RESUMO
Generation of patient-specific induced pluripotent cells (iPSCs) holds great promise for regenerative medicine. Epstein-Barr virus immortalized lymphoblastoid B-cell lines (LCLs) can be generated from a minimal amount of blood and are banked worldwide as cellular reference material for immunologic or genetic analysis of pedigreed study populations. We report the generation of iPSCs from 2 LCLs (LCL-iPSCs) via a feeder-free episomal method using a cocktail of transcription factors and small molecules. LCL-derived iPSCs exhibited normal karyotype, expressed pluripotency markers, lost oriP/EBNA-1 episomal vectors, generated teratomas, retained donor identity, and differentiated in vitro into hematopoietic, cardiac, neural, and hepatocyte-like lineages. Significantly, although the parental LCLs express viral EBNA-1 and other Epstein-Barr virus latency-related elements for their survival, their presence was not detectable in LCL-iPSCs. Thus, reprogramming LCLs could offer an unlimited source for patient-specific iPSCs.
Assuntos
Linfócitos B/citologia , Linfócitos B/virologia , Herpesvirus Humano 4/isolamento & purificação , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/virologia , Linfócitos B/metabolismo , Diferenciação Celular , Linhagem Celular , Antígenos Nucleares do Vírus Epstein-Barr/genética , Vetores Genéticos/genética , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Fatores de Transcrição/metabolismo , TransfecçãoRESUMO
Epstein-Barr virus (EBV) infects cells in latent or lytic forms, but the role of lytic infection in EBV-induced lymphomas is unclear. Here, we have used a new humanized mouse model, in which both human fetal CD34(+) hematopoietic stem cells and thymus/liver tissue are transplanted, to compare EBV pathogenesis and lymphoma formation following infection with a lytic replication-defective BZLF1-deleted (Z-KO) virus or a lytically active BZLF1(+) control. Both the control and Z-KO viruses established long-term viral latency in all infected animals. The infection appeared well controlled in some animals, but others eventually developed CD20(+) diffuse large B cell lymphomas (DLBCL). Animals infected with the control virus developed tumors more frequently than Z-KO virus-infected animals. Specific immune responses against EBV-infected B cells were generated in mice infected with either the control virus or the Z-KO virus. In both cases, forms of viral latency (type I and type IIB) were observed that are less immunogenic than the highly transforming form (type III) commonly found in tumors of immunocompromised hosts, suggesting that immune pressure contributed to the outcome of the infection. These results point to an important role for lytic EBV infection in the development of B cell lymphomas in the context of an active host immune response.
Assuntos
Infecções por Vírus Epstein-Barr/imunologia , Infecções por Vírus Epstein-Barr/patologia , Herpesvirus Humano 4/patogenicidade , Linfoma de Células B/patologia , Linfoma de Células B/virologia , Proteínas Virais/metabolismo , Animais , Antígenos CD34/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Infecções por Vírus Epstein-Barr/virologia , Transplante de Células-Tronco Hematopoéticas , Herpesvirus Humano 4/genética , Humanos , Transplante de Fígado , Linfoma de Células B/imunologia , Camundongos , Camundongos Knockout , Linfócitos T/imunologia , Timo/transplante , Transativadores/genética , Transplante Heterólogo , Proteínas Virais/genética , Latência ViralRESUMO
Macrophages are innate immune cells that play critical roles in tissue homeostasis, inflammation, and immune oncology. Macrophages differentiated from human induced pluripotent stem cells (iPSCs) overcome many limitations of using peripheral blood derived macrophages. The ability to scale up and cryopreserve a large amount of end stage macrophages from single clonal iPSCs from normal and disease specific donors offers a unique opportunity for genomic analysis and drug screening. The present study describes the step wise generation and characterization of macrophages from iPSCs using a defined serum free method amenable to scale up to generate a large batch of pure end stage cryopreservable macrophages expressing CD68, CD33, CD11c, CD11b, CD1a, HLA-DR, CD86, CD64, CD80, CD206, CD169, CD47, HLA-ABC, and CX3CR. The end stage macrophages pre and post cryopreservation retain purity, morphology, responsiveness to stimuli and display robust phagocytic function coming right out of cryopreservation. The same differentiation process was used to generate end stage macrophages from isogenic iPSCs engineered to mimic mutations associated with Parkinson's disease (SNCA A53T), neuronal ceroid lipofuscinosis (GRN2/GRN R493X), and Rett syndrome (MECP2-Knockout). End stage macrophages from isogenic engineered clones displayed differential macrophage-specific purity markers, phagocytic function, and response to specific stimuli. Thus, generating a panel of functional, physiologically relevant iPSC-derived macrophages can potentially facilitate the understanding of neural inflammatory responses associated with neurodegeneration.
Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Pluripotentes Induzidas/citologia , Macrófagos/citologia , Antígenos CD/metabolismo , Biomarcadores/metabolismo , Criopreservação , Humanos , Macrófagos/metabolismoRESUMO
The failure of engraftment in human cases of in utero hematopoietic cell transplantation (IUHCT) in which no immunodeficiency exists suggests the presence of an unrecognized fetal immune barrier. A similar barrier in murine IUHCT appears to be dependent on the chimerism level and is poorly explained by a lack of T-cell tolerance induction. Therefore, we studied the effect of the chimerism level on engraftment and host natural killer (NK)-cell education in a murine model of IUHCT. The dose of transplanted cells was found to exhibit a strong correlation with both the engraftment rate and chimerism level. More specifically, a threshold level of initial chimerism (> 1.8%) was identified that predicted durable engraftment for allogeneic IUHCT, whereas low initial chimerism (< 1.8%) predicted a loss of engraftment. NK cells taken from chimeras above the "chimerism threshold" displayed durable calibration of alloresponsive Ly49A receptors and tolerance to donor antigens. Depletion of recipient NK cells stabilized engraftment in low-level chimeras (< 1.8%). These studies illustrate the importance of the early chimerism threshold in predicting long-term engraftment and host NK-cell tolerance after in utero transplantation.
Assuntos
Feto/cirurgia , Sobrevivência de Enxerto , Transplante de Células-Tronco Hematopoéticas/métodos , Tolerância Imunológica , Células Matadoras Naturais/imunologia , Quimeras de Transplante , Animais , Feminino , Doenças Fetais/terapia , Camundongos , Modelos Animais , Subfamília A de Receptores Semelhantes a Lectina de Células NK , Gravidez , Transplante HomólogoRESUMO
Non-alcoholic fatty liver disease (NAFLD) affects 30-40% of adults and 10% of children in the US. About 20% of people with NAFLD develop non-alcoholic steatohepatitis (NASH), which may lead to cirrhosis and liver cancer, and is projected to be a leading cause of liver transplantation in the near future. Human induced pluripotent stem cells (iPSC) from NASH patients are useful for generating a large number of hepatocytes for NASH modeling applications and identification of potential drug targets. We developed a novel defined in vitro differentiation process to generate cryopreservable hepatocytes using an iPSC panel of NASH donors and apparently healthy normal (AHN) controls. iPSC-derived hepatocytes displayed stage specific phenotypic markers, hepatocyte morphology, with bile canaliculi. Importantly, both fresh and cryopreserved definitive endoderm and hepatoblasts successfully differentiated to pure and functional hepatocytes with increased CYP3A4 activity in response to rifampicin and lipid accumulation upon fatty acid (FA) treatment. End-stage hepatocytes integrated into three-dimensional (3D) liver organoids and demonstrated increased levels of albumin secretion compared to aggregates consisting of hepatocytes alone. End-stage hepatocytes derived from NASH donors demonstrated spontaneous lipidosis without FA supplementation, recapitulating a feature of NASH hepatocytes in vivo Cryopreserved hepatocytes generated by this protocol across multiple donors will provide a critical cell source to facilitate the fundamental understanding of NAFLD/NASH biology and potential high throughput screening applications for preclinical evaluation of therapeutic targets.
Assuntos
Descoberta de Drogas , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/citologia , Modelos Moleculares , Biomarcadores , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Criopreservação/métodos , Descoberta de Drogas/métodos , Endoderma/citologia , Citometria de Fluxo , Hepatócitos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Hepatopatia Gordurosa não AlcoólicaRESUMO
PURPOSE: We present preclinical data showing the in vitro intranuclear uptake of motexafin gadolinium by glioblastoma multiforme cells, which could serve as a prelude to the future development of radiosensitizing techniques, such as gadolinium synchrotron stereotactic radiotherapy (GdSSR), a new putative treatment for glioblastoma multiforme. EXPERIMENTAL DESIGN: In this approach, administration of a tumor-seeking Gd-containing compound would be followed by stereotactic external beam radiotherapy with 51-keV photons from a synchrotron source. At least two criteria must be satisfied before this therapy can be established: Gd must accumulate in cancer cells and spare the normal tissue; Gd must be present in almost all the cancer cell nuclei. We address the in vitro intranuclear uptake of motexafin gadolinium in this article. We analyzed the Gd distribution with subcellular resolution in four human glioblastoma cell lines, using three independent methods: two novel synchrotron spectromicroscopic techniques and one confocal microscopy. We present in vitro evidence that the majority of the cell nuclei take up motexafin gadolinium, a drug that is known to selectively reach glioblastoma multiforme. RESULTS: With all three methods, we found Gd in at least 90% of the cell nuclei. The results are highly reproducible across different cell lines. The present data provide evidence for further studies, with the goal of developing GdSSR, a process that will require further in vivo animal and future clinical studies.
Assuntos
Neoplasias Encefálicas/metabolismo , Núcleo Celular/metabolismo , Glioblastoma/metabolismo , Metaloporfirinas/farmacocinética , Radiossensibilizantes/farmacocinética , Neoplasias Encefálicas/diagnóstico , Linhagem Celular Tumoral , Glioblastoma/diagnóstico , Humanos , Técnicas In Vitro , Imageamento por Ressonância Magnética , Microscopia Confocal , Microscopia Eletrônica de Transmissão e VarreduraRESUMO
The poor prognosis of malignant glioma patients highlights the need to develop low toxicity, tumor specific agents with the ability to synergize with proven efficacious treatment modalities, e.g., ionizing irradiation. This paper investigates the potential of BNP1350 (karenitecin), a topoisomerase I-targeting anticancer agent, and flavopridol a cyclin-dependent kinase inhibitor as radiosensitizers at clinically relevant doses in glioblastoma cell lines. A clonogenic survival and apoptosis assays were performed to test the effect of karenitecin (0.1 nM to 10 nM), flavopridol, (50 nM to 500 nM), radiation (1 Gy to 5.5 Gy) and a combination of radiation and karenitecin or radiation and flavopridol on the glioma cell lines T986 and M059K. Cells were stained for cyclins B and D using antibodies followed by flow cytometry. Propidium Iodide staining was used to reveal the various phases of the cell cycle; cyclin staining in the G0/G1 and G2/M phase of the cell cycle was estimated as the Mean Fluorescence Intensity (MFI) after subtracting the MFI recorded by the isotype controls. Results demonstrated that in irradiated cells, pretreatment with karenitecin induced apoptosis, a transient arrest in the G2/M phase of the cell cycle and increased the expression of cyclin B1. Flavopridol treatment also induced apoptosis and a transient block in the G2/M phase of the cell cycle. The combined effects of karenitecin and flavopridol displayed synergistic effects. The unique radiosensitizing activity of orally administrable karenitecin and flavopridol is consistent with continued investigation of these compounds preclinically, as well as in the clinical setting.
RESUMO
OBJECTIVE: Gadolinium neutron capture therapy (GdNCT) is a potential treatment for malignant tumors based on two steps: (1) injection of a tumor-specific (157)Gd compound; (2) tumor irradiation with thermal neutrons. The GdNC reaction can induce cell death provided that Gd is proximate to DNA. Here, we studied the nuclear uptake of Gd by glioblastoma (GBM) tumor cells after treatment with two Gd compounds commonly used for magnetic resonance imaging, to evaluate their potential as GdNCT agents. METHODS: Using synchrotron X-ray spectromicroscopy, we analyzed the Gd distribution at the subcellular level in: (1) human cultured GBM cells exposed to Gd-DTPA or Gd-DOTA for 0-72 hours; (2) intracerebrally implanted C6 glioma tumors in rats injected with one or two doses of Gd-DOTA, and (3) tumor samples from GBM patients injected with Gd-DTPA. RESULTS: In cell cultures, Gd-DTPA and Gd-DOTA were found in 84% and 56% of the cell nuclei, respectively. In rat tumors, Gd penetrated the nuclei of 47% and 85% of the tumor cells, after single and double injection of Gd-DOTA, respectively. In contrast, in human GBM tumors 6.1% of the cell nuclei contained Gd-DTPA. DISCUSSION: Efficacy of Gd-DTPA and Gd-DOTA as GdNCT agents is predicted to be low, due to the insufficient number of tumor cell nuclei incorporating Gd. Although multiple administration schedules in vivo might induce Gd penetration into more tumor cell nuclei, a search for new Gd compounds with higher nuclear affinity is warranted before planning GdNCT in animal models or clinical trials.
Assuntos
Neoplasias Encefálicas/radioterapia , Gadolínio/uso terapêutico , Glioblastoma/radioterapia , Terapia por Captura de Nêutron/métodos , Radioisótopos/uso terapêutico , Animais , Mapeamento Encefálico , Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Gadolínio/farmacocinética , Glioblastoma/mortalidade , Glioblastoma/patologia , Compostos Heterocíclicos com 1 Anel/farmacocinética , Compostos Heterocíclicos com 1 Anel/uso terapêutico , Humanos , Angiografia por Ressonância Magnética/métodos , Imageamento por Ressonância Magnética/métodos , Espectrometria de Massas/métodos , Transplante de Neoplasias/métodos , Ácido Pentético/farmacocinética , Ácido Pentético/uso terapêutico , Radiografia/métodos , Cintilografia , Ratos , Fatores de Tempo , Distribuição TecidualRESUMO
The methodology to create induced pluripotent stem cells (iPSCs) affords the opportunity to generate cells specific to the individual providing the host tissue. However, existing methods of reprogramming as well as the types of source tissue have significant limitations that preclude the ability to generate iPSCs in a scalable manner from a readily available tissue source. We present the first study whereby iPSCs are derived in parallel from multiple donors using episomal, non-integrating, oriP/EBNA1-based plasmids from freshly drawn blood. Specifically, successful reprogramming was demonstrated from a single vial of blood or less using cells expressing the early lineage marker CD34 as well as from unpurified peripheral blood mononuclear cells. From these experiments, we also show that proliferation and cell identity play a role in the number of iPSCs per input cell number. Resulting iPSCs were further characterized and deemed free of transfected DNA, integrated transgene DNA, and lack detectable gene rearrangements such as those within the immunoglobulin heavy chain and T cell receptor loci of more differentiated cell types. Furthermore, additional improvements were made to incorporate completely defined media and matrices in an effort to facilitate a scalable transition for the production of clinic-grade iPSCs.
Assuntos
Antígenos CD34/metabolismo , Doadores de Sangue , Coleta de Amostras Sanguíneas , Vetores Genéticos/genética , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Plasmídeos/genética , Adulto , Proliferação de Células , Separação Celular , Reprogramação Celular , Feminino , Mobilização de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Indicadores e Reagentes , Masculino , Pessoa de Meia-Idade , Transfecção , Transgenes/genéticaRESUMO
Human ESC and iPSC are an attractive source of cells of high quantity and purity to be used to elucidate early human development processes, for drug discovery, and in clinical cell therapy applications. To efficiently differentiate pluripotent cells into a pure population of hematopoietic progenitors we have developed a new 2-dimensional, defined and highly efficient protocol that avoids the use of feeder cells, serum or embryoid body formation. Here we showed that a single matrix protein in combination with growth factors and a hypoxic environment is sufficient to generate from pluripotent cells hematopoietic progenitors capable of differentiating further in mature cell types of different lineages of the blood system. We tested the differentiation method using hESCs and 9 iPSC lines generated from different tissues. These data indicate the robustness of the protocol providing a valuable tool for the generation of clinical-grade hematopoietic cells from pluripotent cells.
Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Pluripotentes/citologia , Meios de Cultura Livres de Soro , Citometria de Fluxo , Humanos , Técnicas In Vitro , Oxigênio/metabolismoRESUMO
CD1 molecules are glycoproteins that present lipids and glycolipids for recognition by T cells. CD1-dependent immune activation has been implicated in a wide range of immune responses, however, our understanding of the role of this pathway in human disease remains limited because of species differences between humans and other mammals: whereas humans express five different CD1 gene products (CD1a, CD1b, CD1c, CD1d, and CD1e), muroid rodents express only one CD1 isoform (CD1d). Here we report that immune deficient mice engrafted with human fetal thymus, liver, and CD34(+) hematopoietic stem cells develop a functional human CD1 compartment. CD1a, b, c, and d isoforms were highly expressed by human thymocytes, and CD1a(+) cells with a dendritic morphology were present in the thymic medulla. CD1(+) cells were also detected in spleen, liver, and lungs. APCs from spleen and liver were capable of presenting bacterial glycolipids to human CD1-restricted T cells. ELISpot analyses of splenocytes demonstrated the presence of CD1-reactive IFN-γ producing cells. CD1d tetramer staining directly identified human iNKT cells in spleen and liver samples from engrafted mice, and injection of the glycolipid antigen α-GalCer resulted in rapid elevation of human IFN-γ and IL-4 levels in the blood indicating that the human iNKT cells are biologically active in vivo. Together, these results demonstrate that the human CD1 system is present and functionally competent in this humanized mouse model. Thus, this system provides a new opportunity to study the role of CD1-related immune activation in infections to human-specific pathogens.
Assuntos
Antígenos CD1/metabolismo , Animais , Antígenos CD1/genética , Citometria de Fluxo , Humanos , Camundongos , Camundongos SCIDRESUMO
We evaluated the immunocompetence of human T cells in humanized NOD-SCID interleukin (IL)-2r-gamma-null (hu-NSG) mice bearing a human thymic organoid, after multilineage reconstitution with isogeneic human leukocytes. Delayed type hypersensitivity (DTH) response was assessed by a direct footpad challenge of the immunized hu-NSG host, or by transfer of splenocytes from immunized hu-NSG, along with antigen, into footpads of C.B-17 scid mice (trans vivo [tv] DTH). Both methods revealed cellular immunity to tetanus toxoid (TT) or collagen type V (ColV). Immunohistochemical analysis of the swollen footpads revealed infiltration of human CD45(+) cells, including CD3(+) T cells, CD68(+) macrophages, and murine Ly6G(+) neutrophils. We observed a significant correlation between the percentage of circulating human CD4(+) cells and the direct DTH swelling response to TT. The tvDTH response to TT was inhibited by anti-interferon-gamma, whereas the tvDTH response to collagen V was inhibited by anti-IL-17 antibody, mimicking the cytokine bias of adult human T cells to these antigens. hu-NSG mice were also capable of mounting a B-cell response (primarily IgM) to TT antigen. The activation of either Th1- or Th17-dependent cellular immune response supports the utility of hu-NSG mice as a surrogate model of allograft rejection and autoimmunity.
Assuntos
Hipersensibilidade Tardia/imunologia , Interleucina-17/metabolismo , Camundongos Endogâmicos NOD , Camundongos SCID , Receptores de Interleucina-2 , Células Th1/metabolismo , Animais , Anticorpos Monoclonais/administração & dosagem , Antígenos CD/biossíntese , Células Cultivadas , Colágeno Tipo V/imunologia , Colágeno Tipo V/metabolismo , Modelos Animais de Doenças , Embrião de Mamíferos , Estudos de Viabilidade , Humanos , Imunocompetência/efeitos dos fármacos , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-17/imunologia , Camundongos , Neutrófilos/imunologia , Receptores de Interleucina-2/genética , Toxoide Tetânico/imunologia , Toxoide Tetânico/metabolismo , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Células Th1/patologiaRESUMO
Induced pluripotent stem cells (iPSCs) hold enormous potential for the development of personalized in vitro disease models, genomic health analyses, and autologous cell therapy. Here we describe the generation of T lymphocyte-derived iPSCs from small, clinically advantageous volumes of non-mobilized peripheral blood. These T-cell derived iPSCs ("TiPS") retain a normal karyotype and genetic identity to the donor. They share common characteristics with human embryonic stem cells (hESCs) with respect to morphology, pluripotency-associated marker expression and capacity to generate neurons, cardiomyocytes, and hematopoietic progenitor cells. Additionally, they retain their characteristic T-cell receptor (TCR) gene rearrangements, a property which could be exploited for iPSC clone tracking and T-cell development studies. Reprogramming T-cells procured in a minimally invasive manner can be used to characterize and expand donor specific iPSCs, and control their differentiation into specific lineages.
Assuntos
Desdiferenciação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Linfócitos T/citologia , Adulto , Diferenciação Celular , Linhagem Celular , Humanos , MasculinoRESUMO
Progress toward clinical application of ESC-derived hematopoietic cellular transplantation will require rigorous evaluation in a large animal allogeneic model. However, in contrast to human ESCs (hESCs), efforts to induce conclusive hematopoietic differentiation from rhesus macaque ESCs (rESCs) have been unsuccessful. Characterizing these poorly understood functional differences will facilitate progress in this area and likely clarify the critical steps involved in the hematopoietic differentiation of ESCs. To accomplish this goal, we compared the hematopoietic differentiation of hESCs with that of rESCs in both EB culture and stroma coculture. Initially, undifferentiated rESCs and hESCs were adapted to growth on Matrigel without a change in their phenotype or karyotype. Subsequent differentiation of rESCs in OP9 stroma led to the development of CD34(+)CD45(-) cells that gave rise to endothelial cell networks in methylcellulose culture. In the same conditions, hESCs exhibited convincing hematopoietic differentiation. In cytokine-supplemented EB culture, rESCs demonstrated improved hematopoietic differentiation with higher levels of CD34(+) and detectable levels of CD45(+) cells. However, these levels remained dramatically lower than those for hESCs in identical culture conditions. Subsequent plating of cytokine-supplemented rhesus EBs in methylcellulose culture led to the formation of mixed colonies of erythroid, myeloid, and endothelial cells, confirming the existence of bipotential hematoendothelial progenitors in the cytokine-supplemented EB cultures. Evaluation of four different rESC lines confirmed the validity of these disparities. Although rESCs have the potential for hematopoietic differentiation, they exhibit a pause at the hemangioblast stage of hematopoietic development in culture conditions developed for hESCs.
Assuntos
Células-Tronco Embrionárias/citologia , Sistema Hematopoético/citologia , Macaca mulatta , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Colágeno/efeitos dos fármacos , Ensaio de Unidades Formadoras de Colônias , Citocinas/farmacologia , Combinação de Medicamentos , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/metabolismo , Células-Tronco Embrionárias/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Fibroblastos , Perfilação da Expressão Gênica , Sistema Hematopoético/efeitos dos fármacos , Humanos , Cariotipagem , Laminina/efeitos dos fármacos , Camundongos , Fenótipo , Proteoglicanas/efeitos dos fármacos , Células Estromais/citologia , Células Estromais/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacosRESUMO
BACKGROUND: The management of hormone-insensitive locally advanced prostate cancer is difficult and complex and there is an urgent need for the development of effective chemotherapeutic agents intended for combination with currently available treatment modalities. METHODS: The present paper demonstrates the effectiveness of the monoterpene perillyl alcohol (POH) as potent radiosensitizer on DU145 and PC3 cell lines by performing clonogenic survival assays, cycle analysis, and assays to detect viability, apoptosis, and Fas receptor/ligand by flow cytometry. RESULTS: POH pretreatment resulted in a dose dependent sensitization to kill cell by radiation. Furthermore, POH treatment induced a transient G(2)/M arrest, enhanced the expression of the membrane bound form of the Fas ligand and sensitized the cells to Fas mediated apoptosis. CONCLUSIONS: The unique manner of radiosensitization in addition to its low toxicity profile makes POH a promising new agent for preclinical evaluation as a potential radiosensitizer in the treatment of prostate cancer.
Assuntos
Antineoplásicos/farmacologia , Monoterpenos/farmacologia , Neoplasias da Próstata , Radiossensibilizantes/farmacologia , Receptor fas/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Ciclina B/metabolismo , Ciclina D , Ciclinas/metabolismo , Fase G2/efeitos dos fármacos , Humanos , Masculino , Proteínas de Membrana/metabolismo , Mitose/efeitos dos fármacos , Células Tumorais Cultivadas/citologia , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismoRESUMO
The prognosis for patients with malignant glioma has not significantly changed in two decades, despite advances in surgery, radiation, and chemotherapy, emphasizing the growing need for novel approaches to glioma therapy. Perillyl alcohol (POH) is a naturally occurring monoterpene that has been shown to possess chemotherapeutic as well as chemopreventive activity in animal tumor models and is currently in Phase I and Phase II clinical trials. In the present study, we have demonstrated that POH is an effective radiosensitizer at clinically relevant doses of radiation using established glioma cell lines. POH caused a transient arrest in the G2/M phase of the cell cycle and induced apoptosis in glioma cells. POH treatment sensitized glioma cells to Fas-mediated apoptosis, which was further augmented in the presence of ionizing radiation and abrogated in the presence of antagonistic antibody. POH-induced radiosensitization was partially inhibited in glioma cells expressing dominant negative Fas-associated death domain and completely inhibited in glioma cells overexpressing the cytokine response modifier A. In addition, POH treatment resulted in a dose-dependent sensitization to cisplatin and doxorubicin induced cytotoxicity in glioma cells, highlighting its usefulness as a potent radio/chemosensitizer in the treatment of malignant glioma.