosphatidic acid.