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OBJECTIVES: To identify the role and mechanism of a male specific gene, SRY, in I/R-induced hepatic injury. BACKGROUND: Males are more vulnerable to I/R injury than females. However, the mechanism of these sex-based differences remains poorly defined. METHODS: Clinicopathologic data of patients who underwent hepatic resection were identified from an international multi-institutional database. Liver specific SRY TG mice were generated, and subjected to I/R insult with their littermate WT controls in vivo. In vitro experiments were performed by treating primary hepatocytes from TG and WT mice with hypoxia/reoxygen-ation stimulation. RESULTS: Clinical data showed that postoperative aminotransferase level, incidence of overall morbidity and liver failure were markedly higher among 1267 male versus 508 female patients who underwent hepatic resection. SRY was dramatically upregulated during hepatic I/R injury. Overexpression of SRY in male TG mice and ectopic expression of SRY in female TG mice exacerbated liver I/R injury compared with WTs as manifested by increased inflammatory reaction, oxidative stress and cell death in vivo and in vitro. Mechanistically, SRY interacts with Glycogen synthase kinase-3ß (GSK-3ß) and ß-catenin, and promotes phosphorylation and degradation of ß-catenin, leading to suppression of the downstream FOXOs, and activation of NF-κBand TLR4 signaling. Furthermore, activation of ß-catenin almost completely reversed the SRYoverexpression-mediated exacerbation of hepatic I/R damage. CONCLUSIONS: SRY is a novel hepatic I/R mediator that promotes hepatic inflammatory reaction, oxidative stress and cell necrosis via inhibiting Wnt/ß-catenin signaling, which accounts for the sex-based disparity in hepatic I/R injuries.
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Hepatopatias , Traumatismo por Reperfusão , Proteína da Região Y Determinante do Sexo/metabolismo , Animais , Apoptose , Feminino , Glicogênio Sintase Quinase 3 beta/metabolismo , Isquemia , Fígado/patologia , Hepatopatias/metabolismo , Masculino , Camundongos , Caracteres Sexuais , beta CateninaRESUMO
Iatrogenic femoral artery pseudoaneurysm caused by invasive procedures is one of the common complications for endovascular interventions. We present a case of a young male with a complex iatrogenic femoral artery pseudoaneurysm as a result of iatrogenic femoral artery puncture. The defective femoral artery was repaired with combined bovine pericardial tube and autologous great saphenous vein grafts. Computed tomography angiography showed the grafts were still patent one year after the surgery.
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Falso Aneurisma/etiologia , Falso Aneurisma/cirurgia , Procedimentos Endovasculares/efeitos adversos , Artéria Femoral/lesões , Artéria Femoral/cirurgia , Doença Iatrogênica , Complicações Intraoperatórias/etiologia , Complicações Intraoperatórias/cirurgia , Pericárdio/transplante , Veia Safena/transplante , Procedimentos Cirúrgicos Vasculares/métodos , Ferimentos Penetrantes/etiologia , Ferimentos Penetrantes/cirurgia , Adulto , Falso Aneurisma/diagnóstico por imagem , Animais , Bovinos , Angiografia por Tomografia Computadorizada , Procedimentos Endovasculares/métodos , Artéria Femoral/diagnóstico por imagem , Humanos , Masculino , Transplante Autólogo , Transplante Heterólogo , Resultado do TratamentoRESUMO
Iatrogenic femoral artery pseudoaneurysm is a common complication of the endovascular procedures. Manual compression and thrombin injection are the conventional techniques to occlude the pseudoaneurysms. However, there are still some failed cases that applied these treatment options. The aim of the study is to seek a potential and alternative method with ProGlide system to close the pseudoaneurysm. During April 2018 to February 2019, 2 patients with iatrogenic pseudoaneurysm of the superficial femoral were treated with the suture-base closure device--ProGlide. After punctured the pseudoaneurysm and placed a 6-F sheath, the guide wire was placed in the right femoral artery via the access of the pseudoaneurysm neck. Then the pseudoaneurysm neck was sutured by ProGlide to occlude the blood supply to the pseudoaneurysm. These 2 patients were cured with no complications and complaints, which revealed that percutaneous suture technique with ProGlide at the neck level of pseudoaneurysm provides a novel method for the management of vascular access pseudoaneurysm, especially in those with a wide and short neck.
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Falso Aneurisma/cirurgia , Artéria Femoral/cirurgia , Complicações Pós-Operatórias/cirurgia , Técnicas de Sutura , Adulto , Procedimentos Endovasculares , Humanos , Doença Iatrogênica , Masculino , Pessoa de Meia-Idade , Resultado do TratamentoRESUMO
Beneficial effects of metformin on cancer risk and mortality have been proved by epidemiological and clinical studies, thus attracting research interest in elucidating the underlying mechanisms. Recently, tumour-associated macrophages (TAMs) appeared to be implicated in metformin-induced antitumour activities. However, how metformin inhibits TAMs-induced tumour progression remains ill-defined. Here, we report that metformin-induced antitumour and anti-angiogenic activities were not or only partially contributed by its direct inhibition of functions of tumour and endothelial cells. By skewing TAM polarization from M2- to M1-like phenotype, metformin inhibited both tumour growth and angiogenesis. Depletion of TAMs by clodronate liposomes eliminated M2-TAMs-induced angiogenic promotion, while also abrogating M1-TAMs-mediated anti-angiogenesis, thus promoting angiogenesis in tumours from metformin treatment mice. Further in vitro experiments using TAMs-conditioned medium and a coculture system were performed, which demonstrated an inhibitory effect of metformin on endothelial sprouting and tumour cell proliferation promoted by M2-polarized RAW264.7 macrophages. Based on these results, metformin-induced inhibition of tumour growth and angiogenesis is greatly contributed by skewing of TAMs polarization in microenvironment, thus offering therapeutic opportunities for metformin in cancer treatment.
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Substantial data from preclinical studies have revealed the biphasic effects of statins on cardiovascular angiogenesis. Although some have reported the anti-angiogenic potential of statins in malignant tumors, the underlying mechanism remains poorly understood. The aim of this study is to elucidate the mechanism by which simvastatin, a member of the statin family, inhibits tumor angiogenesis. Simvastatin significantly suppressed tumor cell-conditioned medium-induced angiogenic promotion in vitro, and resulted in dose-dependent anti-angiogenesis in vivo. Further genetic silencing of hypoxia-inducible factor-1α (HIF-1α) reduced vascular endothelial growth factor and fibroblast growth factor-2 expressions in 4T1 cells and correspondingly ameliorated HUVEC proliferation facilitated by tumor cell-conditioned medium. Additionally, simvastatin induced angiogenic inhibition through a mechanism of post-transcriptional downregulation of HIF-1α by increasing the phosphorylation level of AMP kinase. These results were further validated by the fact that 5-aminoimidazole-4-carboxamide ribonucleotide reduced HIF-1α protein levels and ameliorated the angiogenic ability of endothelial cells in vitro and in vivo. Critically, inhibition of AMPK phosphorylation by compound C almost completely abrogated simvastatin-induced anti-angiogenesis, which was accompanied by the reduction of protein levels of HIF-1α and its downstream pro-angiogenic factors. These findings reveal the mechanism by which simvastatin induces tumor anti-angiogenesis, and therefore identifies the target that explains the beneficial effects of statins on malignant tumors.
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Proteínas Quinases Ativadas por AMP/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Neovascularização Patológica/prevenção & controle , Sinvastatina/farmacologia , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Proliferação de Células , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Feminino , Fator 2 de Crescimento de Fibroblastos/análise , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Camundongos , Camundongos Endogâmicos BALB C , Fator A de Crescimento do Endotélio Vascular/análiseRESUMO
PURPOSE: To compare outcomes of patients who received simultaneous tributary endovenous laser ablation (EVLA) or foam sclerotherapy (FS) with EVLA of the great saphenous vein (GSV) trunk. METHODS AND MATERIALS: This study recruited 418 patients (542 legs) with diagnosed varicose veins. Patients in the EVLA/FS group (255 patients, 327 legs) received concomitant FS for the tributaries with truncal lasering. For the EVLA-alone group (163 patients, 215 legs), tributaries (8W) were ablated with EVLA in addition to the GSV trunk (14W). Complications, Aberdeen Varicose Vein Questionnaire (AVVQ), EuroQol Group 5-Dimension Self-Report Questionnaire (EQ-5D), numerical rating scale (NRS) scores, and condition of residual varicosities were assessed at 3 days, 4 weeks, and 6 months after procedure. All residual varicosities were identified and treated with a staged FS at 6 months. RESULTS: Except for ecchymosis, incidence of other complications was not significantly different between both groups at 6 months. Pain NRS scores of the EVLA/FS group were remarkably elevated at 4 weeks and then, at 6 months, declined to a level similar to the EVLA-alone group. The EVLA/FS group exhibited more significant improvement in both AVVQ and EQ-5D scales than the EVLA group at 6 months, while exhibiting poor improvement at 4 weeks. The EVLA/FS group had a significantly lower rate of residual varicosities than the EVLA group, thus reducing the need for the staged FS. CONCLUSIONS: These results confirm the feasibility and safety of simultaneous tributary EVLA and FS. In addition, they indicate better early quality-of-life improvement and a reduced reoperation rate of simultaneously combined truncal EVLA and tributary FS.
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Terapia a Laser/métodos , Perna (Membro)/irrigação sanguínea , Veia Safena , Escleroterapia/métodos , Varizes/terapia , Terapia Combinada , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Medição da Dor , Estudos Prospectivos , Inquéritos e Questionários , Resultado do Tratamento , Ultrassonografia de IntervençãoRESUMO
OBJECTIVE: To develop a peptide probe that could be used for gastric cancer detection via binding to CD44 protein with specificity and affinity. RESULTS: A 12-mer phage peptide library was screened against immobilized CD44 protein. Bound phage counts using ELISA were performed to identify phage clones carrying the most highly selective peptide, which termed RP-1. Immunofluorescence and flow cytometry analysis indicated that the consensus peptide RP-1 could bind to CD44-positive gastric cancer cells with mean fluorescence intensities significantly higher than that of CD44-negative cells. CD44 knockdown led to decreased binding activity of RP-1 to the same cell line. Tissue array technique was used to identify the relationship (r = 0.556) between peptide binding and CD44 detection on gastric cancer tissues. Further, the hyaluronan-binding domain of CD44 was docked with RP-1 using computer modeling/docking approaches, revealing a RP-1/CD44 interaction with geometrical and energy match (-8.6 kcal/mol). CONCLUSIONS: The RP-1 peptide we screened exhibits affinity and specificity to CD44 on cells and has the potential to be used as a candidate probe for gastric cancer cell targeting.
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Receptores de Hialuronatos/química , Receptores de Hialuronatos/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Neoplasias Gástricas/química , Linhagem Celular Tumoral , Humanos , Imuno-Histoquímica , Simulação de Acoplamento Molecular , Biblioteca de Peptídeos , Peptídeos/genética , Neoplasias Gástricas/metabolismoRESUMO
Triple negative breast cancer (TNBC) is an invasive and metastatic phenotype of breast cancer with limited treatment options. Published studies have demonstrated an inhibitory effect of HIF-α inhibition by its inhibitor YC-1 (lificiguat) on growth and angiogenesis of TNBC. However, the underlying mechanism remains poorly understood. In the current paper, our results show that HIF-1α inhibitor significantly inhibited TNBC growth by increasing cellular apoptosis and decreasing MVD, independent of a cell-autonomous mechanism in both endothelial and tumor cells. Genetic screening and in vivo experiments showed that a large number of M2-polarized TAMs accumulated in the hypoxic peri-necrotic region (PNR), where placental growth factor (PlGF) and its ligand, vascular endothelial growth factor receptor-1 (VEGFR-1) were upregulated. Furthermore, YC-1 skewed the polarization of TAMs away from M2 to M1 phenotype, therefore inhibiting TNBC angiogenesis and growth. This effect was further abrogated by VEGFR-1 neutralization and TAM depletion following clodronate liposome injection. These findings provide preclinical evidence for an indirect mechanism underlying YC-1-induced suppression of TNBC growth and angiogenesis, thereby offering a treatment option for TNBC.
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Neoplasias de Mama Triplo Negativas , Humanos , Feminino , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Fator de Crescimento Placentário , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular , Macrófagos/metabolismo , Linhagem Celular Tumoral , Subunidade alfa do Fator 1 Induzível por HipóxiaRESUMO
The development of chemoresistance which results in a poor prognosis often renders current treatments for colorectal cancer (CRC). In this study, we identified reduced microvessel density (MVD) and vascular immaturity resulting from endothelial apoptosis as therapeutic targets for overcoming chemoresistance. We focused on the effect of metformin on MVD, vascular maturity, and endothelial apoptosis of CRCs with a non-angiogenic phenotype, and further investigated its effect in overcoming chemoresistance. In situ transplanted cancer models were established to compare MVD, endothelial apoptosis and vascular maturity, and function in tumors from metformin- and vehicle-treated mice. An in vitro co-culture system was used to observe the effects of metformin on tumor cell-induced endothelial apoptosis. Transcriptome sequencing was performed for genetic screening. Non-angiogenic CRC developed independently of angiogenesis and was characterized by vascular leakage, immaturity, reduced MVD, and non-hypoxia. This phenomenon had also been observed in human CRC. Furthermore, non-angiogenic CRCs showed a worse response to chemotherapeutic drugs in vivo than in vitro. By suppressing endothelial apoptosis, metformin sensitized non-angiogenic CRCs to chemo-drugs via elevation of MVD and improvement of vascular maturity. Further results showed that endothelial apoptosis was induced by tumor cells via activation of caspase signaling, which was abrogated by metformin administration. These findings provide pre-clinical evidence for the involvement of endothelial apoptosis and subsequent vascular immaturity in the chemoresistance of non-angiogenic CRC. By suppressing endothelial apoptosis, metformin restores vascular maturity and function and sensitizes CRC to chemotherapeutic drugs via a vascular mechanism.
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Immune checkpoint blockade is considered a breakthrough in cancer treatment. However, with the low response rates and therapeutic resistance of patients with hepatocellular carcinoma (HCC), the challenges facing the application of this treatment are tremendous. Liver fibrosis is a key driver of tumor immune escape, the underlying mechanism has never been clarified. This study sought to explore the role of liver fibrosis in regulating tumor-infiltrating lymphocytes (TILs) and inducing tumor immunosuppression. Ninety-nine fixed HCC tissue samples were used to analyze the association between liver fibrosis and immune escape using immunohistochemistry. In HCC patients, low FIB-4 values and high CD8+ T cell infiltration were correlated with prolonged survival. Elevated expression of immune checkpoints and attenuated antitumor immunity were observed in CCl4-induced mice liver fibrosis models and human fibrotic livers compared to control group. GOLM1 levels were increased in livers of patients with fibrosis and mice in response to CCl4-induced liver fibrosis. CD8+ T cell infiltrations were significantly decreased and PD-L1 expression was significantly increased in tumor tissues from hepatocyte-specific GOLM1 transgenic mice (Alb/GOLM1 mice) inducing chemical carcinogenesis compared to their corresponding control WT mice. GOLM1 induced PD-L1 expression via EGFR pathway activation. EGFR inhibitors, especially together with anti-PD-L1 therapy, improved the efficacy of immunotherapy in HCC. These findings illustrate the importance of liver fibrosis-induced immunosuppression as a tumor-promoting mechanism. GOLM1, which is highly upregulated in the fibrotic liver, regulates tumor microenvironmental immune escape via the EGFR/PD-L1 signaling pathway. EGFR blockade may bolster the efficacy of immune checkpoint inhibitors for HCC treatment.
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Antígeno B7-H1/imunologia , Carcinoma Hepatocelular/imunologia , Cirrose Hepática/imunologia , Neoplasias Hepáticas/imunologia , Proteínas de Membrana/imunologia , Animais , Antígeno B7-H1/biossíntese , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Receptores ErbB/metabolismo , Humanos , Cirrose Hepática/patologia , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Transcrição STAT3/metabolismo , Evasão Tumoral , Microambiente Tumoral , Regulação para CimaRESUMO
BACKGROUND: Vascular maturity and functionality are closely associated with tumor progression and chemosensitivity. The antidiabetic agent metformin has shown its ability to inhibit tumor angiogenesis in metastatic breast cancer models. However, it remains unclear if or how metformin remodels the abnormal vasculature of metastatic breast cancer, while inhibiting angiogenesis. METHODS: Metastatic breast cancer models were constructed to compare microvessel density (MVD), vascular maturity and function, lung metastasis and chemosensitivity in metformin-treated or untreated mice. Protein array assay and transcriptome sequencing were performed for genetic screening. Lentiviral shRNA-PDGF-B transfection was used for observing the contribution of PDGF-B knockdown to metformin's vascular effects. RESULTS: Metastatic breast cancers were characterized by an excessively angiogenic, immature and morphologically abnormal vasculature. Compared to control, metformin significantly reduced MVD, leakage and hypoxia, and increased vascular mural cells coverage and perfusion, namely, "vessel normalization". Metformin at human blood concentrations had no direct effect on the migration and proliferation of cancer cells. Based on that, reduced lung metastasis of the primary tumor and improved chemosensitization by metformin were assumed to be mediated via metformin's vascular effects. Further results of genetic screening and in vivo experiments showed that the downregulation of platelet-derived growth factor B (PDGF-B) greatly contributed to the metformin-induced vessel normalization. CONCLUSIONS: These findings provide pre-clinical evidences for the vascular mechanism of metformin-induced metastasis inhibition and the chemosensitization of metastatic breast cancers.
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Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Metformina/farmacologia , Neovascularização Patológica/genética , Proteínas Proto-Oncogênicas c-sis/genética , Animais , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/mortalidade , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Modelos Animais de Doenças , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Humanos , Camundongos , Modelos Biológicos , Estadiamento de Neoplasias , Neovascularização Patológica/tratamento farmacológico , Prognóstico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The anti-diabetic metformin has been demonstrated to be effective in suppression of tumor progression via multiple mechanisms, in which angiogenic inhibition is involved. Hypoxia is a common feather of malignant tumor and promotes angiogenesis via induction of pro-angiogenic factors. However, the effect of metformin on tumor hypoxia and the association with angiogenic inhibition are still unclear. In the current study, we investigated the effects of metformin on both tumor blood perfusion and hypoxia-induced excessive angiogenesis. In the tumor region adjacent to necrosis, aberrantly excessive angiogenesis resulted from hypoperfusion-induced intense hypoxia and greatly contributed to the high average levels of both microvessel density and vascular branch density. Metformin administration increased the percentage of lectin-perfused vessels and reduced hypoxyprobe-positive area. This metformin-induced amelioration of hypoxia was accompanied by a significant reduction in expressions of both HIF-1α and angiogenesis-associated factors (AAFs). Consequently, inhibited excessive angiogenesis in hypoxic peri-necrotic region was observed in metformin-treated tumor. Further stable knockdown of HIF-1α abrogated hypoxia-induced AAFs in vitro and reduced both microvessel density and area of fitc-conjugated dextran that leaked outside the vascular lumen. Taken together, metformin ameliorated tumor hypoxia and restrained HIF-1α-induced expressions of AAFs through elevating tumor blood perfusion, thus suppressing the excessive tumor angiogenesis.
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OBJECTIVE: To construct eukaryotic expression plasmid of porcine CCK gene pIRES2-EGFP/ CCK and express it in COS-7 cells and hamsters. METHODS: The aimed segments were obtained from intermediate vector pMD18-T/CCK by the method of restricted enzymatic resection and were inserted into a eukaryotic expression plasmid pIRES2-EGFP to construct a recombinant expression plasmid pIRES2-EGFP/CCK. The recombinant expression plasmid was transfected into COS-7 cells by liposome-mediated gene transfer method and observed through Fluorescence microscopy. The plasmid was injected into the skeletal muscle of hamsters directly to detect the expression of the recombinant plasmid in vivo. RESULTS: A recombinant eukaryotic expression plasmid pIRES2-EGFP/CCK was successfully constructed. Green fluorescent protein could be detected in the transfected COS-7 cells 24, 48, and 72 hours post transfection and the expression of green fluorescent protein reached its peak 72 h post transfection. The green fluorescent protein could be detected at the injection site on the 4th day post injection and the fluorescence intensity became stronger on the 14th day. The level of fluorescence became ever stronger on the 42nd day. No expression of green fluorescence was detected in the control group. CONCLUSION: Porcine CCK cDNA eukaryotic expression plasmid pIRES2-EGFP/CCK has been successfully constructed and expressed in mammal cells COS-7 and hamster in vivo. The research paved the way for cross immunity therapy of hamster pancreatic carcinoma.
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Colecistocinina/genética , Proteínas de Fluorescência Verde/genética , Proteínas Recombinantes de Fusão/biossíntese , Animais , Células COS , Chlorocebus aethiops , Colecistocinina/biossíntese , Cricetinae , Feminino , Proteínas de Fluorescência Verde/biossíntese , Humanos , Imunoterapia , Mesocricetus , Neoplasias Pancreáticas/terapia , Plasmídeos/genética , Proteínas Recombinantes de Fusão/genética , Suínos , TransfecçãoRESUMO
Caudal-related homeobox protein 2 (CDX2), a tumor suppressor in the adult colon, is overexpressed under a non-cancer specific cytomegalovirus promoter in certain tumor cells; furthermore, non-specific expression of CDX2 may result in aberrant side effects in normal cells. The human telomerase reverse transcriptase (hTERT) promoter is active in the majority of cancer cells but not in normal cells. Hypoxia is a key feature of solid tumors, and targeted genes may be significantly upregulated by five copies of hypoxia-response elements (HREs) under hypoxic conditions. However, the effect of CDX2 overexpression, as controlled by five copies of HREs and the hTERT promoter, on human colorectal cancer (CRC) cell proliferation in vitro remains to be fully elucidated. In the current study, a recombinant lentivirus containing the CDX2 gene under the control of five HREs and the hTERT promoter was generated. An immunofluorescence assay was used to detect CDX2 expression by the 5 HhC lentivirus, whereas an MTT assay was used to detect the effects of CoCl2 on the viability of LoVo cells. Western blot analysis was conducted in order to determine the relative ratios of recombinant CDX2 protein to the internal control ß-actin, following 5 HhC/LoVo cell culture under normoxic and hypoxic conditions (100, 200, 300, 400 or 500 µmol/l CoCl2) for 24 h, then for 12, 24 or 36 h with the optimal concentration (300 µmol/l) of CoCl2. Reverse transcription polymerase chain reaction analysis was used to determine the transcription of recombinant CDX2 mRNA following culture of 5 HhC/LoVo cells under normoxic or hypoxic conditions. Finally, a cloning assay was used to detect the proliferative ability of 5 HhC/LoVo and 5 Hh cells. High CDX2 expression was observed in hTERT-positive LoVo cells under hypoxic conditions, an effect which was mimicked by treatment with CoCl2 to inhibit LoVo cell proliferation in vitro. High expression of CDX2 therefore provides a promising strategy for the development of novel targeted treatments and gene therapy for CRC.
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Vetores Genéticos/metabolismo , Proteínas de Homeodomínio/metabolismo , Lentivirus/genética , Fator de Transcrição CDX2 , Hipóxia Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cobalto/toxicidade , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Vetores Genéticos/genética , Proteínas de Homeodomínio/genética , Humanos , Microscopia de Fluorescência , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Elementos de Resposta/genética , Telomerase/genéticaRESUMO
BACKGROUND: Since transfection of established tumors with immunostimulatory genes can elicit antitumor immunity, we treat mouse HCC with in vivo transfection of superantigen SEA and/or costimulatory molecule CD80 and evaluated the safety and efficacy. METHODS: Mice with HCC were treated with lipid-complexed plasmid DNA encoding staphylococcal enterotoxin A and/or CD80. Then the mice were evaluated for tumor regression, systemic immunologic responses, survival times and treatment-associated toxicity. RESULTS: Of all treated mice, the overall response rates (complete or partial remission) for SEA, CD80 and SEA/CD80 treated mice in this study were 65%, 60% and 75% separately, and were significantly higher than that of untreated mice. Most of the treat mice completed the therapy without any significant reaction. CTL activity increased with time of treatment and correlated temporally with an objective tumor response. Also our results indicated that local intratumoral expression of SEA did not lead to detectable deletion or anergy of SEA-reactive spleen T cells. Survival times for hepatoma mice in this study treated by intratumoral injection of SEA, CD80 and SEA/CD80 were prolonged significantly (P < 0.01) compared with the control mice.
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Antígeno B7-1/genética , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/terapia , Enterotoxinas/genética , Neoplasias Hepáticas Experimentais/terapia , Superantígenos/genética , Animais , Anticorpos Antibacterianos/sangue , Carcinoma Hepatocelular/patologia , Enterotoxinas/imunologia , Neoplasias Hepáticas Experimentais/imunologia , Neoplasias Hepáticas Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Superantígenos/imunologia , Taxa de Sobrevida , Linfócitos T/imunologia , Linfócitos T Citotóxicos/imunologia , TransfecçãoRESUMO
BACKGROUND: Identification of the cytotoxic T lymphocytes (CTL) restricted epitopes of tumor antigens opens up possibilities of developing a new cancer vaccine. For the MAGE-n has been demonstrated closely associated with hepatocellular carcinoma (HCC) and HLA-A2.1 is found in over 50% of HCC patients in China, we aim at identifying MAGE-n-encoded peptide presented by HLA-A2.1. MATERIALS: A HLA-A2.1-restricted CTL epitope was identified by using an improved "reverse immunology" strategy: (a) computer-based epitope prediction from the amino acid sequence of MAGE-n antigen; (b) peptide-binding assay to determine the affinity of the predicted peptide with HLA-A2.1 molecule; (c) stimulation of primary T-cell response against the predicted peptides in vitro; and (d) testing of the induced CTLs toward HCC cells expressing MAGE-n antigen and HLA-A2.1. RESULTS: Of the five tested peptides, effectors induced by a peptide of MAGE-n at residue position 159-167(QLVFGIEVV) lysed HCC cells expressing both MAGE-n and HLA-A2.1. Our results indicated that peptide QLVFGIEVV was a new HLA-A2.1-restricted CTL epitope capable of inducing MAGE-n specific CTLs in vitro. CONCLUSIONS: Identification of the MAGE-n /HLA-A2.1 peptide QLVFGIEVV may facilitate peptide-based specific immunotherapy for HCC. The combination of epitope prediction, epitope reconstruction method and immunological methods can improve the efficiency and accuracy of CTL epitope studies.
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Carcinoma Hepatocelular/genética , Antígeno HLA-A2/genética , Antígeno HLA-A2/imunologia , Neoplasias Hepáticas/genética , Proteínas de Neoplasias/genética , Sequência de Aminoácidos , Antígenos de Neoplasias , Vacinas Anticâncer , Carcinoma Hepatocelular/patologia , Epitopos , Previsões , Humanos , Imunoterapia/métodos , Neoplasias Hepáticas/patologia , Dados de Sequência Molecular , Linfócitos T CitotóxicosRESUMO
MAGE-n is a new member of MAGE gene family and has been demonstrated closely associated with hepatocellular carcinoma (HCC). In this study, MAGE-n-derived peptide-specific cytotoxic T lymphocytes (CTL) were induced from the peripheral blood mononuclear cells of healthy donors by multiple stimulations with HLA-A2-restricted MAGE-n peptide-pulsed T2 cells. The induced CTLs exhibited specific lysis against T2 cells pulsed with the peptide and HLA-A2+ HCC cells expressing MAGE-n, while HLA-A2+ HCC cell lines that did not express MAGE-n could not be recognized by the CTLs. The peptide-specific activity was inhibited by anti-MHC class I monoclonal antibody. These results suggested the MAGE-n peptide could be a potential target of specific immunotherapy for HLA-A2 patients with HCC.
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Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/farmacologia , Carcinoma Hepatocelular/imunologia , Antígeno HLA-A2/imunologia , Neoplasias Hepáticas/imunologia , Ativação Linfocitária , Proteínas de Neoplasias , Linfócitos T Citotóxicos/fisiologia , Anticorpos Monoclonais/imunologia , Linhagem Celular , Humanos , Imunoterapia/métodos , PeptídeosRESUMO
AIM: To construct an eukaryotic superantigen gene expression vector containing the recombinant gene of SEA and CD80 molecule transmembrane region (CD80TM), and to express staphylococcus enterotoxin A (SEA) on the membrane of hepatocellular carcinoma (HCC) cell to form a superantigen gene modified tumor vaccine for HCC. METHODS: SEA and linker-CD80TM gene were amplified through PCR from plasmid containing cDNA of SEA and CD80. Gene fragments were then subcloned into the multiple cloning sites of retroviral vector pLXSN. Recombinant plasmid was transferred into HepG2 cells mediated with lipofectamine, positive clones were selected in culture medium containing G418. RT-PCR and indirect immunofluorescence studies confirmed that SEA was expressed specifically on HCC cell membrane. INFgamma-ELISPOT study demonstrated that SEA protein was expressed on the membrane of HCC cells. Cytotoxicity of HepG2-SEA primed CTLs (SEA-T) was analyzed by (51)Cr release assay. T cells cultured with rhIL-2 (IL-2-T) were used as control. RESULTS: Restriction digestion and sequence analyses confirmed the correctness of length, position and orientation of inserted fusion genes. SEA was expressed on the surface of HepG2 cells, HepG2-SEA had strong stimulating effect on production of HepG2 specific CTL (P<0.001). SEA-T had enhanced cytotoxicity to HepG2 cells (P<0.05). CONCLUSION: Tumor cell membrane expressed superantigen can be used to reinforce the immune effect of tumor cell vaccine for HCC, which provides a new method of the enhanced active immunotherapy for HCC.
Assuntos
Vacinas Anticâncer/genética , Carcinoma Hepatocelular , Enterotoxinas/genética , Neoplasias Hepáticas , Superantígenos/genética , Antígeno B7-1/genética , Linhagem Celular Tumoral , Membrana Celular/fisiologia , Expressão Gênica , Humanos , Técnicas In Vitro , TransfecçãoRESUMO
AIM: To construct a gene modified hepatocellular carcinoma (HCC) specific EGFP expression vector regulated by abbreviated cis-acting element of AFP gene. METHODS: The minimal essential DNA segments of AFP gene enhancer and promoter were synthesized through PCR from Genome DNA of HepG2 cells. Gene fragments were then cloned into the multiple cloning site of non-promoter EGFP vector pEGFP-1. Recombinant plasmid was transferred into positive or negative AFP cell lines by means of lipofectamine. The expression of EGFP was tested by fluorescence microscope and flow cytometry. The effect of all-trans retinoic acid (ATRA) on the expression of EGFP was tested in different concentrations. RESULTS: By the methods of restriction digestion and sequence analyses we confirmed that the length, position and orientation of inserted genes of cis-acting element of AFP were all correct. The transcription of EGFP was under the control of AFP cis-acting element. The expressing EGFP can only been detected in AFP producing hepatoma cells. The expression rate of EGFP in G418 screened cell line was 34.9+/-4.1 %. 48 h after adding 1X10(-7)M retinoic acid, EGFP expression rate was 14.7+/-3.5 %. The activity of AFP gene promoter was significantly suppressed by addition of 1 x 10(-7)M retinoic acid (P<0.05, P=0.003, t=6.488). CONCLUSION: This recombinant expression vector can be used as a gene therapy vector for HCC. The expression of tumor killing gene will be confined within the site of tumor and the activity of which can be regulated by retinoic acid.
Assuntos
Carcinoma Hepatocelular/genética , Terapia Genética/métodos , Neoplasias Hepáticas/genética , alfa-Fetoproteínas/genética , Carcinoma Hepatocelular/terapia , Elementos Facilitadores Genéticos , Marcadores Genéticos , Vetores Genéticos , Proteínas de Fluorescência Verde , Humanos , Neoplasias Hepáticas/terapia , Proteínas Luminescentes/genética , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/análise , Mapeamento por Restrição , Células Tumorais CultivadasRESUMO
OBJECTIVE: To establish a strain of human cervical carcinoma cell line and to provide a cervical carcinoma animal model. METHODS: The cervical carcinoma specimens incised aseptically were cultured in vitro by tissue culture methods, giving a tumor cell growth curve. Morphology of the cells was observed, with cell cycling analysis and chromosome analysis performed. The tumor markers (ER, PR, Keratine, PCNA) expressions of the cell line were detected by immuno-cytochemical technique. RESULTS: A human cervical carcinoma cell line HCC-0214 (H) has been obtained by in vitro tissue culture methods. The cells have been maintained for 16 months through 131 passages, showing a stable growth with a population doubling time of 35.48 h and a tendency to pile up without contact inhibition. The ultrastructure showed typical desmosomes and numerous tonofilaments. Chromosome analysis revealed the number of chromosomes per cell varied from 35-156 with a stem-line number of 58-80 (64.8%). The morphology of chromosomes showed human tumor cell structure. The tumor markers (ER, PR, Keratine, PCNA) of the cells showed a high expression. The DNA index was 1.931 by flow cytometry (FCM). The histopathology of the transplanted tumors in nude mice was the same as the original tumor, though with none successful by serum culture. CONCLUSION: A human cervical carcinoma cell line HCC-0214 established by tissue culture is identical to the primary cancer cell in biological characters. After the cells have been passaged for more than 16 months continually, their characteristics are still retained. Therefore, HCC-0214 may be used as a stable cell line.