RESUMO
Angiogenesis and immunosuppression are closely related pathophysiologic processes. Widely prescribed in malignant tumor and proliferative retinal lesions, VEGF signaling pathway inhibitors may cause hypertension and renal injury in some patients, presenting with proteinuria, nephrotic syndrome, renal failure and thrombotic microangiopathy. VEGF signaling pathway inhibitors block the action of both VEGF-A and VEGF-C. However, VEGF-A and VEGF-C produced by podocytes are vital to maintain the physiological function of glomerular endothelial cells and podocytes. There is still no effective treatment for kidney disease associated with VEGF signaling pathway inhibitors and some patients have progressive renal failure even after withdrawal of the drug. Recent studies reveal that blocking of VEGF-A and VEGF-C can activate CD4 +and CD8+ T cells, augment antigen-presenting function of dendritic cells, enhance cytotoxicity of macrophages and initiate complement cascade activation. VEGF and VEGFR are expressed in immune cells, which are involved in the immunosuppression and cross-talk among immune cells. This review summarizes the expression and function of VEGF-A and VEGF-C in the kidney. The current immunoregulation mechanisms of VEGF signaling pathway inhibitors are reviewed. Finally, combinate strategies are summarized to highlight the proposal for VEGF signaling pathway inhibitors.
Assuntos
Insuficiência Renal , Fator A de Crescimento do Endotélio Vascular , Humanos , Linfócitos T CD8-Positivos/metabolismo , Células Endoteliais/metabolismo , Rim/metabolismo , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator C de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator C de Crescimento do Endotélio Vascular/metabolismoRESUMO
Despite improvement during the last ten years in the longevity of patients with metastatic clear cell renal cell carcinoma (mccRCC) the disease remains incurable. Hence, new therapeutic strategies are urgently needed. Relapse following anti-angiogenic treatment depends on the over-expression of vascular endothelial growth factor C (VEGFC), one of the main drivers of lymphangiogenesis. Therefore, we developed specific mouse monoclonal antibodies and evaluated their therapeutic efficacy in vitro and in vivo. Immunization of mice with the domain of VEGFC that stimulates the VEGF receptor 3 (VEGFR3) led to the selection of one hybridoma producing specific anti-VEGFC monoclonal antibodies. The selected 1E9 antibodies were sequenced, and the corresponding variable light and heavy chains were subcloned into expression vectors in frame with sequences encoding the human IgG1 constant heavy and light chains. CHO cells were stably transfected and cloned to produce chimeric antibodies. These antibodies inhibited the activation of VEGFR3 signaling, and therefore the proliferation and migration of VEGFC-stimulated endothelial cells. Moreover, they inhibited the proliferation of VEGFC-expressing renal cancer cells through NRP2 signaling. 1E9 antibodies inhibited the growth of experimental RCC, and their therapeutic efficacy was enhanced by the anti-VEGF antibody bevacizumab. Hence, our results suggest that targeting VEGFC could have a relevant therapeutic impact on mccRCC that relapse following anti-angiogenic treatment.
Assuntos
Inibidores da Angiogênese/farmacologia , Antineoplásicos Imunológicos/farmacologia , Carcinoma de Células Renais/tratamento farmacológico , Células Endoteliais/efeitos dos fármacos , Neoplasias Renais/tratamento farmacológico , Neovascularização Patológica , Fator C de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Feminino , Humanos , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Vasos Linfáticos/efeitos dos fármacos , Vasos Linfáticos/metabolismo , Vasos Linfáticos/patologia , Camundongos Nus , Transdução de Sinais , Carga Tumoral/efeitos dos fármacos , Fator C de Crescimento do Endotélio Vascular/imunologia , Fator C de Crescimento do Endotélio Vascular/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Accumulating evidence on the association of VEGF-C with lymphangiogenesis and lymph node metastasis implicates lymphatic vessels as a potential target in anti-cancer therapy. To evaluate whether blocking VEGF-C and VEGFR-3 signaling can inhibit multi-organ metastases, a mouse metastatic mammary cancer model was subjected to gene therapy using a soluble VEGFR-3 expression vector (psVEGFR-3). We showed that psVEGFR-3 significantly diminished cell growth in vitro with or without added VEGF-C, and significantly reduced primary tumor growth and tumor metastases to wide-spectrum organs in vivo. Although apoptotic cell death and angiogenesis levels did not differ between the control and psVEGFR-3 groups, cell proliferation and lymphangiogenesis in the mammary tumors were significantly decreased in the psVEGFR-3 group. Furthermore, lymphatic vessel invasion was significantly inhibited in this group. Real-time RT-PCR analysis revealed significantly high expression of the Vegfr3 gene due to gene therapy, and the transcriptional levels of Pcna and Lyve1 tended to decrease in the psVEGFR-3 group. Immunofluorescence staining indicated that phospho-tyrosine expression was considerably lower in tumor cells of psVEGFR-3-treated mammary carcinomas than those of control tumors. Double immunofluorescence staining indicated that phospho-tyrosine+ /LYVE-1+ (a lymphatic vessel marker) tended to decrease in psVEGFR-3-treated mammary carcinomas compared with control mice, indicating a decline in the activity of the VEGF-C/VEGFR-3 axis. These findings showed that a blockade of VEGF-C/VEGFR-3 signaling caused by sVEGFR-3 sequestered VEGF-C and prevented the side-effects of anti-angiogenesis and suppressed overall metastases, suggesting their high clinical significance.
Assuntos
Terapia Genética/métodos , Neoplasias Mamárias Experimentais/terapia , Metástase Neoplásica/terapia , Fator C de Crescimento do Endotélio Vascular/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/genética , Animais , Linhagem Celular Tumoral/transplante , Feminino , Vasos Linfáticos/patologia , Glândulas Mamárias Animais/patologia , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Camundongos , Invasividade Neoplásica/genética , Invasividade Neoplásica/prevenção & controle , Metástase Neoplásica/genética , Fator C de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
A phage-derived human monoclonal antibody against VEGF-C was developed as a potential anti-tumor therapeutic and exhibited fast clearance in preclinical species, with notably faster clearance in serum than in plasma. The purpose of this work was to understand the factors contributing to its fast clearance. In vitro incubations in animal and human blood, plasma, and serum were conducted with radiolabeled anti-VEGF-C to determine potential protein and cell-based interactions with the antibody as well as any matrix-dependent recovery dependent upon the matrix. A tissue distribution study was conducted in mice with and without heparin infusion in order to identify a tissue sink and determine whether heparin could affect antibody recovery from serum and/or plasma. Incubation of radiolabeled anti-VEGF-C in human and animal blood, plasma, or serum revealed that the antibody formed a complex with an endogenous protein, likely VEGF-C. This complex was trapped within the blood clot during serum preparation from blood, but not within the blood cell pellet during plasma preparation. Low level heparin infusion in mice slowed down clot formation during serum preparation and allowed for better recovery of the radiolabeled antibody in serum. No tissue sink was found in mice. Thus, during this characterization, we determined that the blood sampling matrix greatly impacted the amount of antibody recovered in the samples, therefore, altering its derived pharmacokinetic parameters. Target biology should be considered when selecting appropriate sampling matrices for PK analysis.
Assuntos
Anticorpos Monoclonais/farmacocinética , Fator C de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Anticorpos Monoclonais/sangue , Artefatos , Coagulação Sanguínea , Feminino , Humanos , Macaca fascicularis , Camundongos Nus , Ratos Sprague-Dawley , Distribuição Tecidual , Fator C de Crescimento do Endotélio Vascular/imunologiaRESUMO
Androgen is known to regulate microRNA-135a (miR-135a) and can be regulated by androgen, suggesting that it may contribute to polycystic ovary syndrome (PCOS) with hyperandrogenism. However, its roles and mechanisms of action in PCOS are unknown. In this study, the role and molecular mechanisms underlying miR-135a in granulosa cells (GCs) in PCOS were evaluated. miR-135a expression was upregulated in patients with PCOS and in GCs isolated from patients compared with that in the respective controls (P < 0.01), as determined by RT-qPCR. The overexpression of miR-135a inhibited GC proliferation and induced GC apoptosis, as observed by CCK-8 assay and apoptosis assay. Furthermore, miR-135a overexpression increased the expression of double-strand break maker, γH2AX, as confirmed by western blotting. Our results further suggest that these effects were mediated via downregulation of vascular endothelial growth factor C (VEGFC), which was identified as a direct target of miR-135a. Moreover, levels of VEGFC and miR-135a expression showed a negative correlation. These findings indicate that miR-135a promotes apoptosis and the DNA damage response in GCs in PCOS, likely via VEGFC signaling. This study provides novel insights into GC dysregulation in PCOS and suggests that miR-135a is a promising therapeutic target for PCOS treatment.
Assuntos
Apoptose/fisiologia , Células da Granulosa/metabolismo , Células Lúteas/metabolismo , MicroRNAs/biossíntese , Síndrome do Ovário Policístico/metabolismo , Fator C de Crescimento do Endotélio Vascular/biossíntese , Adulto , Células Cultivadas , Feminino , Expressão Gênica , Células da Granulosa/patologia , Humanos , Células Lúteas/patologia , MicroRNAs/genética , Síndrome do Ovário Policístico/patologia , Fator C de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator C de Crescimento do Endotélio Vascular/genética , Adulto JovemRESUMO
PURPOSE: OPT-302 is a novel inhibitor of vascular endothelial growth factor (VEGF)-C and VEGF-D. A phase 1 trial assessed the safety of intravitreal OPT-302 as monotherapy or combined with ranibizumab (Lucentis; Genentech, South San Francisco, CA) in patients with neovascular age-related macular degeneration (nAMD). DESIGN: Open-label, dose escalation followed by a randomized dose expansion. PARTICIPANTS: Fifty-one patients with nAMD who were either treatment naïve (n = 25) or previously were treated with anti-VEGF A therapy (n = 26). METHODS: In the dose escalation, groups of 5 patients in 4 cohorts received ascending doses of OPT-302 (0.3 mg, 1 mg, or 2 mg) in combination with ranibizumab (0.5 mg), or as monotherapy (2 mg). In the dose expansion, 31 patients were randomized (3:1) to OPT-302 (2 mg) in combination with ranibizumab (n = 23) or as monotherapy (n = 8). Participants received three intravitreal treatments of OPT-302 once every 4 weeks either with or without ranibizumab. MAIN OUTCOME MEASURES: Safety and tolerability, OPT-302 pharmacokinetics and immunogenicity, effects on best-corrected visual acuity (BCVA), and anatomic changes. RESULTS: Intravitreal OPT-302 with or without ranibizumab was well tolerated with low systemic exposure, no dose-limiting toxicities and no immunogenicity. In patients receiving OPT-302 monotherapy, 7 of 13 (54%) did not require rescue anti-VEGF-A therapy and the mean change in BCVA from baseline to week 12 was +5.6 letters (range, 0-18 letters). Mean BCVA gains from baseline to week 12 following combination OPT-302 with ranibizumab were +10.8 letters (95% confidence interval [CI], 4-17; n = 18) in treatment-naïve patients and +4.9 letters (95% CI, 3-7; n = 19) in previously treated patients, respectively. Corresponding reductions in mean central subfield thickness at week 12 in both groups were -119 µm (95% CI, -176 to -62 µm) and -54 µm (95% CI, -82 to -26 µm), respectively, whilst 50% of treatment-naïve patients also showed no detectable choroidal neovascularization at week 12 on fluorescein angiography. CONCLUSIONS: Intravitreal OPT-302 inhibition of VEGF-C and -D was well tolerated, and OPT-302 combination therapy may overcome an escape mechanism to VEGF-A suppression in the management of nAMD.
Assuntos
Inibidores da Angiogênese/administração & dosagem , Fator C de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator D de Crescimento do Endotélio Vascular/antagonistas & inibidores , Degeneração Macular Exsudativa/tratamento farmacológico , Idoso , Feminino , Angiofluoresceinografia/métodos , Seguimentos , Fundo de Olho , Humanos , Macula Lutea/patologia , Masculino , Estudos Prospectivos , Tomografia de Coerência Óptica/métodos , Acuidade Visual , Degeneração Macular Exsudativa/diagnóstico , Degeneração Macular Exsudativa/metabolismoRESUMO
Chondrosarcoma is a common primary malignant tumor of the bone that can metastasize through the vascular system to other organs. A key step in the metastatic process, lymphangiogenesis, involves vascular endothelial growth factor-C (VEGF-C). However, the effects of lymphangiogenesis in chondrosarcoma metastasis remain to be clarified. Accumulating evidence shows that resistin, a cytokine secreted from adipocytes and monocytes, also promotes tumor pathogenesis. Notably, chondrosarcoma can easily metastasize. In this study, we demonstrate that resistin enhances VEGF-C expression and lymphatic endothelial cells (LECs)-associated lymphangiogenesis in human chondrosarcoma cells. We also show that resistin triggers VEGF-C-dependent lymphangiogenesis via the c-Src signaling pathway and down-regulating micro RNA (miR)-186. Overexpression of resistin in chondrosarcoma cells significantly enhanced VEGF-C production and LECs-associated lymphangiogenesis in vitro and tumor-related lymphangiogenesis in vivo. Resistin levels were positively correlated with VEGF-C-dependent lymphangiogenesis via the down-regulation of miR-186 expression in clinical samples from chondrosarcoma tissue. This study is the first to evaluate the mechanism underlying resistin-induced promotion of LECs-associated lymphangiogenesis via the upregulation of VEGF-C expression in human chondrosarcomas. We suggest that resistin may represent a molecular target in VEGF-C-associated tumor lymphangiogenesis in chondrosarcoma metastasis.
Assuntos
Neoplasias Ósseas/metabolismo , Condrossarcoma/metabolismo , Linfangiogênese/fisiologia , MicroRNAs/biossíntese , Resistina/biossíntese , Fator C de Crescimento do Endotélio Vascular/biossíntese , Neoplasias Ósseas/genética , Linhagem Celular Transformada , Linhagem Celular Tumoral , Condrossarcoma/genética , Relação Dose-Resposta a Droga , Humanos , Linfangiogênese/efeitos dos fármacos , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Resistina/genética , Resistina/farmacologia , Fator C de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator C de Crescimento do Endotélio Vascular/genéticaRESUMO
Clinical studies have found that the incidence of cancer metastasis through the lymphatic vessels are 3-5 times higher than that through the blood vessels. These findings suggest the potency of anti-lymphangiogenic therapy in reducing the incidence of cancer metastasis. Previously, we reported LHbisD4, which is the conjugate of low molecular weight heparin (LMWH) and four bis-deoxycholates as a potent anti-angiogenic drug with less toxicity and orally active property. Here, we show that LHbisD4 could also suppress the formation of new lymphatic vessels and attenuate the incidence of metastasis by blocking VEGF-C signaling pathway. LHbisD4 significantly enhanced binding affinity with VEGF-C when compared with LMWH, which enables LHbisD4 to suppress the proliferation, migration and formation of tubular structures of human dermal lymphatic endothelial cells(HDLECs) in in vitro condition even in the presence of excessive amounts of VEGF-C. Similarly, we found that the density of lymphatic vessels in the primary tumor tissue in breast cancer bearing mice was significantly diminished when LHbisD4 was administered compared with the control group. Also, the incidence of axillary lymph nodes and distant organ metastasis was significantly reduced in the LHbisD4 administered group, which demonstrates that LHbisD4 could successfully lower the incidence of metastasis through blocking VEGF-C induced lymphangiogenesis. Based on these results, we propose LHbisD4 as a potent anti-cancer drug that can reduce the incidence of metastasis by suppressing lymphangiogenesis through blocking VEGF-C signaling pathway.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Heparina de Baixo Peso Molecular/química , Linfangiogênese/efeitos dos fármacos , Fator C de Crescimento do Endotélio Vascular/antagonistas & inibidores , Inibidores da Angiogênese/química , Inibidores da Angiogênese/farmacologia , Animais , Movimento Celular , Proliferação de Células , Ácido Desoxicólico/química , Células Endoteliais/efeitos dos fármacos , Feminino , Humanos , Linfonodos/efeitos dos fármacos , Linfonodos/patologia , Metástase Linfática , Vasos Linfáticos/efeitos dos fármacos , Vasos Linfáticos/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Neovascularização Patológica/tratamento farmacológicoRESUMO
The Janus kinase-signal transducers and activators of transcription signaling pathway (JAK/STAT pathway) have displayed a critical role in tumor development and progression in multiple malignancies. Previous studies showed that inhibition of JAK/STAT signaling blocked cell growth and metastasis in cancer cells, however, the antitumor effects of JAK inhibitor AG490 on gallbladder cancer (GBC) have not been reported. Our present study aimed to investigate the effects and associated mechanisms of JAK inhibitor AG490 on cell growth, invasive potential and apoptosis in GBC cells (GBC-SD and SGC-996) indicated by MTT, cell colony formation, Transwell and flow cytometry. As a consequence, we found that JAK2 inhibitor AG490 inhibited cell growth and invasion, and induced cell apoptosis and cycle arrest in GBC-SD and SGC-996 cells. Furthermore, the expression levels of p-JAK2, p-STAT3, VEGFC-/-D and cyclinD1 were downregulated, while p53 expression was upregulated in AG490-treated GBC cells indicated by Western blot assay. Therefore, our findings demonstrate that JAK inhibitor AG490 inhibits growth and invasion of GBC cells via blockade of JAK2/STAT3 signaling and provides the potential therapeutic strategy for the treatment of GBC patients.
Assuntos
Antineoplásicos/farmacologia , Células Epiteliais/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Janus Quinase 2/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Fator de Transcrição STAT3/antagonistas & inibidores , Tirfostinas/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ciclina D1/antagonistas & inibidores , Ciclina D1/genética , Ciclina D1/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Vesícula Biliar/efeitos dos fármacos , Vesícula Biliar/metabolismo , Vesícula Biliar/patologia , Humanos , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/agonistas , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Fator C de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator C de Crescimento do Endotélio Vascular/genética , Fator C de Crescimento do Endotélio Vascular/metabolismo , Fator D de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator D de Crescimento do Endotélio Vascular/genética , Fator D de Crescimento do Endotélio Vascular/metabolismoRESUMO
Secondary lymphedema is still a worldwide problem. Symptomatic approaches to lymphedema therapy have been mainly used, with complete decongestive therapy as the cornerstone. Due to a lack of regenerative therapy, researchers have established various animal models to obtain insights into pathomechanisms and to reveal the best therapeutic option. Since the first reproducible and reliable animal model of lymphedema was reported in dogs, the technique of circumferential excision of lymphatic tissue has been translated mainly to rodents to induce secondary lymphedema. In these models, various promising pharmacological and surgical approaches have been investigated to improve secondary lymphedema therapy. Imaging modalities are crucial to detect the extent of lymphatic dysfunction and decide the best therapy. The gold standard of lymphoscintigraphy is currently limited by poor spatial resolution and lack of quantification. Animal models could help to bridge a gap in improving morphological correlation and quantifying lymphatic functionality. This review summarizes the animal models used in lymphatic research and focuses on new therapeutic options and requirements for imaging modalities to visualize the lymphatic system.
Assuntos
Linfedema/diagnóstico , Linfedema/terapia , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Anti-Inflamatórios não Esteroides/uso terapêutico , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Neoplasias da Mama/complicações , Neoplasias da Mama/cirurgia , Terapia Combinada , Citocinas/farmacologia , Citocinas/uso terapêutico , Modelos Animais de Doenças , Feminino , Haplorrinos , Humanos , Hialuronoglucosaminidase/farmacologia , Hialuronoglucosaminidase/uso terapêutico , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/uso terapêutico , Vasos Linfáticos/efeitos dos fármacos , Vasos Linfáticos/metabolismo , Vasos Linfáticos/patologia , Linfedema/etiologia , Linfografia/métodos , Linfocintigrafia , Terapia de Alvo Molecular , Coelhos , Roedores , Ovinos , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Procedimentos Cirúrgicos Operatórios , Suínos , Fator C de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator C de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND AND PURPOSE: Lymphangiogenesis is an important biological process associated with the pathogenesis of several diseases, including metastatic dissemination, graft rejection, lymphoedema and other inflammatory disorders. The development of new drugs that block lymphangiogenesis has become a promising therapeutic strategy. In this study, we investigated the ability of toluquinol, a 2-methyl-hydroquinone isolated from the culture broth of the marine fungus Penicillium sp. HL-85-ALS5-R004, to inhibit lymphangiogenesis in vitro, ex vivo and in vivo. EXPERIMENTAL APPROACH: We used human lymphatic endothelial cells (LECs) to analyse the effect of toluquinol in 2D and 3D in vitro cultures and in the ex vivo mouse lymphatic ring assay. For in vivo approaches, the transgenic Fli1:eGFPy1 zebrafish, mouse ear sponges and cornea models were used. Western blotting and apoptosis analyses were carried out to search for drug targets. KEY RESULTS: Toluquinol inhibited LEC proliferation, migration, tubulogenesis and sprouting of new lymphatic vessels. Furthermore, toluquinol induced apoptosis of LECs after 14 h of treatment in vitro, blocked the development of the thoracic duct in zebrafish and reduced the VEGF-C-induced lymphatic vessel formation and corneal neovascularization in mice. Mechanistically, we demonstrated that this drug attenuates VEGF-C-induced VEGFR-3 phosphorylation in a dose-dependent manner and suppresses the phosphorylation of Akt and ERK1/2. CONCLUSIONS AND IMPLICATIONS: Based on these findings, we propose toluquinol as a new candidate with pharmacological potential for the treatment of lymphangiogenesis-related pathologies. Notably, its ability to suppress corneal neovascularization paves the way for applications in vascular ocular pathologies.
Assuntos
Células Endoteliais/efeitos dos fármacos , Hidroquinonas/farmacologia , Linfangiogênese/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fator C de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Relação Estrutura-Atividade , Peixe-ZebraRESUMO
Allicin, the most abundant organosulfur compound in freshly crushed garlic tissues, has been shown to have various health-promoting effects, including anticancer actions. A better understanding of the effects and mechanisms of allicin on tumorigenesis could facilitate development of allicin or garlic products for cancer prevention. Here we found that allicin inhibited lymphangiogenesis, which is a critical cellular process implicated in tumor metastasis. In primary human lymphatic endothelial cells, allicin at 10 µM inhibited capillary-like tube formation and cell migration, and it suppressed phosphorylation of vascular endothelial growth factor receptor 2 and focal adhesion kinase. Using a Matrigel plug assay in mice, addition of 10 µg allicin in Matrigel plug inhibited 40-50% of vascular endothelial growth factor-C-induced infiltration of lymphatic endothelial cells and leukocytes. S-Allylmercaptoglutathione, a major cellular metabolite of allicin, had no effect on lymphangiogenic responses in lymphatic endothelial cells. Together, these results demonstrate the antilymphangiogenic effect of allicin in vitro and in vivo, suggesting a novel mechanism for the health-promoting effects of garlic compounds.
Assuntos
Linfangiogênese/efeitos dos fármacos , Ácidos Sulfínicos/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Células Cultivadas , Dissulfetos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais , Fator C de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator C de Crescimento do Endotélio Vascular/fisiologiaRESUMO
The high mobility group box 1 (HMGB1), which is a highly conserved and evolutionarily non-histone nuclear protein, has been shown to associate with a variety of biological important processes, such as transcription, DNA repair, differentiation, and extracellular signalling. High HMGB1 expression has been reported in many cancers, such as prostate, kidney, ovarian, and gastric cancer. However, there have been few studies of the function of HMGB1 in the malignant biological behaviour of bladder urothelial carcinoma (BUC), and the potential mechanism of HMGB1 in the pathogenesis of BUC remains unclear. Thus, in this study, we constructed plasmid vectors that are capable of synthesizing specific shRNAs targeting HMGB1 and transfected them into BUC cells to persistently suppress the endogenous gene expression of HMGB1. The expression of HMGB1, the bioactivity of BUC cells, including proliferation, apoptosis, cell cycle distribution, migration and invasion, and the effects of HMGB1 knockdown on downstream signalling pathways were investigated. Our data suggest that HMGB1 promotes the malignant biological behaviour of BUC, and that this effect may be partially mediated by the NF-κB signalling pathway. HMGB1 may serve as a potential therapeutic target for BUC in the future.
Assuntos
Carcinoma de Células de Transição/genética , Regulação Neoplásica da Expressão Gênica , Proteína HMGB1/genética , NF-kappa B/genética , Neoplasias da Bexiga Urinária/genética , Apoptose , Carcinoma de Células de Transição/metabolismo , Carcinoma de Células de Transição/patologia , Ciclo Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Feminino , Proteína HMGB1/antagonistas & inibidores , Proteína HMGB1/metabolismo , Humanos , Proteínas I-kappa B/antagonistas & inibidores , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Masculino , Inibidor de NF-kappaB alfa , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Invasividade Neoplásica , Cultura Primária de Células , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Microambiente Tumoral/genética , Bexiga Urinária/metabolismo , Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologia , Fator C de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator C de Crescimento do Endotélio Vascular/genética , Fator C de Crescimento do Endotélio Vascular/metabolismoRESUMO
MicroRNAs (miRs) have important roles in the pathogenesis of human malignancy. It has previously been suggested that deregulation of miR101 is associated with the progression of intrahepatic cholangiocarcinoma (ICC); however, the exact role of miR101 in the regulation of ICC metastasis remains largely unknown. The present study demonstrated that the expression levels of miR101 were significantly decreased in ICC tissue, as compared with matched adjacent normal tissue. Furthermore, miR101 was downregulated in the ICC9810 human ICC cell line, as compared with in the normal human intrahepatic biliary epithelial cell (HIBEC) line. Vascular endothelial growth factor (VEGF)C was identified as a target gene of miR101 in ICC9810 cells. The expression of VEGFC was negatively regulated by miR101 at the posttranscriptional level in ICC9810 cells. Further investigation demonstrated that overexpression of miR101 markedly suppressed the migration and invasion of ICC9810 cells, and these effects were similar to those observed following VEGFC knockdown. Conversely, restoration of VEGFC reversed the inhibitory effects of miR101 overexpression on ICC9810 cell migration and invasion, thus suggesting that miR101 may suppress ICC9810 cell migration and invasion, at least partly via inhibition of VEGFC. It was also demonstrated that the mRNA and protein expression levels of VEGFC were frequently upregulated in ICC tissue and cells, and its expression level was inversely correlated with that of miR101 in ICC tissue. In conclusion, the present study identified important roles for miR101 and VEGFC in ICC, suggesting that miR101/VEGFC signaling may be a promising diagnostic and/or therapeutic target for ICC.
Assuntos
Neoplasias dos Ductos Biliares/patologia , Colangiocarcinoma/patologia , MicroRNAs/metabolismo , Fator C de Crescimento do Endotélio Vascular/genética , Fator C de Crescimento do Endotélio Vascular/metabolismo , Regiões 3' não Traduzidas , Adulto , Sequência de Bases , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Colangiocarcinoma/genética , Colangiocarcinoma/metabolismo , Regulação para Baixo , Feminino , Humanos , Masculino , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Pessoa de Meia-Idade , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Interferência de RNA , Alinhamento de Sequência , Transdução de Sinais , Regulação para Cima , Fator C de Crescimento do Endotélio Vascular/antagonistas & inibidoresRESUMO
PURPOSE: To explore the inhibitory effects of antivascular endothelial growth factor C (VEGF-C) therapy on corneal lymphangiogenesis and allograft rejection in rats. METHODS: Fischer 344 rat corneas were transplanted into Lewis rat eyes. After corneal transplantation, Lewis rats (the recipients) were randomly and equally divided into 2 groups: anti-VEGF-C treatment (group A) and control (group B). Corneal hemangiogenesis and lymphangiogenesis were characterized using whole-mount immunofluorescence, and the immune rejection of the grafts was examined using a slit lamp and evaluated by scoring the rejection index (RI). In addition, the expression of VEGF-C was examined by immunohistochemistry and real-time polymerase chain reaction. The association of corneal lymphangiogenesis and hemangiogenesis with VEGF-C in transplanted corneas was also characterized. RESULTS: VEGF-C expression was markedly downregulated after anti-VEGF-C therapy. The outgrowth of corneal lymphangiogenesis dramatically decreased in group A. There was a significant relationship between VEGF-C reduction and the decrease in the lymphatic vessel area (r=0.55, P<0.05), whereas the relationship between the reduction of VEGF-C and the decrease in blood vessel area was not significant (r=0.11, P>0.05). In addition, the RI scores were significantly lower in group A compared with group B at 7, 10, and 14 days after transplantation. The graft survival time in group A rats (20.33±1.37 days) was significantly longer than that in group B rats (12.83±1.47 days; P<0.05). CONCLUSIONS: The results suggested that VEGF-C blockade had a significant role in preventing corneal lymphangiogenesis in corneal beds, which resulted in higher allograft survival rates.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Transplante de Córnea/métodos , Linfangiogênese , Fator C de Crescimento do Endotélio Vascular/antagonistas & inibidores , Aloenxertos , Animais , Córnea/metabolismo , Regulação para Baixo , Imunofluorescência , Sobrevivência de Enxerto , Masculino , Ratos , Ratos Endogâmicos Lew , Reação em Cadeia da Polimerase em Tempo Real , Lâmpada de Fenda , Fatores de Tempo , Fator C de Crescimento do Endotélio Vascular/genéticaRESUMO
PURPOSE: The contribution of lymphangiogenesis (LA) to allergy has received considerable attention and therapeutic inhibition of this process via targeting VEGF has been considered. Likewise, certain inflammatory settings affecting the ocular mucosa can trigger pathogenic LA in the naturally avascular cornea. Chronic inflammation in allergic eye disease (AED) impacts the conjunctiva and cornea, leading to sight threatening conditions. However, whether corneal LA is involved is completely unknown. We addressed this using a validated mouse model of AED. METHODS: Allergic eye disease was induced by ovalbumin (OVA) immunization and chronic OVA exposure. Confocal microscopy of LYVE-1-stained cornea allowed evaluation of corneal LA, and qRT-PCR was used to evaluate expression of VEGF-C, -D, and -R3 in these mice. Administration of VEGF receptor (R) inhibitor was incorporated to inhibit corneal LA in AED. Immune responses were evaluated by in vitro OVA recall responses of T cells, and IgE levels in the serum. RESULTS: Confocal microscopy of LYVE-1-stained cornea revealed the distinct presence of corneal LA in AED, and corroborated by increased corneal expression of VEGF-C, -D, and -R3. Importantly, prevention of corneal LA in AED via VEGFR inhibition was associated with decreased T helper two responses and IgE production. Furthermore, VEGFR inhibition led a significant reduction in clinical signs of AED. CONCLUSIONS: Collectively, these data reveal that there is a distinct involvement of corneal LA in AED. Furthermore, VEGFR inhibition prevents corneal LA and consequent immune responses in AED.
Assuntos
Córnea/fisiologia , Modelos Animais de Doenças , Ceratoconjuntivite/fisiopatologia , Linfangiogênese/fisiologia , Vasos Linfáticos/fisiopatologia , Animais , Axitinibe , Feminino , Glicoproteínas/metabolismo , Imidazóis/farmacologia , Imunoglobulina E/sangue , Indazóis/farmacologia , Ceratoconjuntivite/metabolismo , Ceratoconjuntivite/patologia , Proteínas de Membrana Transportadoras , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Ovalbumina/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Fator C de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator C de Crescimento do Endotélio Vascular/metabolismo , Fator D de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator D de Crescimento do Endotélio Vascular/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
The aim of the current study was to elucidate the association between vascular endothelial growth factor C (VEGF-C) and resistance of bladder cancer cells to cisplatin and the underlying mechanism involving maspin. A total of 32 bladder cancer tissue samples from patients (18 males and 14 females with an average age of 65.9 years) were collected from the Fifth Affiliated Hospital of Zhengzhou University (Zhengzhou, China). All patients had undergone cisplatin-based combination chemotherapy. In addition, the BIU87 human bladder cancer cell line was cultured and a cisplatin-resistant subline (BIU87-CisR) was established by continuous exposure to cisplatin. The mRNA expression levels of VEGF-C and maspin in tissue samples, BIU87 cells and BIU87-CisR cells were analyzed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Targeted inhibition of VEGF-C in BIU87-CisR cells was performed using small interfering (si)RNA technology and the alteration in levels of maspin was confirmed by RT-qPCR and western blot analysis. siRNA-treated and -untreated BIU87-CisR cells were divided into the following four groups: Control group (no drug treatment), 3 µM cisplatin treated group, 3 µM cisplatin + siRNA treated group and the siRNA treated group. Cell viability following treatment in each group was evaluated by the cell counting kit 8 assay. The cell cycle and apoptotic rate of BIU87-CisR cells was analyzed by propidium iodide (PI) staining and Annexin V-PI double staining with flow cytometry. Furthermore, pcDNA-maspin transfected BIU78-CisR cells were used to establish the effect of maspin on the sensitivity to cisplatin. VEGF-C expression in chemoresistant patients and BIU87-CisR cells was significantly increased compared with chemosensitive patients and normal BIU87 cells, respectively. By contrast, maspin levels were lower in chemoresistant patients and BIU87-CisR cells. Subsequent to VEGF-C inhibition, maspin expression was markedly increased. Cisplatin (3 µM) resulted in moderate proliferation inhibition of BIU87-CisR cells without siRNA pretreatment; however, significant inhibition was observed in the VEGF-C siRNA treated group. In addition, the cell cycle arrest and apoptosis induced by cisplatin was enhanced by VEGF-C inhibition. Overexpression of maspin was able to improve the sensitivity of BIU87-CisR cells to cisplatin. In conclusion, the resistance of bladder cancer cells to cisplatin may be induced by upregulation of VEGF-C, and inhibition of VEGF-C reverses resistance by elevating maspin expression levels.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Carcinoma/tratamento farmacológico , Cisplatino/farmacologia , Regulação Neoplásica da Expressão Gênica , Serpinas/agonistas , Neoplasias da Bexiga Urinária/tratamento farmacológico , Fator C de Crescimento do Endotélio Vascular/antagonistas & inibidores , Idoso , Apoptose/efeitos dos fármacos , Carcinoma/genética , Carcinoma/patologia , Carcinoma/cirurgia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Serpinas/genética , Serpinas/metabolismo , Transdução de Sinais , Bexiga Urinária/efeitos dos fármacos , Bexiga Urinária/metabolismo , Bexiga Urinária/patologia , Bexiga Urinária/cirurgia , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/cirurgia , Fator C de Crescimento do Endotélio Vascular/genética , Fator C de Crescimento do Endotélio Vascular/metabolismoRESUMO
RATIONALE: Janus kinase/signal transducer and activator of transcription (JAK/STAT) signals and their endogenous inhibitor, suppressor of cytokine signaling 3 (SOCS3), in vascular endothelial cells (ECs) reportedly dominate the pathological angiogenesis. However, how these inflammatory signals are potentiated during pathological angiogenesis has not been fully elucidated. We suspected that an intracellular protease calpain, which composes the multifunctional proteolytic systems together with its endogenous inhibitor calpastatin (CAST), contributes to the JAK/STAT regulations. OBJECTIVE: To specify the effect of EC calpain/CAST systems on JAK/STAT signals and their relationship with pathological angiogenesis. METHODS AND RESULTS: The loss of CAST, which is ensured by several growth factor classes, was detectable in neovessels in murine allograft tumors, some human malignant tissues, and oxygen-induced retinopathy lesions in mice. EC-specific transgenic introduction of CAST caused downregulation of JAK/STAT signals, upregulation of SOCS3 expression, and depletion of vascular endothelial growth factor (VEGF)-C, thereby counteracting unstable pathological neovessels and disease progression in tumors and oxygen-induced retinopathy lesions in mice. Neutralizing antibody against VEGF-C ameliorated pathological angiogenesis in oxygen-induced retinopathy lesions. Small interfering RNA-based silencing of endogenous CAST in cultured ECs facilitated µ-calpain-induced proteolytic degradation of SOCS3, leading to VEGF-C production through amplified interleukin-6-driven STAT3 signals. Interleukin-6-induced angiogenic tube formation in cultured ECs was accelerated by CAST silencing, which is suppressible by pharmacological inhibition of JAK/STAT signals, antibody-based blockage of VEGF-C, and transfection of calpain-resistant SOCS3, whereas transfection of wild-type SOCS3 exhibited modest angiostatic effects. CONCLUSIONS: Loss of CAST in angiogenic ECs facilitates µ-calpain-induced SOCS3 degradation, which amplifies pathological angiogenesis through interleukin-6/STAT3/VEGF-C axis.
Assuntos
Proteínas de Ligação ao Cálcio/fisiologia , Calpaína/metabolismo , Células Endoteliais/metabolismo , Neoplasias/irrigação sanguínea , Proteínas Supressoras da Sinalização de Citocina/antagonistas & inibidores , Adenocarcinoma/irrigação sanguínea , Sequência de Aminoácidos , Animais , Aorta , Proteínas de Ligação ao Cálcio/genética , Carcinoma Pulmonar de Lewis/irrigação sanguínea , Células Cultivadas , Citocinas/fisiologia , Feminino , Glioblastoma/irrigação sanguínea , Humanos , Janus Quinases/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Neovascularização Patológica/fisiopatologia , Proteínas Recombinantes de Fusão/metabolismo , Retinopatia da Prematuridade/fisiopatologia , Fatores de Transcrição STAT/fisiologia , Transdução de Sinais/fisiologia , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/genética , Proteínas Supressoras da Sinalização de Citocina/fisiologia , Fator C de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator C de Crescimento do Endotélio Vascular/fisiologiaRESUMO
BACKGROUND: Graft failure because of immune rejection remains a significant problem in organ transplantation, and lymphatic and blood vessels are important components of the afferent and efferent arms of the host alloimmune response, respectively. We compare the effect of antihemangiogenic and antilymphangiogenic therapies on alloimmunity and graft survival in a murine model of high-risk corneal transplantation. METHODS: Orthotopic corneal transplantation was performed in hemevascularized and lymph-vascularized high-risk host beds, and graft recipients received subconjunctival vascular endothelial growth factor (VEGF)-trap, anti-VEGF-C, sVEGFR-3, or no treatment, beginning at the time of surgery. Fourteen days after transplantation, graft hemeangiogenesis and lymphangiogenesis were evaluated by immunohistochemistry. The frequencies of Th1 cells in regional lymphoid tissue and graft-infiltrating immune cells were evaluated by flow cytometry. Long-term allograft survival was compared using Kaplan-Meier curves. RESULTS: VEGF-trap significantly decreased graft hemangiogenesis as compared to the control group and was most effective in reducing the frequency of graft-infiltrating immune cells. Anti-VEGF-C and sVEGFR3 significantly decreased graft lymphangiogenesis and lymphoid Th1 cell frequencies as compared to control. VEGF-trap (72%), anti-VEGF-C (25%), and sVEGFR-3 (11%) all significantly improved in the 8-week graft survival compared to control (0%), although VEGF-trap was significantly more effective than both anti-VEGF-C (P < 0.05) and sVEGFR-3 (P < 0.05). CONCLUSION: In a clinically relevant model of high-risk corneal transplantation in which blood and lymphatic vessels are present and treatment begins at the time of transplantation, VEGF-trap is significantly more effective in improving long-term graft survival as compared to anti-VEGF-C and sVEGFR-3, but all approaches improve survival when compared to untreated control.
Assuntos
Inibidores da Angiogênese/farmacologia , Transplante de Córnea/efeitos adversos , Rejeição de Enxerto/prevenção & controle , Sobrevivência de Enxerto/efeitos dos fármacos , Receptores de Fatores de Crescimento do Endotélio Vascular/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/metabolismo , Rejeição de Enxerto/fisiopatologia , Linfangiogênese/efeitos dos fármacos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Modelos Animais , Neovascularização Fisiológica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Células Th1/metabolismo , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator C de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator C de Crescimento do Endotélio Vascular/imunologia , Fator C de Crescimento do Endotélio Vascular/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/farmacologiaRESUMO
BACKGROUND: Oral squamous cell carcinoma (OSCC) remains as one of the most difficult malignancies to control because of its high propensity for local invasion and cervical lymph node dissemination. The aim of present study was to evaluate the efficacy of novel pH and temperature sensitive doxorubicin-methotrexate- loaded nanoparticles (DOX-MTX NP) in terms of their potential to change the VEGF-C expression profile in a rat OSCC model. MATERIALS AND METHODS: 120 male rats were divided into 8 groups of 15 animals administrated with 4-nitroquinoline-1-oxide to induce OSCCs. Newly formulated doxorubicin-methotrexate-loaded nanoparticles (DOX-MTX NP) and free doxorubicin were IV and orally administered. RESULTS: RESULTS indicated that both oral and IV forms of DOX-MTX- nanoparticle complexes caused significant decrease in the mRNA level of VEGF-C compared to untreated cancerous rats (p<0.05) . Surprisingly, the VEGF-C mRNA was not affected by free DOX in both IV and oral modalities (p>0.05). Furthermore, in DOX-MTX NP treated group, less tumors characterized with advanced stage and VEGF-C mRNA level paralleled with improved clinical outcome (p<0.05). In addition, compared to untreated healthy rats , the VEGF-C expression was not affected in healthy groups that were treated with IV and oral dosages of nanodrug (p>0.05). CONCLUSIONS: VEGF-C is one of the main prognosticators for lymph node metastasis in OSCC. Down-regulation of this lymph-angiogenesis promoting factor is a new feature acquired in group treated with dual action DOX-MTX-NPs. Beside the synergic apoptotic properties of concomitant use of DOX and MTX on OSCC, DOX-MTX NPs possessed anti-angiogenesis properties which was related to the improved clinical outcome in treated rats. Taking together, we conclude that our multifunctional doxorubicin-methotrexate complex exerts specific potent apoptotic and anti-angiogenesis properties that could ameliorate the clinical outcome presumably via down-regulating dissemination factor-VEGF-C expression in a rat OSCC model.