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Diffuse Midline Gliomas (DMGs) are universally fatal, primarily pediatric malignancies affecting the midline structures of the central nervous system. Despite decades of clinical trials, treatment remains limited to palliative radiation therapy. A major challenge is the coexistence of molecularly distinct malignant cell states with potentially orthogonal drug sensitivities. To address this challenge, we leveraged established network-based methodologies to elucidate Master Regulator (MR) proteins representing mechanistic, non-oncogene dependencies of seven coexisting subpopulations identified by single-cell analysis-whose enrichment in essential genes was validated by pooled CRISPR/Cas9 screens. Perturbational profiles of 372 clinically relevant drugs helped identify those able to invert the activity of subpopulation-specific MRs for follow-up in vivo validation. While individual drugs predicted to target individual subpopulations-including avapritinib, larotrectinib, and ruxolitinib-produced only modest tumor growth reduction in orthotopic models, systemic co-administration induced significant survival extension, making this approach a valuable contribution to the rational design of combination therapy.
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BACKGROUND: Diffuse midline glioma (DMG) is a pediatric tumor with dismal prognosis. Systemic strategies have been unsuccessful and radiotherapy (RT) remains the standard-of-care. A central impediment to treatment is the blood-brain barrier (BBB), which precludes drug delivery to the central nervous system (CNS). Focused ultrasound (FUS) with microbubbles can transiently and non-invasively disrupt the BBB to enhance drug delivery. This study aimed to determine the feasibility of brainstem FUS in combination with clinical doses of RT. We hypothesized that FUS-mediated BBB-opening (BBBO) is safe and feasible with 39 Gy RT. METHODS: To establish a safety timeline, we administered FUS to the brainstem of non-tumor bearing mice concurrent with or adjuvant to RT; our findings were validated in a syngeneic brainstem murine model of DMG receiving repeated sonication concurrent with RT. The brainstems of male B6 (Cg)-Tyrc-2J/J albino mice were intracranially injected with mouse DMG cells (PDGFB+, H3.3K27M, p53-/-). A clinical RT dose of 39 Gy in 13 fractions (39 Gy/13fx) was delivered using the Small Animal Radiation Research Platform (SARRP) or XRAD-320 irradiator. FUS was administered via a 0.5 MHz transducer, with BBBO and tumor volume monitored by magnetic resonance imaging (MRI). RESULTS: FUS-mediated BBBO did not affect cardiorespiratory rate, motor function, or tissue integrity in non-tumor bearing mice receiving RT. Tumor-bearing mice tolerated repeated brainstem BBBO concurrent with RT. 39 Gy/13fx offered local control, though disease progression occurred 3-4 weeks post-RT. CONCLUSION: Repeated FUS-mediated BBBO is safe and feasible concurrent with RT. In our syngeneic DMG murine model, progression occurs, serving as an ideal model for future combination testing with RT and FUS-mediated drug delivery.
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Barreira Hematoencefálica , Glioma , Humanos , Ratos , Criança , Masculino , Camundongos , Animais , Modelos Animais de Doenças , Ratos Sprague-Dawley , Tronco Encefálico , Sistemas de Liberação de Medicamentos/métodos , Imageamento por Ressonância Magnética , Glioma/radioterapia , Microbolhas , EncéfaloRESUMO
PURPOSE: Diffuse midline glioma (DMG) is a fatal tumor traditionally treated with radiation therapy (RT) and previously characterized as having a noninflammatory tumor immune microenvironment (TIME). FLASH is a novel RT technique using ultra-high dose rate that is associated with decreased toxicity and effective tumor control. However, the effect of FLASH and conventional (CONV) RT on the DMG TIME has not yet been explored. METHODS AND MATERIALS: Here, we performed single-cell RNA sequencing (scRNA-seq) and flow cytometry on immune cells isolated from an orthotopic syngeneic murine model of brainstem DMG after the use of FLASH (90 Gy/sec) or CONV (2 Gy/min) dose-rate RT and compared to unirradiated tumor (SHAM). RESULTS: At day 4 post-RT, FLASH exerted similar effects as CONV in the predominant microglial (MG) population, including the presence of two activated subtypes. However, at day 10 post-RT, we observed a significant increase in the type 1 interferon α/ß receptor (IFNAR+) in MG in CONV and SHAM compared to FLASH. In the non-resident myeloid clusters of macrophages (MACs) and dendritic cells (DCs), we found increased type 1 interferon (IFN1) pathway enrichment for CONV compared to FLASH and SHAM by scRNA-seq. We observed this trend by flow cytometry at day 4 post-RT in IFNAR+ MACs and DCs, which equalized by day 10 post-RT. DMG control and murine survival were equivalent between RT dose rates. CONCLUSIONS: Our work is the first to map CONV and FLASH immune alterations of the DMG TIME with single-cell resolution. Although DMG tumor control and survival were similar between CONV and FLASH, we found that changes in immune compartments differed over time. Importantly, although both RT modalities increased IFN1, we found that the timing of this response was cell-type and dose-rate dependent. These temporal differences, particularly in the context of tumor control, warrant further study.
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Glioma , Microglia , Animais , Glioma/radioterapia , Glioma/imunologia , Glioma/patologia , Camundongos , Microglia/efeitos da radiação , Microglia/imunologia , Microambiente Tumoral/imunologia , Neoplasias Encefálicas/radioterapia , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/patologia , Receptor de Interferon alfa e beta/genética , Camundongos Endogâmicos C57BL , Análise de Célula Única/métodos , Células Dendríticas/imunologia , Células Dendríticas/efeitos da radiação , Macrófagos/imunologiaRESUMO
Leptomeningeal disease (LMD) in pediatric brain tumors (PBTs) is a poorly understood and categorized phenomenon. LMD incidence rates, as well as diagnosis, treatment, and screening practices, vary greatly depending on the primary tumor pathology. While LMD is encountered most frequently in medulloblastoma, reports of LMD have been described across a wide variety of PBT pathologies. LMD may be diagnosed simultaneously with the primary tumor, at time of recurrence, or as primary LMD without a primary intraparenchymal lesion. Dissemination and seeding of the cerebrospinal fluid (CSF) involves a modified invasion-metastasis cascade and is often the result of direct deposition of tumor cells into the CSF. Cells develop select environmental advantages to survive the harsh, nutrient poor and turbulent environment of the CSF and leptomeninges. Improved understanding of the molecular mechanisms that underlie LMD, along with improved diagnostic and treatment approaches, will help the prognosis of children affected by primary brain tumors.
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Neoplasias Encefálicas , Neoplasias Cerebelares , Meduloblastoma , Neoplasias Meníngeas , Criança , Humanos , Neoplasias Meníngeas/diagnóstico , Neoplasias Meníngeas/secundário , Neoplasias Encefálicas/patologia , Meduloblastoma/diagnóstico , Meduloblastoma/patologia , Prognóstico , Neoplasias Cerebelares/patologiaRESUMO
BACKGROUND: Safe sampling of central nervous system tumor tissue for diagnostic purposes may be difficult if not impossible, especially in pediatric patients, and an unmet need exists to develop less invasive diagnostic tests. METHODS: We report our clinical experience with minimally invasive molecular diagnostics using a clinically validated assay for sequencing of cerebrospinal fluid (CSF) cell-free DNA (cfDNA). All CSF samples were collected as part of clinical care, and results reported to both clinicians and patients/families. RESULTS: We analyzed 64 CSF samples from 45 pediatric, adolescent and young adult (AYA) patients (pediatric = 25; AYA = 20) with primary and recurrent brain tumors across 12 histopathological subtypes including high-grade glioma (n = 10), medulloblastoma (n = 10), pineoblastoma (n = 5), low-grade glioma (n = 4), diffuse leptomeningeal glioneuronal tumor (DLGNT) (n = 4), retinoblastoma (n = 4), ependymoma (n = 3), and other (n = 5). Somatic alterations were detected in 30/64 samples (46.9%) and in at least one sample per unique patient in 21/45 patients (46.6%). CSF cfDNA positivity was strongly associated with the presence of disseminated disease at the time of collection (81.5% of samples from patients with disseminated disease were positive). No association was seen between CSF cfDNA positivity and the timing of CSF collection during the patient's disease course. CONCLUSIONS: We identified three general categories where CSF cfDNA testing provided additional relevant diagnostic, prognostic, and/or therapeutic information, impacting clinical assessment and decision making: (1) diagnosis and/or identification of actionable alterations; (2) monitor response to therapy; and (3) tracking tumor evolution. Our findings support broader implementation of clinical CSF cfDNA testing in this population to improve care.
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Neoplasias Encefálicas , Ácidos Nucleicos Livres , Neoplasias do Sistema Nervoso Central , Glioma , Adolescente , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Ácidos Nucleicos Livres/líquido cefalorraquidiano , Criança , Glioma/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação , Patologia Molecular , Adulto JovemRESUMO
PURPOSE OF REVIEW: Molecular subtyping in medulloblastoma (MB) has diagnostic and prognostic values which impact therapy. This paper provides guidance for the clinician caring for pediatric and adult patients with medulloblastoma in the modern era. RECENT FINDINGS: Medulloblastoma comprises four molecularly distinct subgroups: wingless activated (WNT), sonic hedgehog activated (SHH), group 3, and group 4. Risk stratification before and after the discovery of molecular subgroups aims at minimizing toxicity by reducing radiation and chemotherapy doses in low-risk patients while maintaining favorable overall survival (OS). The mainstay of newly diagnosed medulloblastoma treatment is surgery, radiation therapy, and chemotherapy, except for children under 6 years of age, where high-dose chemotherapy with autologous stem cell rescue is used to avoid or delay radiotherapy, preventing neurocognitive sequelae. Management of recurrent/refractory medulloblastoma remains a challenge with immunotherapy and small-molecule inhibitors forming the backbone of novel strategies. Recent innovations in medulloblastoma research allow us to better understand pathogenesis and molecular characteristics resulting in advanced risk stratification models, new therapeutic approaches, and overall improved survival and quality of life.
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Neoplasias Cerebelares/terapia , Meduloblastoma/terapia , Neoplasias Cerebelares/diagnóstico , Ensaios Clínicos como Assunto , Humanos , Imunoterapia , Meduloblastoma/diagnóstico , Recidiva Local de Neoplasia , Terapia Viral Oncolítica , Medição de Risco , Transdução de SinaisRESUMO
The syntheses and biological evaluations of new GHRH analogs of Miami (MIA) series with greatly increased anticancer activity are described. In the design and synthesis of these analogs, the following previous substitutions were conserved: D-Arg2, Har9, Abu15, and Nle27. Most new analogs had Ala at position 8. Since replacements of both Lys12 and Lys21 with Orn increased resistance against enzymatic degradation, these modifications were kept. The substitutions of Arg at both positions 11 and 20 by His were also conserved. We kept D-Arg28, Har29 -NH2 at the C-terminus or inserted Agm or 12-amino dodecanoic acid amide at position 30. We incorporated pentafluoro-Phe (Fpa5), instead of Cpa, at position 6 and Tyr(Me) at position 10 and ω-amino acids at N-terminus of some analogs. These GHRH analogs were prepared by solid-phase methodology and purified by HPLC. The evaluation of the activity of the analogs on GH release was carried out in vitro on rat pituitaries and in vivo in male rats. Receptor binding affinities were measured in vitro by the competitive binding analysis. The inhibitory activity of the analogs on tumor proliferation in vitro was tested in several human cancer cell lines such as HEC-1A endometrial adenocarcinoma, HCT-15 colorectal adenocarcinoma, and LNCaP prostatic carcinoma. For in vivo tests, various cell lines including PC-3 prostate cancer, HEC-1A endometrial adenocarcinoma, HT diffuse mixed ß cell lymphoma, and ACHN renal cell carcinoma cell lines were xenografted into nude mice and treated subcutaneously with GHRH antagonists at doses of 1-5µg/day. Analogs MIA-602, MIA-604, MIA-610, and MIA-640 showed the highest binding affinities, 30, 58, 48, and 73 times higher respectively, than GHRH (1-29) NH2. Treatment of LNCaP and HCT-15 cells with 5µM MIA-602 or MIA-690 decreased proliferation by 40%-80%. In accord with previous tests in various human cancer lines, analog MIA-602 showed high inhibitory activity in vivo on growth of PC-3 prostate cancer, HT-mixed ß cell lymphoma, HEC-1A endometrial adenocarcinoma and ACHN renal cell carcinoma. Thus, GHRH analogs of the Miami series powerfully suppress tumor growth, but have only a weak endocrine GH inhibitory activity. The suppression of tumor growth could be induced in part by the downregulation of GHRH receptors levels.
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Proliferação de Células/efeitos dos fármacos , Hormônio Liberador de Hormônio do Crescimento/administração & dosagem , Hormônio do Crescimento/biossíntese , Neoplasias/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Hormônio do Crescimento/metabolismo , Hormônio Liberador de Hormônio do Crescimento/análogos & derivados , Hormônio Liberador de Hormônio do Crescimento/antagonistas & inibidores , Hormônio Liberador de Hormônio do Crescimento/síntese química , Humanos , Camundongos , Neoplasias/patologia , Ratos , Relação Estrutura-AtividadeRESUMO
BACKGROUND: We previously showed that growth hormone-releasing hormone (GHRH) agonists are cardioprotective following myocardial infarction (MI). Here, our aim was to evaluate the in vitro and in vivo activities of highly potent new GHRH agonists, and elucidate their mechanisms of action in promoting cardiac repair. METHODS AND RESULTS: H9c2 cells were cultured in serum-free medium, mimicking nutritional deprivation. GHRH agonists decreased calcium influx and significantly improved cell survival. Rats with cardiac infarction were treated with GHRH agonists or placebo for four weeks. MI size was reduced by selected GHRH agonists (JI-38, MR-356, MR-409); this accompanied an increased number of cardiac c-kit+ cells, cellular mitotic divisions, and vascular density. One week post-MI, MR-409 significantly reduced plasma levels of IL-2, IL-6, IL-10 and TNF-α compared to placebo. Gene expression studies revealed favorable outcomes of MR-409 treatment partially result from inhibitory activity on pro-apoptotic molecules and pro-fibrotic systems, and by elevation of bone morphogenetic proteins. CONCLUSIONS: Treatment with GHRH agonists appears to reduce the inflammatory responses post-MI and may consequently improve mechanisms of healing and cardiac remodeling by regulating pathways involved in fibrosis, apoptosis and cardiac repair. Patients with cardiac dysfunction could benefit from treatment with novel GHRH agonists.
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Insuficiência Cardíaca/tratamento farmacológico , Infarto do Miocárdio/tratamento farmacológico , Receptores de Neuropeptídeos/agonistas , Receptores de Neuropeptídeos/química , Receptores de Hormônios Reguladores de Hormônio Hipofisário/agonistas , Receptores de Hormônios Reguladores de Hormônio Hipofisário/química , Alprostadil/análogos & derivados , Alprostadil/química , Animais , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Hormônio Liberador de Hormônio do Crescimento/análogos & derivados , Hormônio Liberador de Hormônio do Crescimento/química , Humanos , Inflamação , Interleucina-10/sangue , Interleucina-2/sangue , Interleucina-6/sangue , Microscopia de Fluorescência , Mitose , Ratos , Sermorelina/análogos & derivados , Sermorelina/química , Fator de Necrose Tumoral alfa/sangueRESUMO
Malignant melanoma is the deadliest form of skin cancer; the treatment of advanced and recurrent forms remains a challenge. It has recently been reported that growth hormone-releasing hormone (GHRH) receptor is involved in the pathogenesis of melanoma. Therefore, we investigated the effects of our new GHRH antagonists on a human melanoma cancer cell line. Antiproliferative effects of GHRH antagonists, MIA-602, MIA-606 and MIA-690, on the human melanoma cell line, A-375, were studied in vitro using the MTS assay. The effect of MIA-690 (5 µg/day 28 d) was further evaluated in vivo in nude mice bearing xenografts of A-375. Subcellular localization of p27 was detected with Western blot and immunofluorescent staining. MIA-690 inhibited the proliferation of A-375 cells in a dose-dependent manner (33% at 10 µM, and 19.2% at 5 µM, P < 0 .05 vs. control), and suppressed the growth of xenografted tumors by 70.45% (P < 0.05). Flow cytometric analysis of cell cycle effects following the administration of MIA-690 revealed a decrease in the number of cells in G2/M phase (from 19.7% to 12.9%, P < 0.001). Additionally, Western blot and immunofluorescent studies showed that exposure of A-375 cells to MIA-690 triggered the nuclear accumulation of p27. MIA-690 inhibited tumor growth in vitro and in vivo, and increased the translocation of p27 into the nucleus thus inhibiting progression of the cell cycle. Our findings indicate that patients with malignant melanoma could benefit from treatment regimens, which combine existing chemotherapy agents and novel GHRH-antagonists.
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Núcleo Celular/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Hormônio Liberador de Hormônio do Crescimento/antagonistas & inibidores , Melanoma/patologia , Animais , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Hormônio Liberador de Hormônio do Crescimento/metabolismo , Humanos , Melanoma/genética , Camundongos Nus , Receptores de Neuropeptídeos/metabolismo , Receptores de Hormônios Reguladores de Hormônio Hipofisário/metabolismo , Sermorelina/análogos & derivados , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Neoplasias Cutâneas , Ensaios Antitumorais Modelo de Xenoenxerto , Melanoma Maligno CutâneoRESUMO
BACKGROUND: Triple negative breast cancer (TNBC) is a distinct subtype of breast cancer burdened with a dismal prognosis due to the lack of effective therapeutic agents. Receptors for LHRH (luteinizing hormone-releasing hormone) can be successfully targeted with AEZS-108 [AN-152], an analog of LHRH conjugated to doxorubicin. Our study evaluates the presence of this target LHRH receptor in human specimens of TNBC and investigates the efficacy and toxicity of AEZS-108 in vivo. We also studied in vitro activity of AEZS-125, a new LHRH analog conjugated with the highly potent natural compound, Disorazol Z. METHODS: 69 human surgical specimens of TNBC were investigated for LHRH-R expression by immunohistochemistry. Expression of LHRH-R in two TNBC cell lines was evaluated by real time RT-PCR. Cytotoxicity of AEZS-125 was evaluated by Cell Titer Blue cytoxicity assay. LHRH- receptor expression was silenced with an siRNA in both cell lines. For the in vivo experiments an athymic nude mice model xenotransplanted with the cell lines, MDA-MB-231 and HCC 1806, was used. The animals were randomised to three groups receiving solvent only (d 1, 7, 14, i.v.) for control, AEZS-108 (d 1, 7, 14, i.v.) or doxorubicin at an equimolar dose (d 1, 7, 14, i.v.). RESULTS: In human clinical specimens of TNBC, expression of the LHRH-receptor was present in 49% (n = 69).HCC 1806 and MDA-MB-231 TNBC cells expressed mRNA for the LHRH-receptor. Silencing of the LHRH-receptor significantly decreased the cytotoxic effect of AEZS-108. MDA-MB-231 and HCC 1806 tumors xenografted into nude mice were significantly inhibited by treatment with AEZS-108; doxorubicin at equimolar doses was ineffective.As compared to AEZS 108, the Disorazol Z - LHRH conjugate, AEZS-125, demonstrated an increased cytotoxicity in vitro in HCC 1806 and MDA-MB-231 TNBC; this was diminished by receptor blockade with synthetic LHRH agonist (triptorelin) pretreatment. CONCLUSION: The current study confirms that LHRH-receptors are expressed by a significant proportion of TNBC and can be successfully used as homing sites for cytotoxic analogs of LHRH, such as AEZS-108 and AEZS-125.
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Antineoplásicos/administração & dosagem , Doxorrubicina/análogos & derivados , Hormônio Liberador de Gonadotropina/análogos & derivados , Oxazóis/administração & dosagem , Receptores LHRH/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Doxorrubicina/administração & dosagem , Doxorrubicina/farmacologia , Feminino , Hormônio Liberador de Gonadotropina/administração & dosagem , Hormônio Liberador de Gonadotropina/metabolismo , Hormônio Liberador de Gonadotropina/farmacologia , Humanos , Camundongos , Camundongos Nus , Oxazóis/farmacologia , Receptores LHRH/genética , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Management of castration-resistant prostate cancer (CRPC) is challenging due to lack of efficacious therapy. Luteinizing hormone-releasing hormone analogs appear to act directly on cells based on the LHRH receptors on human prostate adenocarcinoma cells. We explored anticancer activity of a cytotoxic analog of LHRH, AEZS-108 consisting of LHRH agonist linked to doxorubicin. Nude mice bearing DU-145 tumors were used to compare antitumor effects of AEZS-108 with its individual constituents or their unconjugated combination. The tumor growth inhibition of conjugate was greatest among treatment groups (90.5% inhibition vs. 41% by [D-Lys(6)]LHRH+DOX). The presence of LHRH receptors on DU-145 cells was confirmed by immunocytochemistry. In vitro, AEZS-108 significantly inhibited cell proliferation (61.2% inhibition) and elevated apoptosis rates (by 46%). By the detection of the inherent doxorubicin fluorescence, unconjugated doxorubicin was seen in the nucleus; the conjugate was perinuclear and at cell membrane. Autophagy, visualized by GFP-tagged p62 reporter, was increased by AEZS-108 (7.9-fold vs. 5.3-fold by DOX+[D-Lys(6)]LHRH. AEZS-108 more effectively increased reactive oxygen species (ROS, 2-fold vs. 1.4-fold by DOX+[D-Lys(6)]LHRH) and levels of the apoptotic regulator p21 in vivo and in vitro. We demonstrate robust inhibitory effects of the targeted cytotoxic LHRH analog AEZS-108 on LHRHR positive castration-resistant prostate cancer cells.
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Doxorrubicina/análogos & derivados , Hormônio Liberador de Gonadotropina/análogos & derivados , Neoplasias de Próstata Resistentes à Castração/genética , Proteínas rho de Ligação ao GTP/genética , Animais , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Doxorrubicina/metabolismo , Hormônio Liberador de Gonadotropina/genética , Hormônio Liberador de Gonadotropina/metabolismo , Humanos , Masculino , Camundongos , Neoplasias de Próstata Resistentes à Castração/metabolismo , Espécies Reativas de Oxigênio , Receptores LHRH/metabolismoRESUMO
The dismal prognosis of malignant brain tumors drives the development of new treatment modalities. In view of the multiple activities of growth hormone-releasing hormone (GHRH), we hypothesized that pretreatment with a GHRH agonist, JI-34, might increase the susceptibility of U-87 MG glioblastoma multiforme (GBM) cells to subsequent treatment with the cytotoxic drug, doxorubicin (DOX). This concept was corroborated by our findings, in vivo, showing that the combination of the GHRH agonist, JI-34, and DOX inhibited the growth of GBM tumors, transplanted into nude mice, more than DOX alone. In vitro, the pretreatment of GBM cells with JI-34 potentiated inhibitory effects of DOX on cell proliferation, diminished cell size and viability, and promoted apoptotic processes, as shown by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide proliferation assay, ApoLive-Glo multiplex assay, and cell volumetric assay. Proteomic studies further revealed that the pretreatment with GHRH agonist evoked differentiation decreasing the expression of the neuroectodermal stem cell antigen, nestin, and up-regulating the glial maturation marker, GFAP. The GHRH agonist also reduced the release of humoral regulators of glial growth, such as FGF basic and TGFß. Proteomic and gene-expression (RT-PCR) studies confirmed the strong proapoptotic activity (increase in p53, decrease in v-myc and Bcl-2) and anti-invasive potential (decrease in integrin α3) of the combination of GHRH agonist and DOX. These findings indicate that the GHRH agonists can potentiate the anticancer activity of the traditional chemotherapeutic drug, DOX, by multiple mechanisms including the induction of differentiation of cancer cells.
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Tratamento Farmacológico/métodos , Glioblastoma/tratamento farmacológico , Hormônio Liberador de Hormônio do Crescimento/análogos & derivados , Hormônio Liberador de Hormônio do Crescimento/agonistas , Fragmentos de Peptídeos/farmacologia , Animais , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Sinergismo Farmacológico , Ensaio de Imunoadsorção Enzimática , Proteína Glial Fibrilar Ácida , Hormônio Liberador de Hormônio do Crescimento/farmacologia , Imuno-Histoquímica , Camundongos , Camundongos Nus , Proteínas do Tecido Nervoso/metabolismo , Nestina/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
In view of the recent findings of stimulatory effects of GHRH analogs, JI-34, JI-36 and JI-38, on cardiomyocytes, pancreatic islets and wound healing, three series of new analogs of GHRH(1-29) have been synthesized and evaluated biologically in an endeavor to produce more potent compounds. "Agmatine analogs", MR-356 (N-Me-Tyr(1)-JI-38), MR-361(N-Me-Tyr(1), D-Ala(2)-JI-38) and MR-367(N-Me-Tyr(1), D-Ala(2), Asn(8)-JI-38), in which Dat in JI-38 is replaced by N-Me-Tyr(1), showed improved relative potencies on GH release upon subcutaneous administration in vivo and binding in vitro. Modification with N-Me-Tyr(1) and Arg(29)-NHCH3 as in MR-403 (N-Me-Tyr(1), D-Ala(2), Arg(29)-NHCH3-JI-38), MR-406 (N-Me-Tyr(1), Arg(29)-NHCH3-JI-38) and MR-409 (N-Me-Tyr(1), D-Ala(2), Asn(8), Arg(29)-NHCH3-JI-38), and MR-410 (N-Me-Tyr(1), D-Ala(2), Thr(8), Arg(29)-NHCH3-JI-38) resulted in dramatically increased endocrine activities. These appear to be the most potent GHRH agonistic analogs so far developed. Analogs with Apa(30)-NH2 such as MR-326 (N-Me-Tyr(1), D-Ala(2), Arg(29), Apa(30)-NH2-JI-38), and with Gab(30)-NH2, as MR-502 (D-Ala(2), 5F-Phe(6), Ser(28), Arg(29),Gab(30)-NH2-JI-38) also exhibited much higher potency than JI-38 upon i.v. administration. The relationship between the GH-releasing potency and the analog structure is discussed. Fourteen GHRH agonists with the highest endocrine potencies were subjected to cardiologic tests. MR-409 and MR-356 exhibited higher potency than JI-38 in activating myocardial repair in rats with induced myocardial infarction. As the previous class of analogs, exemplified by JI-38, had shown promising results in multiple fields including cardiology, diabetes and wound healing, our new, more potent, GHRH agonists should manifest additional efficacy for possible medical applications.
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Agmatina , Sistema Endócrino/metabolismo , Hormônio Liberador de Hormônio do Crescimento/agonistas , Peptídeos , Animais , Hormônio Liberador de Hormônio do Crescimento/metabolismo , Peptídeos/síntese química , Peptídeos/química , Peptídeos/farmacologia , Estrutura Secundária de Proteína , Ratos , Ratos Sprague-Dawley , Relação Estrutura-AtividadeRESUMO
Pancreatic carcinoma is one of the cancers with the worse prognosis, thus any therapeutic improvement is imperative. Cytotoxic LH-RH analog, AN-152 (proprietary designation, AEZS-108), consisting of doxorubicin (DOX) conjugated to D-Lys6LH-RH, is now in clinical trials for targeted therapy of several sex hormone-dependent tumors that express LH-RH receptors. We investigated LH-RH receptors in human pancreatic carcinoma and the effects of AN-152 (AEZS-108) on experimental pancreatic cancers. We determined LH-RH receptor presence in human pancreatic cancer samples by immunohistochemistry and, in three human pancreatic cancer lines (SW-1990, Panc-1 and CFPAC-1), by binding assays and Western blotting. The effects of the cytotoxic LH-RH analog were investigated on growth of these same cancer lines xenografted into nude mice. We also analyzed differences between the antitumor effects of the cytotoxic analog and its cytotoxic radical alone, doxorubicin (DOX), on the expression of cancer-related genes by PCR arrays. LH-RH receptors were expressed in two randomly selected surgically removed human pancreatic cancer samples and in all three cancer lines. Cytotoxic LH-RH analogs powerfully inhibited growth of all three tumor lines in nude mice; AN-152 was significantly stronger than DOX on Panc-1 and CFPAC-1 cancers. PCR array showed that cytotoxic LH-RH analog AN-152 affected the expression of genes associated with cellular migration, invasion, metastasis and angiogenesis more favorably than DOX, however the changes in gene expression varied considerably among the three cancer lines. Cytotoxic LH-RH analog, AEZS-108, may be a useful agent for the treatment of LH-RH receptor positive advanced pancreatic carcinoma.
Assuntos
Doxorrubicina/análogos & derivados , Hormônio Liberador de Gonadotropina/análogos & derivados , Neoplasias Pancreáticas/tratamento farmacológico , Receptores LHRH/metabolismo , Animais , Adesão Celular/genética , Linhagem Celular Tumoral , Doxorrubicina/uso terapêutico , Feminino , Hormônio Liberador de Gonadotropina/uso terapêutico , Humanos , Camundongos , Camundongos Nus , Transplante de NeoplasiasRESUMO
Glioblastoma multiforme is the most frequent tumor of the central nervous system in adults and has a dismal clinical outcome, which necessitates the development of new therapeutic approaches. We investigated in vivo the action of the targeted cytotoxic analog of luteinizing hormone releasing hormone, AN-152 (AEZS-108) in nude mice (Ncr nu/nu strain) bearing xenotransplanted U-87 MG glioblastoma tumors. We evaluated in vitro the expression of LHRH receptors, proliferation, apoptosis and the release of oncogenic and tumor suppressor cytokines. Clinical and U-87 MG samples of glioblastoma tumors expressed LHRH receptors. Treatment of nude mice with AN-152, once a week at an intravenous dose of 413 nmol/20 g, for six weeks resulted in 76 % reduction in tumor growth. AN-152 nearly completely abolished tumor progression and elicited remarkable apoptosis in vitro. Genomic (RT-PCR) and proteomic (ELISA, Western blot) studies revealed that AN-152 activated apoptosis, as reflected by the changes in p53 and its regulators and substrates, inhibited cell growth, and elicited changes in intermediary filament pattern. AN-152 similarly reestablished contact regulation as demonstrated by expression of adhesion molecules and inhibited vascularization, as reflected by the transcription of angiogenic factors. Our findings suggest that targeted cytotoxic analog AN-152 (AEZS-108) should be considered for a treatment of glioblastomas.
Assuntos
Doxorrubicina/análogos & derivados , Glioblastoma/tratamento farmacológico , Hormônio Liberador de Gonadotropina/análogos & derivados , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/genética , Glioblastoma/patologia , Hormônio Liberador de Gonadotropina/farmacologia , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Nus , Receptores LHRH/genética , Receptores LHRH/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Resultado do Tratamento , Carga Tumoral/efeitos dos fármacos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Five-year survival of patients afflicted with glioblastoma multiforme (GBM) is rare, making this cancer one of the most feared malignancies. Previously, we reported that growth hormone-releasing hormone (GHRH) is a potent growth factor in cancers. The present work evaluated the effects of two antagonistic analogs of GHRH (MIA-604 and MIA-690) on the proliferation of U-87 MG GBM tumors, in vivo as well as in vitro. Both analogs were administered subcutaneously and dose-dependently inhibited the growth of tumors transplanted into nude mice (127 animals in seven groups). The analogs also inhibited cell proliferation in vitro, decreased cell size, and promoted apoptotic and autophagic processes. Both antagonists stimulated contact inhibition, as indicated by the expression of the E-cadherin-ß-catenin complex and integrins, and decreased the release of humoral regulators of glial growth such as FGF, PDGFß, and TGFß, as revealed by genomic or proteomic detection methods. The GHRH analogs downregulated other tumor markers (Jun-proto-oncogene, mitogen-activated protein kinase-1, and melanoma cell adhesion molecule), upregulated tumor suppressors (p53, metastasis suppressor-1, nexin, TNF receptor 1A, BCL-2-associated agonist of cell death, and ifκBα), and inhibited the expression of the regulators of angiogenesis and invasion (angiopoetin-1, VEGF, matrix metallopeptidase-1, S100 calcium binding protein A4, and synuclein-γ). Our findings indicate that GHRH antagonists inhibit growth of GBMs by multiple mechanisms and decrease both tumor cell size and number.
Assuntos
Glioblastoma/tratamento farmacológico , Hormônio Liberador de Hormônio do Crescimento/análogos & derivados , Hormônio Liberador de Hormônio do Crescimento/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Glioblastoma/metabolismo , Glioblastoma/patologia , Hormônio Liberador de Hormônio do Crescimento/metabolismo , Humanos , Camundongos , Camundongos Nus , Terapia de Alvo Molecular , Proto-Oncogene Mas , Distribuição Aleatória , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Gastrin releasing-peptide (GRP) is a potent growth factor in many malignancies. Benign prostatic hyperplasia (BPH) is a progressive age-related proliferation of glandular and stromal tissues; various growth factors and inflammatory processes are involved in its pathogenesis. We have demonstrated that potent antagonists of GRP inhibit growth of experimental human tumors including prostate cancer, but their effect on models of BPH has not been studied. Here, we evaluated the effects of GRP antagonist RC-3940-II on viability and cell volume of BPH-1 human prostate epithelial cells and WPMY-1 prostate stromal cells in vitro, and in testosterone-induced BPH in Wistar rats in vivo. RC-3940-II inhibited the proliferation of BPH-1 and WPMY-1 cells in a dose-dependent manner and reduced prostatic cell volume in vitro. Shrinkage of prostates was observed after 6 wk of treatment with RC-3940-II: a 15.9% decline with 25 µg/d; and a 18.4% reduction with 50 µg/d (P < 0.05 for all). Significant reduction in levels of proliferating cell nuclear antigen, NF-κß/p50, cyclooxygenase-2, and androgen receptor was also seen. Analysis of transcript levels of genes related to growth, inflammatory processes, and signal transduction showed significant changes in the expression of more than 90 genes (P < 0.05). In conclusion, GRP antagonists reduce volume of human prostatic cells and lower prostate weight in experimental BPH through direct inhibitory effects on prostatic GRP receptors. GRP antagonists should be considered for further development as therapy for BPH.
Assuntos
Bombesina/análogos & derivados , Tamanho Celular/efeitos dos fármacos , Peptídeo Liberador de Gastrina/antagonistas & inibidores , Fragmentos de Peptídeos/farmacologia , Próstata/citologia , Hiperplasia Prostática/tratamento farmacológico , Análise de Variância , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Bombesina/farmacologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Ciclo-Oxigenase 2/sangue , Relação Dose-Resposta a Droga , Perfilação da Expressão Gênica , Humanos , Masculino , NF-kappa B/sangue , Antígeno Nuclear de Célula em Proliferação/sangue , Próstata/efeitos dos fármacos , Hiperplasia Prostática/induzido quimicamente , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Receptores Androgênicos/sangue , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Testosterona/toxicidade , Sais de Tetrazólio , TiazóisRESUMO
BACKGROUND: Benign prostatic hyperplasia (BPH) affects aging men. Combined therapy with antagonists of growth hormone-releasing hormone (GHRH) and of luteinizing hormone-releasing hormone (LHRH or GnRH) induces prostate shrinkage in rat models. We investigated the mechanisms of action of this combination on cell cycle traverse and expression of prostatic genes. METHODS: Effects of GHRH antagonist, JMR-132 (40 µg/day), the LHRH antagonist, cetrorelix (0.625 mg/kg), and their combination were evaluated on testosterone-induced benign prostatic hyperplasia in male Wistar rats. Influence of JMR-132, cetrorelix, and their combinations on cell viability was assessed by MTS assay in BPH-1 human prostate epithelial cells and WPMY-1 normal prostate stromal cells. Cell cycle was analyzed by laser flow cytometry. Real-time PCR arrays were performed. RESULTS: The combination of antagonists caused marked shrinkage of rat prostate (29.5%). In vitro, JMR-132 plus cetrorelix (both 5µM) produced synergistic (57.4%) inhibition of growth of BPH-1 cells, but a lesser inhibition (46%) of WPMY-1 cells. Co-treatment of with JMR-132 plus cetrorelix induced a significant increase of BPH-1 cells blocked in S-phase plus cells with lower G0 /G1 and G2 /M DNA content. Significant changes in expression of >40 gene transcripts related to growth factors, inflammatory cytokines, and signal transduction were identified. CONCLUSIONS: GHRH antagonist and LHRH antagonist combination potentiates rat prostate weight reduction and synergistically inhibits of growth of BPH-1 leading to cell cycle arrest in S-phase. These effects were lesser in normal stromal prostate cell line, WPMY-1. Our findings suggest that GHRH antagonists could be useful for BPH therapy, possibly in combination with LHRH antagonists.
Assuntos
Pontos de Checagem do Ciclo Celular/fisiologia , Hormônio Liberador de Gonadotropina/análogos & derivados , Hormônio Liberador de Gonadotropina/antagonistas & inibidores , Hormônio Liberador de Hormônio do Crescimento/antagonistas & inibidores , Hiperplasia Prostática/tratamento farmacológico , Sermorelina/análogos & derivados , Animais , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Sinergismo Farmacológico , Perfilação da Expressão Gênica , Hormônio Liberador de Gonadotropina/farmacologia , Humanos , Masculino , Tamanho do Órgão , Hiperplasia Prostática/patologia , Ratos , Ratos Wistar , Sermorelina/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Previously, we have shown that the targeted cytotoxic somatostatin (sst) analogue AN-162 [AZSE-124] inhibits the growth of MDA-MB-231 human breast cancers xenografted into nude mice. In this study, we examined the trafficking of AN-162 into the cell, the expression of the somatostatin receptors (sstr) in specimens of human triple-negative breast cancers (TNBC), and the effect of AN-162 on HCC 1806 human TNBC xenografts. The expression of sstr in TNBC tumor samples was investigated by immunohistochemical staining. The expression of sstr in HCC 1806 was evaluated by reverse transcription PCR. Internalization studies with I-labeled AN-162 were carried out and the autofluorescence sign of doxorubicin moiety in the cell nucleus after incubation with AN-162 was measured using a fluorescence assay. The effects of AN-162 on the growth of HCC 1806 xenografted into nude mice were studied. A fluorescence microscopy cytotoxicity assay in vitro to detect cell death after treatment with AN-162 was also carried out. About 28% of TNBC tumor specimens showed a positive staining for sstr subtype 2a. HCC 1806 expresses all five subtypes of sstr. In the fluorescence cytotoxicity assay, dead HCC 1806 cells were found 24 h after incubation with AN-162. The growth of HCC 1806 tumors in nude mice was significantly inhibited by treatment with AN-162. AN-162 was internalized into the HCC 1806 cells and doxorubicin moiety was detected in the cell nuclei. This study is the first to show that the trafficking of the cytotoxic sst analogue AN-162 into the cell is mediated by sstr. Our work shows that the growth of xenografted HCC 1806 TNBCs can be effectively inhibited in vivo with AN-162. This investigation provides information on the mechanism of action and efficacy of this new targeted cytotoxic sst analogue and identifies in this relation the sstr as a favorable therapeutic target in TNBC.
Assuntos
2-Hidroxifenetilamina/análogos & derivados , Compostos de Anilina/farmacologia , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Receptores de Somatostatina/metabolismo , 2-Hidroxifenetilamina/farmacologia , Animais , Morte Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Doxorrubicina/farmacologia , Feminino , Humanos , Camundongos , Camundongos Nus , Receptor ErbB-2/metabolismo , Receptores de Estrogênio/metabolismo , Receptores de Progesterona , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE OF REVIEW: We provide new viewpoints of hormonal control of benign prostatic hyperplasia (BPH). The latest treatment findings with 5-alpha reductase inhibitors (5-ARIs) finasteride and dutasteride, refined indications, efficacy, and safety are discussed and compared. We also discuss potential new 5-ARIs and other hormonal treatments. RECENT FINDINGS: Finasteride and dutasteride have equal efficacy and safety for the treatment and prevention of progression of BPH. 5-ARIs are especially recommended for prostates greater than 40âml and PSA greater than 1.5âng/ml. Combination therapy is the treatment of choice in these patients, but with prostate volume greater than 58âml or International Prostate Symptom Score of at least 20, combinations have no advantage over 5-ARI monotherapy. Updates on the recent developments on BPH therapy with luteinizing hormone-releasing hormone (LHRH) antagonist are also reviewed and analyzed. Preclinical studies suggest that growth hormone-releasing hormone (GHRH) antagonists effectively shrink experimentally enlarged prostates alone or in combination with LHRH antagonists. SUMMARY: New 5-ARIs seem to be the promising agents that need further study. Preclinical studies revealed that GHRH and LHRH antagonists both can cause a reduction in prostate volume. Recent data indicate that prostate shrinkage is induced by the direct inhibitory action of GHRH and of LHRH antagonists exerted through prostatic receptors. The adverse effects of 5ARIs encourage alternative therapy.