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1.
Blood Adv ; 8(8): 1869-1879, 2024 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-38330193

RESUMO

ABSTRACT: Fc gamma receptor (FcγR) IIIA is an important receptor for immunoglobulin G (IgG) and is involved in immune defense mechanisms as well as tissue destruction in some autoimmune diseases including immune thrombocytopenia (ITP). FcγRIIIA on macrophages can trigger phagocytosis of IgG-sensitized platelets, and prior pilot studies observed blockade of FcγRIIIA increased platelet counts in patients with ITP. Unfortunately, although blockade of FcγRIIIA in patients with ITP increased platelet counts, its engagement by the blocking antibody drove serious adverse inflammatory reactions. These adverse events were postulated to originate from the antibody's Fc and/or bivalent nature. The blockade of human FcγRIIIA in vivo with a monovalent construct lacking an active Fc region has not yet been achieved. To effectively block FcγRIIIA in vivo, we developed a high affinity monovalent single-chain variable fragment (scFv) that can bind and block human FcγRIIIA. This scFv (17C02) was expressed in 3 formats: a monovalent fusion protein with albumin, a 1-armed human IgG1 antibody, and a standard bivalent mouse (IgG2a) antibody. Both monovalent formats were effective in preventing phagocytosis of ITP serum-sensitized human platelets. In vivo studies using FcγR-humanized mice demonstrated that both monovalent therapeutics were also able to increase platelet counts. The monovalent albumin fusion protein did not have adverse event activity as assessed by changes in body temperature, whereas the 1-armed antibody induced some changes in body temperature even though the Fc region function was impaired by the Leu234Ala and Leu235Ala mutations. These data demonstrate that monovalent blockade of human FcγRIIIA in vivo can potentially be a therapeutic strategy for patients with ITP.


Assuntos
Púrpura Trombocitopênica Idiopática , Trombocitopenia , Humanos , Camundongos , Animais , Púrpura Trombocitopênica Idiopática/tratamento farmacológico , Receptores de IgG/metabolismo , Modelos Animais de Doenças , Imunoglobulina G/uso terapêutico , Albuminas/uso terapêutico
2.
Mol Ther Oncolytics ; 28: 334-348, 2023 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-36938544

RESUMO

VG2025 is a recombinant oncolytic herpes simplex virus type 1 (HSV-1) that uses transcriptional and translational dual regulation (TTDR) of critical viral genes to enhance virus safety and promote tumor-specific virus replication without reducing virulence. The TTDR platform is based on transcriptional control of the essential HSV-1 immediate-early protein ICP27 using a tumor-specific carcinoembryonic antigen (CEA) promoter, coupled with translational control of the neurovirulence factor ICP34.5 using multiple microRNA (miR)-binding sites. VG2025 further incorporates IL-12 and the IL-15/IL-15 receptor alpha subunit complex to enhance the antitumor and immune stimulatory properties of oncolytic HSVs. The TTDR strategy was verified in vitro and shown to be highly selective. Strong in vivo antitumor efficacy was observed following both intratumoral and intravenous administration. Clear abscopal and immune memory effects were also evident, indicating a robust antitumor immune response. Gene expression profiling of treated tumors revealed increased immune cell infiltration and activation of multiple immune-signaling pathways when compared with the backbone virus. Absence of neurotoxicity was verified in mice and in rhesus monkeys. Taken together, the enhanced tumor clearance, excellent safety profile, and positive correlation between CEA levels and viral replication efficiency may provide an opportunity for using biomarker-based precision medicine in oncolytic virotherapy.

3.
Viruses ; 14(11)2022 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-36366425

RESUMO

Oncolytic viruses (OVs) can specifically replicate in the host and cause cancer cell lysis while inducing an antitumor immune response. The aim of this study is to investigate the impact of either pre-existing immunity against herpes simplex virus type-1 (HSV-1) or multicycle treatment with OVs on anticancer efficacy of VG161, an HSV-1 OV in phase 2 clinical trial. VG161 efficacy was tested in CT26 mouse models by comparing the efficacy and immune response in naïve mice or in mice that were immunized with VG161. Moreover, VG161 efficacy in HLA-matched CD34+ humanized intrahepatic cholangiocarcinoma (ICC) patient-derived xenograft (PDX) models was also tested in multicycle treatment and was compared to standard chemotherapy for this type of cancer (gemcitabine). The HSV-1-immunized mice significantly inhibited tumor growth in VG161-treated mice compared to control naïve treated mice. RNA expression profiling and ELISPOT analyses indicated changes in the tumor's immune profile in the immunized and treated group compared to naïve and treated mice, as well as enhanced T cell function depicted by higher numbers of tumor specific lymphocytes, which was enhanced by immunization. In the ICC PDX model, repeated treatment of VG161 with 2 or 3 cycles seemed to increase the anticancer efficacy of VG161. In conclusion, the anticancer efficacy of VG161 can be enhanced by pre-immunization with HSV-1 and multicycle administration when the virus is given intratumorally, indicating that pre-existing antiviral immunity might enhance OV-induced antitumor immunity. Our results suggest potential clinical benefits of HSV-1-based OV therapy in HSV-1-seropositive patients and multicycle administration of VG161 for long-term maintenance treatment.


Assuntos
Herpesvirus Humano 1 , Neoplasias , Terapia Viral Oncolítica , Vírus Oncolíticos , Humanos , Camundongos , Animais , Vírus Oncolíticos/fisiologia , Herpesvirus Humano 1/genética , Neoplasias/terapia , Imunidade , Modelos Animais de Doenças , Terapia Viral Oncolítica/métodos
4.
Front Oncol ; 12: 856424, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35600398

RESUMO

Podocalyxin (Podxl) is a CD34-related cell surface sialomucin that is normally highly expressed by adult vascular endothelia and kidney podocytes where it plays a key role in blocking adhesion. Importantly, it is also frequently upregulated on a wide array of human tumors and its expression often correlates with poor prognosis. We previously showed that, in xenograft studies, Podxl plays a key role in metastatic disease by making tumor initiating cells more mobile and invasive. Recently, we developed a novel antibody, PODO447, which shows exquisite specificity for a tumor-restricted glycoform of Podxl but does not react with Podxl expressed by normal adult tissue. Here we utilized an array of glycosylation defective cell lines to further define the PODO447 reactive epitope and reveal it as an O-linked core 1 glycan presented in the context of the Podxl peptide backbone. Further, we show that when coupled to monomethyl auristatin E (MMAE) toxic payload, PODO447 functions as a highly specific and effective antibody drug conjugate (ADC) in killing ovarian, pancreatic, glioblastoma and leukemia cell lines in vitro. Finally, we demonstrate PODO447-ADCs are highly effective in targeting human pancreatic and ovarian tumors in xenografted NSG and Nude mouse models. These data reveal PODO447-ADCs as exquisitely tumor-specific and highly efficacious immunotherapeutic reagents for the targeting of human tumors. Thus, PODO447 exhibits the appropriate characteristics for further development as a targeted clinical immunotherapy.

5.
Biomedicines ; 8(11)2020 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-33182232

RESUMO

Oncolytic virotherapy is a promising new tool for cancer treatment, but direct lytic destruction of tumor cells is not sufficient and must be accompanied by strong immune activation to elicit anti-tumor immunity. We report here the creation of a novel replication-competent recombinant oncolytic herpes simplex virus type 1 (VG161) that carries genes coding for IL-12, IL-15, and IL-15 receptor alpha subunit, along with a peptide fusion protein capable of disrupting PD-1/PD-L1 interactions. The VG161 virus replicates efficiently and exhibits robust cytotoxicity in multiple tumor cell lines. Moreover, the encoded cytokines and the PD-L1 blocking peptide work cooperatively to boost immune cell function. In vivo testing in syngeneic CT26 and A20 tumor models reveals superior efficacy when compared to a backbone virus that does not express exogenous genes. Intratumoral injection of VG161 induces abscopal responses in non-injected distal tumors and grants resistance to tumor re-challenge. The robust anti-tumor effect of VG161 is associated with T cell and NK cell tumor infiltration, expression of Th1 associated genes in the injection site, and increased frequency of splenic tumor-specific T cells. VG161 also displayed a superb safety profile in GLP acute and repeated injection toxicity studies performed using cynomolgus monkeys. Overall, we demonstrate that VG161 can induce robust oncolysis and stimulate a robust anti-tumor immune response without sacrificing safety.

6.
J Immunother Cancer ; 8(2)2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33243933

RESUMO

BACKGROUND: The success of new targeted cancer therapies has been dependent on the identification of tumor-specific antigens. Podocalyxin (Podxl) is upregulated on tumors with high metastatic index and its presence is associated with poor outcome, thus emerging as an important prognostic and theragnostic marker in several human cancers. Moreover, in human tumor xenograft models, Podxl expression promotes tumor growth and metastasis. Although a promising target for immunotherapy, the expression of Podxl on normal vascular endothelia and kidney podocytes could hamper efforts to therapeutically target this molecule. Since pathways regulating post-translational modifications are frequently perturbed in cancer cells, we sought to produce novel anti-Podxl antibodies (Abs) that selectively recognize tumor-restricted glycoepitopes on the extracellular mucin domain of Podxl. METHODS: Splenic B cells were isolated from rabbits immunized with a Podxl-expressing human tumor cell line. Abs from these B cells were screened for potent reactivity to Podxl+ neoplastic cell lines but not Podxl+ primary endothelial cells. Transcripts encoding heavy and light chain variable regions from promising B cells were cloned and expressed as recombinant proteins. Tumor specificity was assessed using primary normal tissue and an ovarian cancer tissue microarray (TMA). Mapping of the tumor-restricted epitope was performed using enzyme-treated human tumor cell lines and a glycan array. RESULTS: One mAb (PODO447) showed strong reactivity with a variety of Podxl+ tumor cell lines but not with normal primary human tissue including Podxl+ kidney podocytes and most vascular endothelia. Screening of an ovarian carcinoma TMA (219 cases) revealed PODO447 reactivity with the majority of tumors, including 65% of the high-grade serous histotype. Subsequent biochemical analyses determined that PODO447 reacts with a highly unusual terminal N-acetylgalactosamine beta-1 (GalNAcß1) motif predominantly found on the Podxl protein core. Finally, Ab-drug conjugates showed specific efficacy in killing tumor cells in vitro. CONCLUSIONS: We have generated a novel and exquisitely tumor-restricted mAb, PODO447, that recognizes a glycoepitope on Podxl expressed at high levels by a variety of tumors including the majority of life-threatening high-grade serous ovarian tumors. Thus, tumor-restricted PODO447 exhibits the appropriate specificity for further development as a targeted immunotherapy.


Assuntos
Anticorpos Monoclonais/imunologia , Neoplasias Ovarianas/imunologia , Sialoglicoproteínas/imunologia , Animais , Anticorpos Monoclonais/farmacologia , Biomarcadores Tumorais/imunologia , Células CHO , Linhagem Celular Tumoral , Cricetulus , Epitopos/imunologia , Feminino , Células HEK293 , Humanos , Neoplasias Ovarianas/terapia , Coelhos
7.
Cancer Med ; 9(9): 2943-2959, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32130786

RESUMO

Traditional therapies have limited efficacy in hepatocellular carcinoma, pancreatic cancer, and biliary tract cancer, especially for advanced and refractory cancers. Through a deeper understanding of antitumor immunity and the tumor microenvironment, novel immunotherapies are becoming available for cancer treatment. Oncolytic virus (OV) therapy is an emerging type of immunotherapy that has demonstrated effective antitumor efficacy in many preclinical studies and clinical studies. Thus, it may represent a potential feasible treatment for hard to treat gastrointestinal (GI) tumors. Here, we summarize the research progress of OV therapy for the treatment of hepato-bilio-pancreatic cancers. In general, most OV therapies exhibits potent, specific oncolysis both in cell lines in vitro and the animal models in vivo. Currently, several clinical trials have suggested that OV therapy may also be effective in patients with refractory hepato-bilio-pancreatic cancer. Multiple strategies such as introducing immunostimulatory genes, modifying virus capsid and combining various other therapeutic modalities have been shown enhanced specific oncolysis and synergistic anti-cancer immune stimulation. Combining OV with other antitumor therapies may become a more effective strategy than using virus alone. Nevertheless, more studies are needed to better understand the mechanisms underlying the therapeutic effects of OV, and to design appropriate dosing and combination strategies.


Assuntos
Neoplasias do Sistema Biliar/terapia , Neoplasias Hepáticas/terapia , Terapia Viral Oncolítica/métodos , Neoplasias Pancreáticas/terapia , Animais , Neoplasias do Sistema Biliar/patologia , Humanos , Neoplasias Hepáticas/patologia , Neoplasias Pancreáticas/patologia
8.
J Immunother ; 42(5): 162-174, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30933043

RESUMO

Herein we demonstrate that ultraviolet light-inactivated Herpes Simplex Virus-1 (UV-HSV-1) stimulates peripheral blood mononuclear cells (PBMCs) to lyse both androgen-sensitive and androgen-independent prostate cancer (PrCA) cell lines, but not the benign prostatic hyperplastic epithelial cell line, BPH-1, and is 1000-10,000-fold more potent at stimulating this killing than ultraviolet light-inactivated Vesicular Stomatitis Virus, adenovirus, reovirus or cytomegalovirus. Among PBMCs, natural killer (NK) cells appear to be a major cell type involved in this killing and UV-HSV-1 appears to directly and potently stimulate NK cell expression of CD69, degranulation, cytokine production, and migration to IL-8 in PC3 conditioned medium. We also found that UV-HSV-1 stimulates glycolysis in PBMCs and NK cells, and that 2-deoxyglucose and the protein kinase C inhibitor, Go6976, and the NFκB inhibitor, Bay 11-7082, all abrogate UV-HSV-1 activated killing of PC3 cells by PBMCs and NK cells. Using neutralizing anti-Toll-like receptor 2 (TLR2) we found that UV-HSV-1, like HSV-1, activates NK cells via TLR2. Taken together, these results are consistent with Toll-like receptor 2 ligands on UV-HSV-1 stimulating TLR2 on NK cells to activate protein kinase C, leading to enhanced glycolysis and NFκB activation, both of which play a critical role in this anti-PrCA innate immune response. Importantly, UV-HSV-1 synergizes with IL-15 to increase the cytolytic activity of PBMCs against PC3 cells and there was considerable donor-to-donor variation in killing ability. These results support the preclinical development of UV-HSV-1 as an adjuvant, in combination with IL-15, for cell infusions of healthy, preselected NK cells to treat PrCA.


Assuntos
Citotoxicidade Imunológica , Herpesvirus Humano 1/imunologia , Herpesvirus Humano 1/efeitos da radiação , Células Matadoras Naturais/imunologia , Raios Ultravioleta , Inativação de Vírus/efeitos da radiação , Biomarcadores , Linhagem Celular Tumoral , Citocinas/metabolismo , Glicólise , Humanos , Imunofenotipagem , Células Matadoras Naturais/metabolismo , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Ativação Linfocitária/imunologia , Masculino , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/metabolismo , Receptor 2 Toll-Like/metabolismo
9.
Expert Rev Proteomics ; 15(3): 259-275, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29343144

RESUMO

INTRODUCTION: Cancer is the second most common cause of death worldwide and its heterogeneity complicates therapy. Standard cytotoxic regiments disrupt rapidly dividing cells, regardless of their neoplastic status. The introduction of less toxic targeted therapies has partially contributed to the observed decrease in cancer-related mortality. Cell-surface proteins represent attractive targets for therapy, due to their easily-accessible localization and their involvement in essential signaling pathways, often dysregulated in cancer. Despite their clinical appeal, cell-surface proteins are often underrepresented in standard proteomic data sets, due to their poor solubility and lower expression levels compared to intracellular proteins. Areas covered: This review will summarize some of the available techniques for enriching the cell-surface proteome, and discuss their advantages, limitations and applicability to clinical sample-testing. Moreover, we discuss currently available strategies for the development of novel targeted therapies in cancer. Expert commentary: The interest in elucidating the cancer-associated surfaceome is growing and will likely benefit from recent advancements in instrument sensitivity, method development, and a growing body of high-quality proteomics databases. Multiomics studies, in combination with functional validations (e.g. dropout screens), and evaluation of the healthy surfaceome, will likely aid in the selection of relevant targets for future therapy development.


Assuntos
Antígenos de Neoplasias/química , Biomarcadores Tumorais/química , Terapia de Alvo Molecular/métodos , Proteômica/métodos , Animais , Antígenos de Neoplasias/metabolismo , Biomarcadores Tumorais/metabolismo , Humanos , Espectrometria de Massas/métodos
10.
Haematologica ; 102(9): 1537-1548, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28659338

RESUMO

The bone marrow microenvironment is known to provide a survival advantage to residual acute myeloid leukemia cells, possibly contributing to disease recurrence. The mechanisms by which stroma in the microenvironment regulates leukemia survival remain largely unknown. Using reverse-phase protein array technology, we profiled 53 key protein molecules in 11 signaling pathways in 20 primary acute myeloid leukemia samples and two cell lines, aiming to understand stroma-mediated signaling modulation in response to the targeted agents temsirolimus (MTOR), ABT737 (BCL2/BCL-XL), and Nutlin-3a (MDM2), and to identify the effective combination therapy targeting acute myeloid leukemia in the context of the leukemia microenvironment. Stroma reprogrammed signaling networks and modified the sensitivity of acute myeloid leukemia samples to all three targeted inhibitors. Stroma activated AKT at Ser473 in the majority of samples treated with single-agent ABT737 or Nutlin-3a. This survival mechanism was partially abrogated by concomitant treatment with temsirolimus plus ABT737 or Nutlin-3a. Mapping the signaling networks revealed that combinations of two inhibitors increased the number of affected proteins in the targeted pathways and in multiple parallel signaling, translating into facilitated cell death. These results demonstrated that a mechanism-based selection of combined inhibitors can be used to guide clinical drug selection and tailor treatment regimens to eliminate microenvironment-mediated resistance in acute myeloid leukemia.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Leucemia Mieloide Aguda/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos , Animais , Linhagem Celular , Feminino , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Masculino , Camundongos , Células Tumorais Cultivadas
12.
PLoS One ; 11(12): e0168072, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27977740

RESUMO

In previous studies we found that macrophages (MФs) from SH2-containing inositol-5'-phosphatase (SHIP) deficient mice are M2 polarized while their wild type (WT) counterparts are M1 polarized and that this difference in MФ phenotype can be recapitulated during in vitro derivation from bone marrow if mouse plasma (MP), but not fetal calf serum, is added to standard M-CSF-containing cultures. In the current study we investigated the mechanism by which MP skews SHIP-/- but not +/+ MФs to an M2 phenotype. Our results suggest that SHIP-/- basophils constitutively secrete higher levels of IL-4 than SHIP+/+ basophils and this higher level of IL-4 is sufficient to skew both SHIP+/+ and SHIP-/- MФs to an M2 phenotype, but only when MP is present to increase the sensitivity of the MФs to this level of IL-4. MP increases the IL-4 sensitivity of both SHIP+/+ and -/- MФs not by increasing cell surface IL-4 or CD36 receptor levels, but by triggering the activation of Erk and Akt and the production of ROS, all of which play a critical role in sensitizing MФs to IL-4-induced M2 skewing. Studies to identify the factor(s) in MP responsible for promoting IL-4-induced M2 skewing suggests that all-trans retinoic acid (ATRA), TGFß and prostaglandin E2 (PGE2) all play a role. Taken together, these results indicate that basophil-secreted IL-4 plays an essential role in M2 skewing and that ATRA, TGFß and PGE2 within MP collaborate to dramatically promote M2 skewing by acting directly on MФs to increase their sensitivity to IL-4.


Assuntos
Basófilos/metabolismo , Dinoprostona/farmacologia , Interleucina-4/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Plasma/química , Fator de Crescimento Transformador beta/farmacologia , Tretinoína/farmacologia , Animais , Células Cultivadas , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/genética
13.
Oncotarget ; 7(48): 79722-79735, 2016 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-27806325

RESUMO

Metabolic reprogramming has been described as a hallmark of transformed cancer cells. In this study, we examined the role of the glutamine (Gln) utilization pathway in acute myeloid leukemia (AML) cell lines and primary AML samples. Our results indicate that a subset of AML cell lines is sensitive to Gln deprivation. Glutaminase (GLS) is a mitochondrial enzyme that catalyzes the conversion of Gln to glutamate. One of the two GLS isoenzymes, GLS1 is highly expressed in cancer and encodes two different isoforms: kidney (KGA) and glutaminase C (GAC). We analyzed mRNA expression of GLS1 splicing variants, GAC and KGA, in several large AML datasets and identified increased levels of expression in AML patients with complex cytogenetics and within specific molecular subsets. Inhibition of glutaminase by allosteric GLS inhibitor bis-2-(5-phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethyl sulfide or by novel, potent, orally bioavailable GLS inhibitor CB-839 reduced intracellular glutamate levels and inhibited growth of AML cells. In cell lines and patient samples harboring IDH1/IDH2 (Isocitrate dehydrogenase 1 and 2) mutations, CB-839 reduced production of oncometabolite 2-hydroxyglutarate, inducing differentiation. These findings indicate potential utility of glutaminase inhibitors in AML therapy, which can inhibit cell growth, induce apoptosis and/or differentiation in specific leukemia subtypes.


Assuntos
Antineoplásicos/farmacologia , Benzenoacetamidas/farmacologia , Metabolismo Energético/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Glutaminase/antagonistas & inibidores , Glutamina/metabolismo , Leucemia Mieloide Aguda/tratamento farmacológico , Sulfetos/farmacologia , Tiadiazóis/farmacologia , Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Glutamato Desidrogenase/antagonistas & inibidores , Glutamato Desidrogenase/genética , Glutamato Desidrogenase/metabolismo , Ácido Glutâmico/metabolismo , Glutaminase/genética , Glutaminase/metabolismo , Glutaratos/metabolismo , Humanos , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Mutação , Isoformas de Proteínas
14.
Oncotarget ; 7(32): 51435-51449, 2016 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-27283492

RESUMO

Metformin displays antileukemic effects partly due to activation of AMPK and subsequent inhibition of mTOR signaling. Nevertheless, Metformin also inhibits mitochondrial electron transport at complex I in an AMPK-independent manner, Here we report that Metformin and rotenone inhibit mitochondrial electron transport and increase triglyceride levels in leukemia cell lines, suggesting impairment of fatty acid oxidation (FAO). We also report that, like other FAO inhibitors, both agents and the related biguanide, Phenformin, increase sensitivity to apoptosis induction by the bcl-2 inhibitor ABT-737 supporting the notion that electron transport antagonizes activation of the intrinsic apoptosis pathway in leukemia cells. Both biguanides and rotenone induce superoxide generation in leukemia cells, indicating that oxidative damage may sensitize toABT-737 induced apoptosis. In addition, we demonstrate that Metformin sensitizes leukemia cells to the oligomerization of Bak, suggesting that the observed synergy with ABT-737 is mediated, at least in part, by enhanced outer mitochondrial membrane permeabilization. Notably, Phenformin was at least 10-fold more potent than Metformin in abrogating electron transport and increasing sensitivity to ABT-737, suggesting that this agent may be better suited for targeting hematological malignancies. Taken together, our results suggest that inhibition of mitochondrial metabolism by Metformin or Phenformin is associated with increased leukemia cell susceptibility to induction of intrinsic apoptosis, and provide a rationale for clinical studies exploring the efficacy of combining biguanides with the orally bioavailable derivative of ABT-737, Venetoclax.


Assuntos
Apoptose/efeitos dos fármacos , Biguanidas/farmacologia , Compostos de Bifenilo/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Transporte de Elétrons/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Nitrofenóis/farmacologia , Sulfonamidas/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linhagem Celular Tumoral , Sinergismo Farmacológico , Humanos , Metformina/farmacologia , Mitocôndrias/metabolismo , Fenformin/farmacologia , Piperazinas/farmacologia , Rotenona/farmacologia , Células U937
15.
PLoS One ; 11(3): e0152538, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27031833

RESUMO

Dimethyl sulfoxide (DMSO) is currently used as an alternative treatment for various inflammatory conditions as well as for cancer. Despite its widespread use, there is a paucity of data regarding its safety and efficacy as well as its mechanism of action in human cells. Herein, we demonstrate that DMSO has ex-vivo anti-inflammatory activity using Escherichia coli- (E. coli) and herpes simplex virus-1 (HSV-1)-stimulated whole human blood. Specifically, we found that between 0.5%-2%, DMSO significantly suppressed the expression of many pro-inflammatory cytokines/chemokines and prostaglandin E2 (PGE2). However, a significant reduction in monocyte viability was also observed at 2% DMSO, suggesting a narrow window of efficacy. Anti-inflammatory concentrations of DMSO suppressed E. coli-induced ERK1/2, p38, JNK and Akt phosphorylation, suggesting DMSO acts on these signaling pathways to suppress inflammatory cytokine/chemokine production. Although DMSO induces the differentiation of B16/F10 melanoma cells in vitro, topical administration of DMSO to mice subcutaneously implanted with B16 melanoma cells was ineffective at reducing tumor growth, DMSO was also found to block mouse macrophages from polarizing to either an M1- or an M2-phenotype, which may contribute to its inability to slow tumor growth. Topical administration of DMSO, however, significantly mitigated K/BxN serum-induced arthritis in mice, and this was associated with reduced levels of pro-inflammatory cytokines in the joints and white blood cell levels in the blood. Thus, while we cannot confirm the efficacy of DMSO as an anti-cancer agent, the use of DMSO in arthritis warrants further investigation to ascertain its therapeutic potential.


Assuntos
Artrite/patologia , Células Sanguíneas/metabolismo , Citocinas/metabolismo , Dimetil Sulfóxido/farmacologia , Animais , Artrite/induzido quimicamente , Artrite/tratamento farmacológico , Células Sanguíneas/citologia , Células Sanguíneas/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Quimiocinas/análise , Quimiocinas/genética , Quimiocinas/metabolismo , Citocinas/análise , Citocinas/genética , Dimetil Sulfóxido/uso terapêutico , Dinoprostona/metabolismo , Escherichia coli/metabolismo , Feminino , Herpesvirus Humano 1/fisiologia , Humanos , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Masculino , Melanoma/tratamento farmacológico , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Transdução de Sinais/efeitos dos fármacos
16.
Blood ; 127(21): 2575-86, 2016 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-26941401

RESUMO

Herein we demonstrate that oncolytic herpes simplex virus-1 (HSV-1) potently activates human peripheral blood mononuclear cells (PBMCs) to lyse leukemic cell lines and primary acute myeloid leukemia samples, but not healthy allogeneic lymphocytes. Intriguingly, we found that UV light-inactivated HSV-1 (UV-HSV-1) is equally effective in promoting PBMC cytolysis of leukemic cells and is 1000- to 10 000-fold more potent at stimulating innate antileukemic responses than UV-inactivated cytomegalovirus, vesicular stomatitis virus, reovirus, or adenovirus. Mechanistically, UV-HSV-1 stimulates PBMC cytolysis of leukemic cells, partly via Toll-like receptor-2/protein kinase C/nuclear factor-κB signaling, and potently stimulates expression of CD69, degranulation, migration, and cytokine production in natural killer (NK) cells, suggesting that surface components of UV-HSV-1 directly activate NK cells. Importantly, UV-HSV-1 synergizes with interleukin-15 (IL-15) and IL-2 in inducing activation and cytolytic activity of NK cells. Additionally, UV-HSV-1 stimulates glycolysis and fatty acid oxidation-dependent oxygen consumption in NK cells, but only glycolysis is required for their enhanced antileukemic activity. Last, we demonstrate that T cell-depleted human PBMCs exposed to UV-HSV-1 provide a survival benefit in a murine xenograft model of human acute myeloid leukemia (AML). Taken together, our results support the preclinical development of UV-HSV-1 as an adjuvant, alone or in combination with IL-15, for allogeneic donor mononuclear cell infusions to treat AML.


Assuntos
Herpesvirus Humano 1/imunologia , Imunidade Celular , Células Matadoras Naturais/imunologia , Leucemia/imunologia , Raios Ultravioleta , Inativação de Vírus/efeitos da radiação , Degranulação Celular/imunologia , Movimento Celular/imunologia , Feminino , Humanos , Interleucina-15/imunologia , Interleucina-2/imunologia , Células Jurkat , Masculino , NF-kappa B/imunologia , Proteína Quinase C/imunologia , Transdução de Sinais/imunologia , Receptor 2 Toll-Like/imunologia
17.
Blood ; 126(16): 1874-5, 2015 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-26472736

RESUMO

In this issue of Blood, Ricciardi et al report a novel fatty acid oxidation (FAO) inhibitor, ST1326, that effectively inhibits proliferation, survival, and chemoresistance in leukemia cell lines and primary samples.


Assuntos
Carnitina O-Palmitoiltransferase/antagonistas & inibidores , Carnitina/análogos & derivados , Sistemas de Liberação de Medicamentos , Ácidos Graxos/metabolismo , Leucemia Mieloide Aguda/tratamento farmacológico , Proteínas de Neoplasias/antagonistas & inibidores , Humanos
18.
Nat Commun ; 6: 7530, 2015 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-26179207

RESUMO

Extensive reprogramming of cellular energy metabolism is a hallmark of cancer. Despite its importance, the molecular mechanism controlling this tumour metabolic shift remains not fully understood. Here we show that 14-3-3σ regulates cancer metabolic reprogramming and protects cells from tumorigenic transformation. 14-3-3σ opposes tumour-promoting metabolic programmes by enhancing c-Myc poly-ubiquitination and subsequent degradation. 14-3-3σ demonstrates the suppressive impact on cancer glycolysis, glutaminolysis, mitochondrial biogenesis and other major metabolic processes of tumours. Importantly, 14-3-3σ expression levels predict overall and recurrence-free survival rates, tumour glucose uptake and metabolic gene expression in breast cancer patients. Thus, these results highlight that 14-3-3σ is an important regulator of tumour metabolism, and loss of 14-3-3σ expression is critical for cancer metabolic reprogramming. We anticipate that pharmacologically elevating the function of 14-3-3σ in tumours could be a promising direction for targeted anticancer metabolism therapy development in future.


Assuntos
Proteínas 14-3-3/genética , Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Metabolismo Energético/genética , Exorribonucleases/genética , Regulação Neoplásica da Expressão Gênica , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas 14-3-3/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Intervalo Livre de Doença , Exorribonucleases/metabolismo , Feminino , Técnicas de Inativação de Genes , Glutamina/metabolismo , Glicólise/genética , Células HCT116 , Humanos , Pessoa de Meia-Idade , Biogênese de Organelas , Prognóstico , Proteólise , Ubiquitinação/genética , Adulto Jovem
19.
BMC Musculoskelet Disord ; 16: 153, 2015 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-26092588

RESUMO

BACKGROUND: Platelet-rich plasma (PRP) preparations are a common treatment in osteoarthritis (OA) and inflammatory synovitis. However, there is ambiguity regarding the ideal concentration of leukocytes and platelets in these preparations necessary to induce an adequate anti-inflammatory and anabolic response in joint tissues, such as the synovial membrane. This research aimed to study, in normal synovial membrane explants (SME) challenged with lipopolysaccharide (LPS), the temporal effects (at 48 and 96h) of leukocyte- and platelet-rich gel (L-PRG) and pure platelet-rich gel (P-PRG) supernatants on the production and degradation of platelet associated growth factors (GF) (platelet derived GF isoform BB (PDGF-BB) and transforming growth factor beta-1 (TGF-ß1)), pro-inflammatory (tumour necrosis factor alpha (TNF-α)) and anti-inflammatory cytokines (interleukin 4 (IL-4) and IL-1 receptor antagonists (IL-1ra)) and hyaluronan (HA). METHODS: Synovial membrane explants (SMEs) from 6 horses were challenged with LPS and cultured for 96h with L-PRG and P-PRG supernatants at concentrations of 25 and 50 %, respectively. The SME culture medium was changed every 48h and used for determination by ELISA of PDGF-BB, TGF-ß1, TNF-α, IL-4, IL-1ra and HA. These molecules were also determined in synovial fluid from the horses. RESULTS: Both the 25 and 50 % PRG supernatants produced a molecular profile in the culture media unlike that of the SME challenged with LPS only. They presented GF, cytokine and HA concentrations very near to the concentrations of these molecules in normal synovial fluid when compared with the SME control groups (either with LPS or without LPS). However, in comparison with the rest of the SME treated groups, the 25 % L-PRG produced the most IL-1ra, and the 50 % P-PRG induced the sustained production of IL-4 and HA. CONCLUSIONS: These in vitro findings suggest that anabolic and anti-inflammatory joint responses depend on the leukocyte and platelet concentration of the PRP preparation and on the volume of this substance injected. Moreover, it is possible, that leukoreduced PRP preparations are more effective for the medical treatment of patients with OA and inflammatory synovitis.


Assuntos
Plaquetas/metabolismo , Citocinas/metabolismo , Ácido Hialurônico/metabolismo , Mediadores da Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Lipopolissacarídeos/farmacologia , Plasma Rico em Plaquetas/metabolismo , Membrana Sinovial/efeitos dos fármacos , Animais , Plaquetas/imunologia , Fracionamento Celular , Meios de Cultura/metabolismo , Géis , Cavalos , Leucócitos/efeitos dos fármacos , Leucócitos/imunologia , Leucócitos/metabolismo , Plasma Rico em Plaquetas/imunologia , Líquido Sinovial/imunologia , Líquido Sinovial/metabolismo , Membrana Sinovial/imunologia , Membrana Sinovial/metabolismo , Fatores de Tempo , Técnicas de Cultura de Tecidos
20.
Cancer Microenviron ; 7(1-2): 79-90, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25112275

RESUMO

Here we report that leukemia cell lines and primary CD34+ leukemic blasts exposed to platelet rich plasma (PRP) or platelet lysates (PL) display increased resistance to apoptosis induced by mitochondria-targeted agents ABT-737 and CDDO-Me. Intriguingly, leukemia cells exposed to platelet components demonstrate a reduction in mitochondrial membrane potential (ΔΨM) and a transient increase in oxygen consumption, suggestive of mitochondrial uncoupling. Accompanying the ranolazine-sensitive increase in oxygen consumption, a reduction in triglyceride content was also observed in leukemia cells cultured with platelet components indicating that lipolysis and fatty acid oxidation may support the molecular reduction of oxygen in these cells. Mechanistically, platelet components antagonized Bax oligomerization in accordance with previous observations supporting an antiapoptotic role for fatty acid oxidation in leukemia cells. Lastly, substantiating the notion that mitochondrial uncoupling reduces oxidative stress, platelet components induced a marked decrease in basal and rotenone-induced superoxide levels in leukemia cells. Taken together, the decrease in ΔΨM, the transient increase in ranolazine-sensitive oxygen consumption, the reduction in triglyceride levels, and the reduced generation of superoxide, all accompanying the increased resistance to mitochondrial apoptosis, substantiate the hypothesis that platelets may contribute to the chemoprotective sanctuary of the bone marrow microenvironment via promotion of mitochondrial uncoupling.

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