RESUMO
Pulmonary fibrosis (PF) is chronic and irreversible damage to the lung characterized by fibroblast activation and matrix deposition. Although recently approved novel anti-fibrotic agents can improve the lung function and survival of patients with PF, the overall outcomes remain poor. In this study, a novel imidazopurine compound, 3-(2-chloro-6-fluorobenzyl)-1,6,7-trimethyl-1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione (IM-1918), markedly inhibited transforming growth factor (TGF)-ß-stimulated reporter activity and reduced the expression of representative fibrotic markers, such as connective tissue growth factor, fibronectin, collagen and α-smooth muscle actin, on human lung fibroblasts. However, IM-1918 neither decreased Smad-2 and Smad-3 nor affected p38MAPK and JNK. Instead, IM-1918 reduced Akt and extracellular signal-regulated kinase 1/2 phosphorylation increased by TGF-ß. Additionally, IM-1918 inhibited the phosphorylation of fibroblast growth factor receptors 1 and 3. In a bleomycin-induced murine lung fibrosis model, IM-1918 profoundly reduced fibrotic areas and decreased collagen and α-smooth muscle actin accumulation. These results suggest that IM-1918 can be applied to treat lung fibrosis.
Assuntos
Inibidores Enzimáticos/farmacologia , Imidazóis/química , Fibrose Pulmonar/tratamento farmacológico , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Fator de Crescimento Transformador beta/metabolismo , Animais , Antibióticos Antineoplásicos/toxicidade , Bleomicina/toxicidade , Fibronectinas/genética , Fibronectinas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , Fator de Crescimento Transformador beta/genéticaRESUMO
Radiotherapy is one of the most common treatments for cancer, but radioresistance and injury to normal tissue are considered major obstacles to successful radiotherapy. Thus, there is an urgent need to develop radiosensitizers to improve the therapeutic outcomes of radiotherapy in cancer patients. Our previous efforts to identify novel radiosensitizers, using high-throughput screening targeting p53 and Nrf2 revealed a promising N-phenylpyrimidin-2-amine (PPA) lead compound. In the present study, 17 derivatives of this lead compound were examined, and it was found that 4-(4-fluorophenyl)-N-(4-nitrophenyl)-6-phenylpyrimidin-2-amine (PPA5), 4-((4-(4-fluorophenyl)pyrimidin-2-yl)amino)-3-methoxy-N-methyl -benzamide (PPA13), 4-((4-(4-fluorophenyl)pyrimidin-2-yl)amino)benzenesulfonamide (PPA14), 4-((4-(2-chlorophenyl)pyrimidin-2-yl)amino)benzenesulfonamide (PPA15), and 4-((4-(2-chlorophenyl)pyrimidin-2-yl)amino)-N-methylbenzamide (PPA17) inhibited cell viability by more than 50%, with a marked increase in the proportion of cells arrested at the G2/M phase of cell cycle. Among these compounds, PPA15 markedly increased the sub-G1 cell population and increased the levels of cyclin B1 and the phosphorylation levels of cyclin-dependent kinase (CDK) 1. Combined treatment with radiation and PPA14 or PPA15 significantly decreased clonogenic survival. An in vitro kinase assay revealed that PPA15 inhibited multiple CDKs involved in cell cycle regulation. Compared with drug or radiation treatment alone, combined treatment with PPA15 and radiation resulted in the suppression of A549 tumor growth in mice by 59.5% and 52.7%, respectively. Treatment with PPA15 alone directly inhibited tumor growth by 25.7%. These findings suggest that the novel pan CDK inhibitor, PPA15, may be a promising treatment to improve the effectiveness of radiotherapy for the treatment of cancer. SIGNIFICANCE STATEMENT: Several inhibitors of CDK have been successfully evaluated in combination with other chemotherapeutics in clinical trials, but negative side effects have partially restricted their clinical use. In this study, we identified a novel pan-CDK inhibitor to increase radiosensitivity, and we hope this work will encourage the development of promising small-molecule radiosensitizers.
Assuntos
Ciclo Celular/efeitos dos fármacos , Neoplasias Pulmonares/patologia , Pirimidinas/química , Pirimidinas/farmacologia , Radiossensibilizantes/química , Radiossensibilizantes/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Ciclo Celular/efeitos da radiação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Feminino , Humanos , Camundongos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Ionizing radiation (IR) has been widely used in the treatment of cancer. Radiation-induced DNA damage triggers the DNA damage response (DDR), which can confer radioresistance and early local recurrence by activating DNA repair pathways. Since karyopherin-α2 (KPNA2), playing an important role in nucleocytoplasmic transport, was significantly increased by IR in our previous study, we aimed to determine the function of KPNA2 with regard to DDR. Exposure to radiation upregulated KPNA2 expression in human colorectal cancer HT29 and HCT116 cells and breast carcinoma MDA-MB-231 cells together with the increased expression of DNA repair protein BRCA1. The knockdown of KPNA2 effectively increased apoptotic cell death via inhibition of BRCA1 nuclear import following IR. Therefore, we propose that KPNA2 is a potential target for overcoming radioresistance via interruption to DDR.
Assuntos
Proteína BRCA1/metabolismo , Morte Celular/efeitos da radiação , Sobrevivência Celular/fisiologia , alfa Carioferinas/metabolismo , Apoptose/efeitos da radiação , Proteína BRCA1/genética , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/genética , Ensaio Cometa , Dano ao DNA/efeitos da radiação , Reparo do DNA/efeitos da radiação , Células HCT116 , Células HT29 , Humanos , Imunoprecipitação , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Radiação IonizanteRESUMO
Although favorable immune responses to low-dose irradiation (LDI) have been observed in normal mice, i.e., a hormesis effect, little is known about the effects of LDI in infectious diseases. In this study, we examined the effects of LDI on mice with sepsis, a severe and often lethal hyperinflammatory response to bacteria. Female C57BL/6 mice were whole-body irradiated with 10cGy 48h before Escherichia coli infection, and survival, bacterial clearance, cytokines, and antioxidants were quantified. LDI pretreatment significantly increased survival from 46.7% in control mice to 75% in mice with sepsis. The bacterial burden was significantly lower in the blood, spleen, and kidney of LDI-treated mice than in those of control septic mice. The levels of pro-inflammatory cytokines, e.g., IL-1ß and IL-6, as well as anti-inflammatory IL-10 were markedly reduced in pre-LDI septic mice. Nitric oxide production by peritoneal macrophages was also reduced in pre-LDI septic mice. Immune cells in the spleen increased and Nrf2 and HO-1 were induced in pre-LDI septic mice. LDI stimulates the immune response and minimizes lethality in septic mice via enhanced bacterial clearance and reduced initial proinflammatory responses.
Assuntos
Infecções por Escherichia coli/radioterapia , Sepse/radioterapia , Irradiação Corporal Total , Animais , Contagem de Colônia Microbiana , Citocinas/sangue , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/sangue , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Feminino , Rim/microbiologia , Rim/efeitos da radiação , Macrófagos Peritoneais/metabolismo , Macrófagos Peritoneais/efeitos da radiação , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico/metabolismo , Células RAW 264.7 , Sepse/sangue , Sepse/imunologia , Sepse/microbiologia , Baço/microbiologia , Baço/efeitos da radiaçãoRESUMO
Rab GTPases regulate various types of intracellular membrane trafficking in all eukaryotes. Since Rab27a and its multiple effectors are involved in exocytosis of lysosome-related organelles and play a major role in malignancy, compounds targeting Rab27a could be likely used to inhibit invasive growth and tumor metastasis. Thus, we designed and synthesized several compounds based on the previously reported Rab27a-targeting synthetic compounds identified by virtual screening, and investigated their anti-metastatic effects in MDA-MB231 and A375 cells. Among the synthesized compounds, (E)-N-(3-chlorophenyl)-6-(2-(3,4-dihydroxy benzylidene)hydrazinyl)pyridine-3-sulfonamide (3d) and (E)-N-benzyl-6-(2-(3,4-dihydroxy benzylidene)hydrazinyl)-N-methylpyridine-3-sulfonamide (3f) significantly inhibited the invasiveness of both tumor cell lines. Compounds 3d and 3f also decreased the levels of signature extracellular matrix marker proteins (fibronectin, collagen, and α-smooth muscle actin) and representative mesenchymal cell markers (N-cadherin and vimentin). Taken together, our results suggest that novel sulfonamide analogs have anti-metastatic activity in breast and melanoma cancer cell lines and may be used as therapeutic agents to treat malignant cancer.
Assuntos
Antineoplásicos/química , Sulfonamidas/química , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Sítios de Ligação , Caderinas/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Proteínas da Matriz Extracelular/metabolismo , Humanos , Simulação de Acoplamento Molecular , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/farmacologia , Vimentina/metabolismo , Proteínas rab de Ligação ao GTP/antagonistas & inibidores , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab27 de Ligação ao GTPRESUMO
Numerous studies have attempted to develop biological markers for the response to radiation for broad and straightforward application in the field of radiation. Based on a public database, the present study selected several molecules involved in the DNA damage repair response, cell cycle regulation and cytokine signaling as promising candidates for lowdose radiationsensitive markers. The HuT 78 and IM9 cell lines were irradiated in a concentrationdependent manner, and the expression of these molecules was analyzed using western blot analysis. Notably, the activation of ataxia telangiectasia mutated (ATM), checkpoint kinase 2 (CHK2), p53 and H2A histone family member X (H2AX) significantly increased in a concentrationdependent manner, which was also observed in human peripheral blood mononuclear cells. To determine the radioprotective effects of cinobufagin, as an ATM and CHK2 activator, an in vivo model was employed using sublethal and lethal doses in irradiated mice. Treatment with cinobufagin increased the number of bone marrow cells in sublethal irradiated mice, and slightly elongated the survival of lethally irradiated mice, although the difference was not statistically significant. Therefore, KU60019, BML277, pifithrinα, and nutlin3a were evaluated for their ability to modulate radiationinduced cell death. The use of BML277 led to a decrease in radiationinduced pCHK2 and γH2AX levels and mitigated radiationinduced apoptosis. On the whole, the present study provides a novel approach for developing drug candidates based on the profiling of biological radiationsensitive markers. These markers hold promise for predicting radiation exposure and assessing the associated human risk.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia , Dano ao DNA , Radiação Ionizante , Transdução de Sinais , Dano ao DNA/efeitos da radiação , Dano ao DNA/efeitos dos fármacos , Humanos , Animais , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiação , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Camundongos , Quinase do Ponto de Checagem 2/metabolismo , Quinase do Ponto de Checagem 2/genética , Histonas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Masculino , Imidazóis/farmacologia , Protetores contra Radiação/farmacologia , Linhagem Celular Tumoral , Relação Dose-Resposta à RadiaçãoRESUMO
Radiotherapy (RT) is a commonly used treatment option for patients with cancer because it can effectively control tumor growth and kill tumor cells. However, the impact of RT goes beyond direct tumor cell killing because it can change the tumor microenvironment by altering surrounding tissues and infiltrating cells and modulating the expression of immune checkpoints. Poliovirus receptor (PVR, cluster of differentiation (CD)155), a member of the nectin-like molecule family, is overexpressed in many human cancers. However, its role in the tumor growth and T-cell immune responses of triple-negative breast cancer (TNBC) remains unclear. In the present study, we observe that radiation exposure increases PVR expression in MDA-MB-231 and BT549 cells. Silencing PVR not only inhibited the proliferation of breast cancer cells but also significantly enhanced the cytotoxicity of cytotoxic T lymphocytes (CTLs) compared with the control or RT groups. Treatment of T cells with PVR decreased CD8+ T cells, increased CD4+ T cells, and induced PVR ligands such as T cell immunoreceptor with immunoglobulin and ITIM domain, CD226, and CD96. However, after treatment with PVR, CTL responses decreased and secretion of interferon-γ, tumor necrosis factor-α, interleukin (IL)-2, IL-6, and IL-10 was significantly inhibited. In contrast, PVR knockdown increased the production of these cytokines, illustrating the immunosuppressive function of PVR. Suppression of PVR using an anti-PVR antibody inhibited 4T1 tumor growth by increasing immune cell infiltration. These results provide new insights into the role of PVR in TNBC and highlight its potential as a target for T cell-mediated immunotherapy in breast cancer.
RESUMO
Cells under hypoxic stress either activate an adaptive response or undergo cell death. Although some mechanisms have been reported, the exact mechanism behind hypoxic cell death remains unclear. Recently, increased expression of fatty acid synthase (FASN) has been observed in various human cancers. In highly proliferating cells, tumor-associated FASN is considered necessary for both membrane lipids production and post-translational protein modification, but the exact mechanisms are not fully understood. Further, FASN overexpression is associated with aggressive and malignant cancer diseases and FASN inhibition induces apoptosis in cancer cells. For this reason, FASN is emerging as a key target for the potential diagnosis and treatment of various cancers. Here, we observed decreased FASN expression under hypoxic cell death conditions in HepG2 cells. Thus, we examined the effect of decreased FASN expression on hypoxia-induced cell death in HepG2 cells and also investigated the mechanism responsible for reduction of FASN expression under hypoxic cell death conditions. As a result, reduction of FASN expression resulted in hypoxic cell death via malonyl-CoA accumulation. In addition, SREBP-1 restored FASN reduction and hypoxia-induced apoptosis. Taken together, we suggest that hypoxic cell death is promoted by the reduced expression of FASN through SREBP-1 down-regulation.
Assuntos
Regulação para Baixo , Ácido Graxo Sintase Tipo I/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Contagem de Células , Morte Celular , Hipóxia Celular , Proliferação de Células , Sobrevivência Celular/efeitos dos fármacos , Fragmentação do DNA , Eletroforese em Gel Bidimensional , Ácido Graxo Sintase Tipo I/genética , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Glucose/farmacologia , Células Hep G2 , Humanos , Malonil Coenzima A/genética , Malonil Coenzima A/metabolismo , Regiões Promotoras Genéticas , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , TransfecçãoRESUMO
The survival and death of eukaryotic cells are tightly controlled by a variety of proteins in response to the cellular environment. Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) is a receptor-interacting Ser/Thr kinase that has recently been reported as an important regulator of cell survival, apoptosis, and necroptosis; however, its role in liver cancer remains unclear. In this study, we examined the effect of siRNA-mediated RIPK1 knockdown on the survival and death of liver cancer cells. Treatment with siRIPK1 decreased the growth rate of liver cancer cells and increased apoptotic, but not necrotic cell death, which was higher in wild-type p53 (wt-p53) cells than in mutant-type p53 (mt-p53) cells. In addition, RIPK1 knockdown increased p53 expression and G1 phase arrest in wt-p53 cells. Although suppressing p53 did not alter RIPK1 expression, it did attenuate siRIPK1-induced cell death. Interestingly, RIPK1 knockdown also increased the generation of reactive oxygen species and DNA damage by inhibiting signal transduced and activator of transcription 3 (STAT3) and ATM and RAD3-related (ATR) in wt-p53 cells but not in mt-p53 cells. Moreover, STAT3 or ATR inhibition in p53 mutant cells restored siRIPK1-mediated cell death. Together, the results of this study suggest that RIPK1 suppression induces apoptotic cell death by inhibiting the STAT3/ATR axis in a p53-dependent manner. Furthermore, these findings suggest that RIPK1, alone or in combination, may be a promising target for treating liver cancer.
RESUMO
Our previous work demonstrated that (E)-N-benzyl-6-(2-(3, 4-dihydroxybenzylidene) hydrazinyl)-N-methylpyridine-3-sulfonamide (BHMPS), a novel synthetic inhibitor of Rab27aSlp(s) interaction, suppresses tumor cell invasion and metastasis. Here, we aimed to further investigate the mechanisms of action and biological significance of BHMPS. BHMPS decreased the expression of epithelial-mesenchymal transition transcription factors through inhibition of focal adhesion kinase and c-Jun N-terminal kinase activation, thereby reducing the migration and invasion of breast cancer. Additionally, knockdown of Rab27a inhibited tumor migration, with changes in related signaling molecules, whereas overexpression of Rab27a reversed this phenomenon. BHMPS effectively prevented the interaction of Rab27a and its effector Slp4, which was verified by co-localization, immunoprecipitation, and in situ proximity ligation assays. BHMPS decreased the secretion of epidermal growth factor receptor and fibronectin by interfering with vesicle trafficking, as indicated by increased perinuclear accumulation of CD63-positive vesicles. Moreover, administration of BHMPS suppressed tumor growth in Rab27a-overexpressing MDA-MB-231 xenograft mice. These findings suggest that BHMPS may be a promising candidate for attenuating tumor migration and invasion by blocking Rab27a-mediated exocytosis.
RESUMO
Low-dose irradiation (LDI) has recently been shown to have various beneficial effects on human health, such as on cellular metabolic activities, DNA repair, antioxidant activity, homeostasis potency, and immune activation. Although studies on the immunogenic effects of LDI are rapidly accumulating, clinical trials for cancer treatment are considered premature owing to the lack of available preclinical results and protocols. Here, we aim to investigate anti-tumor and anti-metastatic effects of whole-body LDI in several tumor-bearing mouse models. Mice were exposed to single or fractionated whole-body LDI prior to tumor transplantation, and tumor growth and metastatic potential were determined, along with analysis of immune cell populations and expression of epithelial-mesenchymal transition (EMT) markers. Whole-body fractionated-LDI decreased tumor development and lung metastasis not only by infiltration of CD4+, CD8+ T-cells, and dendritic cells (DCs) but also by attenuating EMT. Moreover, a combination of whole-body LDI with localized high-dose radiation therapy reduced the non-irradiated abscopal tumor growth and increased infiltration of effector T cells and DCs. Therefore, whole-body LDI in combination with high-dose radiation therapy could be a potential therapeutic strategy for treating cancer.
RESUMO
Hepatitis B virus (HBV) encoding the HBV x protein (HBx) is a known causative agent of hepatocellular carcinoma (HCC). Its pathogenic activities in HCC include interference with several signaling pathways associated with cell proliferation and apoptosis. Mutant C-terminal-truncated HBx isoforms are frequently found in human HCC and have been shown to enhance proliferation and invasiveness leading to HCC malignancy. We investigated the molecular mechanism of the reduced doxorubicin cytotoxicity by C-terminal truncated HBx. Cells transfected with C-terminal truncated HBx exhibited reduced cytotoxicity to doxorubicin compared to those transfected with full-length HBx. The doxorubicin resistance of cells expressing C-terminal truncated HBx correlated with upregulation of the ATP binding cassette subfamily B member 1(ABCB1) transporter, resulting in the enhanced efflux of doxorubicin. Inhibiting the activity of ABCB1 and silencing ABCB1 expression by small interfering ribonucleic acid (siRNA) increased the cytotoxicity of doxorubicin. These results indicate that elevated ABCB1 expression induced by C-terminal truncation of HBx was responsible for doxorubicin resistance in HCC. Hence, co-treatment with an ABCB1 inhibitor and an anticancer agent may be effective for the treatment of patients with liver cancer containing the C-terminal truncated HBx. [BMB Reports 2019; 52(5): 330-335].
Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/metabolismo , Doxorrubicina/farmacologia , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Transativadores/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Antibióticos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Humanos , Neoplasias Hepáticas/genética , Transdução de Sinais , Transativadores/genética , Ativação Transcricional , Regulação para Cima , Proteínas Virais Reguladoras e AcessóriasRESUMO
OBJECTIVE: Tumor-treating fields are currently used to successfully treat various cancers; however, the specific pathways associated with its efficacy remain unknown in the immune responses. Here, we evaluated tumor-treating fields-mediated initiation of the macrophage-specific immune response. MATERIALS AND METHODS: We subjected RAW 264.7 mouse macrophages to clinically relevant levels of tumor-treating fields (0.9 V/cm, 150 kHz) and evaluated alterations in cytokine expression and release, as well as cell viability. Additionally, we investigated the status of immunomodulatory pathways to determine their roles in tumor-treating fields-mediated immune activation. RESULTS AND DISCUSSION: Our results indicated that tumor-treating fields treatment at 0.9 V/cm decreased cell viability and increased cytokine messenger RNA/protein levels, as well as levels of nitric oxide and reactive oxygen species, relative to controls. The levels of tumor necrosis factor α, interleukin 1ß, and interleukin 6 were markedly increased in tumor-treating fields-treated RAW 264.7 cells cocultured with 4T1 murine mammary carcinoma cells compared with those in 4T1 or RAW 264.7 cells with or without tumor-treating fields treatment. Moreover, the viability of 4T1 cells treated with the conditioned medium of tumor-treating fields-stimulated RAW 264.7 cells decreased, indicating that macrophage activation by tumor-treating fields effectively killed the tumor cells. Moreover, tumor-treating fields treatment activated the nuclear factor κB and mitogen-activated protein kinase pathways involved in immunomodulatory signaling. CONCLUSION: These results provide critical insights into the mechanisms through which tumor-treating fields affect macrophage-specific immune responses and the efficacy of this method for cancer treatment.
Assuntos
Antígenos de Histocompatibilidade Classe II/imunologia , Ativação de Macrófagos/imunologia , Magnetoterapia , Neoplasias/radioterapia , Animais , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/efeitos da radiação , Humanos , Sistema de Sinalização das MAP Quinases/efeitos da radiação , Ativação de Macrófagos/genética , Ativação de Macrófagos/efeitos da radiação , Macrófagos/imunologia , Macrófagos/efeitos da radiação , Camundongos , NF-kappa B/genética , Neoplasias/imunologia , Neoplasias/patologia , Células RAW 264.7 , Transdução de Sinais/imunologia , Transdução de Sinais/efeitos da radiaçãoRESUMO
Angiogenesis is important in the development and progression of cancer, therefore the therapeutic approach based on anti-angiogenesis may represent a promising therapeutic option. KR-31831 is a novel anti-ischemic agent. Previously, we reported the anti-angiogenic activity of KR-31831. In the present study we investigated the molecular mechanisms underlying anti-angiogenic activity of KR-31831. We show that KR-31831 inhibits vascular endothelial growth factor (VEGF)-induced proliferation and tube formation via release of intracellular Ca2+ and phosphorylation of extra-cellular regulated kinase 1/2 (Erk 1/2) in human umbilical vein endothelial cells (HUVECs). Moreover, the expression of VEGF receptor 2 (VEGFR2, known as Flk-1 or KDR) was reduced by the treatment of KR-31831. These results suggest that KR-31831 may have inhibitory effects on tumor angiogenesis through down-regulation of KDR expression.
Assuntos
Benzopiranos/farmacologia , Endotélio Vascular/efeitos dos fármacos , Imidazóis/farmacologia , Neovascularização Patológica/prevenção & controle , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Colágeno/metabolismo , Combinação de Medicamentos , Humanos , Immunoblotting , Laminina/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Proteoglicanas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Veias Umbilicais/citologia , Fator A de Crescimento do Endotélio Vascular/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
Focal adhesion kinase 1 (FAK1) is known to promote tumor progression and metastasis by controlling cell movement, invasion, survival and the epithelial-to-mesenchymal transition in the tumor microenvironment. As recent reports imply that FAK1 is highly associated with tumor cell development and malignancy, the inhibition of FAK1 activity could be an effective therapeutic approach for inhibiting the growth and metastasis of tumor cells. In this study, we aimed to determine the effect of a novel synthetic FAK1 inhibitor 2-[2-(2-methoxy-4-morpholin-4-yl-phenylamino)-5-trifluoromethyl-pyrimidin-4-ylamino]-N-methyl-benzamide, (MPAP) on lung cancer cells. MPAP suppressed cancer cell proliferation and the phosphorylation of FAK1. Combined treatment with MPAP and irradiation (IR) showed enhanced suppression of cancer cell proliferation in wild-type p53 cells and more intense suppression in p53-null cells. In addition, the combination treatment effectively induced G1 cell cycle arrest in a p53-independent manner. In an in vivo tumor xenograft mouse model, treatment with both MPAP and IR reduced tumor growth more than the treatment with IR or MPAP alone. Overall, these data demonstrate that the radiosensitizing effect of MPAP is mediated by the regulation of retinoblastoma protein (RB) phosphorylation in a p53-independent manner.
Assuntos
Quinase 1 de Adesão Focal/antagonistas & inibidores , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/radioterapia , Tolerância a Radiação/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Benzamidas/administração & dosagem , Benzamidas/síntese química , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/efeitos da radiação , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos da radiação , Quinase 1 de Adesão Focal/genética , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos da radiação , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Radiação , Tolerância a Radiação/genética , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/efeitos da radiação , Proteína Supressora de Tumor p53/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The kringle domain is a triple loop structure present in angiostatin and endostatin. The disulfide bond-linked kringle architectures have been known to be essential for anti-angiogenic activity. Plasma hyaluronan binding protein (PHBP) is a novel serine protease which consists of three epidermal growth factor (EGF) domains, a kringle domain, and a serine protease domain. PHBP can be cleaved autocatalytically to generate activity and is highly expressed in the human blood and liver. To determine the anti-angiogenic activities of PHBP, we purified recombinant mouse PHBP from stable cell line overexpressing PHBP and used protein in vivo and in vitro angiogenesis assays. We found that recombinant PHBP inhibits not only angiogenesis in vivo in chorioallantoic membrane (CAM) assay but also the basic fibroblast growth factor (bFGF)-induced proliferation, invasion and tube formation of human umbilical vein endothelial cells (HUVECs) in a dose-dependant manner. Moreover, we found that the kringle domain of PHBP was essential for the anti-angiogenic action of PHBP by the deletion mutants. These findings unravel a new function of PHBP as an inhibitor of the proangiogenic phenotype of vascular endothelial cells and demonstrate that the kringle domain of PHBP might be a potent novel inhibitor of activated endothelial cells in vitro and in vivo.
Assuntos
Inibidores da Angiogênese/farmacologia , Células Endoteliais/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Serina Endopeptidases/farmacologia , Animais , Movimento Celular , Proliferação de Células , Células Cultivadas , Embrião de Galinha , Membrana Corioalantoide/irrigação sanguínea , Membrana Corioalantoide/efeitos dos fármacos , Colágeno , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Células Endoteliais/citologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Humanos , Kringles/genética , Laminina , Mutação , Proteoglicanas , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Serina Endopeptidases/química , Serina Endopeptidases/genética , Veias Umbilicais/citologia , Veias Umbilicais/efeitos dos fármacosRESUMO
Although many studies have examined the roles of hypoxia and transforming growth factor- (TGF-) ß separately in the tumor microenvironment, the effects of simultaneous treatment with hypoxia/reoxygenation and TGF-ß on tumor malignancy are unclear. Here, we investigated the effects of redox signaling and oncogenes on cell proliferation and radioresistance in A549 human lung cancer cells in the presence of TGF-ß under hypoxia/reoxygenation conditions. Combined treatment with TGF-ß and hypoxia activated epidermal growth factor receptor (EGFR) and nuclear factor (erythroid-derived 2)-like 2 (Nrf2), a redox-sensitive transcription factor. Interestingly, Nrf2 knockdown suppressed the effects of combined treatment on EGFR phosphorylation. In addition, blockade of EGFR signaling also suppressed induction of Nrf2 following combined treatment with hypoxia and TGF-ß, indicating that the combined treatment induced positive crosstalk between Nrf2 and EGFR. TGF-ß and hypoxia/reoxygenation increased the accumulation of reactive oxygen species (ROS), while treatment with N-acetyl-l-cysteine abolished the activation of Nrf2 and EGFR. Treatment with TGF-ß under hypoxic conditions increased the proliferation of A549 cells compared with that after vehicle treatment. Moreover, cells treated with the combined treatment exhibited resistance to ionizing radiation (IR), and knockdown of Nrf2 increased IR-induced cell death under these conditions. Thus, taken together, our findings suggested that TGF-ß and hypoxia/reoxygenation promoted tumor progression and radioresistance of A549 cells through ROS-mediated activation of Nrf2 and EGFR.
Assuntos
Receptores ErbB/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Oxigênio/farmacologia , Tolerância a Radiação/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia , Células A549 , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Hipóxia Celular/efeitos dos fármacos , Humanos , Ligantes , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacosRESUMO
Accumulating evidence suggests the potential for radiation therapy to generate antitumor immune responses against tumor cells by inducing immunogenic cell death and phenotypic changes. We recently found that ionizing radiation upregulated karyopherin α2 (KPNA2) in HT-29 colorectal tumor cells using quantitative proteomic analysis. To determine whether this increased KPNA2 could function as a damage-associated molecular pattern to induce antitumor immune responses, mouse bone-marrow-derived dendritic cells (BMDCs) were treated with KPNA2. KPNA2 enhanced the surface expression of CD40, CD54, CD80, CD86, and MHC class I/II on BMDCs. DCs treated with KPNA2 exhibited increased secretion of pro-inflammatory cytokines such as IL-1ß, IL-6, IL-12, IL-23, and TNF-α. Co-culture of CD4(+) T cells and KPNA2-treated DCs resulted in induction of Th1/17 cytokines (IFN-γ and IL-17) and reduction of TGF-ß production. Moreover, KPNA2-treated DCs were capable of increasing granzyme B and perforin expression in cytotoxic T lymphocytes. These results demonstrated that radiation-induced dying colorectal cancer cells released considerable amounts of KPNA2 that induce the maturation and activation of DCs for synergistic antitumor effect of radiation.
Assuntos
Neoplasias Colorretais/imunologia , Neoplasias Colorretais/radioterapia , Células Dendríticas/imunologia , Proteínas Nucleares/imunologia , alfa Carioferinas/imunologia , Animais , Morte Celular/fisiologia , Morte Celular/efeitos da radiação , Diferenciação Celular/fisiologia , Diferenciação Celular/efeitos da radiação , Proliferação de Células/fisiologia , Proliferação de Células/efeitos da radiação , Neoplasias Colorretais/patologia , Células Dendríticas/efeitos da radiação , Feminino , Células HT29 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Nucleares/metabolismo , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/efeitos da radiação , Regulação para Cima/efeitos da radiação , alfa Carioferinas/metabolismoRESUMO
Mitochondrial dysfunction has been found to be associated with various pathological conditions, particularly cancer. However, the mechanisms underlying tumor malignancy induced by mitochondrial dysfunction are not fully understood. In the present study, the effects of mitochondrial dysfunction on epithelial-mesenchymal transition (EMT), were investigated using mitochondrial-depleted ρ(0) cells derived from the Hep3B hepatocarcinoma cell line. The Hep3B/ρ(0) cells displayed the EMT phenotype with more aggressive migration and higher invasiveness compared to their parental cells. The Hep3B/ρ(0) cells also showed typical expression pattern of EMT markers such as vimentin and E-cadherin. These phenotypes in Hep3B/ρ(0) cells were mediated by increased transforming growth factor-ß (TGF-ß) through the canonical Smad-dependent signaling pathway. Additionally, TGF-ß signaling was activated via induction of c-Jun/AP-1 expression and activity. Therefore, mitochondrial dysfunction induces EMT through TGF-ß/Smad/Snail signaling via c-Jun/AP-1 activation. These results indicate that mitochondrial dysfunction plays an important role in the EMT process and could be a novel therapeutic target for malignant cancer therapy.
Assuntos
Carcinoma Hepatocelular/genética , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Neoplasias Hepáticas/genética , Fator de Transcrição AP-1/genética , Fator de Crescimento Transformador beta/genética , Caderinas/biossíntese , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/genética , Humanos , Neoplasias Hepáticas/patologia , Mitocôndrias/genética , Mitocôndrias/patologia , Transdução de Sinais , Proteínas Smad/genética , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: Radiotherapy is widely used to treat cancer alone or in combination with surgery, chemotherapy, and immunotherapy. However, damage to normal tissues and radioresistance of tumor cells are major obstacles to successful radiotherapy. Furthermore, the immune network around tumors appears to be connected to tumor progression and recurrence. METHODS: We investigated the cytosolic proteins produced by irradiated tumor cells by using a quantitative proteomic approach based on stable isotope labeling by amino acids in cell culture. MDA-MB-231 breast cancer cells were treated with a single or fractionated 10 Gray dose of (137)Cs γ-radiation, which was selected based on cell viability. RESULTS: Radiation-induced proteins were differentially expressed based on the fractionated times of radiation and were involved in multiple biological functions, including energy metabolism and cytoskeleton organization. We identified 46 proteins increased by at least 1.3-fold, and high ranks were determined for cathepsin D, gelsolin, arginino-succinate synthase 1, peroxiredoxin 5, and C-type mannose receptor 2. CONCLUSION: These results suggest that a number of tumor-derived factors upregulated by γ-radiation are promising targets for modulation of the immune response during radiation treatment.