RESUMO
This work develops a sustainable and global strategy to enhance fruit preservation efficacy. The dual-use composite coating or film comprises silk fibroin/cellulose nanocrystals (SF/CNC) with superior ductility through a synergistic plasticizing effect of glycerol and natural aloe-emodin powder (AE) as antimicrobial agents. To confirm our strategy, two common fruit preservation materials (edible surface coating-SCA-CS; packaging film-SCA-PF) and five different fruits (strawberries, bananas, apples, blueberries, and guavas) have been used. Moreover, SCA-CS coating with antibacterial and antioxidant activities formed an ultrathin layer on the fruit's surfaces with a thickness of 7.7 µm and could be easily washable. Therefore, bananas and strawberries' shelf-life with SCA-CS coating can be extended for 9 days and 6 days, respectively. The discharge water of SCA-CS has excellent biosafety in an indoor environment with no threat to plant health (microgreens bean sprouts germination as a case study). The plant exhibited positive results within 15 days, and leaves maintained their green color with a germination rate of 97.6 %. The toughness of SCA-PF film increased by 14,685.7 % with a water vapor transmission rate (WPTR) of 17 g mm m-2 day-1, which confirms that the concept of SCA-PF film and SCA-CS coating are feasible to be used for fruit preservation/packaging.
Assuntos
Anti-Infecciosos , Quitosana , Filmes Comestíveis , Frutas/microbiologia , Contenção de Riscos Biológicos , Antibacterianos , Conservação de Alimentos/métodos , Quitosana/química , Embalagem de AlimentosRESUMO
SARS-CoV-2 pandemic has profound impacts on human life and global economy since the outbreak in 2019. With the new variants continue to emerge with greater immune escaping capability, the protectivity of the available vaccines is compromised. Therefore, development a vaccine that is capable of inducing immunity against variants including omicron strains is in urgent need. In this study, we developed a protein-based vaccine BCVax that is consisted of antigen delta strain spike protein and QS21-based adjuvant AB801 in nanoparticle immune stimulation complex format (AB801-ISCOM). Results from animal studies showed that high level of anti-S protein IgG was induced after two doses of BCVax and the IgG was capable of neutralizing multiple variants of pseudovirus including omicron BA.1 or BA.2 strains. In addition, strong Th1 response was stimulated after BCVax immunization. Furthermore, BCvax with AB801-ISCOM as the adjuvant showed significant stronger immunity compared with the vaccine using aluminum hydroxide plus CpG 1018 as the adjuvant. BCVax was also evaluated as a booster after two prior vaccinations, the IgG titers and pseudovirus neutralization activities against BA.2 or BA.4/BA.5 were further enhanced suggesting BCVax is a promising candidate as booster. Taken together, the pre-clinical data warrant BCVax for further development in clinic.
Assuntos
COVID-19 , ISCOMs , Animais , Humanos , Vacinas contra COVID-19 , SARS-CoV-2 , Subunidades Proteicas , COVID-19/prevenção & controle , Glicoproteína da Espícula de Coronavírus/genética , Adjuvantes Imunológicos , Adjuvantes Farmacêuticos , Animais de Laboratório , Imunoglobulina G , Anticorpos Antivirais , Anticorpos NeutralizantesRESUMO
Globo H (GH), a hexasaccharide, is expressed at low levels in normal tissues but is highly expressed in multiple cancer types, rendering it a promising target for cancer immunotherapy. OBI-999, a novel antibody-drug conjugate, is derived from a conjugation of a GH-specific mAb with a monomethyl auristatin E (MMAE) payload through a site-specific ThioBridge and a cleavable linker. OBI-999 high homogeneity with a drug-to-antibody ratio of 4 (>95%) was achieved using ThioBridge. OBI-999 displayed GH-dependent cellular internalization and trafficked to endosome and lysosome within 1 and 5 hours, respectively. Furthermore, OBI-999 showed low nanomolar cytotoxicity in the assay with high GH expression on tumor cells and exhibited a bystander killing effect on tumor cells with minimal GH expression. Tissue distribution indicated that OBI-999 and free MMAE gradually accumulated in the tumor, reaching maximum level at 168 hours after treatment, whereas OBI-999 and free MMAE decreased quickly at 4 hours after treatment in normal organs. Maximum MMAE level in the tumor was 16-fold higher than in serum, suggesting that OBI-999 is stable during circulation and MMAE is selectively released in the tumor. Excellent tumor growth inhibition of OBI-999 was demonstrated in breast, gastric, and pancreatic cancer xenograft or lung patient-derived xenograft models in a dose-dependent manner. The highest nonseverely toxic dose in cynomolgus monkeys is 10 mg/kg determined by a 3-week repeated-dose toxicology study demonstrating an acceptable safety margin. Taken together, these results support further clinical development of OBI-999, which is currently in a phase I/II clinical study in multiple solid tumors (NCT04084366). OBI-999, the first GH-targeting ADC, displayed excellent tumor inhibition in animal models across multiple cancer types, including breast, gastric, pancreatic, and lung cancers, warranting further investigation in the treatment of solid tumors.
Assuntos
Imunoconjugados/uso terapêutico , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Imunoconjugados/farmacologia , CamundongosRESUMO
Relative to several other p21-activated kinase (PAK) family members, the role of PAK3 in regulating cancer cell functions remains unclear. Our study obtained evidence that PAK3 regulates the Akt-GSK3ß-ß-catenin signaling by acting as Ser473-Akt kinase in several pancreatic cancer cell lines. Specifically, knockdown of PAK3 or overexpression of dominant-negative PAK3 inhibited the phosphorylation of Ser473-Akt and GSK3ß, resulting in the proteasomal degradation of ß-catenin. Conversely, overexpression of PAK3 led to activation of Akt signaling and increased ß-catenin expression. These changes, however, were not noted with the silencing and/or overexpression of PAK1, PAK2, or PAK4, which underlies the impetus of PAK3 as a key effector in governing malignant phenotypes in these pancreatic cancer cells, including cancer stem cell (CSC) expansion. Accordingly, PAK3 depletion effectively suppresses tumorsphere formation, ALDH activity, and the expression of CSC surface markers. Moreover, we used a stable knockdown clone of AsPC-1â¯cells to demonstrate the in vivo efficacy of PAK3 inhibition in suppressing tumorigenesis and xenograft tumor growth. Together, these findings suggest the potential role of PAK3 as a target for pancreatic cancer therapy, which warrants further investigations.
Assuntos
Proliferação de Células , Glicogênio Sintase Quinase 3 beta/metabolismo , Células-Tronco Neoplásicas/enzimologia , Neoplasias Pancreáticas/enzimologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , beta Catenina/metabolismo , Quinases Ativadas por p21/metabolismo , Animais , Linhagem Celular Tumoral , Feminino , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos Nus , Células-Tronco Neoplásicas/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Fosforilação , Serina , Transdução de Sinais , Esferoides Celulares , Carga Tumoral , beta Catenina/genética , Quinases Ativadas por p21/genéticaRESUMO
Chronic pancreatitis (CP) is a fibro-inflammatory disease leading to pain, maldigestion, and pancreatic insufficiency. No therapeutic options exist due to a limited understanding of the biology of CP pathology. Recent findings implicate pancreatic stellate cells (PSC) as prominent mediators of inflammatory and fibrotic processes during CP. Here, we utilized primary and immortalized PSC obtained from mice and patients with CP or pancreatic cancer to examine the effect of Jak/STAT and MAPK pathway inhibition in vitro. The well-characterized caerulein model of CP was used to assess the therapeutic efficacy of Jak1/2 inhibition in vivo. Treatment of cultured PSC with the Jak1/2 inhibitor ruxolitinib reduced STAT3 phosphorylation, cell proliferation, and expression of alpha-smooth muscle actin (α-SMA), a marker of PSC activation. Treatment with the MAPK inhibitor, MEK162, had less consistent effects on PSC proliferation and no impact on activation. In the caerulein-induced murine model of CP, administration of ruxolitinib for one week significantly reduced biomarkers of inflammation and fibrosis. These data suggest that the Jak/STAT pathway plays a prominent role in PSC proliferation and activation. In vivo treatment with the Jak1/2 inhibitor ruxolitinib reduced the severity of experimental CP, suggesting that targeting Jak/STAT signaling may represent a promising therapeutic strategy for CP.
Assuntos
Ceruletídeo/efeitos adversos , Janus Quinases/metabolismo , Células Estreladas do Pâncreas/metabolismo , Pancreatite Crônica/etiologia , Pancreatite Crônica/metabolismo , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais , Animais , Linhagem Celular , Proliferação de Células , Citocinas/metabolismo , Modelos Animais de Doenças , Humanos , Mediadores da Inflamação/metabolismo , Camundongos , Pancreatite Crônica/patologia , Inibidores de Proteínas Quinases/farmacologiaRESUMO
Substantial evidence has clearly demonstrated the role of the IL-6-NF-κB signaling loop in promoting aggressive phenotypes in breast cancer. However, the exact mechanism by which this inflammatory loop is regulated remains to be defined. Here, we report that integrin-linked kinase (ILK) acts as a molecular switch for this feedback loop. Specifically, we show that IL-6 induces ILK expression via E2F1 upregulation, which, in turn, activates NF-κB signaling to facilitate IL-6 production. shRNA-mediated knockdown or pharmacological inhibition of ILK disrupted this IL-6-NF-κB signaling loop, and blocked IL-6-induced cancer stem cells in vitro and estrogen-independent tumor growth in vivo Together, these findings establish ILK as an intermediary effector of the IL-6-NF-κB feedback loop and a promising therapeutic target for breast cancer.
Assuntos
Neoplasias da Mama/metabolismo , Interleucina-6/metabolismo , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Transdução de Sinais , HumanosRESUMO
BACKGROUND: Integrin-linked kinase (ILK) is a serine-threonine kinase that regulates interactions between the cell and the extracellular matrix. In many cancers, overexpression of ILK leads to increased cell proliferation, motility, and invasion. We hypothesized that ILK functions as a regulator of viability and migration in thyroid cancer cells. METHODS: Eleven human thyroid cancer cell lines were screened for ILK protein expression. The cell lines with the greatest expression were treated with either ILK small interfering RNA (siRNA) or a novel ILK inhibitor, T315, and the effects were evaluated via Western blot and migration assay. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assays were performed to assess cell viability. RESULTS: siRNA against ILK decreased phosphorylation of downstream effectors Akt and MLC, as well as decreased migration. Treatment with T315 showed a dose-related decrease in both Akt and MLC phosphorylation, as well as decreased migration. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assays showed T315 to have an half maximal inhibitory concentration of less than 1 µM in cell lines with high ILK expression. CONCLUSION: ILK is expressed differentially in thyroid cancer cell lines. Both ILK siRNA and T315 inhibit motility of thyroid cancer cell lines, and T315 is shown to be cytotoxic at low concentrations. Altogether, our study suggests that ILK may represent an important kinase in aggressive thyroid cancers.
Assuntos
Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases , RNA Interferente Pequeno/farmacologia , Neoplasias da Glândula Tireoide/tratamento farmacológico , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Serina-Treonina Quinases/genética , Transdução de SinaisRESUMO
BACKGROUND: Cancer stem cells (CSCs) are considered responsible for the recurrence and chemoresistance of cancer. Dysregulated autophagy is highly prevalent in many types of cancer including pancreatic cancer and has been implicated in cytoprotection and tumor promotion. This study aimed to investigate the role of autophagy in regulating cancer stemness and chemoresistance of pancreatic cancer. METHODS: The correlation between autophagy and CSCs and its clinical significance were analyzed using pancreatic cancer tissue microarrays. Genetic and pharmacological approaches were applied to explore the function of autophagy on CSC activity and gemcitabine resistance of pancreatic cancer cells in vitro and in vivo. RESULTS: LC3 expression positively correlated with the expression of CSC markers aldehyde dehydrogenase 1 (ALDH1), CD44, and CD133 in pancreatic cancer tissues. High coexpression of LC3/ALDH1 was associated with both poor overall survival and progression-free survival. In pancreatic cancer cell lines, higher LC3-II expression was observed in the sphere-forming cells than in the bulk cells. Blockade of autophagy by silencing ATG5, ATG7, and BECN1 or the administration of autophagy inhibitor chloroquine markedly reduced the CSC populations, ALDH1 activity, sphere formation, and resistance to gemcitabine in vitro and in vivo. Furthermore, osteopontin (OPN) was found to stimulate LC3-II, ALDH1, CD44, and CD133 expression in PANC-1 cells, whereas this effect could be prevented by OPN knockdown and autophagy blockade. After treatment with various inhibitors against the major signaling pathways downstream of OPN, only the inhibitor of NF-κB activation, BAY 1170-82, could effectively counteract OPN-induced autophagy and CSC activity. According to the histochemical results, pancreatic cancer patients manifesting high levels of OPN/LC3/ALDH1 and OPN/CD44/CD133 had poor survival. CONCLUSIONS: Induction of autophagy mediated by OPN/NF-κB signaling is required for maintenance of pancreatic CSC activity. Combination of gemcitabine with pharmacological autophagy inhibitors is a promising therapeutic strategy for pancreatic cancer.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Desoxicitidina/análogos & derivados , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Neoplasias Pancreáticas/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Desoxicitidina/farmacologia , Humanos , Masculino , Pessoa de Meia-Idade , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas , GencitabinaRESUMO
A technique for synthesizing biocompatible hydrogels by cross-linking calcium-form poly(γ-glutamic acid), alginate sodium, and Pluronic F-127 was created, in which alginate can be cross-linked by Ca(2+) from Ca-γ-PGA directly and γ-PGA molecules introduced into the alginate matrix to provide pH sensitivity and hemostasis. Mechanical properties, swelling behavior, and blood compatibility were investigated for each hydrogel compared with alginate and for γ-PGA hydrogel with the sodium form only. Adding F-127 improves mechanical properties efficiently and influences the temperature-sensitive swelling of the hydrogels but also has a minor effect on pH-sensitive swelling and promotes anticoagulation. MG-63 cells were used to test biocompatibility. Gelation occurred gradually through change in the elastic modulus as the release of calcium ions increased over time and caused ionic cross-linking, which promotes the elasticity of gel. In addition, the growth of MG-63 cells in the gel reflected nontoxicity. These results showed that this biocompatible scaffold has potential for application in bone materials.
Assuntos
Alginatos/química , Substitutos Ósseos/síntese química , Substitutos Ósseos/farmacologia , Osteoblastos/citologia , Ácido Poliglutâmico/análogos & derivados , Alicerces Teciduais , Alginatos/farmacologia , Materiais Biocompatíveis/síntese química , Materiais Biocompatíveis/farmacologia , Linhagem Celular , Sobrevivência Celular/fisiologia , Ácido Glucurônico/química , Ácido Glucurônico/farmacologia , Ácidos Hexurônicos/química , Ácidos Hexurônicos/farmacologia , Humanos , Teste de Materiais , Osteoblastos/efeitos dos fármacos , Osteoblastos/fisiologia , Osteogênese/efeitos dos fármacos , Osteogênese/fisiologia , Ácido Poliglutâmico/química , Ácido Poliglutâmico/farmacologia , Engenharia Tecidual/instrumentação , Engenharia Tecidual/métodosRESUMO
Liver fibrosis is a risk factor for hepatoma. Activated hepatic stellate cells (HSCs) play a critical role in progression of hepatoma. Resected hepatoma patients with high α-SMA+HSCs infiltration had worse survival, OR: 2.2 and p=0.0434. We hypothesized that HSCs could increase the epithelial-mesenchymal transition (EMT) ability of hepatoma cells. In murine model of liver fibrosis with injection of ML1 mice HCC cell line, E-cadherin was lost at the margin of tumor nodule around α-SMA+HSC sites. In subcutaneous tumor model, HSCs could increase the metastatic nodules in the lung, and the expression of E-cadherin was decreased and the Slug was induced. To elucidate the effect of HSCs on hepatoma cells, HSC-T6 was co-cultured with ML1 and the condition medium of HSC-T6 can trigger ML1 cell morphological change, down-expression of E-cadherin, induction of Slug expression, and cell migration. Proteomic analysis of the condition medium showed that collagen I was the target molecule. Collagen type I alone also induced EMT of ML1 cells. Knockdown of collagen type I in HSC-T6 could decrease its induction of EMT on ML1 cells. In conclusion, HSC can secrete collagen type I to trigger hepatoma cells to undergo EMT for metastasis.
RESUMO
Interferon-gamma (IFN-γ), a potent Th1 cytokine with multiple biological functions, can induce autophagy to enhance the clearance of the invading microorganism or cause cell death. We have reported that Concanavalin A (Con A) can cause autophagic cell death in hepatocytes and induce both T cell-dependent and -independent acute hepatitis in immunocompetent and immunodeficient mice, respectively. Although IFN-γ is known to enhance liver injury in Con A-induced hepatitis, its role in autophagy-related hepatocyte death is not clear. In this study we report that IFN-γ can enhance Con A-induced autophagic flux and cell death in hepatoma cell lines. A necrotic cell death with increased lysosomal membrane permeabilization (LMP) is observed in Con A-treated hepatoma cells in the presence of IFN-γ. Cathepsin B and L were released from lysosomes to cause cell death. Furthermore, IFN-γ induces immunity related GTPase family M member 1(IRGM1) translocation to lysosomes and prolongs its activity in Con A-treated hepatoma cells. Knockdown of IRGM1 inhibits the IFN-γ/Con A-induced LMP change and cell death. Furthermore, IFN-γ(-/-) mice are resistant to Con A-induced autophagy-associated necrotic hepatocyte death. We conclude that IFN-γ enhances Con A-induced autophagic flux and causes an IRGM1-dependent lysosome-mediated necrotic cell death in hepatocytes.
Assuntos
Autofagia , Concanavalina A/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Hepatócitos/citologia , Animais , Carcinoma Hepatocelular/metabolismo , Catepsina B/metabolismo , Catepsina L/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Endocitose , Células Hep G2 , Humanos , Interferon gama/metabolismo , Neoplasias Hepáticas/metabolismo , Lisossomos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Necrose , PermeabilidadeRESUMO
Liver cirrhosis and hepatocellular carcinomas are two major causes of morbidity and mortality worldwide, and can synergistically interact to expedite the tumor progression. How fibrosis promotes the hepatoma growth remains completely unexplained. Using an in situ murine hepatoma model together with fibrosis induction by thioacetamide (TAA), the hepatoma growth and the immune factors in the fibrotic liver were analyzed. We found that TAA-fibrosis induction enhanced hepatoma cell growth in the liver and increased the mortality of hepatoma-bearing mice. The tumor-infiltrating CD4(+) or CD8(+) T cells are downregulated by fibrosis induction. The Foxp3(+) regulatory T cells (Treg) cells were induced. We conclude that fibrosis induction causes further immunosuppression, in which Treg cells exert a downregulation effect on the antitumor immunity.
Assuntos
Carcinoma Hepatocelular/patologia , Cirrose Hepática/imunologia , Neoplasias Hepáticas Experimentais/patologia , Neoplasias/patologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Carcinógenos , Carcinoma Hepatocelular/induzido quimicamente , Carcinoma Hepatocelular/imunologia , Linhagem Celular Tumoral , Proliferação de Células , Fatores de Transcrição Forkhead/imunologia , Tolerância Imunológica , Cirrose Hepática/patologia , Neoplasias Hepáticas Experimentais/imunologia , Contagem de Linfócitos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias/imunologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologia , Tioacetamida , Evasão TumoralRESUMO
We have reported both T-cell-dependent and -independent hepatitis in immunocompetent and immunodeficiency mice, respectively, after intravenous injection of Con A in mice. The mode of hepatocyte cell death is different: autophagy for T-cell-independent hepatitis in contrast to apoptosis for T-cell-dependent one. In this study, we further demonstrate that liver blood vessels are the first target in both modes. The infused Con A bond to the hepatic vascular endothelial cells and cause its damage with autophagy. Before the elevation of the serum alanine aminotransferase at 6 h post-injection, the plasma leakage and hemorrhage occur at 1-3 h without inflammation. Con A induces autophagy of endothelial cells and hemorrhage that is enhanced by IFN-gamma. Using the endothelial cell line HMEC-1, a dose- and time-dependent cell death with autophagic LC3-II (microtubule-associated protein light chain 3) conversion was induced by Con A and was enhanced by IFN-gamma. In conclusion, Con A induced autophagy on hepatic endothelial cells; the damage of liver blood vessel occurs before the induction of T-cell-dependent hepatitis via apoptosis or T-cell-independent hepatitis via autophagy.
Assuntos
Autofagia , Doença Hepática Induzida por Substâncias e Drogas/patologia , Concanavalina A , Células Endoteliais/patologia , Hepatócitos/patologia , Interferon gama/imunologia , Animais , Western Blotting , Sobrevivência Celular , Células Cultivadas , Doença Hepática Induzida por Substâncias e Drogas/fisiopatologia , Células Endoteliais/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos SCID , Fatores de TempoRESUMO
alpha-Lipoic acid (LA) has been intensely investigated as a therapeutic agent for several diseases, including hepatic disorder and diabetic polyneuropathy. However, the effects of LA or its reduced form, dihydrolipoic acid (DHLA), on cancer chemoprevention has seldom been studied. Tetrachlorohydroquinone (TCHQ) is a toxic metabolite of pentachlorophenol (PCP) that was proven to be a tumor promoter in our previous study. We recently reported that DHLA can inhibit DMBA/TPA-induced skin tumor formation through its anti-inflammatory and anti-oxidizing functions. In the present study, we further examined the effects of DHLA on DMBA/TCHQ-induced skin tumor formation and the possible mechanisms. We found that DHLA significantly inhibited tumor incidence and tumor multiplicity in DMBA/TCHQ-induced skin tumor formation. Administration of DHLA prevented ROS generation, cytotoxicity, genotoxicity and apoptotic cell death in cells treated with TCHQ. In addition, activation of JNK and p38 MAPK may be involved in TCHQ-mediated apoptosis. Nonetheless, the detailed mechanisms of DHLA in attenuating TCHQ-induced skin tumor promotion are still unclear and need to be further investigated. We conclude that DHLA may be a useful protective agent against TCHQ-induced toxicity in epithelial cells, and for reversing TCHQ-induced damage in mouse skin.
Assuntos
Anticarcinógenos , Antioxidantes/farmacologia , Carcinógenos/antagonistas & inibidores , Carcinógenos/toxicidade , Hidroquinonas/antagonistas & inibidores , Hidroquinonas/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Ácido Tióctico/análogos & derivados , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Caspases/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Ensaio Cometa , Epididimo/patologia , Citometria de Fluxo , Imuno-Histoquímica , Masculino , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Células NIH 3T3 , Antígeno Nuclear de Célula em Proliferação/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/prevenção & controle , Ácido Tióctico/farmacologiaRESUMO
Extensive researches have found that the mutation of p53 tumor suppressor gene is the most frequent event in many human cancers and associated with a poor clinical outcome in lung cancer patients. Because the p53 molecular mutation involved in tumorigenesis of patients with lung cancer in Taiwan remains poorly defined, the aim of this study was to assess the p53 mutation spectrum and possible etiological factors of Taiwan's patients with Non-Small Cell Lung Cancer (NSCLC). Cancer specimens were obtained surgically from 61 patients with pathologically proven NSCLC. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and direct sequencing were used to study p53 mutations in exon 4-8. We also performed immunohistochemistry (IHC) to detect p53 protein expression. Our results provided that 34 mutations of p53 gene were found in 27 cases with a mutation rate of 44% (27/61). There were six cases having more than two p53 mutations. Among the 34 mutations, 19 were point mutations (56%, 19/34) consisted of a majority of missense mutations including transversion (13/19, 68%) and transitions (6/19, 32%) with four cases (4/6, 67%) occurring in the CpG sequence. One of the most important finding in our study was the high frequency of frameshift (44%, 15/34) which included 11 insertions and 4 deletions of p53 in NSCLC in Taiwan. Surprisingly, our results disclosed distinct novel mutations at codon 181, 185, 208 (Exon 5-6) of p53. Especially, 4 cases with mutation at codon181 and codon 185 seemed to have more advanced clinical outcome with survival time less than 6 months. In addition, there were two recurring mutations at codon 168 and three at condon193. The different mutation spectrum in our series, including a high frequency of frameshift mutations and distinctly novel hot spots suggested the heterogenous entity of exogenous mutagens in NSCLC in Taiwan.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Mutação da Fase de Leitura , Genes p53 , Neoplasias Pulmonares/genética , Idoso , Carcinoma Pulmonar de Células não Pequenas/etiologia , Transformação Celular Neoplásica , Análise Mutacional de DNA , Feminino , Humanos , Neoplasias Pulmonares/etiologia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , Prognóstico , TaiwanRESUMO
TCHQ is a major carcinogenic metabolite of the widely used wood preservative PCP. Recently, we found that TCHQ was a promoter in a mouse skin carcinogenesis model. However, the mechanism is still not clear. In this study, we showed that overexpression of Bcl-2 effectively suppressed TCHQ-induced apoptosis in NIH3T3 cells, as evidenced by morphological changes and DNA fragmentation. Although production of ROS contributes to TCHQ-induced apoptosis, Bcl-2 failed to attenuate TCHQ-elicited increase of intracellular ROS level. In addition, overexpressed Bcl-2 provides only partial protection against TCHQ-induced cellular DNA damage. We also found that TCHQ induced a change in mitochondrial transmembrane potential, and that caspase-9 and subsequent caspase-3 can be activated during TCHQ-induced acute apoptosis. Interestingly, TCHQ induced a significant upregulation of Bcl-2 expression, and over-expressed Bcl-2 can dramatically inhibit the change of mitochondria membrane potential and activation of both caspase-9 and -3. Thus, our results suggest TCHQ-induced tumor promotion may be through a mechanism of upregulation of Bcl-2 protein and subsequent apoptosis inhibition.