RESUMO
Through Smad3-dependent signalings, transforming growth factor-ß (TGF-ß) suppresses the development, maturation, cytokine productions and cytolytic functions of NK cells in cancer. Silencing Smad3 remarkably restores the cytotoxicity of NK-92 against cancer in TGF-ß-rich microenvironment, but its effects on the immunoregulatory functions of NK cells remain obscure. In this study, we identified Smad3 functioned as a transcriptional repressor for CSF2 (GM-CSF) in NK cells. Therefore, disrupting Smad3 largely mitigated TGF-ß-mediated suppression on GM-CSF production by NK cells. Furthermore, silencing GM-CSF in Smad3 knockout NK cells substantially impaired their anti-lung carcinoma effects. In-depth study demonstrated that NK-derived GM-CSF strengthened T cell immune responses by stimulating dendritic cell differentiation and M1 macrophage polarization. Meanwhile, NK-derived GM-CSF promoted the survival of neutrophils, which in turn facilitated the terminal maturation of NK cells, and subsequently boosted NK-cell mediated cytotoxicity against lung carcinoma. Thus, Smad3-silenced NK-92 (NK-92-S3KD) may serve as a promising immunoadjuvant therapy with clinical translational value given its robust cytotoxicity against malignant cells and immunostimulatory functions to reinforce the therapeutic effects of other immunotherapies.
Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos , Células Matadoras Naturais , Neoplasias Pulmonares , Proteína Smad3 , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Proteína Smad3/metabolismo , Proteína Smad3/genética , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/genética , Animais , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Linhagem Celular Tumoral , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Diferenciação Celular , Macrófagos/metabolismo , Macrófagos/imunologia , Transdução de SinaisRESUMO
Monitoring hydrogen sulfide (H2S) in living organisms is very important because H2S acts as a regulator in many physiological and pathological processes. Upregulation of endogenous H2S concentration has been shown to be closely related to the occurrence and development of tumors, atherosclerosis, neurodegenerative diseases and diabetes. Herin, a novel fluorescent probe HND with aggregation-induced emission was designed. Impressively, HND exhibited a high selectivity, fast response (1 min) and low detection limit (0.61 µM) for H2S in PBS buffer (10 mM, pH = 7.42). Moreover, the reaction mechanism between HND and H2S was conducted by Job's plot, HR-MS, and DFT. In particular, HND was successfully employed to detect H2S in HeLa cells.
Assuntos
Corantes Fluorescentes , Sulfeto de Hidrogênio , Sulfeto de Hidrogênio/análise , Humanos , Corantes Fluorescentes/química , Células HeLa , Imagem Óptica/métodos , Espectrometria de Fluorescência/métodos , Limite de DetecçãoRESUMO
Exosomal long noncoding RNAs (lncRNA) derived from cancer cells are implicated in various processes, including cancer cell proliferation, metastasis, and immunomodulation. We investigated the role and underlying mechanism of exosome-transmitted lncRNA NEAT1 in the immune escape of multiple myeloma cells from natural killer (NK) cells. Multiple myeloma cells and samples from patients with multiple myeloma were obtained. The effects of multiple myeloma cell-derived exosomes (multiple myeloma exosomes) and exosomal NEAT1 on the functions of NK cells were evaluated using EdU staining, CCK-8, flow cytometry, and ELISA. Chromatin and RNA immunoprecipitation were performed to identify interactions between NEAT1, enhancer of Zeste Homolog 2 (EZH2), and pre-B-cell leukemia transcription factor 1 (PBX1). A xenograft tumor model was constructed to verify the effects of exosomal NEAT1 on tumor growth. qRT-PCR, Western blot analysis, and IHC were conducted to detect related genes. NEAT1 levels were upregulated in multiple myeloma tumor tissues, multiple myeloma cells, and multiple myeloma exosomes. Multiple myeloma exosomes suppressed cell proliferation, promoted apoptosis, reduced natural killer group 2, member D (NKG2D)-positive cells, and the production of TNFα) and interferon-gamma (IFN-γ) in NK cells, whereas NEAT1-silenced exosomes had little effect. NEAT1 silenced PBX1 by recruiting EZH2. PBX1 knockdown abrogated the effects of NEAT1-silenced exosomes on NK and multiple myeloma cells. NEAT1-silenced exosomes inhibited tumor growth in mice, decreased Ki67 and PD-L1, and increased NKG2D, TNFα, and IFNγ in tumor tissues. In summary, multiple myeloma cell-derived exosomal NEAT1 suppressed NK-cell activity by downregulating PBX1, promoting multiple myeloma cell immune escape. This study suggests a potential strategy for treating multiple myeloma. IMPLICATIONS: This study reveals that exosomal NEAT1 regulates EZH2/PBX1 axis to inhibit NK-cell activity, thereby promoting multiple myeloma cell immune escape, which offers a novel therapeutic potential for multiple myeloma.
Assuntos
Exossomos , MicroRNAs , Mieloma Múltiplo , RNA Longo não Codificante , Animais , Humanos , Camundongos , Linhagem Celular Tumoral , Proliferação de Células , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Exossomos/genética , Células Matadoras Naturais , MicroRNAs/genética , Mieloma Múltiplo/genética , Subfamília K de Receptores Semelhantes a Lectina de Células NK , Fator de Transcrição 1 de Leucemia de Células Pré-B , RNA Longo não Codificante/genética , Fator de Necrose Tumoral alfaAssuntos
Mieloma Múltiplo , Insuficiência Renal , Humanos , Mieloma Múltiplo/complicações , Mieloma Múltiplo/diagnóstico , Mieloma Múltiplo/tratamento farmacológico , Bortezomib/efeitos adversos , Talidomida/efeitos adversos , Insuficiência Renal/complicações , Insuficiência Renal/diagnóstico , Dexametasona/efeitos adversosRESUMO
BACKGROUND: Chronic myeloid leukemia (CML) is a malignant hematologic malignancy that can progress to blast phase with a myeloid or lymphoid phenotype. Some patients with CML can also progress to blast crisis phase; however, the transformation of CML into Philadelphia-positive lymphoma is extremely rare. CASE SUMMARY: We present a patient with CML who experienced a sudden transformation to anaplastic large-cell lymphoma (ALCL) after 7 mo of treatment with imatinib, during which she had achieved partial cytogenetic response as well as early molecular response. The patient noticed a mass in her left shoulder, the biopsy data of which were consistent with ALCL; moreover, her lymphoma cells exhibited BCR-ABL gene fusion. The patient was diagnosed with Philadelphia-positive ALCL that progressed from CML, and was thus treated with the second generation tyrosine kinase inhibitor nilotinib. Six months later, the mass had totally disappeared and the BCR-ABL fusion gene was undetectable in the peripheral blood. To our knowledge, this is the first patient known to have developed Philadelphia-positive ALCL transformed from CML. CONCLUSION: Unexplained lymphadenopathy or an extramedullary mass in a patient with CML may warrant a biopsy and testing for BCR-ABL fusion.
RESUMO
Transforming growth factor ß (TGF-ß) is a well-known key mediator for the progression and metastasis of lung carcinoma. However, cost-effective anti-TGF-ß therapeutics for lung cancer remain to be explored. Specifically, the low efficacy in drug delivery greatly limits the clinical application of small molecular inhibitors of TGF-ß. In the present study, specific inhibitor of Smad3 (SIS3) is developed into a self-carried nanodrug (SCND-SIS3) using the reprecipitation method, which largely improves its solubility and bioavailability while reduces its nephrotoxicity. Compared to unmodified-SIS3, SCND-SIS3 demonstrates better anti-cancer effects through inducing tumor cell apoptosis, inhibiting angiogenesis, and boosting NK cell-mediated immune responses in syngeneic Lewis Lung Cancer (LLC) mouse model. Better still, it could achieve comparable anti-cancer effect with just one-fifth the dose of unmodified-SIS3. Mechanistically, RNA-sequencing analysis and cytokine array results unveil a TGF-ß/Smad3-dependent immunoregulatory landscape in NK cells. In particular, SCND-SIS3 promotes NK cell cytotoxicity by ameliorating Smad3-mediated transcriptional inhibition of Ndrg1. Furthermore, improved NK cell cytotoxicity by SCND-SIS3 is associated with higher expression of activation receptor Nkp46, and suppressed levels of Trib3 and TSP1 as compared with unmodified-SIS3. Taken together, SCND-SIS3 possesses superior anti-cancer effects with enhanced bioavailability and biocompatibility, therefore representing as a novel therapeutic strategy for lung carcinoma with promising clinical potential.
Assuntos
Carcinoma , Neoplasias Pulmonares , Nanopartículas , Animais , Carcinoma/tratamento farmacológico , Linhagem Celular Tumoral , Isoquinolinas/farmacologia , Isoquinolinas/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Camundongos , Nanopartículas/uso terapêutico , Piridinas/farmacologia , Pirróis/uso terapêutico , Transdução de Sinais , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
RATIONALE: Significant concerns about the adverse effects following chimeric antigen receptor T cell (CAR-T) therapy are still remained including cytokine release syndrome (CRS). In rare circumstances, CRS may be refractory to tocilizumab and/or corticosteroids, a new treatment is needed for the management of CRS. PATIENT CONCERNS: We present a case of a 20-year-old male patient with acute lymphoblastic leukemia developed CRS after CD19/CD22 bispecific CAR-T treatment. DIAGNOSIS: The patient was diagnosed with BCR-ABL(P210) positive B-ALL and developed CRS after CD19/CD22 bispecific CAR-T treatment. INTERVENTIONS: Tocilizumab and methylprednisolone were administered, unfortunately the patient's symptoms of CRS were still not resolved. Another methylprednisolone and ruxolitinib were administered. OUTCOMES: The persistent fever and hypotension of this patient achieved a rapid clinical remission within hours after ruxolitinib administration. LESSONS: Ruxolitinib can be used as an alternative therapeutic approach for severe and refractory CRS without impairing CAR-T amplification and anti-tumor effect.
Assuntos
Síndrome da Liberação de Citocina/tratamento farmacológico , Imunoterapia Adotiva/efeitos adversos , Inibidores de Janus Quinases/uso terapêutico , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Pirazóis/uso terapêutico , Receptores de Antígenos Quiméricos/uso terapêutico , Síndrome da Liberação de Citocina/etiologia , Humanos , Imunoterapia Adotiva/métodos , Masculino , Nitrilas , Pirimidinas , Adulto JovemRESUMO
Transforming growth factor ß (TGF-ß) has been shown to promote tumor invasion and metastasis by activating the matrix metalloproteinases (MMPs); however, signaling mechanisms remain controversial and therapies targeting MMPs are still suboptimal. In the present study, we found that combined therapy with Asiatic acid (AA), a Smad7 agonist, and Naringenin (NG), a Smad3 inhibitor, effectively retrieved the balance between Smad3 and Smad7 signaling in the TGF-ß-rich tumor microenvironment and thus significantly suppressed tumor invasion and metastasis in mouse models of melanoma and lung carcinoma. Mechanistically, we unraveled that Smad3 acted as a transcriptional activator of MMP2 and as a transcriptional suppressor of tissue inhibitors of metalloproteinase-2 (TIMP2) via binding to 5' UTR of MMP2 and 3' UTR of TIMP2, respectively. Treatment with NG inhibited Smad3-mediated MMP2 transcription while increasing TIMP, whereas treatment with AA enhanced Smad7 to suppress TGF-ß/Smad3 signaling, as well as the activation of MMP2 by targeting the nuclear factor-κB (NF-κB)-membrane-type-1 MMP (MT1-MMP) axis. Therefore, the combination of AA and NG additively suppressed invasion and metastasis of melanoma and lung carcinoma by targeting TGF-ß/Smad-dependent MMP2 transcription, post-translational activation, and function.
RESUMO
Tumor-associated macrophages (TAM) have important roles in cancer progression, but the signaling behind the formation of protumoral TAM remains understudied. Here, by single-cell RNA sequencing, we revealed that the pattern recognition receptor Mincle was highly expressed in TAM and significantly associated with mortality in patients with non-small cell lung cancer. Cancer cells markedly induced Mincle expression in bone marrow-derived macrophages (BMDM), thus promoting cancer progression in invasive lung carcinoma LLC and melanoma B16F10 in vivo and in vitro Mincle was predominately expressed in the M2-like TAM in non-small cell lung carcinoma and LLC tumors, and silencing of Mincle unexpectedly promoted M1-like phenotypes in vitro Mechanistically, we discovered a novel Mincle/Syk/NF-κB signaling pathway in TAM needed for executing their TLR4-independent protumoral activities. Adoptive transfer of Mincle-silenced BMDM significantly suppressed TAM-driven cancer progression in the LLC-bearing NOD/SCID mice. By modifying our well-established ultrasound microbubble-mediated gene transfer protocol, we demonstrated that tumor-specific silencing of Mincle effectively blocked Mincle/Syk/NF-κB signaling, therefore inhibiting the TAM-driven cancer progression in the syngeneic mouse cancer models. Thus, our findings highlight the function of Mincle as a novel immunotherapeutic target for cancer via blocking the Mincle/Syk/NF-κB circuit in TAM.
Assuntos
Lectinas Tipo C/metabolismo , Neoplasias Pulmonares/imunologia , Melanoma/imunologia , Proteínas de Membrana/metabolismo , NF-kappa B/metabolismo , Receptores Imunológicos/metabolismo , Quinase Syk/metabolismo , Macrófagos Associados a Tumor/imunologia , Idoso , Animais , Linhagem Celular Tumoral , Citocinas/imunologia , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Lectinas Tipo C/imunologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Macrófagos/imunologia , Macrófagos/patologia , Masculino , Melanoma/metabolismo , Melanoma/patologia , Proteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , NF-kappa B/imunologia , Receptores Imunológicos/imunologia , Transdução de Sinais , Quinase Syk/imunologia , Macrófagos Associados a Tumor/patologiaRESUMO
Aims: To investigate roles of miR-29a-DNMT1-SOCS1 axis in cervical cancer invasion and migration. Materials & methods: The methylation level of SOCS1 was determined by methylation specific PCR. The cell apoptosis, proliferation, migration and invasion were examined by Annexin-V/PI staining, MTT and colony formation assays, plus scratch and transwell assays respectively. The expressions of epithelial-mesenchymal transition and NF-κB related proteins were determined by western blotting. Results: MiR-29a was downregulated, accompanied with DNMT1 upregulation and SOCS1 downregulation in cervical cancer tissues. MiR-29a suppressed DNMT1, inhibited SOCS1 promoter methylation and upregulated its expression. Moreover, miR-29a promoted cell apoptosis, suppressed proliferation, inhibited migration and invasion via inactivation of NF-κB signaling pathway in cervical cancer cells. Conclusion: MiR-29a-DNMT1-SOCS1 axis plays an important role on invasion and metastasis in cervical cancer via NF-κB signaling pathway.
Assuntos
Metilação de DNA , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Interferência de RNA , Proteína 1 Supressora da Sinalização de Citocina/genética , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia , Adulto , Idoso , Apoptose/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células , DNA (Citosina-5-)-Metiltransferase 1/genética , Decitabina/farmacologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Humanos , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Invasividade Neoplásica , Metástase Neoplásica , Estadiamento de NeoplasiasRESUMO
BACKGROUND: PD-L1 enhanced the tumorigenesis and immune escape abilities of cancers. The upstream mechanisms of PD-L1 in regulating tumorigenesis and immune escape of diffuse large B cell lymphoma (DLBCL) remained unclear. METHODS: Human DLBCL cell line OCI-Ly10 and DLBCL patient samples were used in this study. MALAT1 was knocked down by shRNA. MiR-195 was inhibited by miR-195 inhibitor. Levels of MALAT1, PD-L1, miR-195 and CD8 were detected by RT-qPCR. Protein levels of PD-L1, Ras, p-ERK1/2, ERK1/2, Slug, E-cadherin, N-cadherin, Vimentin were detected by western blotting. The interaction between MALAT1 and miR-195, miR-195 and PD-L1 were detected by luciferase assay. OCI-Ly10 cell proliferation and apoptosis were detected by MTT and Annexin V/PI assays, respectively. Migration was detected by transwell assay. Cytotoxicity of CD8+ T cells was detected by LDH cytotoxicity kit. Proliferation and apoptosis of CD8+ T cell co-cultured with OCI-Ly10 cells were analyzed by CFSE and Annexin V/PI staining. RESULTS: MALAT1, PD-L1 and CD8 were up-regulated in DLBCL tissues while miR-195 was down-regulated. MiR-195 was negatively correlated with MALAT1 and PD-L1. MALAT1 could sponge miR-195 to regulate the expression of PD-L1. shMALAT1 treatment increased miR-195 level and decreased PD-L1 level. It also inhibited cell proliferation, migration and immune escape ability while increased apoptosis ratio of OCI-Ly10 cells. shMALAT1 treatment in OCI-Ly10 cells also promoted proliferation and inhibited apoptosis of CD8+ T cells. Knocking down of MALAT1 also suppressed EMT-like process via Ras/ERK signaling pathway. These effects were all rescued by miR-195 inhibitor. CONCLUSION: Long non-coding RNA MALAT1 sponged miR-195 to regulate proliferation, apoptosis and migration and immune escape abilities of DLBCL by regulation of PD-L1.
Assuntos
Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/imunologia , MicroRNAs/imunologia , RNA Longo não Codificante/imunologia , Evasão Tumoral/genética , Apoptose/fisiologia , Antígeno B7-H1/biossíntese , Antígeno B7-H1/genética , Antígeno B7-H1/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Carcinogênese , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Transformação Celular Neoplásica , Humanos , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/patologia , MicroRNAs/genética , RNA Longo não Codificante/genética , RNA Interferente Pequeno/genética , Transdução de Sinais , Evasão Tumoral/imunologiaRESUMO
Understanding the molecular mechanisms for the development of non-Hodgkin lymphoma (NHL) will improve our ability to cure the patients. qRT-PCR was applied for the examination of the efficiency of shRNA for DNMT1, the expression of suppressor genes, miRNA-152. The MTT analysis, cell cycle analysis, clonal formation, and apoptotic analysis were used to examine the functions of DNMT1 and miR-152 in lymphoma cells. Methylation-specific polymerase chain reaction (MSP) was used to examine the methylation of tumor suppressor genes. The dual luciferase assay and western blot were used to validate if DNMT1 is the target of miR-152. For the in vivo experiments, the lymphoma cells were injected into the nude mice for quantification of the tumor growth after transfection of miR-152 mimics. Knockdown of DNMT1 by shRNA (sh-DNMT1) in OCI-Ly10 and Granta-159 cells significantly upregulated the expression of tumor suppressor genes (SOCS3, BCL2L10, p16, p14, and SHP-1) via decreasing their methylation level. At the cellular level, we found sh-DNMT1 inhibited the proliferation, clonal formation and cell cycle progression and induced the cell apoptosis of lymphoma cells. Furthermore, we found miR-152 can downregulates the expression of DNMT1 via directly targeting the gene. Overexpression of miR-152 also increased the expression of tumor suppressor genes SOCS3 and SHP-1. And miR-152 also can inhibit the cell proliferation and induce the cell apoptosis. Moreover, we found overexpression of miR-152 significantly repressed the tumor growth with decreased DNMT1 expression and increased expression of tumor suppressor genes in vivo. Our study demonstrates that miR-152 can inhibit lymphoma growth via suppressing DNMT1-mediated silencing of SOCS3 and SHP-1. These data demonstrate a new mechanism for the development of NHL and this may provide a new therapeutic target for NHL.
Assuntos
DNA (Citosina-5-)-Metiltransferase 1/genética , Linfoma não Hodgkin/genética , MicroRNAs/genética , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Proteína 3 Supressora da Sinalização de Citocinas/genética , Animais , Apoptose/fisiologia , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Regulação para Baixo , Técnicas de Silenciamento de Genes , Genes Supressores de Tumor , Células HEK293 , Xenoenxertos , Humanos , Linfoma não Hodgkin/metabolismo , Linfoma não Hodgkin/patologia , Camundongos , MicroRNAs/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Transdução de Sinais , Proteína 3 Supressora da Sinalização de Citocinas/metabolismo , TransfecçãoRESUMO
Transforming growth factor ß1 (TGF-ß1) plays a promoting role in tumor growth via a mechanism associated with hyperactive Smad3 and suppressed Smad7 signaling in the tumor microenvironment. We report that retrieving the balance between Smad3 and Smad7 signaling with asiatic acid (AA, a Smad7 inducer) and naringenin (NG, a Smad3 inhibitor) effectively inhibited tumor progression in mouse models of invasive melanoma (B16F10) and lung carcinoma (LLC) by promoting natural killer (NK) cell development and cytotoxicity against cancer. Mechanistically, we found that Smad3 physically bound Id2 and IRF2 to suppress NK cell production and NK cell-mediated cytotoxicity against cancer. Treatment with AA and NG greatly inhibited Smad3 translation and phosphorylation while it restored Smad7 expression, and, therefore, it largely promoted NK cell differentiation, maturation, and cytotoxicity against cancer via Id2/IRF2-associated mechanisms. In contrast, silencing Id2 or IRF2 blunted the protective effects of AA and NG on NK cell-dependent anti-cancer activities. Thus, treatment with AA and NG produced an additive effect on inactivating TGF-ß1/Smad3 signaling, and, therefore, it suppressed melanoma and lung carcinoma growth by promoting NK cell immunity against cancer via a mechanism associated with Id2 and IRF2.
Assuntos
Flavanonas/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Triterpenos Pentacíclicos/farmacologia , Proteína Smad3/metabolismo , Proteína Smad7/metabolismo , Animais , Western Blotting , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Proteína 2 Inibidora de Diferenciação/metabolismo , Fator Regulador 2 de Interferon/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/efeitos dos fármacosRESUMO
Natural killer (NK) cells, early effectors in anticancer immunity, are paralyzed by TGFß1, an immunosuppressive cytokine produced by cancer cells. Development and activity of NK cells are largely inhibited in the Smad3-dependent tumor microenvironment. Here, we used genetic engineering to generate a stable SMAD3-silencing human NK cell line, NK-92-S3KD, whose cancer-killing activity and cytokine production were significantly enhanced under TGFß1-rich condition compared with the parental cell line. Interestingly, we identified that the IFNG gene is a direct E4BP4 target gene. Thus, silencing of SMAD3 allows upregulation of E4BP4 that subsequently promoting interferon-γ (IFNγ) production in the NK-92-S3KD cells. More importantly, NK-92-S3KD immunotherapy increases the production of not only IFNγ, but also granzyme B and perforin in tumors; therefore, inhibiting cancer progression in two xenograft mouse models with human hepatoma (HepG2) and melanoma (A375). Thus, the NK-92-S3KD cell line may be useful for the clinical immunotherapy of cancer. Cancer Immunol Res; 6(8); 965-77. ©2018 AACR.
Assuntos
Imunoterapia/métodos , Células Matadoras Naturais/imunologia , Neoplasias Hepáticas Experimentais/terapia , Melanoma Experimental/terapia , Proteína Smad3/genética , Animais , Linhagem Celular , Proteínas da Matriz Extracelular/imunologia , Inativação Gênica , Engenharia Genética/métodos , Humanos , Interferon gama/biossíntese , Neoplasias Hepáticas Experimentais/imunologia , Melanoma Experimental/imunologia , Camundongos Endogâmicos NOD , Camundongos Knockout , Proteína Smad3/imunologia , Fator de Crescimento Transformador beta/imunologia , Microambiente Tumoral/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.
Assuntos
Técnicas Citológicas/métodos , Células Matadoras Naturais/citologia , Animais , Fatores de Transcrição de Zíper de Leucina Básica/antagonistas & inibidores , Células da Medula Óssea/citologia , Diferenciação Celular/fisiologia , Linhagem Celular , Inativação Gênica , Células Matadoras Naturais/fisiologia , Camundongos , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genéticaRESUMO
Src activation has been associated with fibrogenesis after kidney injury. Macrophage-myofibroblast transition is a newly identified process to generate collagen-producing myofibroblasts locally in the kidney undergoing fibrosis in a TGF-ß/Smad3-dependent manner. The potential role of the macrophage-myofibroblast transition in Src-mediated renal fibrosis is unknown. In studying this by RNA sequencing at single-cell resolution, we uncovered a unique Src-centric regulatory gene network as a key underlying mechanism of macrophage-myofibroblast transition. A total of 501 differentially expressed genes associated with macrophage-myofibroblast transition were identified. However, Smad3-knockout largely reduced the transcriptome diversity. More importantly, inhibition of Src largely suppresses ureteral obstruction-induced macrophage-myofibroblast transition in the injured kidney in vivo along with transforming growth factor-ß1-induced elongated fibroblast-like morphology, α-smooth muscle actin expression and collagen production in bone marrow derived macrophages in vitro. Unexpectedly, we further uncovered that Src serves as a direct Smad3 target gene and also specifically up-regulated in macrophages during macrophage-myofibroblast transition. Thus, macrophage-myofibroblast transition contributes to Src-mediated tissue fibrosis. Hence, targeting Src may represent as a precision therapeutic strategy for macrophage-myofibroblast transition-driven fibrotic diseases.
Assuntos
Transdiferenciação Celular , Cicatriz/enzimologia , Nefropatias/enzimologia , Rim/enzimologia , Macrófagos/enzimologia , Miofibroblastos/enzimologia , Quinases da Família src/metabolismo , Animais , Transdiferenciação Celular/efeitos dos fármacos , Transdiferenciação Celular/genética , Células Cultivadas , Cicatriz/genética , Cicatriz/patologia , Cicatriz/prevenção & controle , Modelos Animais de Doenças , Fibrose , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Rim/efeitos dos fármacos , Rim/patologia , Nefropatias/genética , Nefropatias/patologia , Nefropatias/prevenção & controle , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/patologia , Inibidores de Proteínas Quinases/farmacologia , Análise de Sequência de RNA , Transdução de Sinais , Análise de Célula Única , Proteína Smad3/genética , Proteína Smad3/metabolismo , Obstrução Ureteral/tratamento farmacológico , Obstrução Ureteral/enzimologia , Obstrução Ureteral/genética , Quinases da Família src/genéticaRESUMO
TGF-ß is known to influence tumour progression. Here we report an additional role of Smad3 in the tumour microenvironment regulating cancer progression. Deletion or inhibition of Smad3 in the tumour microenvironment suppresses tumour growth, invasion and metastasis in two syngeneic mouse tumour models. Smad3-/- bone marrow gives rise to an expanded NK cell population with enhanced tumour-suppressive activities in vivo, and promotes differentiation of NK cells ex vivo. We identify E4BP4/NFIL3 as a direct Smad3 target gene critical for NK cell differentiation. Smad3 suppresses transcription of IFN-γ via E4BP4 in a T-bet independent manner. Therefore disruption of Smad3 enhances both the E4BP4-mediated NK cell differentiation and anti-cancer effector functions in vivo and in vitro. Furthermore, systemic treatment with a Smad3 inhibitor SIS3 effectively suppresses cancer progression. In summary, suppression of NK cell-mediated immunosurveillance via the Smad3-E4BP4 axis contributes to cancer progression. We propose targeting Smad3-dependent tumour microenvironment may represent an effective anti-cancer strategy.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Células Matadoras Naturais/metabolismo , Neoplasias Experimentais/metabolismo , Proteína Smad3/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina Básica/genética , Diferenciação Celular/imunologia , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Isoquinolinas/farmacologia , Estimativa de Kaplan-Meier , Células Matadoras Naturais/imunologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Camundongos Transgênicos , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/genética , Piridinas/farmacologia , Pirróis/farmacologia , Interferência de RNA , Proteína Smad3/antagonistas & inibidores , Proteína Smad3/genéticaRESUMO
PURPOSE: Idiopathic Asthenozoospermia (AZS) is a common symptom of male infertility described as reduced forward motility or absence of sperm motility. The PATE1 is generally expressed in male genital tract and related to sperm development, maturation and fertilization. However, the single nucleotide polymorphisms (SNPs) of the PATE1 gene which contribute to AZS were still unknown. For this reason, the possible association between the single nucleotide polymorphisms of the PATE1 gene and idiopathic asthenozoospermia was investigated in this research. METHODS: 108 idiopathic asthenozoospermia were screened by karyotype analysis, detection of Y microdeletions and mutations in 5 other genes from 140 clinical AZS. The sequence analyses of the PATE1 gene were conducted in 108 idiopathic asthenozoospermia and 106 fertile men with normospermic parameters in Sichuan, China. RESULTS: In this study, a total 108 patients without chromosomal abnormalities, Y microdeletions and selected genes mutation were confirmed. The 1423G (odds ratio [OR] 1.939, 95% confidence interval [CI] 1.320-2.848, P=0.001) was found to be increased significantly in idiopathic asthenozoospermic patients compared with their fertile counterparts. This mutation substitutes a highly conserved glutamic to arginine at the position of the 47th amino acid which was shown to be located on the flank of the pleated sheet domain in PATE1 protein by the 3D model given by the Protein Model Portal (PMP). Moreover, PolyPhen-2 analysis predicted that this variant was "probably damaging". CONCLUSIONS: These results suggested that PATE1 variant (A1423G) was probably one of the high risk genetic factors for idiopathic asthenozoospermia among males in Sichuan, China.
Assuntos
Astenozoospermia/genética , Proteínas de Membrana/genética , Deleção de Sequência/genética , Adulto , China , Simulação por Computador , Análise Mutacional de DNA , Estudos de Associação Genética , Testes Genéticos , Humanos , Masculino , Polimorfismo de Nucleotídeo Único , Conformação Proteica , Motilidade dos Espermatozoides , Espermatogênese/genéticaRESUMO
AIM: Preparation of monoclonal antibody (mAb) to HCCR, which is a candidate biomarker for human hepatocellular carcinoma (HCC). METHODS: The recombinant protein HCCR-1(167-360); was expressed and was used as immunogen to immunize mouse for generation of mAb against HCCR. The protein Ep-HCCR, which displayed a epitope of HCCR, was also expressed and purified to use to detect serum antibody titer and to screen the positive clones of hybridmas. The properties of HCCR antibody were analyzed by ELISA, Western blot, immunofluorescence and immunohistochemistry. RESULTS: A hybridmas clone, which secreted anti-HCCR mAb, was obtained. The affinity constant (Kaff) of the mAb is 5.4×10(6); L/mol analyzed by ELISA; Western blot showed that the mAb could specifically recognize HCCR-1 and HCCR-2 expressed in HepG2 cells; The mAb was also used to detect the expression of HCCR proteins in hepatoma cells and HCC tissues. The results of immunofluorescence indicated that HCCR proteins mainly localized on the plasma membrane and cytoplasm of HepG2 cells. In addition, HCCR was found high-expressed in HCC tissues but not in normal liver tissue detected by Immunohistochemistry. CONCLUSION: A specific mAb against HCCR was successfully generated, which laid the foundation for establishing HCC detection method based on HCCR.