RESUMO
ABSTRACT Background: Cancer gene therapy using a nonviral vector is expected to be repeatable, safe, and inexpensive, and to have long-term effectiveness. Gene therapy using the E3 and C1 (E3C1) domain of developmental endothelial locus-1 (Del1) has been shown to improve prognosis in a mouse transplanted tumor model. Objective: In this study, we examined how this treatment affects angiogenesis in mouse transplanted tumors. Materials and methods: Mouse transplanted tumors (SCCKN human squamous carcinoma cell line) were injected locally with a nonviral plasmid vector encoding E3C1 weekly. Histochemical analysis of the transplanted tumors was then performed to assess the effects of E3C1 on prognosis. Results: All mice in the control group had died or reached an endpoint within 39 days. In contrast, one of ten mice in the E3C1 group had died by day 39, and eight of ten had died or reached an endpoint by day 120 (p < 0.01). Enhanced apoptosis in tumor stroma was seen on histochemical analyses, as was inhibited tumor angiogenesis in E3C1-treated mice. In addition, western blot analysis showed decreases in active Notch and HEY1 proteins. Conclusion: These findings indicate that cancer gene therapy using a nonviral vector encoding E3C1 significantly improved life-span by inhibiting tumor angiogenesis. (REV INVEST CLIN. 2021;73(1):39-51)
Assuntos
Animais , Coelhos , Proteínas de Ligação ao Cálcio/uso terapêutico , Carcinoma de Células Escamosas/irrigação sanguínea , Carcinoma de Células Escamosas/terapia , Moléculas de Adesão Celular/uso terapêutico , Fator de Crescimento Epidérmico/uso terapêutico , Domínio Discoidina/genética , Proteínas de Ligação ao Cálcio/genética , Células Tumorais Cultivadas , Terapia Genética , Moléculas de Adesão Celular/genética , Motivos de Aminoácidos , Fator de Crescimento Epidérmico/genética , Camundongos Nus , Transplante de Neoplasias , Neovascularização Patológica/terapiaRESUMO
BACKGROUND: Cancer gene therapy using a nonviral vector is expected to be repeatable, safe, and inexpensive, and to have longterm effectiveness. Gene therapy using the E3 and C1 (E3C1) domain of developmental endothelial locus-1 (Del1) has been shown to improve prognosis in a mouse transplanted tumor model. OBJECTIVE: In this study, we examined how this treatment affects angiogenesis in mouse transplanted tumors. MATERIALS AND METHODS: Mouse transplanted tumors (SCCKN human squamous carcinoma cell line) were injected locally with a nonviral plasmid vector encoding E3C1 weekly. Histochemical analysis of the transplanted tumors was then performed to assess the effects of E3C1 on prognosis. RESULTS: All mice in the control group had died or reached an endpoint within 39 days. In contrast, one of ten mice in the E3C1 group had died by day 39, and eight of ten had died or reached an endpoint by day 120 (p < 0.01). Enhanced apoptosis in tumor stroma was seen on histochemical analyses, as was inhibited tumor angiogenesis in E3C1-treated mice. In addition, western blot analysis showed decreases in active Notch and HEY1 proteins. CONCLUSION: These findings indicate that cancer gene therapy using a nonviral vector encoding E3C1 significantly improved life-span by inhibiting tumor angiogenesis.
Assuntos
Proteínas de Ligação ao Cálcio/uso terapêutico , Carcinoma de Células Escamosas/irrigação sanguínea , Carcinoma de Células Escamosas/terapia , Moléculas de Adesão Celular/uso terapêutico , Domínio Discoidina , Fator de Crescimento Epidérmico/uso terapêutico , Terapia Genética , Neovascularização Patológica/terapia , Motivos de Aminoácidos , Animais , Proteínas de Ligação ao Cálcio/genética , Moléculas de Adesão Celular/genética , Domínio Discoidina/genética , Fator de Crescimento Epidérmico/genética , Camundongos , Camundongos Nus , Transplante de Neoplasias , Células Tumorais CultivadasRESUMO
BACKGROUND: The objectives of the present study were to evaluate angiogenesis and mast cell density in oral epithelial dysplasia and oral squamous cell carcinoma (OSCC). MATERIALS AND METHODS: This was an observational, retrospective and quantitative study. The samples consisted of 60 tissue specimens from patients with squamous cell carcinoma, epithelial dysplasia and controls (n=20/group). Immunohistochemistry was performed using an anti-tryptase antibody to mast cells and anti-CD31 and anti-CD34 for blood vessels and we count the number of mast cells and determine the percentage of CD31 and CD34 antibody staining (vascular density). RESULTS: The mast cells had lower density in OSCC compared to control and dysplasia (p = 0.009). In angiogenesis, the expression of CD31 showed a higher percentage of blood vessels in OSCC (p < 0.001), however, CD34 showed no difference between groups (p=0.092). The CD31 antibody presented as a high immunostaining in oral mucosa than CD34. CONCLUSIONS: The increased vascularity in squamous cell carcinoma suggests that angiogenesis begins when malignant transformation starts that seems to be inversely associated with the number of mast cells.
Assuntos
Biomarcadores Tumorais/análise , Vasos Sanguíneos/patologia , Carcinogênese/patologia , Carcinoma de Células Escamosas/complicações , Mastócitos/patologia , Neoplasias Bucais/complicações , Neovascularização Patológica/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinogênese/imunologia , Carcinoma de Células Escamosas/irrigação sanguínea , Carcinoma de Células Escamosas/imunologia , Carcinoma de Células Escamosas/patologia , Estudos de Casos e Controles , Feminino , Seguimentos , Humanos , Masculino , Mastócitos/imunologia , Pessoa de Meia-Idade , Neoplasias Bucais/irrigação sanguínea , Neoplasias Bucais/imunologia , Neoplasias Bucais/patologia , Neovascularização Patológica/etiologia , Prognóstico , Estudos RetrospectivosRESUMO
Recently, exosomes secreted by menstrual mesenchymal stem cells have been identified as inhibitory agents of tumor angiogenesis and modulators of the tumor cell secretome in prostate and breast cancer. However, their direct effect on endothelial cells and paracrine mediators have not yet been investigated. Using a carrier-based cell culture system to test the scalability for exosome production, we showed that different types of endothelial cells present specific kinetics for exosomes internalization. Exosome-treatment of endothelial cells increased cytotoxicity and reduced VEGF secretion and angiogenesis in a dose-dependent manner. Using the hamster buccal pouch carcinoma as a preclinical model for human oral squamous cell carcinoma, we demonstrated a significant antitumor effect of intra-tumoral injection of exosomes associated with a loss of tumor vasculature. These results address up-scaling of exosome production, a relevant issue for their clinical application, and also assess menstrual stem cell exosomes as potential anti-angiogenic agents for the treatment of neoplastic conditions.
Assuntos
Carcinoma de Células Escamosas/irrigação sanguínea , Carcinoma de Células Escamosas/patologia , Exossomos/metabolismo , Neoplasias Bucais/irrigação sanguínea , Neoplasias Bucais/patologia , Neovascularização Patológica , Células-Tronco/citologia , Animais , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Cricetinae , Células Endoteliais/patologia , Feminino , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND: Tumor-associated neutrophils (TAN), matrix metalloproteinase-9 (MMP-9), interleukin-17 (IL-17), and angiogenesis have been proposed as prognostic biomarkers of malignant tumors. The purpose of this study was to investigate these inflammatory markers as prognostic factors for oral squamous cell carcinoma (OSCC). METHODS: Specimens of OSCC (n = 30), healthy oral mucosa (negative control, n = 10), oral leukoplakia (n = 10), and apical granuloma with abscess (positive inflammatory controls, n = 10) were immunostained for CD66b (neutrophils), MMP-9, IL-17, and CD105 (neoformed microvessels). Semiquantitative (IL-17) and quantitative (CD66b, IL-17, MMP-9, and CD105) analyses were performed. Clinical information (TNM stage, metastasis, recurrence, and survival) and tumor histological grade were also obtained. RESULTS: Positivity for TAN, MMP-9, IL-17, and CD105 was higher in OSCC than in the negative control (P < 0.05) and oral leukoplakia, but similar to the positive inflammatory control. Coincident high counts of inflammatory markers (CD66b, MMP-9, IL-17, and CD105) were associated with lymph node metastasis of OSCC. Associations between high numbers of neoformed microvessels and advanced clinical stage and a higher degree of malignancy were also demonstrated. CONCLUSIONS: Combined positivity for TAN, MMP-9, IL-17, and CD105 appears to be associated with the metastasis-prone phenotype of OSCC.
Assuntos
Biomarcadores Tumorais/análise , Carcinoma de Células Escamosas/diagnóstico , Imuno-Histoquímica , Interleucina-17/análise , Metaloproteinase 9 da Matriz/análise , Neoplasias Bucais/diagnóstico , Neovascularização Patológica , Neutrófilos/patologia , Adulto , Antígenos CD/análise , Carcinoma de Células Escamosas/sangue , Carcinoma de Células Escamosas/irrigação sanguínea , Moléculas de Adesão Celular/análise , Endoglina/análise , Feminino , Proteínas Ligadas por GPI/análise , Humanos , Imuno-Histoquímica/métodos , Masculino , Pessoa de Meia-Idade , Neoplasias Bucais/irrigação sanguínea , PrognósticoRESUMO
This study aims to examine the effects of a new 1,4-dihydropyridine derivative, VdiE-2N, on cell signaling pathways and mitochondrial events in head and neck squamous cell carcinoma (HNSCC) cells, and on a mice model of xenograft tumor growth/cell proliferation. Four HNSCC cell lines (HN13, HN12, HN6, and CAL27), HEK293 cells (human embryonic kidney 293 cells), and human oral healthy mucosa fibroblasts (OHMF) were used for in vitro assessment of cell viability (resazurin assay) and invasion capacity (modified Boyden chamber assay), and mitochondrial membrane potential (JC-1 fluorescence assay), morphology (transmission electron microscopy), and number of mitochondria (MitoTracker® imaging). SET and pDRP1 proteins were analyzed by immunofluorescence, and proteins involved in cell death/survival pathways were analyzed by Western blotting. HN12 xenograft tumors were established in the flank of Balb/c nude mice, and their characteristics and sensitivity to VdiE-2N were determined by immunohistochemistry and histology. VdiE-2N decreased cell viability in HNSCC cells (IC50 = 9.56 and 22.45µM for HN13 and HN12 cells, respectively) more strongly than it decreased cell viability in OHMF and HEK293 cells (IC50 = 32.90 and > 50µM, respectively). In HN13 cells, VdiE-2N dissipated mitochondrial membrane potential and altered the mitochondria size, shape, and number in a concentration-dependent manner, as well as it induced apoptosis and reduced their invasion capacity. Treatment of mice bearing xenograft tumors with VdiE-2N significantly diminished proliferation of cancer cells. Therefore, VdiE-2N induces HNSCC cell death in vitro through mitochondria-mediated apoptotic pathways and dampens tumor growth in vivo, thus supporting a potential anti-cancer effect.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Carcinoma de Células Escamosas/patologia , Di-Hidropiridinas/química , Di-Hidropiridinas/farmacologia , Neoplasias de Cabeça e Pescoço/patologia , Animais , Apoptose/efeitos dos fármacos , Carcinoma de Células Escamosas/irrigação sanguínea , Carcinoma de Células Escamosas/tratamento farmacológico , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes myc/genética , Neoplasias de Cabeça e Pescoço/irrigação sanguínea , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Tamanho Mitocondrial/efeitos dos fármacos , Invasividade Neoplásica , Neovascularização Patológica/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
ABSTRACT INTRODUCTION: Tumors of the lip and oral cavity differ in various aspects; therefore a clarification of the distinctions among these sites may help to better understand the biologic behavior of neoplasms occurring in these locations. OBJECTIVE: Considering that angiogenesis and lymphangiogenesis are two major elements that can influence various aspects of tumor biology, we aimed to compare these factors between squamous cell carcinoma of the lower lip and oral cavity. METHODS: A total of 84 primary squamous cell carcinomas including 45 oral and 39 lower lip tumors were selected and immunohistochemically stained with monoclonal antibody against D2-40 and CD105. Mean microvessel density was assessed in tumoral tissue, while lymphatic vessel density was calculated in both neoplastic tissue and invasion front. Data were statistically analyzed using t-test and p-values of <0.05 were considered significant. RESULTS: We found a mean microvessel density ± standard deviation of 31.94 ± 18.9 in oral cavity and 27.54 ± 20.8 in lower lip squamous cell carcinomas, with no significant difference (p = 0.32). Mean lymphatic vessel density ± standard deviation was 13.05 ± 8.2 and 16.57 ± 10.79 in of oral cavity and lower lip neoplastic tissue, respectively. The corresponding values were 9.94 ± 5.59 and 12.50 ± 7.8 in the invasive front. Significant differences were not observed in either of the lymphatic vessel density variables between the two sites. CONCLUSION: According to our results, it seems that the search for additional factors other than those related to the vasculature should continue, to help clarify the differences in biologic behavior between lower lip and oral cavity squamous cell carcinomas.
Resumo Introdução: Os tumores de lábio e da cavidade oral diferem em vários aspectos; portanto, o conhecimento das diferenças entre eles pode ajudar na melhor compreensão do comportamento biológico das neoplasias que ocorrem nesses locais. Objetivo: Considerando que a angiogênese e a linfangiogênese são dois elementos importantes que podem influenciar diversos aspectos da biologia dos tumores, objetivamos comparar esses fatores entre o carcinoma de células escamosas (CCE) de lábio inferior e da cavidade oral. Método: No total, foram selecionados 84 CCEs primários (45 tumores da cavidade oral e 39 tumores de lábio). Esses tumores foram corados por processo imunohistoquímico com anticorpo monoclonal anti-D2-40 e CD105. Avaliamos a densidade média de microvasos (DMV) no tecido tumoral, enquanto que a densidade vascular linfática (DVL) foi calculada tanto no tecido neoplásico como no front de invasão. Os dados foram estatisticamente analisados com o uso do teste t e valores de p < 0,05 foram considerados significantes. Resultados: Chegamos a uma média para DMV ± DP de 31,94 ± 18,9 para CCEs na cavidade oral e de 27,54 ± 20,8 no lábio inferior, sem diferença significante (p = 0,32). As médias para DVL ± DP foram de 13,05 ± 8,2 e 16,57 ± 10,79 no tecido neoplásico da cavidade oral e lábio inferior, respectivamente. Os valores correspondentes foram 9,94 ± 5,59 e 12,50 ± 7,8 no front invasivo. Não foram observadas diferenças significantes nas duas variáveis DVL entre os dois locais. Conclusão: De acordo com os nossos resultados, a pesquisa por fatores adicionais, além daqueles relacionados à vasculatura, deve ter continuidade, para auxiliar no esclarecimento das diferenças do comportamento biológico entre CCEs no lábio inferior e na cavidade oral.
Assuntos
Humanos , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Idoso , Idoso de 80 Anos ou mais , Adulto Jovem , Neoplasias Labiais/patologia , Neoplasias Bucais/patologia , Carcinoma de Células Escamosas/patologia , Linfangiogênese , Neovascularização Patológica/patologia , Neoplasias Labiais/irrigação sanguínea , Neoplasias Bucais/irrigação sanguínea , Imuno-Histoquímica , Carcinoma de Células Escamosas/irrigação sanguínea , Estudos Retrospectivos , Vasos Linfáticos , Microvasos , Anticorpos Monoclonais Murinos/metabolismoRESUMO
In this study, the existence of vasculogenic mimicry (VM) in cervical squamous cell carcinoma was investigated. To this end, the relationship between hypoxia-inducible factor 1α (HIF-1α) and the development, infiltration, and metastasis of cervical squamous cell carcinoma was studied. Between January 2010 and December 2010, 67 human cervical squamous carcinoma tissue samples were collected and stained by CD34/periodic acid-Schiff double staining to detect the existence of VM. HIF-1α expression was analyzed by immunohistochemistry. The relationship between VM and HIF-1α was also analyzed. Normal cervical tissues (20 cases) from patients who had uterine surgeries in the same period were collected as controls. In the cervical squamous carcinoma tissues, positive rates of VM and HIF-1α were 38.81% (26/67) and 64.18% (43/67), respectively. This was significantly higher than those in the normal cervical tissues [0 (0/20); P < 0.05]. VM rates in cervical squamous carcinoma tissues from patients with different pathological grades, Federation of Gynecology and Obstetrics (FIGO) stages, and lymph node metastasis states were also significantly different (P < 0.05). In addition, significant differences in HIF-1α positivity rates were observed among patients with varying tumor sizes and lymph node metastasis states (P < 0.05). Positive correlation was found between VM and HIF-1α (r = 0.339, P < 0.05). To summarize, we found VM in cervical squamous carcinoma; high expression of HIF-1α may promote VM formation, as well as cervical squamous cell infiltration and metastasis.
Assuntos
Carcinoma de Células Escamosas/irrigação sanguínea , Expressão Gênica , Oxigenases de Função Mista/genética , Neovascularização Patológica , Proteínas Repressoras/genética , Neoplasias do Colo do Útero/irrigação sanguínea , Adulto , Idoso , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/secundário , Feminino , Humanos , Metástase Linfática , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologia , Adulto JovemRESUMO
Precancerous lesions have been studied because of their carcinogenic potential and their association with squamous cell carcinoma (SCC) has been reported. In the tumour microenvironment, the processes of angiogenesis and tissue remodelling are regulated by a family of proteins (Hedgehog) described as being able to modulate epithelial/mesenchymal interactions. The objective of this study was to perform a comparative study of precancerous lesions and SCCs by immunohistochemistry for the presence of Sonic, Gli2, SMO and Patched proteins, members of the Hedgehog pathway. Sixteen cases diagnosed as actinic cheilitis associated with SCC were compared to normal oral mucosa. The sections were subjected to immunohistochemistry and the positively stained cells were counted by morphometric analysis. There was a significant progressive increase in expression of all proteins of the Hedgehog pathway, both in the epithelium and in the connective tissue, when sections of normal mucosa, dysplasia and carcinoma were compared (P < 0.05). Thus, one may suggest that the Hedgehog pathway in tumour transformation influences SCC, and more studies should be conducted to expand the understanding of the role of these proteins in neoplastic transformation.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Proteínas Hedgehog/metabolismo , Carcinoma de Células Escamosas/irrigação sanguínea , Neoplasias de Cabeça e Pescoço/irrigação sanguínea , Proteínas Hedgehog/biossíntese , Humanos , Imuno-Histoquímica , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Projetos Piloto , Transdução de Sinais , Carcinoma de Células Escamosas de Cabeça e PescoçoRESUMO
BACKGROUND: New blood vessel formation events are essential for tumor clonal expansion and progression. Despite the importance of vascular endothelial growth factor A (VEGFA) for tumor angiogenesis, few studies have evaluated the expression profile of this gene in oral squamous cell carcinoma (OSCC) and tumor margins (TM). This study aimed to evaluate the expression of the VEGFA gene and its protein in OSCC and TM. METHODS: Gene expression was evaluated in cryopreserved samples of OSCCs (n = 44), TM (n = 7), and normal mucosa from healthy patients (n = 3; NM). Quantitative PCRs were performed using the TaqMan system for the VEGFA gene, and gene expression was determined using the 2(-∆∆CQ) method. For immunohistochemical evaluation, 27 OSCC samples were embedded in a tissue microarray (TMA) block and reactions were performed using the Advance system. RESULTS: VEGFA transcript levels were 1.7-fold higher in OSCC than in NM samples. VEGFA mRNA was overexpressed in TM samples compared to NM (3.4-fold) and OSCC (2.0-fold) samples. VEGFA transcript level was overexpressed in T3-T4 tumors, metastatic lymph nodes, and stage III-IV OSCCs. Immunoreactivity of the VEGFA protein was detected in the cytoplasm of parenchymal and stromal cells, mainly in endothelial cells and fibroblasts. CONCLUSION: Our results show that VEGFA was overexpressed in aggressive OSCCs and that VEGFA expression may be an important prognostic factor in this type of cancer. Finally, tumor margins may be involved in the production of angiogenic molecules.