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1.
Life Sci ; 350: 122751, 2024 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-38797363

RESUMO

AIM: To understand the mechanism of prostaglandin E2 (PGE2)-mediated immunosuppression in dendritic cells (DCs). MAIN METHODS: In vivo experiments were conducted on 4T1 tumor bearing mice (TBM). In vitro experiments were performed in bone marrow-derived DCs (BMDCs), or spleen cells. Cytokines were monitored by ELISA/ELIspot. Gene expression was monitored by RT-PCR/flow cytometry. KEY FINDINGS: In silico, in vitro, and in vivo experiments in 4T1 TBM revealed that PGE2 induced IL-6/pSTAT3 signaling through EP4 receptors in DCs, resulting in their dysfunction. These effects were reversed by EP4 antibody neutralization, EP4 antagonist, and STAT3 inhibitory peptides. PGE2 induced IL-6 was regulated by miR-365, as its mimic inhibited PGE2 induced IL-6 and the inhibitor increased lL-6 levels in DC. Bio-informatic analysis in human mammary cancers also revealed a strong compared co-relation between PGE2 and IL-6 (Correlation AnalyzeR) (R = 0.94). Mice bearing PTGS-2 KD 4T1 tumors had decreased tumor burden, PGE2, EP4, IL-6, and pSTAT3 signaling, along with improved DCs and T cell functions. Treatment of mice with a cyclooxygenase-2 (COX-2) inhibitor or EP4 antagonist decreased tumor burden, and this effect of EP4 antagonist was abrogated upon in vivo depletion of CD11c cells, indicating the crucial role of PGE2 signaling in DCs in tumor progression. SIGNIFICANCE: In summary, our data highlights the importance of dendritic cells in mediating PGE2-mediated immunosuppression and the use of EP4 or STAT3 inhibitors or miR365 mimics can restore immunogenicity in cancer.


Assuntos
Células Dendríticas , Dinoprostona , Interleucina-6 , MicroRNAs , Fator de Transcrição STAT3 , Transdução de Sinais , Animais , Fator de Transcrição STAT3/metabolismo , Células Dendríticas/metabolismo , Células Dendríticas/imunologia , MicroRNAs/genética , MicroRNAs/metabolismo , Camundongos , Dinoprostona/metabolismo , Interleucina-6/metabolismo , Feminino , Humanos , Camundongos Endogâmicos BALB C , Linhagem Celular Tumoral , Receptores de Prostaglandina E Subtipo EP4/metabolismo
2.
Biochim Biophys Acta Gen Subj ; 1867(4): 130312, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36690186

RESUMO

BACKGROUND: Tinospora cordifolia polysaccharide G1-4A activates antigen-presenting cells, but its effect on natural killer (NK) cells is not known. The objective of this study is to assess the effect of G1-4A on NK cells; direct effects as well as through dendritic cell (DC) cross-talk. METHODS: NK cell phenotype and function were assessed in spleen cells treated in vitro with G1-4A or isolated from mice administered with G1-4A. Following treatment with G1-4A in vitro or in cells isolated from G1-4A treated mice (in vivo), activated NK cell phenotype was characterized as CD3-NKp46+CD69+ cells by flow cytometry; NK cell function was evaluated by IFN-γ secretion (ELISA) and cytotoxicity assay (calcein release by target cells in effector: target cells co-culture assay). RESULTS: Both in vitro as well as in vivoG1-4A treatment increased phenotypic and functional activation of NK cells. So, we wanted to determine if this was through NK-DC crosstalk or direct activation of NK cells. There was increased NK cell activation following co-culture with bone marrow derived DC matured withG1-4A in vitro or splenic DC isolated from G1-4A administered mice indicating crosstalk. G1-4A also increased activation of NK cells in (a) CD11c depleted splenic cells that was contact dependent and (b) purified NKp46+ cells that was abrogated by PKC/mTOR inhibitors indicating direct effects on NK cells. CONCLUSION: In summary, treatment with G1-4A results in phenotypic and functional activation of NK cells directly as well as through NK-DC cross talk and has the potential to be used as an immunotherapeutic agent.


Assuntos
Tinospora , Animais , Camundongos , Células Dendríticas , Células Matadoras Naturais , Serina-Treonina Quinases TOR , Proteína Quinase C
3.
Biochim Biophys Acta Gen Subj ; 1866(11): 130215, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-35905921

RESUMO

BACKGROUND: Breast cancer is a heterogenous disease composed of multiple clonal populations and the mechanism by which the tumor microenvironment induces cancer stem cell plasticity is not fully understood. METHODS: MCF7 breast cancer cells were treated with macrophage conditioned medium (MɸCM). PD98059 and SB431542 were used for ERK and TGF-ßR inhibition respectively. Epithelial-mesenchymal transition (EMT) and cancer stem cell markers (CSC) were studied using qRT-PCR and flowcytometry. SCID mice were used for animal experiments. RESULTS: MɸCM- induced ERK/TGF-ß1 signaling led to enrichment of CSC and EMT in MCF7 cells and mammospheres. These effects were abrogated by both MEK inhibitor PD98059 (TGF-ß1 synthesis) and SB431542 (TGF-ß1 signaling). The increase in CSC was both hybrid (ALDH1+) and mesenchymal (CD44+ CD24- cells). Increase in hybrid E/M state was at a single cell level as confirmed by the increase in both claudin-1 (E) and vimentin (M). This did not have any growth advantage in SCID mice and monitoring of CSC and EMT markers before and after growth in SCID mice indicated reversal of these markers in tumor cells recovered from mice. Removal of MɸCM and neutralization of TNF-α, IL-6 and IL-1ß in MɸCM abrogated ERK phosphorylation, TGF-ß and CSC enrichment indicating the requirement of continuous signaling for maintenance. CONCLUSIONS: ERK signaling plays an important role in MɸCM- induced EMT and CSC plasticity which is completely reversible upon withdrawal of signals. GENERAL SIGNIFICANCE: Our experimental observations support the semi-independent nature of EMT-stemness connection which is very dynamic and reversible depending on the microenvironment.


Assuntos
Transição Epitelial-Mesenquimal , Neoplasias , Animais , Humanos , Células MCF-7 , Macrófagos , Camundongos , Camundongos SCID , Células-Tronco Neoplásicas , Fator de Crescimento Transformador beta1
4.
Life Sci ; 301: 120643, 2022 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-35569570

RESUMO

AIMS: Regulatory T cells play an essential role in immune tolerance and homeostasis. Their long-term stability depends on epigenetic modifications and identification of small molecule modulators of Treg differentiation therefore has many applications. In this study, we performed a novel functional screen of an epigenetic compound library to identify compounds that can modulate generation of TGF-ß1 induced T regulatory cells. MAIN METHODS: A screening strategy based on IFN-γ ELISA was designed to screen epigenetic compound library. Effect of hit compounds on Treg phenotype and function was assessed by RT-PCR, flow cytometry and suppression assays. TGF-ß signalling proteins were assayed by western blotting. Chromatin Immunoprecipitation (ChIP) assay was used to assess epigenetic modifications at Foxp3 gene locus. KEY FINDINGS: We screened 160 compounds to identify hits capable of reversing TGF-ß induced inhibition of IFN-γ production in activated spleen cells and CD4+ T cells. Two compounds EPZ004777 and FG-2216 consistently reversed TGF-ß1 iTregs in terms of (a) differentiation of naïve T cells into CD4+CD25+Foxp3+Treg cells, (b) Foxp3 target gene expression and (c) Treg suppressive function without affecting TGF-ß downstream signalling. ChIP assay revealed that the compounds were able to reverse - TGF- ß mediated decrease in epigenetic marks H3K27me3 and 5-mC and an increase in epigenetic marks H3K4me3 and H3K27Ac in the promoter and conserved non coding sequence (CNS1) regions of the Foxp3 gene. SIGNIFICANCE: EPZ004777 and FG-2216 have been identified as potent epigenetic modulators that can reverse TGF-ß1 induced T regulatory cells and may be used to treat diverse immune disorders.


Assuntos
Linfócitos T Reguladores , Fator de Crescimento Transformador beta1 , Adenosina/análogos & derivados , Epigênese Genética , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Compostos de Fenilureia , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta1/metabolismo
5.
Int J Radiat Biol ; : 1-15, 2022 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-35446183

RESUMO

PURPOSE: We have earlier characterized increased TGF-ß signaling in radioresistant breast cancer cells. In this study, we wanted to determine the effect of prolonged treatment of TGF-ßR inhibitor SB431542 on radiation-induced signaling, viz., genes regulating apoptosis, EMT, anti and pro-inflammatory cytokines. MATERIALS AND METHODS: Breast cancer cells were pretreated with TGF-ßR inhibitor (SB 431542) followed by exposure to 6 Gy and recovery period of 7 days (D7-6G). We assessed cell survival by MTT assay, cytokines by ELISA and expression analysis by RT-PCR, flow cytometry, and western blot. We carried out migration assays using trans well inserts. We performed bioinformatics analyses of human cancer database through cBioportal. RESULTS: There was an upregulation of TGF-ß1 and 3 and downregulation of TGF-ß2, TGF-ßR1, and TGF-ßR2 in invasive breast carcinoma samples compared to normal tissue. TGF-ß1 and TNF-α was higher in radioresistant D7-6G cells with upregulation of pSMAD3, pNF-kB, and ERK signaling. Pretreatment of D7-6G cells with TGF-ßR inhibitor SB431542 abrogated pSMAD3, increased proliferation, and migration along with an increase in apoptosis and pro-apoptotic genes. This was associated with hybrid E/M phenotype and downregulation of TGF-ß downstream genes, HMGA2 and Snail. There was complete agreement in the expression of mRNA and protein data in genes like vimentin, Snail and HMGA2 in different treatment groups. However, there was disagreement in expression of mRNA and protein in genes like Bax, Bcl-2, E-cadherin, Zeb-1 among the different treatment groups indicating post-transcriptional and post-translational processing of these proteins. Treatment of cells with only SB431542 also increased expression of some E/M genes indicating TGF-ß independent effects. Increased IL-6 and IL-10 secretion by SB431542 along with increase in pSTAT3 and pCREB1 could probably explain these TGF-ß/Smad3 independent effects. CONCLUSION: These results highlight that TGF-ß-pSMAD3 and TNF-α-pNF-kB are the predominant signaling pathways in radioresistant cells and possibility of some TGF-ß/Smad3 independent effects on prolonged treatment with the drug SB431542.

6.
Cell Biol Int ; 45(4): 804-819, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33325135

RESUMO

Our previous studies have shown that MCF-7 breast cancer cell line exposed to 6 Gy and allowed to recover for 7 days (D7-6G) developed radio-resistance. In this study, we have tested the ability of these cells to form tumors in severe combined immunodeficiency (SCID) mice and characterized these tumors by proteomic analyses. Untreated (MCF-C) and D7-6G cells (MCF-R) were injected s.c. in SCID mice and tumor growth monitored. On Day 18, the mice were killed and tumor tissues were fixed in formalin or RNA later. Expression of genes was assessed by reverse transcription-polymerase chain reaction and proteins by enzyme-linked immunosorbent assay/antibody labeling and flow cytometry. Label free proteomic analyses was carried out by liquid chromatography-mass spectrometry. Metabolic analysis was carried out using Seahorse analyzer. MCF-R cells had a shorter latency and formed larger tumors. These tumors were characterized by an increased expression of transforming growth factor ß (TGF-ß) isoforms; its downstream genes pSMAD3, Snail-1, Zeb-1, HMGA2; hybrid epithelial/mesenchymal phenotype; migration, enrichment of cancer stem cells and radioresistance following challenge dose of radiation. Proteomic analysis of MCF-7R tumors resulted in identification of a total of 649 differentially expressed proteins and pathway analyses using protein annotation through evolutionary relationship indicated enrichment of genes involved in metabolism. Data are available via ProteomeXchange with identifier PXD022506. Seahorse analyzer confirmed increased metabolism in these cells with increased oxidative phosphorylation as well as glycolysis. Increased uptake of 2-NBDG further confirmed increased glycolysis. In summary, we demonstrate that radioresistant breast cancer cells had an enrichment of TGF-ß signaling and increased metabolism.


Assuntos
Neoplasias da Mama/radioterapia , Tolerância a Radiação , Animais , Transição Epitelial-Mesenquimal , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Camundongos , Camundongos SCID , Transcriptoma , Fator de Crescimento Transformador beta/metabolismo
7.
Colloids Surf B Biointerfaces ; 197: 111445, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33166931

RESUMO

Biohybrids (a combination of biological material and inorganic nanoparticles) offer a number of advantages like improved functionality over conventional materials.Thus, to understand the practical application of biohybrids as drug carriers, a biohybrid drug carrier of colloidal silica nanoparticles (NP)-sodium alginate loaded with doxorubicin (Dox-biohybrid) was synthesized by evaporation induced self-assembly (EISA) using spray drying technique. Further, the morphology, size and interactions between various components of the biohybrid were studied through SEM, DLS and FTIR techniques. The drug loading efficiency, release profile, cellular uptake and cytotoxicity of Dox-biohybrid was investigated and compared with free Dox. The drug loading efficiencies of Dox-biohybrid, Dox-silica NP and Dox-sodium alginate were 93.7 %, 96.4 % and 88.3 % respectively. In vitro release study showed a slow release of entrapped Dox from Dox-biohybrid as compared to other carriers. This release was also pH-responsive with significantly higher cumulative drug release at pH 5.5 (cancer microenvironment) in comparison to pH 7.4 (physiological conditions). The empty biohybrid carrier did not show cytotoxicity to normal mouse lymphocytes upto a concentration of 25 µg/mL which was used further. The uptake of Dox from Dox-biohybrid by A549 cells was more than 2fold as compared to uptake from free Dox. in vitro viability assay revealed that treatment of lung carcinoma A549 cells with Dox-biohybrid resulted in 50 % loss of cell viability at 500 nM, compared to only 12 % loss with free Dox. Thus, we report the synthesis of a novel biohybrid drug delivery system by means of spray drying process that has promising applications in cancer treatment.


Assuntos
Portadores de Fármacos , Nanopartículas , Alginatos , Animais , Doxorrubicina/farmacologia , Sistemas de Liberação de Medicamentos , Liberação Controlada de Fármacos , Concentração de Íons de Hidrogênio , Camundongos , Dióxido de Silício , Secagem por Atomização
8.
Cancer Immunol Immunother ; 70(1): 153-168, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32661685

RESUMO

The contribution of immune cells in soft tissue sarcomas (STS) is not completely known and understanding their role is very essential for employing immunotherapy strategies. Here, we show that murine fibrosarcoma-conditioned medium promoted total spleen cell proliferation but inhibited T cell responses to mitogenic and allo-antigen-mediated stimulation. This increased proliferation was found to be in B cells resulting in generation of Breg further leading to Treg population. This was found to be the same in vitro and in vivo. The phenotype of these B cells was CD19+CD81+CD27+CD25+PD-L1hi and they secreted both IL-10 and TGF-ß. These tumor evoked Bregs (tBreg), when co-cultured with B depleted T cells, suppressed their proliferation in response to anti-CD3/CD28 stimulation. tBreg-induced suppression of T cell responses was not abrogated by the inhibition or neutralization of IL-10 but by the small molecule inhibitor of TGFß Receptor type I, SB431542. While SB531542 per se was not cytotoxic to tumor cells, administration of SB431542 in tumor-bearing mice (TBM) significantly reduced the tumor burden. In addition, the treatment significantly reduced Treg cells and rescued proliferation of T cells in response to mitogen and allo-antigen. Collectively, our results identify that tumor evoked Breg cells mediate T cell immune suppression through TGFß-mediated pathway and that targeting the Breg-Treg axis can be potentially used as an immunotherapy agent.


Assuntos
Linfócitos B Reguladores/efeitos dos fármacos , Benzamidas/farmacologia , Dioxóis/farmacologia , Fibrossarcoma/tratamento farmacológico , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Linfócitos T Reguladores/efeitos dos fármacos , Carga Tumoral/efeitos dos fármacos , Animais , Antígenos CD/metabolismo , Linfócitos B Reguladores/imunologia , Proliferação de Células/efeitos dos fármacos , Feminino , Fibrossarcoma/imunologia , Fibrossarcoma/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Interleucina-10/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Baço/efeitos dos fármacos , Baço/imunologia , Baço/metabolismo , Linfócitos T Reguladores/imunologia , Fator de Crescimento Transformador beta/metabolismo , Carga Tumoral/imunologia
9.
Int J Radiat Biol ; 96(8): 988-998, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32396024

RESUMO

Purpose: To evaluate the effect of ionizing radiation (IR) exposure on differentiation and maturation of dendritic cells (DC).Materials and methods: Bone marrow progenitor cells irradiated in vitro or isolated from mice exposed to whole body or localized tumor irradiation were differentiated into DC. Phenotypic maturation of DC was characterized by labeling with specific antibodies and flow cytometry analysis. Cytokines were estimated by ELISA.Results: Splenic and bone marrow-derived DC (BMDC) from tumor-bearing mice exposed to localized irradiation showed abrogation of tumor-induced immunosuppression. This was not due to the effect of radiation on tumor cells as DC derived from normal mice exposed to whole-body irradiation (WBI) also showed increase in immune-activating potential of DC. This was observed in terms of increased phenotypic and functional activation of DCs. This phenomenon was also recapitulated if DC were differentiated from in vitro irradiated progenitor cells and was found to be due to STAT5/Zbtb46 signaling mediated by the irradiation-induced apoptotic bodies (ABs). When these ABs were depleted using annexin-beads, these effects were reversed confirming the involvement of this pathway. The role of ABs was further validated in DC derived from mice exposed to WBI in adaptive response experiments with 0.1 Gy priming dose prior to 2 Gy challenge dose. A corresponding reduction in DC maturation markers was observed with decrease in apoptosis in vivo. Further, these DCs derived from irradiated progenitors (IP) could resist the suppressive effects of tumor conditioned medium (TCM) and had increased immune-activating potential as seen in the tumor-bearing mice.Conclusions: Though radiation is the most commonly used therapeutic modality for cancer, its effects on dendritic cell differentiation is not completely understood. We demonstrate here for the first time that exposure to select doses of IR can increase immune-activating potential of DC through ABs. This can have implications in selection of appropriate doses of IR during radiotherapy of cancer patients.


Assuntos
Apoptose/efeitos da radiação , Células Dendríticas/citologia , Células Dendríticas/efeitos da radiação , Vesículas Extracelulares/efeitos da radiação , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais/efeitos da radiação , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular Tumoral , Células Dendríticas/metabolismo , Vesículas Extracelulares/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação/efeitos da radiação
10.
Int J Radiat Biol ; 95(6): 655-666, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30676176

RESUMO

Purpose: Our earlier studies demonstrated that transient radio-adaptive responses (RAR) in BALB/c mice were due to MAPK hyperactivation. The objective of this study was to determine the time duration of this low dose induced MAPK activation in BALB/c mice and to find out if similar adaptive responses are observed in C57BL/6 mice. Materials and methods: Mice were irradiated with 0.1 Gy priming dose (PD), 2 Gy challenge dose (CD) with an interval of 4 h (P + CD) and radiation induced immunosuppression in splenic lymphocytes was monitored as the endpoint for RAR. Results: Time kinetics following 0.1 Gy demonstrated persistence of MAPK hyperactivation till 48 h. Similar experiments in C57BL/6 mice indicated absence of RAR at 24 h following CD, in spite of MAPK activation which was also confirmed by time kinetics. Therefore, upstream activators of MAPK, viz., reactive oxygen and nitrogen species (ROS, RNS) and calcium levels were estimated. There was increased intracellular calcium (Ca2+) and nitric oxide (NO) in BALB/c and an increase in intracellular ROS in C57BL/6 mice 24 h after PD. Inhibition of NO and calcium chelation abrogated RAR in BALB/c mice. In vitro treatment of spleen cells with combination of NO donor and Ca2+ ionophore mimicked the effect of PD and induced adaptive response after 2 Gy not only in BALB/c but also in C57BL/6 mice confirming their crucial role in RAR. Conclusions: These results suggest that low dose induced differential induction of Ca2+ and NO signaling along with MAPK was responsible for contrasting RAR with respect to immune system of BALB/c and C57BL/6 mice. Abbreviations [3H]-TdR: 3H-methyl-thymidine; BAPTA: 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; CD: Challenge Dose; CFSE: Carboxy Fluorescein Succinamidyl Ester; on A: Concanavalin A; DAF-FM: 4-amino-5-methylamino-2',7'-difluorescein; DCF-DA: 2',7'-dichlorofluorescein diacetate; DSB: Double Strand Break; ELISA: Enzyme Linked ImmunoSorbent Assay; ERK: Extracellular signal-Regulated protein Kinase; FBS: Fetal Bovine Serum; HIF-1A: Hypoxia-Inducible Factor 1-alpha; LDR: Low Dose Radiation; MAPK: Mitogen Activated Protein Kinase; MAPKK/MKK: MAPK Kinase; MAPKKK: MAPK Kinase Kinase; NO: Nitric Oxide; NOS: Nitric Oxide Synthase; P + CD: Priming + Challenge dose; PBS: Phosphate Buffered Saline; PBST: Phosphate Buffered Saline-Tween 20; PD: Priming Dose; PI3K: Phosphatidyl Inositol 3-Kinase; PKC: Protein Kinase C; RAR: Radio Adaptive Response; RNS: Reactive Nitrogen Species; ROS: Reactive Oxygen Species; RPMI-1640: Roswell Park Memorial Institute-1640 medium; SAPK/JNK: Stress-Activated Protein Kinase/ c-Jun NH2-terminal Kinase; SEM: Standard Error of Mean; SNAP: S-nitro amino penicillamine; TP53: Tumor Protein 53; γ-H2AX: Gamma- H2A histone family member X; Th1: Type 1 helper T cell responses; Th2: Type 2 helper T cell responses.


Assuntos
Cálcio/metabolismo , Óxido Nítrico/metabolismo , Tolerância a Radiação , Transdução de Sinais/efeitos da radiação , Animais , Relação Dose-Resposta à Radiação , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/efeitos da radiação , Inibidores Enzimáticos/farmacologia , Feminino , Cinética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Nigericina/análogos & derivados , Nigericina/farmacologia , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos
11.
Biomed Pharmacother ; 111: 119-130, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30579251

RESUMO

AIMS: A major obstacle for effective cancer treatment by radiation therapy is the development of radio-resistance and identification of underlying mechanisms and activated pathways will lead to better combination therapies. MAIN METHODS: Irradiated MCF-7 and MDA-MB-231 breast cancer cell lines were characterised following different recovery periods. Proliferation was assessed by MTT, BrdU and clonogenic assays and apoptosis by Annexin V/ propidium iodide staining and flow cytometry. Gene expression was monitored by real time PCR/ELISA/antibody labelling and migration using transwell inserts. KEY FINDINGS: Breast cancer cell lines exposed to 6 Gy followed by recovery period for 7 days (D7-6 G) had increased ability for proliferation as well as apoptosis. D7-6 G from both cell lines had increased expression of transforming growth factor isoforms (TGF)-ß1, ß2 and ß3, their receptors TGF-ßR1 and TGF-ßR2 which are known for such dual effects. The expression of downstream transcription factors Snail, Zeb-1 and HMGA2 also showed a differential pattern in D7-6 G cells with upregulation of at least two of these transcription factors. D7-6 G cells from both cell lines displayed hybrid epithelial-mesenchymal (E/M) phenotype with increased expression of E/M markers and migration. D7-6 G cells had increased expression of cancer stem cells markers Oct4, Sox2, and Nanog; aldehyde dehydrogenase expression and activity; proportion of CD44+CD24-cells. This was accompanied by radio resistance when exposed to a challenge dose of radiation. Treatment with TGF-ßRI inhibitor abrogated the increase in proliferation of D7-6 G cells. SIGNIFICANCE: Blocking of TGF-ß signalling may therefore be an effective strategy for overcoming radio resistance induced by radiation exposure.


Assuntos
Neoplasias da Mama/metabolismo , Transição Epitelial-Mesenquimal/fisiologia , Células-Tronco Neoplásicas/metabolismo , Tolerância a Radiação/fisiologia , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/metabolismo , Neoplasias da Mama/patologia , Neoplasias da Mama/radioterapia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Sobrevivência Celular/efeitos da radiação , Transição Epitelial-Mesenquimal/efeitos da radiação , Feminino , Humanos , Células MCF-7 , Células-Tronco Neoplásicas/patologia , Células-Tronco Neoplásicas/efeitos da radiação , Fenótipo , Doses de Radiação , Tolerância a Radiação/efeitos dos fármacos , Transdução de Sinais/efeitos da radiação , Fator de Crescimento Transformador beta/efeitos da radiação
12.
Cytokine ; 101: 89-98, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-27344406

RESUMO

The mechanism by which tumor microenvironment derived cytokine network modulates therapy response is of great concern in lung cancer but is not completely understood. In this study, we evaluated the effects of tumor necrosis factor α (TNF-α) and insulin-like growth factor 1 (IGF-1) on response of lung cancer cell lines to ionizing radiation (IR). While TNF-α increased radio sensitivity and inhibited cell migration, treatment with IGF-1 promoted cell growth and increased migration. These effects of TNF- α were mediated by increased immediate activation of stress-activated protein kinases (SAPK)/jun amino-terminal kinases (JNK) and p38. IR induced DNA damage was increased by TNF- α and not altered by IGF-1. However, in IGF-1 treated cells, there was decreased γ- H2AX along with an increase in mitotic index, resulting in abnormal chromosomal segregation in the cells. Bio informatics analysis of 982 lung cancer patients revealed that higher expression of TNF- α was associated with low risk of cancer progression while overexpression of IGF-1 was correlated with high risk. Collectively, these results reveal that the cytokines in the tumor microenvironment differentially modulate radiation therapy through a variety of signaling mechanisms.


Assuntos
Fator de Crescimento Insulin-Like I/farmacologia , Neoplasias Pulmonares/imunologia , Radiação Ionizante , Microambiente Tumoral/efeitos da radiação , Fator de Necrose Tumoral alfa/farmacologia , Células A549 , Biologia Computacional , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/imunologia , Dano ao DNA/efeitos da radiação , Progressão da Doença , Humanos , Fator de Crescimento Insulin-Like I/genética , Quinases de Proteína Quinase Ativadas por Mitógeno , Índice Mitótico , Transdução de Sinais/imunologia , Transdução de Sinais/efeitos da radiação , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia , Fator de Necrose Tumoral alfa/genética
13.
Biochim Biophys Acta Gen Subj ; 1862(3): 669-683, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29248526

RESUMO

BACKGROUND: Tumor microenvironment is composed of a largely altered extracellular matrix with different cell types. The complex interplay between macrophages and tumor cells through several soluble factors and signaling is an important factor in breast cancer progression. METHODS: We have extended our earlier studies on monocyte and macrophage conditioned medium (MϕCM) and have carried out proteomic analysis to identify its constituents as well as validation. The 8-gene signature identified through macrophage-breast cancer cell interactions was queried in cBioportal for bioinformatic analyses. RESULTS: Proteomic analysis (MALDI-TOF and LC-MS/MS) revealed integrin and matrix metalloproteinases in MϕCM which activated TGF-ß1, IL-6, TGF- ßRII and EGFR as well as its downstream STAT and SMAD signaling in breast cancer cells. Neutralization of pro-inflammatory cytokines (TNF-α. Il-1ß, IL-6) abrogated the MϕCM induced migration but invasion to lesser extent. The 8- gene signature identified by macrophage-tumor interactions (TNF-α, IL-1ß, IL-6, MMP1, MMP9, TGF-ß1, TGF-ßRII, EGFR) significantly co-occurred with TP53 mutation, WTAPP1 deletion and SLC12A5 amplification along with differential expression of PSAT1 and ESR1 at the mRNA level and TPD52and PRKCD at the protein level in TCGA (cBioportal). Together these genes form a novel 15 gene signature which is altered in 63.6% of TCGA (1105 samples) data and was associated with high risk and poor survival (p<0.05) in many breast cancer datasets (SurvExpress). CONCLUSIONS: These results highlight the importance of macrophage signaling in breast cancer and the prognostic role of the15-gene signature. GENERAL SIGNIFICANCE: Our study may facilitate novel prognostic markers based on tumor-macrophage interaction.


Assuntos
Adenocarcinoma/genética , Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica , Macrófagos/metabolismo , Proteínas de Neoplasias/genética , Transcriptoma , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Diferenciação Celular , Movimento Celular , Meios de Cultivo Condicionados/farmacologia , Citocinas/biossíntese , Citocinas/genética , Citocinas/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Estimativa de Kaplan-Meier , Células MCF-7 , Metaloproteinases da Matriz/biossíntese , Metaloproteinases da Matriz/genética , Metaloproteinases da Matriz/metabolismo , Monócitos/metabolismo , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/metabolismo , Proteômica , Risco , Transcriptoma/efeitos dos fármacos , Células U937
14.
Immunol Lett ; 184: 23-33, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28161224

RESUMO

The interaction between the immune and tumor cells in the microenvironment is an important factor deciding the progression of cancer. Though many of the soluble mediators in the microenvironment that mediate immunosuppression are known, the mechanism by which the tumor affects the distal progenitors is not known. We report that the tumor derived prostanoids down regulated classical dendritic cells DC (cDC) lineage specific transcription factor Zbtb46 in the progenitor cells which affects its differentiation. Prostanoids also induced ERK/CREB/IL-10 signaling pathway in DC that is more important for maturation of DC. This was observed under in vitro as well as in vivo conditions leading to phenotypic and functional impairment of DC. siRNA mediated knockdown of Zbtb46 and not exogenous IL-10 mimicked the effects of tumor conditioned medium (TCM) on suppression of maturation markers. Treatment of tumor cells with COX-2 inhibitor NS-398 averted TCM induced phenotypic impairment of DC in vitro. Treatment of tumor bearing mice with NS-398 prevented tumor induced down regulation of Zbtb46 resulting in immunocompetent DC which in turn led to a decrease in tumor burden. The effects of NS-398 was indeed through immunomodulation was corroborated by no such response in SCID mice. Our study provides novel insight into the distal regulation of progenitor cells by tumor and the importance of Zbtb46 expression in anti-tumor immunity. These results identify Zbtb46 expression as an indicator of immunocompetent DC in tumor and also highlights that COX-2 inhibitors could be useful in cancer immunotherapy.


Assuntos
Inibidores de Ciclo-Oxigenase 2/farmacologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias/genética , Neoplasias/imunologia , Fatores de Transcrição/genética , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Linhagem Celular Tumoral , Células Dendríticas/citologia , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Interleucina-10/metabolismo , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Inibidores de Proteínas Quinases/farmacologia , Interferência de RNA , Carga Tumoral , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Cancer Microenviron ; 9(2-3): 77-84, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27106232

RESUMO

Apo2L/tumor necrosis factor (TNF)-α-related apoptosis-inducing ligand (TRAIL, TNFSF10) is an important cytokine in the tumor microenvironment and plays a major role in the balance of cell survival/death pathways. Bioinformatic analyses of 839 adenocarcinoma (AC) and 356 squamous cell lung carcinoma patient data (SCC) by cBioPortal (genomic analyses) shows that TRAIL expression leads to differential outcomes of disease free survival in AC and SCC. Oncomine datamining (transcript analyses) reveal that TRAIL is upregulated in 167 SCC as compared to 350 AC patients from six data sets. Genomic analyses using cBioPortal revealed high rates of KRAS mutation in AC accompanied by higher incidence of metastasis and increased amplifications of TRAIL gene in SCC. Bioinformatic analyses of an additional lung cancer patient database also showed that risk of disease progression was significantly increased with high TRAIL expression in AC (461 samples). In vitro studies demonstrated that TRAIL increased phosphorylation of ERK only in adenocarcinoma cell lines with mutant KRAS. This was associated with increased migration that was abrogated by MEK inhibitor PD98059. Effects of increased migration induced by TRAIL persisted even after exposure to ionizing radiation with suppression of DNA damage response. These results help understand the role of TRAIL signaling in metastasis which is essential to develop strategies to revert these signals into pro-apoptotic pathways.

16.
Int J Radiat Biol ; 92(5): 249-62, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26926139

RESUMO

To investigate low dose ionizing radiation (LDIR)-induced adaptive response in lymphocytes of BALB/c mice and to elucidate related molecular mechanisms. Mice were exposed to a priming dose (PD) of 0.1 Gy and challenge dose (CD) of 2 Gy ionizing radiation. Proliferation response to mitogen concanavalin A was assessed using (3)H thymidine incorporation and carboxyfluoresceinsuccinamidylester (CFSE) dye dilution. Early activation markers were assessed by flow cytometry, cytokines by ELISA, DNA damage by comet assay and mitogen activated protein kinase (MAPK) signaling by Western blotting. Radioadaptive response was observed in lymphocytes of mice exposed to PD prior to CD of ionizing radiation in terms of DNA damage, early activation markers CD69, CD71, cytokines IL-2, IFN-γ as well as proliferation. This effect was transient and observed 24 h after CD and not after 0 h or 72 h. Hyper activation of MAPK signaling pathways in lymphocytes from LDIR-exposed mice and abrogation by ERK and p38 inhibitors suggests the involvement of MAPK signaling in radioadaptive response. Our study demonstrates that LDIR-induced transient adaptive response was due to hyper activation of MAPK signaling. Our findings contribute towards the understanding of LDIR-induced adaptive response.


Assuntos
Linfócitos/fisiologia , Linfócitos/efeitos da radiação , Sistema de Sinalização das MAP Quinases/efeitos da radiação , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Tolerância a Radiação/fisiologia , Radiação Ionizante , Adaptação Fisiológica/fisiologia , Adaptação Fisiológica/efeitos da radiação , Animais , Células Cultivadas , Citocinas/metabolismo , Relação Dose-Resposta à Radiação , Feminino , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Doses de Radiação , Irradiação Corporal Total/métodos
17.
Life Sci ; 126: 19-27, 2015 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-25721293

RESUMO

AIMS: The goal of this study is to investigate the tumor cytotoxic effects of sulforaphane (SFN) and ionizing radiation (IR) as well as their ability to up-regulate natural killer group 2, member D (NKG2D) ligands and modulate the susceptibility of tumor cells to natural killer (NK) cell-mediated killing. MAIN METHODS: Expression of MHC class I-related chain molecules A and B (MICA/MICB) and total reactive oxygen species (ROS) were assessed by flow cytometry following labeling with appropriate dyes or antibodies. NK cell cytotoxicity was determined by calcein release of target cells. KEY FINDINGS: The expression of NKG2D ligands MICA/MICB was found to vary in all the four tumor cell lines tested (MCF7 < A549 < MDA-MB-231 < U937). Exposure of these cells to IR and SFN resulted in a differential induction of these ligands. IR induced an increase in expression of MICA/MICB in MCF7 cells and SFN induced MICA/MICB expression in A549 and MDA-MB-231 cells. This SFN induced increase in receptor expression resulted in increased susceptibility to NK cell mediated killing of tumor cells which was abrogated by blocking with anti-MICA/MICB antibody. SFN induced increase in MICA/MICB expression as well as increased susceptibility to NK cell mediated killing was abrogated by N-acetyl cysteine in A549 and MDA-MB-231 cells suggesting a ROS mediated mechanism. SIGNIFICANCE: Our results indicate that SFN has an immunotherapeutic potential to be used in cancer therapy.


Assuntos
Anticarcinógenos/farmacologia , Isotiocianatos/farmacologia , Células Matadoras Naturais/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Neoplasias/imunologia , Espécies Reativas de Oxigênio/imunologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/fisiologia , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Imunidade Celular/efeitos dos fármacos , Células Matadoras Naturais/patologia , Masculino , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Sulfóxidos , Células U937
18.
Int Immunopharmacol ; 23(2): 480-8, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25278461

RESUMO

Dendritic cells (DC) play a central role in the development of an adaptive immune response against tumor. In addition to its role in antigen presentation, DC also possesses cytotoxic activity against tumor cells. We have earlier shown phenotypic and functional maturation of bone marrow derived dendritic cells (BMDC) by G1-4A, an arabinogalactan derived from Tinospora cordifolia. In this study, we have investigated the killer phenotype of BMDC matured in the presence of G1-4A, [mBMDC (G1-4A)] on tumor cells. We have observed several fold increase in killing of tumor cells by mBMDC (G1-4A). The tumoricidal activity was not specific to syngeneic tumors cells but could kill xenogenic tumors also. Nitric oxide released by mBMDC (G1-4A) generates peroxynitrite in tumor cells and is responsible for killing of target cells. This killing was completely abrogated by inducible nitric oxide synthase (iNOS) inhibitor 1400W and NADPH oxidase inhibitor apocyanin. The killed target cells are phagocytosed by BMDC which further activate syngeneic cytotoxic T cells. These results thus show that G1-4A treated mBMDC acquire killer phenotype along with maturation which plays an important role in activation of cytotoxic T cells.


Assuntos
Células Dendríticas/efeitos dos fármacos , Polissacarídeos/farmacologia , Tinospora/química , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Técnicas de Cocultura , Células Dendríticas/fisiologia , Humanos , Camundongos Endogâmicos C57BL , Ácido Peroxinitroso/metabolismo
19.
Cell Signal ; 26(7): 1604-15, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24705025

RESUMO

Macrophages in the tumor microenvironment play an important role in tumor cell survival. They influence the tumor cell to proliferate, invade into surrounding normal tissues and metastasize to local and distant sites. In this study, we evaluated the effect of conditioned medium from monocytes and macrophages on growth and migration of breast cancer cells. Macrophage conditioned medium (MϕCM) containing elevated levels of cytokines TNF-α, IL-1ß and IL-6 had a differential effect on non-invasive (MCF7) and highly invasive (MDA-MB-231) breast cancer cell lines. MϕCM induced the secretion of TGF-ß1 in MCF7 cells. This was associated with apoptosis in a fraction of cells and generation of reactive oxygen and nitrogen species (ROS and RNS) and DNA damage in the remaining cells. This, in turn, increased expression of cAMP response element binding protein (CREB) and vimentin resulting in migration of cells. These effects were inhibited by neutralization of TNF-α, IL-1ß and IL-6, inhibition of ROS and RNS, DNA damage and siRNA mediated knockdown of ATM. In contrast, MDA-MB-231 cells which had higher basal levels of pCREB were not affected by MϕCM. In summary, we have found that pro-inflammatory cytokines secreted by macrophages induce TGF-ß1 in tumor cells, which activate pCREB signaling, epithelial-mesenchymal-transition (EMT) responses and enhanced migration.


Assuntos
Neoplasias da Mama/patologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Transição Epitelial-Mesenquimal/imunologia , Macrófagos/imunologia , Fator de Crescimento Transformador beta1/metabolismo , Anticorpos/imunologia , Anticorpos/farmacologia , Apoptose/imunologia , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular Tumoral , Movimento Celular/imunologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Meios de Cultivo Condicionados/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/biossíntese , Dano ao DNA/imunologia , Feminino , Humanos , Interleucina-1beta/antagonistas & inibidores , Interleucina-1beta/imunologia , Interleucina-6/antagonistas & inibidores , Interleucina-6/imunologia , Células MCF-7 , Monócitos/imunologia , Interferência de RNA , RNA Interferente Pequeno , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Microambiente Tumoral/imunologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/imunologia , Vimentina/biossíntese
20.
Cytokine ; 64(1): 196-207, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23972545

RESUMO

Cytokines in tumor microenvironment play an important role in the success or failure of molecular targeted therapies. We have chosen tumor necrosis factor α (TNF-α), TNF related apoptosis inducing ligand (TRAIL), insulin-like growth factor 1 (IGF-1) and transforming growth factor ß (TGF-ß) as representative pro-inflammatory, pro-apoptotic, anti-apoptotic and anti-inflammatory tumor derived cytokines. Analysis of Oncomine database revealed the differential expression of these cytokines in a subset of cancer patients. The effects of these cytokines on cytotoxicity of FDA approved drugs - cisplatin and taxol and inhibitors of epidermal growth factor receptor - AG658, Janus kinase - AG490 and SIRT1 - sirtinol were assessed in A549 lung cancer cells. TRAIL augmented cytotoxicity of sirtinol and IGF-1 had a sparing effect. Since TRAIL and IGF-1 differentially modulated sirtinol cytotoxicity, further studies were carried out to identify the mechanisms. Sirtinol or knockdown of SIRT1 increased the expression of death receptors DR4 and DR5 and sensitized A549 cells to TRAIL. Increased cell death in presence of TRAIL and sirtinol was caspase independent and demonstrated classical features of necroptosis. Inhibition of iNOS increased caspase activity and switched the mode of cell death to caspase mediated apoptosis. Interestingly, sirtinol or SIRT1 knockdown did not increase IGF-1R expression. Instead, it abrogated ligand induced downregulation of IGF-1R and increased cell survival through PI3K-AKT pathway. In conclusion, these findings reveal that the tumor microenvironment contributes to modulation of cytotoxicity of drugs and that combination therapy, with agents that increase TRAIL signaling and suppress IGF-1 pathway may potentiate anticancer effect.


Assuntos
Apoptose/efeitos dos fármacos , Benzamidas/farmacologia , Citocinas/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Naftóis/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/farmacologia , Receptores ErbB/antagonistas & inibidores , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Janus Quinases/antagonistas & inibidores , Terapia de Alvo Molecular , Paclitaxel/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Receptor IGF Tipo 1/biossíntese , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/biossíntese , Receptores do Fator de Necrose Tumoral/biossíntese , Sirtuína 1/antagonistas & inibidores , Sirtuína 1/genética , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Microambiente Tumoral , Fator de Necrose Tumoral alfa/metabolismo , Tirfostinas/farmacologia
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