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BACKGROUND Emerging evidence suggests the involvement of Reelin in chemoresistance in various cancers. However, its function in cisplatin (DDP) sensitivity of non-small cell lung cancer (NSCLC) needs to be investigated. MATERIAL AND METHODS Reelin expression in cisplatin-sensitive A549 cells and cisplatin-resistant NSCLC (A549/DDP) cells was analyzed by western blot analysis. qRT-PCR, western blotting, immunofluorescence, CCK-8 assays, Annexin V/propidium iodide apoptosis assay, and Transwell migration assays were carried out to determine the function of Reelin on DDP resistance. RESULTS Reelin was markedly increased in A549/DDP cells relative to A549 cells. Knockdown of Reelin enhanced DDP chemosensitivity of A549/DDP cells, whereas overexpression of Reelin enhanced DDP resistance of A549, H1299, and H460 cells. Reelin induced DDP resistance in NSCLC cells via facilitating epithelial-mesenchymal transition (EMT). Furthermore, Reelin modulated p38/GSK3ß signal transduction and promoted Snail (EMT-associated transcription factor) expression. Suppression of p38/Snail reversed Reelin-induced EMT and resistance of NSCLC cells to DDP. CONCLUSIONS These data indicated that Reelin induces DDP resistance of NSCLC by regulation of the p38/GSK3ß/Snail/EMT signaling pathway and provide evidence that Reelin suppression can be an effective strategy to suppress DDP resistance in NSCLC.
Assuntos
Antineoplásicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Moléculas de Adesão Celular Neuronais/fisiologia , Cisplatino/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/fisiologia , Transição Epitelial-Mesenquimal/fisiologia , Proteínas da Matriz Extracelular/fisiologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Proteínas do Tecido Nervoso/fisiologia , Serina Endopeptidases/fisiologia , Transdução de Sinais/fisiologia , Fatores de Transcrição da Família Snail/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Proteína ReelinaRESUMO
BACKGROUND: Thrombocytopenia is a common side effect of tumor chemotherapy, the main management approach to which is based on platelet (PLT) transfusion. However, PLTs, containing angiogenesis regulators, play a major role in boosting tumor growth and metastasis. The purpose of the study was to determine whether PLTs have the capacity to overexpress tumstatin by modified megakaryocyte (MK) and PLT precursors using lentivirus-mediated gene transfer, which might lead to alteration in proangiogenic effect of PLTs. STUDY DESIGN AND METHODS: CD34+ hematopoietic stem cells (HSCs) were transduced with recombinant lentivirus carrying tumstatin and induced to produce MKs and PLTs in the culture medium containing a cytokine cocktail. Flow cytometry and aggregation test were used to detect the generation and function of MKs and PLTs. Western blot analysis and confocal microscopy were applied to examine the expression and distribution of tumstatin in transgenic MKs and PLTs. Capillary tube formation of human umbilical vein endothelial cells (HUVECs) was used to evaluate the inhibitory effect of transgenic PLTs. RESULTS: CD34+ HSCs can be efficiently transduced with lentivirus vectors and successfully differentiated into MKs and PLTs. Large amounts of functional MKs and PLTs could be generated and had correct biologic characteristics. The tests demonstrated the feasibility of tumstatin expression in MKs and PLTs under control of the cytomegalovirus promoter, that thus tumstatin was stored in the α-granules of PLTs, and that the releasate of thrombin or A543 cell-stimulated transgenic PLTs obviously inhibited the growth of capillary tube network structures of HUVECs. CONCLUSION: Gene-modified CD34+ HSCs not only successfully differentiated into MKs and PLTs but also expressed tumstatin protein. Release of tumstatin in transgenic PLT granules led to antiangiogenic effect of PLTs.
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Autoantígenos/fisiologia , Plaquetas/fisiologia , Colágeno Tipo IV/fisiologia , Neovascularização Fisiológica/fisiologia , Autoantígenos/biossíntese , Autoantígenos/genética , Capilares/ultraestrutura , Colágeno Tipo IV/biossíntese , Colágeno Tipo IV/genética , Grânulos Citoplasmáticos/metabolismo , Genes Reporter , Vetores Genéticos/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Lentivirus/genética , Megacariócitos/metabolismo , Ativação Plaquetária , Agregação Plaquetária/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo , Trombina/farmacologia , Trombopoese , Transdução Genética , TransgenesRESUMO
Tumstatin is a candidate tumor suppressor that plays an important role in tumor growth and angiogenesis. The purpose of this study was to evaluate the correlation between tumstatin-mRNA expression and the clinicopathologic characteristics, tumor angiogenesis, outcome of patients with non-small cell lung cancer (NSCLC). Specimens from 68 patients with NSCLC were recruited in this study. Tumstatin-mRNA expression and protein level in tumor tissues were quantified respectively by quantitative RT-PCR and ELISA. Microvessel density (MVD) was determined by CD34 immunostaining. The correlation of tumstatin-mRNA expression levels with clinicopathologic variables, tumor angiogenesis, and prognosis was analyzed. Tumstatin-mRNA expression levels were decreased in tumor. Tumstatin-mRNA expression level was significantly correlated with its protein level in tumor (r = 0.562; P = 0.001). Tumstatin-mRNA expression levels in poorly differentiated tumor tissues were significantly lower than in well-differentiated tumor tissues (P < 0.004). Furthermore, tumor tumstatin-mRNA expression were also significantly related to tumor pathologic stage (P = 0.032) and MVD (r = -0.77, P < 0.0001). Overall survival (OS) and disease-free survival (DFS) analysis indicated that NSCLC patients with low tumstatin-mRNA expression had poorer OS and DFS than those with high expression (P = 0.015 and 0.037; respectively). Multivariable Cox regression analysis revealed that the tumstatin-mRNA expression could be an independent prognostic indicators in both DFS and OS. Tumstatin-mRNA expression levels are down-regulated in NSCLC tissues. Tumstatin-mRNA expression level correlates with prognosis, which suggests that tumstatin-mRNA is a new potential independent marker of favorable prognosis in NSCLC.
Assuntos
Autoantígenos/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Colágeno Tipo IV/metabolismo , Neoplasias Pulmonares/metabolismo , RNA Mensageiro/metabolismo , Idoso , Autoantígenos/genética , Carcinoma Pulmonar de Células não Pequenas/irrigação sanguínea , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Carcinoma Pulmonar de Células não Pequenas/patologia , Colágeno Tipo IV/genética , Intervalo Livre de Doença , Regulação para Baixo , Feminino , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/irrigação sanguínea , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Masculino , Microvasos/patologia , Pessoa de Meia-Idade , Análise Multivariada , Prognóstico , Modelos de Riscos Proporcionais , RNA Mensageiro/genética , Transcrição GênicaRESUMO
Non-small cell lung cancer (NSCLC) is one of the most malignant cancer types. N6-methyladenosine (m6A), an abundant eukaryotic mRNA modification, has been observed in multiple diseases, particularly cancer. Methyltransferase-like 14 (METTL14) is a central component of the m6A methyltransferase complex and has been reported to promote tumor development in several cancer types. The present study aimed to investigate the role of METTL14 in NSCLC. Relevant clinical and mRNA sequencing data for m6A-related genes were downloaded from The Cancer Genome Atlas database. R software was used to evaluate the expression of m6A regulators in NSCLC. The biological functions of METTL14 were evaluated using Cell Counting Kit-8, colony formation, Transwell migration and western blot analyses. The results demonstrated that METTL14 expression was upregulated in NSCLC tissues and cell lines, and its expression was high in cancer tissues from patients with NSCLC with all four stages (I, II, III and IV) of disease. METTL14 downregulation inhibited cell proliferation and migration in A549 and SK-MES-1 lung cancer cell lines. Knockdown of METTL14 in lung cancer cell lines increased E-cadherin expression and suppressed N-cadherin expression. Furthermore, METTL14 downregulation reduced the expression levels of the transcription factor Twist and the p-AKT/AKT ratio. In conclusion, the present findings revealed that silencing of METTL14 suppressed NSCLC malignancy by inhibiting Twist-mediated activation of AKT signaling. These data suggest that METTL14 may be a potential therapeutic target for NSCLC.
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Oxaliplatin (Oxa)based chemotherapy is widely used as the firstline treatment for colorectal cancer (CRC). However, Oxaresistance is common for many postoperative CRC patients. To explore drug resistance in CRC, an Oxaresistant cell line, HCT116/Oxa, was established from parental HCT116 cells. These Oxaresistant cells exhibited characteristics of epithelialmesenchymal transition (EMT) and a higher migratory capacity than parental cells. Protein profiles of HCT116/Oxa and HCT116 cells were compared using a tandem mass tagbased quantitative proteomics technique. The protein dehydrogenase/reductase SDR family member 2 (DHRS2) was revealed to be highly expressed in HCT116/Oxa cells. Silencing of DHRS2 in HCT116/Oxa cells effectively restored Oxasensitivity by suppressing the expression of excision repair crosscomplementing group 1 protein via a p53dependent pathway, and reversed the EMT phenotype. Overall, the suppression of DHRS2 expression may be a promising strategy for the prevention of Oxaresistance in CRC.
Assuntos
Carbonil Redutase (NADPH)/antagonistas & inibidores , Neoplasias Colorretais/metabolismo , Proteínas de Ligação a DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos , Endonucleases/metabolismo , Antígenos CD/metabolismo , Antineoplásicos/farmacologia , Caderinas/metabolismo , Carbonil Redutase (NADPH)/genética , Movimento Celular/efeitos dos fármacos , Cromatografia Líquida , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Regulação para Baixo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Células HCT116 , Humanos , Oxaliplatina/farmacologia , Proteômica , Transdução de Sinais/efeitos dos fármacos , Espectrometria de Massas em TandemRESUMO
Tumstatin, a 28-kDa C-terminal fragment of collagen IV, is a potent anti-angiogenic protein and inhibitor of tumour growth. Recombinant tumstatin was prepared from Escherichia coli deposited as insoluble, inactive inclusion bodies. In the present study, we produced soluble and biologically active recombinant human tumstatin in E. coli by the coding region of tumstatin being linked to the 3'-end of the maltose-binding protein (MBP) gene. The fusion protein was expressed as the soluble form after induction by isopropylthio-beta-D-galactoside (IPTG). MBP-tumstatin was purified by amylose affinity chromatography. MBP can be removed by digestion with factor Xa. Expression could represent 20% of the total soluble protein in E. coli, allowing approximately 8.6 mg of highly purified protein to be obtained per litre of bacterial culture. The purified tumstatin specifically inhibited the proliferation of endothelial cells in a dose-dependent manner. Annexin V-FITC apoptotic assay showed that recombinant tumstatin induced significant increase of apoptotic endothelial cells after 20 h of exposure to 20 microg/ml tumstatin, and when tumstatin was incubated on the chicken embryo, chorioallantoic membrane at doses of 1-15 microg, there was a dramatic decrease in the microvasculature allantoids of chicken embryos neovascular vessel test in vivo demonstrated that tumstatin treatment at doses of 1-15 microg gives rise to dramatically decrease the number of neovascular vessel. Our study provides a feasible and convenient approach to produce soluble and biologically active tumstatin.
Assuntos
Autoantígenos/metabolismo , Autoantígenos/farmacologia , Colágeno Tipo IV/metabolismo , Colágeno Tipo IV/farmacologia , Escherichia coli/metabolismo , Expressão Gênica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/farmacologia , Animais , Autoantígenos/genética , Autoantígenos/isolamento & purificação , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Embrião de Galinha , Colágeno Tipo IV/genética , Colágeno Tipo IV/isolamento & purificação , Células Endoteliais/efeitos dos fármacos , Escherichia coli/genética , Vetores Genéticos/genética , Humanos , Proteínas Ligantes de Maltose , Neovascularização Fisiológica/efeitos dos fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , SolubilidadeRESUMO
This article has been withdrawn at the request of the author(s) and/or editor. The Publisher apologizes for any inconvenience this may cause. The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy.
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BACKGROUND: Tumstatin, an angiogenesis inhibitor with anti-tumor activity in mice, is the bioactive NC1 domain of Col IVa3, the potential significance of tumstatin as an endogenous angiogenesis inhibitor in human is not yet completely understood. This study aimed to develop an enzyme-linked immunoassay (ELISA) for tumstatin to accurately measure its concentrations in biological samples. METHODS: Recombinant tumstatin as an immunogen was expressed by E.coli. The purified tumstatin was injected into mice for antibody generation. An ELISA was developed with the monoclonal antibodies. RESULTS: The detection limit of the ELISA was 1.4ng/ml, and the intra- and inter- assay CVs were within 10%. Recovery of tumstatin added to sera was 92.7-115%. Patients with metastases had serum tumstatin levels 50% lower than patients without metastases (P=0.039). Tumstatin levels in poorly differentiated tumor tissues were significantly lower than in nontumorous tissues and well-differentiated tumor tissues (P<0.001). CONCLUSIONS: The development of a highly sensitive and reliable ELISA method capable of quantifying tumstatin in human serum samples and tissue extracts is reported. This assay for tumstatin in biological samples may be helpful in evaluating the therapeutic and/or prognostic value of tumstatin in cancer patients.
Assuntos
Autoantígenos/análise , Autoantígenos/sangue , Colágeno Tipo IV/análise , Colágeno Tipo IV/sangue , Ensaio de Imunoadsorção Enzimática/métodos , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/metabolismo , Adulto , Idoso , Animais , Autoantígenos/isolamento & purificação , Células CHO , Colágeno Tipo IV/isolamento & purificação , Cricetinae , Cricetulus , Feminino , Humanos , Neoplasias Pulmonares/diagnóstico , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Proteínas Recombinantes/sangue , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
The anti-angiogenic activity of tumstatin45-132 is mediated by binding to alphaVbeta3 on endothelial cells and tumor vascular endothelium showing increased expression of alphaVbeta3. Tumor necrosis factor alpha (TNF-alpha) is known to not only possess direct cytotoxicity against tumor cells, but also induces tumor vessel disruption, however, clinical use of TNF-alpha as an anticancer drug is hampered by severe systemic toxicity. In this study, we explore the possibility of fusing tumstatin45-132 with human TNF-alpha in the hope of generating a targeting, bi-functional protein in tumor treatment. Tumstatin45-132-TNF was constructed and expressed in E. coli. The recombinant fusion protein was shown to be insoluble and in an inclusion body form. An effective strategy for refolding and purification of tumstatin45-132-TNF resulted in final purified yields of 3 mg purified fusion protein recovered from 1 liter of E. coli culture. The refolded tumstatin45-132-TNF with a purity of 98% assessed by denaturing SDS - PAGE showed a single band on gels. Endothelial cell proliferation assay and standard cytolytic assays against L929 indicated that the fusion protein maintains tumstatin45-132 and TNF-alpha activity. More importantly, tumstatin45-132-TNF inhibits endothelial cell proliferation more than tumstatin45-132 alone. Cell adhesion assays and competitive binding experiments with anti-integrin antibodies showed that the tumstatin45-132 moiety specifically interacts with alphaVbeta3 integrin. These results lay the solid foundation for further investigation of antitumor activity of tumstatin45-132-TNF in vivo.
Assuntos
Autoantígenos/química , Colágeno Tipo IV/química , Proteínas Recombinantes de Fusão/biossíntese , Animais , Proliferação de Células/efeitos dos fármacos , Humanos , Integrina alfaVbeta3/metabolismo , Camundongos , Dobramento de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacologiaRESUMO
AIM: To clone tumstatin(45-132) gene and to express recombinant human tumstatin(45-132) in E. coli BL21. METHODS: Tumstatin gene was cloned by RT-PCR and then tumstatin(45-132) gene amplified from tumstatin gene was cloned into pBV220. The recombinant plasmid pBV220-tumstatin(45-132) was sequenced and transformed into E.coli BL21. E. coli BL21 transformed with the recombinant plasmid pBV220-tumstatin(45-132) was induced at 42 degrees Celsius. After the recombinant tumstatin(45-132) was purified, its bioactivity was detected by endothelia cell proliferation test. RESULTS: 264 bp tumstatin(45-132) fragment was cloned and its sequence was identical with that in GenBank. The tumstatin(45-132) was expressed in E. coli BL21. Expressed product accounted for about 10% of total bacterial proteins and its relative molecular mass (M(r)) was 9,600. The purified protein showed inhibitory effect on proliferation of endothelia cells ECV304 in vitro. CONCLUSION: tumstatin(45-132) gene has been cloned and expressed successfully in E.coli BL21. Expressed tumstatin(45-132) can inhibit the proliferation of endothelial cell ECV304.