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Glycans, which are widely distributed on most proteins and cell surfaces, are a class of important biomolecules playing crucial roles in various biological processes such as immune response and cellular communication. Modern mass spectrometry (MS) coupled with novel chemical probes greatly facilitates routine analysis of glycans. However, the requirement of high-throughput analysis still calls for advanced tools to be developed. Recently, we devised isobaric multiplex reagents for carbonyl-containing compound (SUGAR) tags for 4-plex N-glycan analysis. To further improve the throughput, we utilized the subtle mass differences among different isotopologues and expanded the multiplexing capacity to 12 channels, a 3-fold throughput improvement for the original SUGAR tag design and achieved high-throughput N-glycan analysis in a single LC-MS/MS injection. We then applied 12-plex SUGAR tags to profile the N-glycans in four subtypes of human Immunoglobulin G (IgG) and to investigate the N-glycan changes in the endometrial cancer cells (ECC1) treated with Atovaquone, a quinone antimicrobial medication, and a dihydroorotate dehydrogenase (DHODH) inhibitor. Data are available via ProteomeXchange with the identifier PXD038501.
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Glicômica , Espectrometria de Massas em Tandem , Humanos , Espectrometria de Massas em Tandem/métodos , Glicômica/métodos , Cromatografia Líquida/métodos , Indicadores e Reagentes , Polissacarídeos/químicaRESUMO
In brief: Developing novel therapies to cure and manage endometriosis is a major unmet need that will benefit over 180 million women worldwide. Results from the current study suggest that inhibitors of oxidative phosphorylation may serve as novel agents for the treatment of endometriosis. Abstract: Current therapeutic strategies for endometriosis focus on symptom management and are not curative. Here, we provide evidence supporting the inhibition of oxidative phosphorylation (OXPHOS) as a novel treatment strategy for endometriosis. Additionally, we report an organotypic organ-on-a-chip luminal model for endometriosis. The OXPHOS inhibitors, curcumin, plumbagin, and the FDA-approved anti-malarial agent, atovaquone, were tested against the endometriosis cell line, 12Z, in conventional as well as the new organotypic model. The results suggest that all three compounds inhibit proliferation and cause cell death of the endometriotic cells by inhibiting OXPHOS and causing an increase in intracellular oxygen radicals. The oxidative stress mediated by curcumin, plumbagin, and atovaquone causes DNA double-strand breaks as indicated by the elevation of phospho-γH2Ax. Mitochondrial energetics shows a significant decrease in oxygen consumption in 12Z cells. These experiments also highlight differences in the mechanism of action as curcumin and plumbagin inhibit complex I whereas atovaquone blocks complexes I, II, and III. Real-time assessment of cells in the lumen model showed inhibition of migration in response to the test compounds. Additionally, using two-photon lifetime imaging, we demonstrate that the 12Z cells in the lumen show decreased redox ratio (NAD(P)H/FAD) and lower fluorescence lifetime of NAD(P)H in the treated cells confirming major metabolic changes in response to inhibition of mitochondrial electron transport. The robust chemotoxic responses observed with atovaquone suggest that this anti-malarial agent may be repurposed for the effective treatment of endometriosis.
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Antimaláricos , Antineoplásicos , Curcumina , Endometriose , Feminino , Humanos , Curcumina/farmacologia , Atovaquona/farmacologia , Fosforilação Oxidativa , Endometriose/tratamento farmacológico , NAD , Proliferação de CélulasRESUMO
OBJECTIVE: MUC16, the mucin that contains the CA125 epitopes, suppresses the cytolytic responses of human NK cells and inhibits the efficacy of therapeutic antibodies. Here, we provide further evidence of the regulatory role of MUC16 on human and murine NK cells and macrophages. METHODS: Target cell cytolysis and doublet formation assays were performed to assess effects of MUC16 on human NK cells. The effect of MUC16 on ovarian tumor growth was determined in a mouse model by monitoring survival and ascites formation. Innate immune cells from spleens and peritoneal cavities of mice were isolated and stimulated in vitro with anti-CD40 antibody, lipopolysaccharide and IFN-γ and their ability to cytolyse MUC16 expressing and non-expressing cells was determined. RESULTS: We confirm that MUC16 inhibits cytolysis by human NK cells as well as the formation of NK-tumor conjugates. Mice implanted with MUC16-knockdown OVCAR-3 show >2-fold increase in survival compared to controls. Murine NK cells and macrophages are more efficient at lysing MUC16-knockdown cells. In vitro cytotoxicity assays with NK cells and macrophages isolated from mice stimulated with anti-CD40 antibody showed 2-3-fold increased activity against the MUC16-knockdown cells as compared to matching target cells expressing this mucin. Finally, knockdown of MUC16 increased the susceptibility of cancer cells to ADCC by murine splenocytes. CONCLUSIONS: For the first time, we demonstrate the immunoregulatory effects of MUC16 on murine NK cells and macrophages. Our study implies that the immunoregulatory role of MUC16 on murine NK cells and macrophages should be considered when examining the biology of MUC16 in mouse models.
Assuntos
Antígeno Ca-125/imunologia , Proteínas de Membrana/imunologia , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Imunidade Inata , Células Matadoras Naturais/imunologia , Macrófagos/imunologia , Melanoma Experimental/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Neoplasias Ovarianas/imunologiaRESUMO
PURPOSE: Citral is composed of a random mixture of two geometric stereoisomers geranial (trans-citral) and neral (cis-citral) yet few studies have directly compared their in vivo antitumor properties. A micelle formulation was therefore developed. METHODS: Geranial and neral were synthesized. Commercially-purchased citral, geranial, and neral were formulated in PEG-b-PCL (block sizes of 5000:10,000, Mw/Mn 1.26) micelles. In vitro degradation, drug release, cytotoxicity, flow cytometry, and western blot studies were conducted. The antitumor properties of drug formulations (40 and 80 mg/kg based on MTD studies) were evaluated on the 4T1 xenograft mouse model and tumor tissues were analyzed by western blot. RESULTS: Micelles encapsulated drugs with >50% LE at 5-40% drug to polymer (w/w), displayed sustained release (t1/2 of 8-9 h), and improved drug stability at pH 5.0. The IC50 of drug formulations against 4T1 cells ranged from 1.4 to 9.9 µM. Western blot revealed that autophagy was the main cause of cytotoxicity. Geranial at 80 mg/kg was most effective at inhibiting tumor growth. CONCLUSIONS: Geranial is significantly more potent than neral and citral at 80 mg/kg (p < 0.001) and western blot of tumor tissues confirms that autophagy and not apoptosis is the major mechanism of tumor growth inhibition in p53-null 4T1 cells.
Assuntos
Antineoplásicos/química , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Inibidores Enzimáticos/química , Inibidores Enzimáticos/uso terapêutico , Monoterpenos/química , Monoterpenos/uso terapêutico , Monoterpenos Acíclicos , Animais , Antineoplásicos/administração & dosagem , Sobrevivência Celular , Química Farmacêutica , Inibidores Enzimáticos/administração & dosagem , Feminino , Humanos , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Camundongos , Camundongos Endogâmicos BALB C , Micelas , Monoterpenos/administração & dosagem , Estereoisomerismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Over three decades have passed since the first report on the expression of CA125 by ovarian tumors. Since that time our understanding of ovarian cancer biology has changed significantly to the point that these tumors are now classified based on molecular phenotype and not purely on histological attributes. However, CA125 continues to be, with the recent exception of HE4, the only clinically reliable diagnostic marker for ovarian cancer. Many large-scale clinical trials have been conducted or are underway to determine potential use of serum CA125 levels as a screening modality or to distinguish between benign and malignant pelvic masses. CA125 is a peptide epitope of a 3-5 million Da mucin, MUC16. Here we provide an in-depth review of the literature to highlight the importance of CA125 as a prognostic and diagnostic marker for ovarian cancer. We focus on the increasing body of literature describing the biological role of MUC16 in the progression and metastasis of ovarian tumors. Finally, we consider previous and on-going efforts to develop therapeutic approaches to eradicate ovarian tumors by targeting MUC16. Even though CA125 is a crucial marker for ovarian cancer, the exact structural definition of this antigen continues to be elusive. The importance of MUC16/CA125 in the diagnosis, progression and therapy of ovarian cancer warrants the need for in-depth research on the biochemistry and biology of this mucin. A renewed focus on MUC16 is likely to culminate in novel and more efficient strategies for the detection and treatment of ovarian cancer.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/genética , Antígeno Ca-125/genética , Imunoterapia , Proteínas de Membrana/genética , Neoplasias Ovarianas/terapia , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/imunologia , Antígeno Ca-125/imunologia , Ensaios Clínicos como Assunto , Progressão da Doença , Feminino , Expressão Gênica , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/imunologia , Metástase Neoplásica , Neoplasias Ovarianas/diagnóstico , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/patologia , Prognóstico , Proteínas/genética , Proteínas/imunologia , Proteína 2 do Domínio Central WAP de Quatro DissulfetosRESUMO
The transition of naive T lymphocytes into antigenically activated effector cells is associated with a metabolic shift from oxidative phosphorylation to aerobic glycolysis. This shift facilitates production of the key anti-tumor cytokine interferon (IFN)-γ; however, an associated loss of mitochondrial efficiency in effector T cells ultimately limits anti-tumor immunity. Memory phenotype (MP) T cells are a newly recognized subset that arises through homeostatic activation signals following hematopoietic transplantation. We show here that human CD4+ MP cell differentiation is associated with increased glycolytic and oxidative metabolic activity, but MP cells retain less compromised mitochondria compared to effector CD4+ T cells, and their IFN-γ response is less dependent on glucose and more reliant on glutamine. MP cells also produced IFN-γ more efficiently in response to weak T cell receptor (TCR) agonism than effectors and mediated stronger responses to transformed B cells. MP cells may thus be particularly well suited to carry out sustained immunosurveillance against neoplastic cells.
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BACKGROUND: Overcoming platinum resistance is a major obstacle in the treatment of Epithelial Ovarian Cancer (EOC). In our previous work Decoy Receptor 3 (DcR3) was found to be related to platinum resistance. The major objective of this work was to define the cellular interaction of DcR3 with EOC and to explore its effects on platinum responsiveness. METHODS: We studied cell lines and primary cultures for the expression of and the cells ability to bind DcR3. Cells were cultured with DcR3 and then exposed to platinum. Cell viability was determined by MTT assay. Finally, the cells molecular response to DcR3 was studied using real time RT-PCR based differential expression arrays, standard RT-PCR, and Western blot. RESULTS: High DcR3 in the peritoneal cavity of women with EOC is associated with significantly shorter time to first recurrence after platinum based therapy (p = 0.02). None-malignant cells contribute DcR3 in the peritoneal cavity. The cell lines studied do not secrete DcR3; however they all bind exogenous DcR3 to their surface implying that they can be effected by DcR3 from other sources. DcR3s protein binding partners are minimally expressed or negative, however, all cells expressed the DcR3 binding Heparan Sulfate Proteoglycans (HSPGs) Syndecans-2, and CD44v3. DcR3 binding was inhibited by heparin and heparinase. After DcR3 exposure both SKOV-3 and OVCAR-3 became more resistant to platinum with 15% more cells surviving at high doses. On the contrary CaOV3 became more sensitive to platinum with 20-25% more cell death. PCR array analysis showed increase expression of BRCA1 mRNA in SKOV-3 and OVCAR-3 and decreased BRCA1 expression in CaOV-3 after exposure to DcR3. This was confirmed by gene specific real time PCR and Western blot analysis. CONCLUSIONS: Non-malignant cells contribute to the high levels of DcR3 in ovarian cancer. DcR3 binds readily to EOC cells via HSPGs and alter their responsiveness to platinum chemotherapy. The paradoxical responses seen were related to the expression pattern of HSPGs available on the cells surface to interact with. Although the mechanism behind this is not completely known alterations in DNA repair pathways including the expression of BRCA1 appear to be involved.
Assuntos
Regulação Neoplásica da Expressão Gênica , Genes BRCA1 , Proteoglicanas de Heparan Sulfato/metabolismo , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Epiteliais e Glandulares/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Platina/farmacologia , Membro 6b de Receptores do Fator de Necrose Tumoral/metabolismo , Antineoplásicos/farmacologia , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Ligantes , Ligação Proteica , Membro 6b de Receptores do Fator de Necrose Tumoral/genéticaRESUMO
Oxidative phosphorylation is an active metabolic pathway in cancer. Atovaquone is an oral medication that inhibits oxidative phosphorylation and is FDA-approved for the treatment of malaria. We investigated its potential anti-cancer properties by measuring cell proliferation in 2D culture. The clinical formulation of atovaquone, Mepron, was given to mice with ovarian cancers to monitor its effects on tumor and ascites. Patient-derived cancer stem-like cells and spheroids implanted in NSG mice were treated with atovaquone. Atovaquone inhibited the proliferation of cancer cells and ovarian cancer growth in vitro and in vivo. The effect of atovaquone on oxygen radicals was determined using flow and imaging cytometry. The oxygen consumption rate (OCR) in adherent cells was measured using a Seahorse XFe96 Extracellular Flux Analyzer. Oxygen consumption and ATP production were inhibited by atovaquone. Imaging cytometry indicated that the majority of the oxygen radical flux triggered by atovaquone occurred in the mitochondria. Atovaquone decreased the viability of patient-derived cancer stem-like cells and spheroids implanted in NSG mice. NMR metabolomics showed shifts in glycolysis, citric acid cycle, electron transport chain, phosphotransfer, and metabolism following atovaquone treatment. Our studies provide the mechanistic understanding and preclinical data to support the further investigation of atovaquone's potential as a gynecologic cancer therapeutic.
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The huKS-IL2 immunocytokine (IC) consists of IL2 fused to a mAb against EpCAM, while the hu14.18-IL2 IC recognizes the GD2 disialoganglioside. They are under evaluation for treatment of EpCAM(+) (ovarian) and GD2(+) (neuroblastoma and melanoma) malignancies because of their proven ability to enhance tumor cell killing by antibody-dependent cell-mediated cytotoxicity (ADCC) and by antitumor cytotoxic T cells. Here, we demonstrate that huKS-IL2 and hu14.18-IL2 bind to tumor cells via their antibody components and increase adhesion and activating immune synapse (AIS) formation with NK cells by engaging the immune cells' IL-2 receptors (IL2R). The NK leukemia cell line, NKL (which expresses high affinity IL2Rs), shows fivefold increase in binding to tumor targets when treated with IC compared to matching controls. This increase in binding is effectively inhibited by blocking antibodies against CD25, the α-chain of the IL2R. NK cells isolated from the peritoneal environment of ovarian cancer patients, known to be impaired in mediating ADCC, bind to huKS-IL2 via CD25. The increased binding between tumor and effector cells via ICs is due to the formation of AIS that are characterized by the simultaneous polarization of LFA-1, CD2 and F-actin at the cellular interface. AIS formation of peritoneal NK and NKL cells is inhibited by anti-CD25 blocking antibody and is 50-200% higher with IC versus the parent antibody. These findings demonstrate that the IL-2 component of the IC allows IL2Rs to function not only as receptors for this cytokine but also as facilitators of peritoneal NK cell binding to IC-coated tumor cells.
Assuntos
Sinapses Imunológicas/imunologia , Imunoterapia/métodos , Subunidade alfa de Receptor de Interleucina-2/imunologia , Interleucina-2/análogos & derivados , Células Matadoras Naturais/imunologia , Anticorpos Monoclonais , Linhagem Celular Tumoral , Separação Celular , Feminino , Citometria de Fluxo , Gangliosídeos/imunologia , Humanos , Interleucina-2/imunologia , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Ativação Linfocitária/imunologia , Microscopia Confocal , Neoplasias Ovarianas/imunologia , Transporte Proteico/imunologia , Proteínas Recombinantes de FusãoRESUMO
OBJECTIVE: Treatment of high-grade serous ovarian cancer (HGSOC) will benefit from early detection of cancer. Here, we provide proof-of-concept data supporting the hypothesis that circulating immune cells, because of their early recognition of tumors and the tumor microenvironment, can be considered for biomarker discovery. METHODS: Longitudinal blood samples from C57BL/6 mice bearing syngeneic ovarian tumors and peripheral blood mononuclear cells (PBMC) from healthy postmenopausal women and newly diagnosed for HGSOC patients were subjected to RNASeq. The results from human immune cells were validated using Affymetrix microarrays. Differentially expressed transcripts in immune cells from tumor-bearing mice and HGSOC patients were compared to matching controls. RESULTS: A total of 1282 transcripts (798 and 484, up- and downregulated, respectively) were differentially expressed in the tumor-bearing mice as compared with controls. Top 100 genes showing longitudinal changes in gene expression 2, 4, 7, and 18 days after tumor implantation were identified. Analysis of the PBMC from healthy post-menopausal women and HGSOC patients identified 4382 differentially expressed genes and 519 of these were validated through Affymetrix microarray analysis. A total of 384 genes, including IL-1R2, CH3L1, Infitm1, FP42, CXC42, Hdc, Spib, and Sema6b, were differentially expressed in the human and mouse datasets. CONCLUSION: The PBMC transcriptome shows longitudinal changes in response to the progressing tumor. Several potential biomarker transcripts were identified in HGSOC patients and mouse models. Monitoring their expression in individual PBMC subsets can serve as additional discriminator for the diagnosis of HGSOC.
Assuntos
Cistadenocarcinoma Seroso/diagnóstico , Neoplasias Ovarianas/diagnóstico , Microambiente Tumoral , Animais , Biomarcadores Tumorais , Linhagem Celular , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/metabolismo , Cistadenocarcinoma Seroso/patologia , Feminino , Perfilação da Expressão Gênica , Humanos , Leucócitos Mononucleares/metabolismo , Camundongos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Estudo de Prova de Conceito , TranscriptomaRESUMO
BACKGROUND: Cancer cells utilize a variety of mechanisms to evade immune detection and attack. Effective immune detection largely relies on the formation of an immune synapse which requires close contact between immune cells and their targets. Here, we show that MUC16, a heavily glycosylated 3-5 million Da mucin expressed on the surface of ovarian tumor cells, inhibits the formation of immune synapses between NK cells and ovarian tumor targets. Our results indicate that MUC16-mediated inhibition of immune synapse formation is an effective mechanism employed by ovarian tumors to evade immune recognition. RESULTS: Expression of low levels of MUC16 strongly correlated with an increased number of conjugates and activating immune synapses between ovarian tumor cells and primary naïve NK cells. MUC16-knockdown ovarian tumor cells were more susceptible to lysis by primary NK cells than MUC16 expressing controls. This increased lysis was not due to differences in the expression levels of the ligands for the activating receptors DNAM-1 and NKG2D. The NK cell leukemia cell line (NKL), which does not express KIRs but are positive for DNAM-1 and NKG2D, also conjugated and lysed MUC16-knockdown cells more efficiently than MUC16 expressing controls. Tumor cells that survived the NKL challenge expressed higher levels of MUC16 indicating selective lysis of MUC16(low) targets. The higher csMUC16 levels on the NKL resistant tumor cells correlated with more protection from lysis as compared to target cells that were never exposed to the effectors. CONCLUSION: MUC16, a carrier of the tumor marker CA125, has previously been shown to facilitate ovarian tumor metastasis and inhibits NK cell mediated lysis of tumor targets. Our data now demonstrates that MUC16 expressing ovarian cancer cells are protected from recognition by NK cells. The immune protection provided by MUC16 may lead to selective survival of ovarian cancer cells that are more efficient in metastasizing within the peritoneal cavity and also at overcoming anti-tumor innate immune responses.
Assuntos
Antígeno Ca-125/imunologia , Citoproteção/imunologia , Sinapses Imunológicas/imunologia , Células Matadoras Naturais/imunologia , Proteínas de Membrana/imunologia , Neoplasias Ovarianas/imunologia , Antígenos de Diferenciação de Linfócitos T/metabolismo , Adesão Celular , Contagem de Células , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Sobrevivência Celular , Citotoxicidade Imunológica , Feminino , Técnicas de Silenciamento de Genes , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Células Matadoras Naturais/patologia , Ligantes , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Neoplasias Ovarianas/patologiaRESUMO
BACKGROUND: MUC16 is a cell surface mucin expressed at high levels by epithelial ovarian tumors. Following proteolytic cleavage, cell surface MUC16 (csMUC16) is shed in the extracellular milieu and is detected in the serum of cancer patients as the tumor marker CA125. csMUC16 acts as an adhesion molecule and facilitates peritoneal metastasis of ovarian tumors. Both sMUC16 and csMUC16 also protect cancer cells from cytotoxic responses of natural killer (NK) cells. In a previous study we demonstrated that sMUC16 binds to specific subset of NK cells. Here, we identify the csMUC16/sMUC16 binding partner expressed on immune cells. RESULTS: Analysis of immune cells from the peripheral blood and peritoneal fluid of ovarian cancer patients indicates that in addition to NK cells, sMUC16 also binds to B cells and monocytes isolated from the peripheral blood and peritoneal fluid. I-type lectin, Siglec-9, is identified as the sMUC16 receptor on these immune cells. Siglec-9 is expressed on approximately 30-40% of CD16pos/CD56dim NK cells, 20-30% of B cells and >95% of monocytes. sMUC16 binds to the majority of the Siglec-9pos NK cells, B cells and monocytes. sMUC16 is released from the immune cells following neuraminidase treatment. Siglec-9 transfected Jurkat cells and monocytes isolated from healthy donors bind to ovarian tumor cells via Siglec-9-csMUC16 interaction. CONCLUSIONS: Recent studies indicate that csMUC16 can act as an anti-adhesive agent that blocks tumor-immune cell interactions. Our results demonstrate that similar to other mucins, csMUC16 can also facilitate cell adhesion by interacting with a suitable binding partner such as mesothelin or Siglec-9. Siglec-9 is an inhibitory receptor that attenuates T cell and NK cell function. sMUC16/csMUC16-Siglec-9 binding likely mediates inhibition of anti-tumor immune responses.
Assuntos
Antígenos CD/metabolismo , Linfócitos B/metabolismo , Antígeno Ca-125/metabolismo , Células Matadoras Naturais/metabolismo , Lectinas/metabolismo , Proteínas de Membrana/metabolismo , Monócitos/metabolismo , Neoplasias Ovarianas/metabolismo , Antígenos CD/imunologia , Linfócitos B/imunologia , Western Blotting , Antígeno Ca-125/imunologia , Separação Celular , Feminino , Citometria de Fluxo , Humanos , Células Jurkat , Células Matadoras Naturais/imunologia , Lectinas/imunologia , Proteínas de Membrana/imunologia , Monócitos/imunologia , Neoplasias Ovarianas/imunologia , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido SiálicoRESUMO
Oxidative stress inhibits Na+/K+-ATPase (NKA), the ion channel that maintains membrane potential. Here, we investigate the role of oxidative stress-mediated by plumbagin and atovaquone in the inhibition of NKA activity. We confirm that plumbagin and atovaquone inhibit the proliferation of three human (OVCAR-3, SKOV-3, and TYKNu) and one mouse (ID8) ovarian cancer cell lines. The oxygen radical scavenger, N-acetylcysteine (NAC), attenuates the chemotoxicity of plumbagin and atovaquone. Whole-cell patch clamping demonstrates that plumbagin and atovaquone inhibit outward and the inward current flowing through NKA in SKOV-3 and OVCAR-3. Although both drugs decrease cellular ATP; providing exogenous ATP (5 mM) in the pipet solution used during patch clamping did not recover NKA activity in the plumbagin or atovaquone treated SKOV-3 and OVCAR-3 cells. However, pretreatment of the cells with NAC completely abrogated the NKA inhibitory activity of plumbagin and atovaquone. Exposure of the SKOV-3 cells to either drug significantly decreases the expression of NKA. We conclude that oxidative stress caused by plumbagin and atovaquone degrades NKA, resulting in the inability to maintain ion transport. Therefore, when evaluating compounds that induce oxidative stress, it is important to consider the contribution of NKA inhibition to their cytotoxic effects on tumor cells.
Assuntos
Atovaquona/farmacologia , Naftoquinonas/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Estresse Oxidativo/efeitos dos fármacos , ATPase Trocadora de Sódio-Potássio/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Antineoplásicos Fitogênicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Transporte de Íons/efeitos dos fármacos , Camundongos , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Estresse Oxidativo/fisiologia , Técnicas de Patch-ClampRESUMO
Although levels of the circulating ovarian cancer marker (CA125) can distinguish ovarian masses that are likely to be malignant and correlate with severity of disease, serum CA125 has not proved useful in general population screening. Recently, cell culture studies have indicated that MUC16 may bind to the Siglec-9 receptor on natural killer (NK) cells where it downregulates the cytotoxicity of NK cells, allowing ovarian cancer cells to evade immune surveillance. We present evidence that the presence of MUC16 can be locally visualized and imaged on the surface of peripheral blood mononuclear cells (PBMCs) in ovarian cancer via a novel "digital" cytometry technique that incorporates: (i) OC125 monoclonal antibody-conjugated gold nanoparticles as optical nanoprobes, (ii) a high contrast dark-field microscopy system to detect PBMC-bound gold nanoparticles, and (iii) a computational algorithm for automatic counting of these nanoparticles to estimate the quantity of surface-bound MUC16. The quantitative detection of our technique was successfully demonstrated by discriminating clones of the ovarian cancer cell line, OVCAR3, based on low, intermediate, and high expression levels of MUC16. Additionally, PBMC surface-bound MUC16 was tracked in an ovarian cancer patient over a 17 month period; the results suggest that the binding of MUC16 on the surface of immune cells may play an early indicator for recurrent metastasis 6 months before computational tomography-based clinical diagnosis. We also demonstrate that the levels of surface-bound MUC16 on PBMCs from five ovarian cancer patients were greater than those from five healthy controls.
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Nanopartículas Metálicas , Neoplasias Ovarianas , Apoptose , Antígeno Ca-125 , Linhagem Celular Tumoral , Feminino , Ouro , Humanos , Leucócitos Mononucleares , Proteínas de MembranaRESUMO
PROBLEM: We hypothesize that activated peritoneal immune cells can be redirected to target ovarian tumors. Here, we obtain fundamental knowledge of the peritoneal immune environment through deep immunophenotyping of T cells, dendritic cells (DC), and innate lymphoid cells (ILC) of ovarian cancer patients. METHOD OF STUDY: T cells, DC, and ILC from ascites of ovarian cancer patients (n = 15) and peripheral blood of post-menopausal healthy donors (n = 6) were immunophenotyped on a BD Fortessa cytometer using three panels-each composed of 16 antibodies. The data were analyzed manually and by t-SNE/DensVM. CA125 levels were obtained from patient charts. RESULTS: We observed decreased CD3+ T cells and a higher proportion of activated CD4+ and effector memory CD4+ /CD8+ T cells, plasmacytoid DC, CD1c+ and CD141+ myeloid DC and CD56Hi NK cells in ascites. t-SNE/DensVM identified eight T cell, 17 DC, and 17 ILC clusters that were unique in the ascites compared to controls. Hierarchical clustering of cell frequency distinctly segregated the T-cell and ILC clusters from controls. Increased CA125 levels were associated with decreased CD8+ /CD45RA+ /CD45RO- /CCR7- T cells. CONCLUSION: The identified immune clusters serve as the basis for interrogation of the peritoneal immune environment and the development of novel immunologic modalities against ovarian cancer.
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Líquido Ascítico/imunologia , Células Dendríticas/imunologia , Linfócitos/imunologia , Neoplasias Ovarianas/imunologia , Adulto , Idoso , Feminino , Humanos , Imunidade Inata , Pessoa de Meia-IdadeRESUMO
The Na+/K+-ATPase (NKA) complex is the master regulator of membrane potential and a target for anti-cancer therapies. Here, we investigate the effect of drug-induced oxidative stress on NKA activity. The natural product, plumbagin increases oxygen radicals through inhibition of oxidative phosphorylation. As a result, plumbagin treatment results in decreased production of ATP and a rapid increase in intracellular oxygen radicals. We show that plumbagin induces apoptosis in canine cancer cells via oxidative stress. We use this model to test the effect of oxidative stress on NKA activity. Using whole-cell patch-clamp electrophysiology we demonstrate that short-term exposure (4 min) to plumbagin results in 48% decrease in outward current at +50 mV. Even when exogenous ATP was supplied to the cells, plumbagin treatment resulted in 46% inhibition of outward current through NKA at +50 mV. In contrast, when the canine cancer cells were pre-treated with the oxygen radical scavenger, N-acetylcysteine, the NKA inhibitory activity of plumbagin was abrogated. These experiments demonstrate that the oxidative stress-causing agents such as plumbagin and its analogues, are a novel avenue to regulate NKA activity in tumors.
Assuntos
Potenciais da Membrana/efeitos dos fármacos , Naftoquinonas/farmacologia , Neoplasias/tratamento farmacológico , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Acetilcisteína/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cães , Ensaios de Seleção de Medicamentos Antitumorais , Naftoquinonas/uso terapêutico , Neoplasias/patologia , Fosforilação Oxidativa/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Técnicas de Patch-Clamp , Espécies Reativas de Oxigênio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
Aerobic glycolysis is an important metabolic adaptation of cancer cells. There is growing evidence that oxidative phosphorylation is also an active metabolic pathway in many tumors, including in high grade serous ovarian cancer. Metastasized ovarian tumors use fatty acids for their energy needs. There is also evidence of ovarian cancer stem cells privileging oxidative phosphorylation (OXPHOS) for their metabolic needs. Metformin and thiazolidinediones such as rosiglitazone restrict tumor growth by inhibiting specific steps in the mitochondrial electron transport chain. These observations suggest that strategies to interfere with oxidative phosphorylation should be considered for the treatment of ovarian tumors. Here, we review the literature that supports this hypothesis and describe potential agents and critical control points in the oxidative phosphorylation pathway that can be targeted using small molecule agents. In this review, we also discuss potential barriers that can reduce the efficacy of the inhibitors of oxidative phosphorylation.
RESUMO
Plumbagin, an anti-cancer agent, is toxic to cells of multiple species. We investigated if plumbagin targets conserved biochemical processes. Plumbagin induced DNA damage and apoptosis in cells of diverse mutational background with comparable potency. A 3-5 fold increase in intracellular oxygen radicals occurred in response to plumbagin. Neutralization of the reactive oxygen species by N-acetylcysteine blocked apoptosis, indicating a central role for oxidative stress in plumbagin-mediated cell death. Plumbagin docks in the ubiquinone binding sites (Q0 and Qi) of mitochondrial complexes I-III, the major sites for oxygen radicals. Plumbagin decreased oxygen consumption rate, ATP production and optical redox ratio (NAD(P)H/FAD) indicating interference with electron transport downstream of mitochondrial Complex II. Oxidative stress induced by plumbagin triggered an anti-oxidative response via activation of Nrf2. Plumbagin and the Nrf2 inhibitor, brusatol, synergized to inhibit cell proliferation. These data indicate that while inhibition of electron transport is the conserved mechanism responsible for plumbagin's chemotoxicity, activation of Nrf2 is the resulting anti-oxidative response that allows plumbagin to serve as a chemopreventive agent. This study provides the basis for designing potent and selective plumbagin analogs that can be coupled with suitable Nrf2 inhibitors for chemotherapy or administered as single agents to induce Nrf2-mediated chemoprevention.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Antioxidantes/farmacologia , Transporte de Elétrons/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Naftoquinonas/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Antineoplásicos Fitogênicos/química , Antioxidantes/química , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sinergismo Farmacológico , Humanos , Modelos Moleculares , Conformação Molecular , Fator 2 Relacionado a NF-E2/antagonistas & inibidores , Naftoquinonas/química , Oxirredução/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
The goal of this study was to investigate the anti-cancer effects of Trans10,cis12 conjugated linoleic acid (t10,c12 CLA). MTT assays and QCM™ chemotaxis 96-wells were used to test the effect of t10,c12 CLA on the proliferation and migration and invasion of cancer cells. qPCR and Western Blotting were used to determine the expression of specific factors. RNA sequencing was conducted using the Illumina platform and apoptosis was measured using a flow cytometry assay. t10,c12 CLA (IC50, 7 µM) inhibited proliferation of ovarian cancer cell lines SKOV-3 and A2780. c9,t11 CLA did not attenuate the proliferation of these cells. Transcription of 165 genes was significantly repressed and 28 genes were elevated. Genes related to ER stress, ATF4, CHOP, and GADD34 were overexpressed whereas EDEM2 and Hsp90, genes required for proteasomal degradation of misfolded proteins, were downregulated upon treatment. While apoptosis was not detected, t10,c12 CLA treatment led to 9-fold increase in autophagolysosomes and higher levels of LC3-II. G1 cell cycle arrest in treated cells was correlated with phosphorylation of GSK3ß and loss of ß-catenin. microRNA miR184 and miR215 were upregulated. miR184 likely contributed to G1 arrest by downregulating E2F1. miR215 upregulation was correlated with increased expression of p27/Kip-1. t10,c12 CLA-mediated inhibition of invasion and migration correlated with decreased expression of PTP1b and decreased Src activation by inhibiting phosphorylation at Tyr416. Due to its ability to inhibit proliferation and migration, t10,c12 CLA should be considered for treatment of ovarian cancer.
Assuntos
Autofagia/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Ácidos Linoleicos Conjugados/farmacologia , Neoplasias Ovarianas/patologia , Quinases da Família src/metabolismo , Linhagem Celular Tumoral , Feminino , HumanosRESUMO
Protein transduction with cell penetrating peptides over the past several years has been shown to be an effective way of delivering proteins in vitro and now several reports have also shown valuable in vivo applications in correcting disease states. An impressive bioinspired phenomenon of crossing biological barriers came from HIV transactivator Tat protein. Specifically, the protein transduction domain of HIV Tat has been shown to be a potent pleiotropic peptide in protein delivery. Various approaches such as molecular modeling, arginine guanidinium head group structural strategy, multimerization of PTD sequence and phage display system have been applied for taming of the PTD. This has resulted in identification of PTD variants which are efficient in cell membrane penetration and cytoplasmic delivery. In spite of these state of the art technologies, the dilemma of low protein transduction efficiency and target specific delivery of PTD fusion proteins remains unsolved. Moreover, some misconceptions about PTD of Tat in the literature require considerations. We have assembled critical information on secretory, plasma membrane penetration and transcellular properties of Tat and PTD using molecular analysis and available experimental evidences.