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Lung cancer is one of the most commonly diagnosed cancer and despite therapeutic advances, mortality remains high. The long period of clinical latency associated with lung cancer provides an ideal window of opportunity to administer vaccines to at-risk individuals that can prevent tumor progression and initiate long-term anti-tumor immune surveillance. Here we describe a personalized vaccination regime that could be applied for both therapeutic and prophylactic prevention of lung cancer, based on the derivation of lung cancer cells from induced pluripotent stem cells. Stem cells from healthy mice were modified to express Cre-dependent KRASG12D and Trp53R172H prior to differentiation to lung progenitor cells. Subsequent viral delivery of Cre caused activation of exogenous driver mutations, resulting in transformation and development of lung cancer cells. iPSC-derived lung cancer cells were highly antigenically related to lung cancer cells induced in LSL-KRASG12D/+; Trp53R172H/+ transgenic mice and were antigenically unrelated to original pluripotent stem cells or pancreatic cancer cells derived using the same technological platform. For vaccination, induced lung cancer cells were infected with oncolytic Adenovirus or Vaccinia virus, to act as vaccine adjuvants, prior to delivery of vaccines sequentially to a murine inducible transgenic model of lung cancer. Application of this Virus-Infected, Reprogrammed Somatic cell-derived Tumor cell (VIReST) regime primed tumor-specific T cell responses that significantly prolonged survival in both subcutaneous post-vaccine challenge models and induced transgenic models of lung cancer, demonstrating that stem cell-derived prophylactic vaccines may be a feasible intervention for treatment or prevention of lung cancer development in at-risk individuals.
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Vacinas Anticâncer/imunologia , Vacinas Anticâncer/uso terapêutico , Neoplasias Pulmonares/terapia , Animais , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/administração & dosagem , Modelos Animais de Doenças , Expressão Gênica , Vetores Genéticos/genética , Imunização , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/prevenção & controle , Masculino , Camundongos , Camundongos Transgênicos , Vírus Oncolíticos/genética , Sobrevida , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Transdução Genética , Resultado do Tratamento , Carga TumoralRESUMO
BACKGROUND: Local recurrence and remote metastasis are major challenges to overcome in order to improve the survival of patients with cancer after surgery. Oncolytic viruses are a particularly attractive option for prevention of postsurgical disease as they offer a non-toxic treatment option that can directly target residual tumor deposits and beneficially modulate the systemic immune environment that is suppressed post surgery and allows residual disease escape from control. Here, we report that a novel Vaccinia virus (VV), VVΔTKΔN1L (with deletion of both thymidine kinase (TK) and N1L genes) armed with interleukin 12 (IL-12), can prolong postoperative survival when used as a neoadjuvant treatment in different murine and hamster surgical models of cancer. METHODS: A tumor-targeted replicating VV with deletion of TK gene and N1L gene (VVΔTKΔN1L) was created. This virus was armed rationally with IL-12. The effect of VVΔTKΔN1L and VVΔTKΔN1L-IL12 on modulation of the tumor microenvironment and induction of tumor-specific immunity as well the feasibility and safety as a neoadjuvant agent for preventing recurrence and metastasis after surgery were assessed in several clinically relevant models. RESULTS: VVΔTKΔN1L can significantly prolong postoperative survival when used as a neoadjuvant treatment in three different surgery-induced metastatic models of cancer. Efficacy was critically dependent on elevation of circulating natural killer cells that was achieved by virus-induced cytokine production from cells infected with N1L-deleted, but not N1L-intact VV. This effect was further enhanced by arming VVΔTKΔN1L with IL-12, a potent antitumor cytokine. Five daily treatments with VVΔTKΔN1L-IL12 before surgery dramatically improved postsurgical survival. VVΔTKΔN1L armed with human IL-12 completely prevented tumor recurrence in surgical models of head and neck cancer in Syrian hamsters. CONCLUSIONS: These data provide a proof of concept for translation of the regime into clinical trials. VVΔTKΔN1L-IL12 is a promising agent for use as an adjuvant to surgical treatment of solid tumors.
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Imunidade/imunologia , Neoplasias Pulmonares/prevenção & controle , Recidiva Local de Neoplasia/prevenção & controle , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/genética , Neoplasias Pancreáticas/prevenção & controle , Vaccinia virus/genética , Adjuvantes Imunológicos/administração & dosagem , Animais , Apoptose , Proliferação de Células , Feminino , Humanos , Interleucina-12/administração & dosagem , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/secundário , Neoplasias Pulmonares/cirurgia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Recidiva Local de Neoplasia/imunologia , Recidiva Local de Neoplasia/patologia , Recidiva Local de Neoplasia/cirurgia , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/cirurgia , Células Tumorais Cultivadas , Microambiente Tumoral/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Our previous studies have shown that Docetaxel (DTX) and Tamoxifen (TMX) loaded nanoparticles(Co-NPs) could exhibit a synergistic effect on estrogen receptor positive cell lines. In the current studyï¼we have studied the synergistic effect of Co-NPs and underlying possible molecular mechanism. METHODS: Cell apoptosis assay, pharmacokinetic experiment and immunohistochemistry experiment were used to explore the synergistic effect and underlying possible mechanism in vitro and in vivo. RESULTS: Cell apoptosis assay revealed that Co-NPs could mediate cell sensitization to a cytotoxic agent, resulting in remarkable cell apoptosis. In addition, pharmacokinetic experiment research showed that Co-NPs have longer circulation time in vivo, which could prolong the treatment time of the chemotherapeutic drugs. Immunohistochemistry experiment revealed that the Co-NPs could downregulate the expression of P-gp level to reduce the drugs' efflux. CONCLUSION: The possible mechanism of the synergistic effect of DTX and TMX by Co-NPs was attributed to the longer in vivo circulation time, significantly increased rate of cell apoptosis and downregulated expression of P-gp level to the tumor cells.
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Antineoplásicos/farmacologia , Docetaxel/farmacologia , Portadores de Fármacos/química , Nanopartículas/química , Tamoxifeno/farmacologia , Células A549 , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/sangue , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Docetaxel/administração & dosagem , Docetaxel/sangue , Sinergismo Farmacológico , Eritrócitos/efeitos dos fármacos , Receptor alfa de Estrogênio/genética , Hemólise/efeitos dos fármacos , Humanos , Células MCF-7 , Camundongos , Ratos , Tamoxifeno/administração & dosagem , Tamoxifeno/sangue , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Rationale: Cardenolides have potential as anticancer drugs. 3'-epi-12ß-hydroxyfroside (HyFS) is a new cardenolide structure isolated by our research group, but its molecular mechanisms remain poorly understood. This study investigates the relationship between its antitumor activities and autophagy in lung cancer cells. Methods: Cell growth and proliferation were detected by MTT, lactate dehydrogenase (LDH) release, 5-ethynyl-20-deoxyuridine (EDU) and colony formation assays. Cell apoptosis was detected by flow cytometry. Autophagic and signal proteins were detected by Western blotting. Markers of autophagy and autophagy flux were also detected by immunofluorescence, transmission electron microscopy and acridine orange staining. Real time RT-PCR was used to analyze the gene expression of Hsp90. Hsp90 ubiquitination was detected by coimmunoprecipitation. The antitumore activities of HyFS were observed in nude mice. Results: HyFS treatment inhibited cell proliferation and induced autophagy in A549 and H460 lung cancer cells, but stronger inhibition of cell proliferation and induction of cell apoptosis were shown when HyFS-mediated autophagy was blocked. The Hsp90/Akt/mTOR axis was found to be involved in the activation of HyFS-mediated autophagy. Evidence of direct interaction between Hsp90 and Akt was observed. HyFS treatment resulted in decreased levels of heat shock protein 90 (Hsp90) and phosphorylated Akt, overexpression of Hsp90 increased activation of autophagy, and inhibition of Hsp90 expression decreased autophagy. In addition, ubiquitin-mediated degradation of Hsp90 and subsequent dephosphorylation of its client protein Akt were also found in HyFS-treated lung cancer cells. Moreover, combination treatment with HyFS and chloroquine showed remarkably increased tumor inhibition in both A549- and H460-bearing mice. Conclusion: Our results demonstrate that HyFS induced cytoprotective autophagy through ubiquitin-mediated degradation of Hsp90, which further blocked the Akt/mTOR pathway in lung cancer cells. Thus, a combination of a HyFS-like cardenolide and an autophagic inhibitor is a potential alternative approach for the treatment of lung cancer.
Assuntos
Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Cardenolídeos/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Animais , Antineoplásicos/administração & dosagem , Western Blotting , Cardenolídeos/administração & dosagem , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Citometria de Fluxo , Imunofluorescência , Formazans/análise , Perfilação da Expressão Gênica , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Microscopia Eletrônica de Transmissão , Transplante de Neoplasias , Proteína Oncogênica v-akt/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Coloração e Rotulagem , Serina-Treonina Quinases TOR/metabolismo , Sais de Tetrazólio/análise , Resultado do TratamentoRESUMO
OBJECTIVES: Coroglaucigenin (CGN), a natural product isolated from Calotropis gigantean by our research group, has been identified as a potential anti-cancer agent. However, the molecular mechanisms involved remain poorly understood. MATERIALS AND METHODS: Cell viability and cell proliferation were detected by MTT and BrdU assays. Flow cytometry, SA-ß-gal assay, western blotting and immunofluorescence were performed to determine CGN-induced apoptosis, senescence and autophagy. Western blotting, siRNA transfection and coimmunoprecipitation were carried out to investigate the mechanisms of CGN-induced senescence and autophagy. The anti-tumour activities of combination therapy with CGN and chloroquine were observed in mice tumour models. RESULTS: We demonstrated that CGN inhibits the proliferation of colorectal cancer cells both in vitro and in vivo. We showed that the inhibition of cell proliferation by CGN is independent of apoptosis, but is associated with cell-cycle arrest and senescence in colorectal cancer cells. Notably, CGN induces protective autophagy that attenuates CGN-mediated cell proliferation. Functional studies revealed that CGN disrupts the association of Hsp90 with both CDK4 and Akt, leading to CDK4 degradation and Akt dephosphorylation, eventually resulting in senescence and autophagy, respectively. Combination therapy with CGN and chloroquine resulted in enhanced anti-tumour effects in vivo. CONCLUSIONS: Our results demonstrate that CGN induces senescence and autophagy in colorectal cancer cells and indicate that combining it with an autophagy inhibitor may be a novel strategy suitable for CGN-mediated anti-cancer therapy.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Produtos Biológicos/farmacologia , Cardenolídeos/farmacologia , Proliferação de Células/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Animais , Calotropis/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cloroquina/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 4 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/metabolismo , Quimioterapia Combinada , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas Associadas aos Microtúbulos/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismoRESUMO
The originally published version of this Article contained errors in Figure 4. In panel b, the square and diamond labels associated with the uppermost survival curve were incorrectly displayed as 'n' and 'u', respectively. These errors have now been corrected in both the PDF and HTML versions of the Article.
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Interleukin-12 (IL-12) has emerged as one of the most potent agents for anti-tumor immunotherapy. However, potentially lethal toxicity associated with systemic administration of IL-12 precludes its clinical application. Here we redesign the molecule in such a way that its anti-tumor efficacy is not compromised, but toxic effects are eliminated. Deletion of the N-terminal signal peptide of IL-12 can effect such a change by preventing IL-12 secretion from cells. We use a newly designed tumor-targeted oncolytic adenovirus (Ad-TD) to deliver non-secreting (ns) IL-12 to tumor cells and examine the therapeutic and toxic effects in Syrian hamster models of pancreatic cancer (PaCa). Strikingly, intraperitoneal delivery of Ad-TD-nsIL-12 significantly enhanced survival of animals with orthotopic PaCa and cured peritoneally disseminated PaCa with no toxic side effects, in contrast to the treatment with Ad-TD expressing unmodified IL-12. These findings offer renewed hope for development of IL-12-based treatments for cancer.
Assuntos
Antineoplásicos/farmacologia , Imunoterapia/métodos , Interleucina-12/imunologia , Interleucina-12/farmacologia , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/genética , Neoplasias Pancreáticas/tratamento farmacológico , Adenoviridae/genética , Animais , Antineoplásicos/efeitos adversos , Antineoplásicos/química , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Cricetinae , Modelos Animais de Doenças , Feminino , Técnicas de Transferência de Genes , Humanos , Interleucina-12/efeitos adversos , Interleucina-12/química , Neoplasias Pancreáticas/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
MicroRNAs (miRNAs), a class of small noncoding RNA molecules, can manipulate the expressions of endogenous tumor-related genes, and are implicated in the development and progression of a wide type of tumors. In this study, the investigation from real-time quantitative PCR revealed that miRNA-16-5p was downregulated in breast carcinoma tissues and cells, coupled with the elevations of HIF-α and VEGFA protein expressions, compared with normal tissues. Lentiviral armed with miR-16-5p markedly increased the miR-16-5p levels in MCF-7 and MDA-MB-231 cells, compared to blank and NC groups, and miR-16-5p overexpression significantly inhibited the proliferation and colony formation in MCF-7 and MDA-MB-231 cells. Besides, miR-16-5p upregulation markedly induced apoptosis and reduced invasion ability in MCF-7 and MDA-MB-231 cells. Notably, VEGFA was direct target of miR-16-5p. Stepwise investigation from in vitro and in vivo experiments demonstrated that miR-16-5p overexpression suppressed tumor growth and reduced HIF-α and VEGFA expressions in breast carcinoma cells and nude mice tumor tissues. These findings provide novel insights into molecular mechanism involved in the roles of miR-16-5p in tumor development and progression of breast carcinoma, and thus manipulation of miR-16-5p may be a novel potential therapeutic target for future therapies of the patients with breast carcinoma.
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Colorectal cancer is the most common cancer. It has high morbidity and mortality worldwide, and more effective treatment strategies need to be developed. Toxicarioside O (TCO), a natural product derived from Antiaris toxicaria, has been shown to be a potential anticancer agent. However, the molecular mechanisms involved remain poorly understood. In this study, our results demonstrated that TCO can induce both apoptosis and autophagy in colorectal cancer cells. Moreover, TCO-induced autophagy was due to the increase of the expression and activity of the enzyme sirtuin-1 (SIRT1), and subsequent inhibition of the Akt/mTOR pathway. Inhibition of SIRT1 activity by its inhibitor, EX-527, attenuated TCO-induced autophagy. Of interest, inhibition of autophagy by chloroguine, an autophagy inhibitor, enhanced TCO-induced apoptotic cell death, suggesting that autophagy plays a protective role in TCO-induced apoptosis. Together, these findings suggest that combination of TCO and autophagy inhibitor may be a novel strategy suitable for potentiating the anticancer activity of TCO for treatment of colorectal cancer.
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Docetaxel (DTX) and tamoxifen (TMX) are first-line drugs used to treat breast cancer. However when used in combination, they produce antagonism because of their differential metabolic pathways. In order to prevent this antagonism, an amphiphilic copolymer, cholesterol modified hyaruronic acid (HA-CHOL), was synthesized for investigating the co-delivery of TMX and DTX. In vitro drug release experiment of the Co-encapsulated (encapsulated DTX+TMX) nanoparticles (Co-NPs) revealed that NPs with unique release mechanism can markedly reduce the release of these drugs in the circulatory system. However, when reaching in cell, TMX can release rapidly to prevent DTX from coming into contact with metabolizing enzymes. In vitro cytotoxicity experiment revealed that the Co-NPs exhibited a significant synergistic effect for inhibiting the proliferation of the cancer cell lines A549, MCF7 and S180. NPs carrying Coumarin-6(Cou6) exhibited increased cellular uptake compared with Cou6 solution at similar drug concentrations. As an in vivo treatment of xenograft tumors involving 180 cells, the Co-NPs displayed a clear tumor-inhibiting effect. This led us to conclude that the reversion of drug antagonism by NPs was attributed to the increased stability of the nanoparticles in the blood circulation, the efficient cellular uptake, the hierarchical drug metabolism in the tumor and the good and orderly delivery of the drugs to the tumor tissue.
Assuntos
Antineoplásicos/farmacologia , Ácido Hialurônico/química , Nanopartículas/química , Polímeros/química , Tamoxifeno/farmacologia , Taxoides/farmacologia , Células A549 , Animais , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cumarínicos/farmacologia , Docetaxel , Feminino , Humanos , Células MCF-7 , Masculino , Camundongos , Tiazóis/farmacologiaRESUMO
Various angiogenesis-related self-molecules have been considered to be therapeutic targets. However, the direct use of self-molecules as vaccines is not recommended because of the inherent ability of the host to develop immune tolerance. Antigen 43 (Ag43) is a surface protein found in E. coli and contains an α and a ß subunits, which contains multiple T epitopes in α subunit. Here we construct a novel Ag43 surface display system (Ag43 system) to express Ag43 chimeric proteins to disrupt immune tolerance against self-molecules. The Ag43 system was constructed from an Escherichia coli strain Tan109, derived from JM109, in which the Ag43 gene was deleted and a recombinant plasmid (pETAg43') expressing a partial Ag43 gene was introduced. The extracellular domain of angiogenesis-related endoglin gene was then subcloned into plasmid pETAg43', resulting in a recombinant plasmid pETAg43'/END(e) which was then used to transform Tan109 for protein expression. We found that Ag43 and endoglin chimeric protein (Ag43'/END(e) ) was expressed on the bacterial surface. The chimeric protein could be separated from the bacterial surface by heating to 60°C and yet retain activity. We used Ag43'/END(e) as a protein vaccine and found that it could disrupt immune tolerance against endoglin by inducing significant antitumor activities and inhibit angiogenesis in several tumor models without significant side effects. These data suggest that Ag43'/END(e) chimeric protein is a potential model vaccine for active tumor immunotherapy, and that Ag43 system could be an effective tool for novel vaccine preparation to break immune tolerance to other angiogenesis-related self-molecules for cancer therapy.
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Adesinas de Escherichia coli/imunologia , Vacinas Anticâncer/imunologia , Carcinoma Pulmonar de Lewis/terapia , Tolerância Imunológica/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Neovascularização Patológica/terapia , Adesinas de Escherichia coli/genética , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Antígenos de Superfície/genética , Antígenos de Superfície/imunologia , Carcinoma Pulmonar de Lewis/imunologia , Endoglina , Epitopos de Linfócito T , Escherichia coli/genética , Escherichia coli/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neovascularização Patológica/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologiaRESUMO
Cytochalasin D (CytD) targets actin, a ubiquitous protein in eukaryotic cells. Previous studies have focused mainly on the antitumor effects of CytD. We previously found CytD to promote lung metastasis in B16 melanoma cells, which we had not anticipated, and, therefore, in the present study we investigated the possible underlying mechanisms. B16 melanoma cells were co-cultured with CytD and other agents and used to establish a lung metastatic model. In this B16 melanoma metastatic model, significantly increased lung metastasis and lung weight were found in CytD-treated mice, which was almost completely suppressed by tissue factor (TF) RNA interference expressed via lentivirus. The results of northern and western blot, and real-time RT-PCR analysis showed that the expression of TF was significantly upregulated in B16 cells treated with CytD but was significantly inhibited by TF RNA interference. In addition, upregulation and phosphorylation of mitogen-activated protein kinase p38 were also found in the metastatic lung tissues treated with CytD and in the B16 cells co-cultured with CytD and factor VIIa (FVIIa), but not in cells cultured with CytD, dimethyl sulfoxide or FVIIa alone. These results indicate that CytD stimulates the expression of TF in B16 melanoma cells, activating both coagulation-dependent and -independent pathways via binding to FVIIa, eventually promoting lung metastasis. TF interference is a potential approach to the prevention of B16 melanoma metastasis.
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Citocalasina D/farmacologia , Neoplasias Pulmonares/secundário , Melanoma Experimental/metabolismo , Tromboplastina/metabolismo , Animais , Linhagem Celular Tumoral , Fator VIIa/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Melanoma Experimental/genética , Camundongos , Camundongos Endogâmicos C57BL , Inibidores da Síntese de Ácido Nucleico/farmacologia , Fosforilação , Interferência de RNA , RNA Interferente Pequeno , Tromboplastina/biossíntese , Tromboplastina/genética , Regulação para Cima , Proteínas Quinases p38 Ativadas por Mitógeno/biossíntese , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Toxicarioside A is a cardenolide isolated mainly from plants and animals. Emerging evidence demonstrate that cardenolides not only have cardiac effects but also anticancer effects. In this study, we used in vivo models to investigate the antitumor activities of toxicarioside A and the potential mechanisms behind them. Murine colorectal carcinoma (CT26) and Lewis lung carcinoma (LL/2) models were established in syngeneic BALB/c and C57BL/6 mice, respectively. We found that the optimum effective dose of toxicarioside A treatment significantly suppressed tumor growth and angiogenesis in CT and LL/2 tumor models in vivo. Northern and Western blot analysis showed significant inhibition of endoglin expression in toxicarioside A-treated human umbilical vein endothelial cells (HUVECs) in vitro and tumor tissues in vivo. Toxicarioside A treatment significantly inhibited cell proliferation, migration and invasion, but did not cause significant cell apoptosis and affected other membrane protein (such as CD31 and MHC I) expression. In addition, TGF-ß expression was also significantly inhibited in CT26 and LL/2 tumor cells treated with toxicarioside A. Western blot analysis indicated that Smad1 and phosphorylated Smad1 but not Smad2/3 and phosphorylated Smad2/3 were attenuated in HUVECs treated with toxicarioside A. Smad1 and Smad2/3 signaling remained unchanged in CT26 and LL/2 tumor cells treated with toxicarioside A. Endoglin knockout by small interfering RNA against endoglin induced alternations in Smad1 and Smad2/3 signaling in HUVECs. Our results indicate that toxicarioside A suppresses tumor growth through inhibition of endoglin-related tumor angiogenesis, which involves in the endoglin/TGF-ß signal pathway.
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Glicosídeos Cardíacos/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neovascularização Patológica , Fator de Crescimento Transformador beta/metabolismo , Alginatos/química , Animais , Apoptose , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Endoglina , Células Endoteliais/citologia , Feminino , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Microcirculação , Modelos Químicos , Invasividade Neoplásica , Transplante de Neoplasias , Neoplasias/patologia , Fosforilação , RNA Interferente Pequeno/metabolismo , Transdução de SinaisRESUMO
Sonic hedgehog (Shh) is a morphogen critically involved in development that is reexpressed in atherosclerotic lesions. It also stimulates proliferation of vascular smooth muscle cells (SMCs). Autophagy in vascular SMCs is known to promote SMC survival and increase plaque stability. The aim of this study was to investigate whether Shh induces autophagy of vascular SMCs. Our study showed that both Shh protein and microtubule-associated protein 1 light chain 3 (LC3)-II were increased in SMCs within neointimal lesions of mouse common carotid arteries. In cultured mouse aortic SMCs, recombinant mouse Shh stimulated LC3-II levels. Overexpression of wild-type mouse Shh through the tetracycline-regulated expression-inducible system in human aortic SMCs time-dependently increased the levels of LC3-II and also stimulated protein kinase B (AKT) phosphorylation. Pretreatment with AKT inhibitor IV (AKTI IV) inhibited AKT phosphorylation and the increase in LC3-II. Shh-induced autophagy was further confirmed by the formation of autophagosomes as detected by immunostaining and transmission electron microscopy, which was inhibited by AKTI IV. Shh further increased SMC LC3-II in the presence of bafilomycin A1, (2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester, and pepstatin A or siRNA for the autophagy-related gene 7 (ATG7). In addition, Shh induced SMC proliferation, which was inhibited not only by AKTI IV but also by cyclopamine, an inhibitor of Shh receptor. Inhibition of autophagy with 3-methyladenine (3-MA), bafilomycin A1, or ATG7 siRNA resulted in inhibition of cell proliferation. Treatment with 3-MA, AKTI IV, or cyclopamine inhibited neointima formation in mouse common carotid arteries. Taken together, our results have shown that Shh induces autophagy of vascular SMCs involving AKT activation, suggesting a role of autophagy in Shh-induced cellular responses.
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Autofagia/fisiologia , Proteínas Hedgehog/metabolismo , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Animais , Autofagia/efeitos dos fármacos , Artérias Carótidas/efeitos dos fármacos , Artérias Carótidas/metabolismo , Artérias Carótidas/patologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Proteínas Hedgehog/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Animais , Músculo Liso Vascular/efeitos dos fármacos , Neointima/metabolismo , Neointima/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/farmacologia , Alcaloides de Veratrum/farmacologiaRESUMO
AIM: To investigate the inhibitory role of toxicarioside A on the gastric cancer cell line human gastric cancer cell line (SGC-7901) and determine the underlying molecular mechanism. METHODS: After SGC-7901 cells were treated with toxicarioside A at various concentrations (0.5, 1.5, 4.5, 9.0 µg/mL) for 24 h or 48 h, cell viability was determined by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay, and the motility and invasion of tumor cells were assessed by the Transwell chamber assay. Immunofluorescence staining, reverse transcription polymerase chain reaction and Western blotting were performed to detect the expression of basic fibroblast growth factor (bFGF) and fibroblast growth factor receptor-1 (FGFR1), and nuclear factor-kappa B (NF-κB) activation was examined by electrophoretic mobility shift assay. RESULTS: The results showed that toxicarioside A was capable of reducing cell viability, inhibiting cell growth, and suppressing cell migration and invasion activities in a time- and dose-dependent manner in SGC-7901 cells. Further analysis revealed that not only the expression of bFGF and its high-affinity receptor FGFR1 but also the NF-κB-DNA binding activity were effectively blocked by toxicarioside A in a dose-dependent manner compared with the control group (P < 0.05 or P < 0.01). Interestingly, application of the NF-κB specific inhibitor, pyrrolidinedithiocarbamate (PDTC), to SGC-7901 cells significantly potentized the toxicarioside A-induced down-regulation of bFGF compared with the control group (P < 0.05). CONCLUSION: These findings suggest that toxicarioside A has an anti-gastric cancer activity and this effect may be achieved partly through down-regulation of NF-κB and bFGF/FGFR1 signaling.
Assuntos
Antineoplásicos/farmacologia , Glicosídeos Cardíacos/farmacologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Neoplasias Gástricas/tratamento farmacológico , Cardenolídeos/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/efeitos dos fármacos , Humanos , NF-kappa B/efeitos dos fármacos , Invasividade Neoplásica , Prolina/análogos & derivados , Prolina/farmacologia , Neoplasias Gástricas/patologia , Tiocarbamatos/farmacologiaRESUMO
OBJECTIVE: To investigate whether cytochalasin D can induce antitumor activities in a tumor model. METHODS: Murine CT26 colorectal carcinoma cells were cultured in vitro and cytochalasin D was used as a cytotoxic agent to detect its capabilities of inhibiting CT26 cell proliferation and inducing cell apoptosis by MTT and a TUNEL-based apoptosis assay. Murine CT26 tumor model was established to observe the tumor growth and survival time. Tumor tissues were used to detect the microvessel density by immunohistochemistry. In addition, alginate encapsulated tumor cell assay was used to quantify the tumor angiogenesis in vivo. RESULTS: Cytochalasin D inhibited CT26 tumor cell proliferation in time and dose dependent manner and induced significant CT26 cell apoptosis, which almost reached the level induced by the positive control nuclease. The optimum effective dose of cytochalasin D for in vivo therapy was about 50 mg/kg. Cytochalasin D in vivo treatment significantly inhibited tumor growth and prolonged the survival times in CT26 tumor-bearing mice. The results of immunohistochemistry analysis and alginate encapsulation assay indicated that the cytochalasin D could effectively inhibited tumor angiogenesis. CONCLUSIONS: Cytochalasin D inhibits CT26 tumor growth potentially through inhibition of cell proliferation, induction of cell apoptosis and suppression of tumor angiogenesis.
Assuntos
Inibidores da Angiogênese/farmacologia , Antineoplásicos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Citocalasina D/farmacologia , Inibidores da Angiogênese/administração & dosagem , Animais , Antineoplásicos/administração & dosagem , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/irrigação sanguínea , Citocalasina D/administração & dosagem , Relação Dose-Resposta a Droga , Marcação In Situ das Extremidades Cortadas , Camundongos , Microvasos/efeitos dos fármacos , Neovascularização Patológica/prevenção & controle , Células Tumorais CultivadasRESUMO
OBJECTIVE: To investigate possible mechanism of toxicarioside A in HS-5 bone stromal cells. METHODS: HS-5 bone stromal cells were cultured in media supplemented with various concentrations of toxicarioside A or control DMSO (not treatment). Endoglin and TGF-ß were detected by Northern and Western blot analysis and quantified in a standard method. Downstream molecules of endoglin and TGF-ß (Smad1, Smad2 and their active phosphorylated counterparts, pSmad1 and pSmad2) were also detected and quantified by Western blot analysis. In addition, cell proliferation assay and small interfering RNA (siRNA) against endoglin were used to certificate the function of endolgin in the HS-5 cells. RESULTS: Compared with the not treated (0 µg/mL) or DMSO treated control HS-5 cells, HS-5 cells treated with toxicarioside A were found significant attenuation of endolgin and TGF-ß expression. Significant inhibition of cell proliferation was also found in the HS-5 cells treated with toxicarioside A. ALK1-related Smad1 and ALK5-related Smad2 were decreased in HS-5 cells treated with toxicarioside A. In addition, phosphorylated Smad1 (pSmad1) and Smad2 (pSmad2) were also found attenuation in toxicarioside A-treated HS-5 cells. RNA interference showed that blockage of endoglin by siRNA also decreased Smad1 and Smad2 expression in HS-5 cells. CONCLUSIONS: Our results indicate that toxicarioside A can influence bone marrow stromal HS-5's function and inhibit HS-5 cell proliferation by alteration of endoglin-related ALK1 (Smad1) and ALK5 (Smad2) signaling.
Assuntos
Antiaris , Células da Medula Óssea/efeitos dos fármacos , Cardenolídeos/farmacologia , Receptores de Superfície Celular/antagonistas & inibidores , Proteína Smad1/metabolismo , Proteína Smad2/metabolismo , Fator de Crescimento Transformador beta/antagonistas & inibidores , Antígenos CD/metabolismo , Northern Blotting , Western Blotting , Células da Medula Óssea/metabolismo , Linhagem Celular , Proliferação de Células , Endoglina , Humanos , Masculino , Receptores de Superfície Celular/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína Smad1/efeitos dos fármacos , Proteína Smad2/efeitos dos fármacos , Células Estromais/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismoRESUMO
Cytochalasin D targets actin and is ubiquitous in eukaryotic cells. When cytochalasin D is used as a cytotoxic agent in cancer therapy, it causes significant side effects. To prevent this, cytochalasin D can be encapsulated in polyethylene liposomes. In this study, high-performance liquid chromatography observation of the biodistribution of pegylated liposomal cytochalasin D in tumour-bearing mice showed that liposomal cytochalasin D could be conveniently dissolved in water for i.v. injection and that it specifically accumulated in tumour tissues, more than natural cytochalasin D did. The half-time of liposomal cytochalasin D in the plasma was also significantly longer than that of natural cytochalasin D (4h versus 10 min). MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that liposomal cytochalasin D treatment could cause significant inhibition of cell proliferation in vitro in a manner similar to that of natural cytochalasin D. The antitumour activities of liposomal cytochalasin D were investigated in B16 melanoma, CT26 colorectal carcinoma and H22 hepatoma models, and the results indicated that liposomal cytochalasin D could significantly inhibit tumour growth and prolong survival in a manner similar to that of cisplatin. TUNEL-based apoptosis assays showed that liposomal cytochalasin D induced significant tumour cell apoptosis. Significant inhibition of tumour angiogenesis was observed in mice treated with liposomal cytochalasin D. In addition, no significant side effects were observed in mice treated with liposomal cytochalasin D. Our results show that liposomal cytochalasin D increases solubility and bioavailability, a lower incidence of side effects and improves antitumour effects, indicating its potential as a chemical agent for cancer therapy.
Assuntos
Antineoplásicos/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Colorretais/tratamento farmacológico , Citocalasina D/farmacologia , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Melanoma Experimental/tratamento farmacológico , Polietilenoglicóis/farmacologia , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacocinética , Antineoplásicos/toxicidade , Apoptose/efeitos dos fármacos , Disponibilidade Biológica , Carcinoma Hepatocelular/irrigação sanguínea , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Química Farmacêutica , Neoplasias Colorretais/irrigação sanguínea , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Citocalasina D/administração & dosagem , Citocalasina D/análogos & derivados , Citocalasina D/farmacocinética , Citocalasina D/toxicidade , Relação Dose-Resposta a Droga , Meia-Vida , Injeções Intravenosas , Lipossomos , Neoplasias Hepáticas Experimentais/irrigação sanguínea , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Hepáticas Experimentais/patologia , Masculino , Melanoma Experimental/irrigação sanguínea , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neovascularização Patológica , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/farmacocinética , Polietilenoglicóis/toxicidade , Solubilidade , Distribuição Tecidual , Carga Tumoral/efeitos dos fármacosRESUMO
Induction of tumor vasculature occlusion by targeting a thrombogen to newly formed blood vessels in tumor tissues represents an intriguing approach to the eradication of primary solid tumors. In the current study, we construct and express a fusion protein containing vascular endothelial growth factor (VEGF) and tissue factor (TF) to explore whether this fusion protein has the capability of inhibiting tumor growth in a colon carcinoma model. The murine cDNA of VEGF A and TF were amplified by reverse transcriptase polymerase chain reaction (RT-PCR), and then cloned into prokaryotic expression plasmid pQE30 with a linker. The expression product recombinant VEGF-TF (rVEGF-TF) was purified and proved to have comparable enzyme activity to a commercial TF and the capability of specific binding to tumor vessels. Significant decrease of tumor growth was found in the mice administered with rVEGF-TF on Day 6 after initiated rVEGF-TF treatment (P<0.05), and the tumor masses in 2 of 10 mice were almost disappeared on Day 14 after the first treatment. In addition, valid thrombogenesis and tumor necrosis were observed in the tumor tissues injected with rVEGF-TF. Our results demonstrate that occlusion of tumor vasculature with rVEGF-TF is potentially an effective approach for cancer therapy.
Assuntos
Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/patologia , Proteínas Recombinantes de Fusão/uso terapêutico , Tromboplastina/uso terapêutico , Trombose/tratamento farmacológico , Fator A de Crescimento do Endotélio Vascular/uso terapêutico , Animais , Linhagem Celular Tumoral , Clonagem Molecular , Neoplasias do Colo/irrigação sanguínea , Modelos Animais de Doenças , Progressão da Doença , Expressão Gênica , Camundongos , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Plasmídeos/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Tromboplastina/genética , Tromboplastina/isolamento & purificação , Tromboplastina/metabolismo , Trombose/patologia , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/isolamento & purificação , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The immunization approaches with DNA vaccine priming and subsequent protein or peptide boosting has been widely tested in various models of infectious diseases. However, these approaches are seldom reported in the areas of cancer immunotherapy. In this study we combined endoglin plasmid DNA and recombinant protein as vaccines and used them to prime and boost, simultaneously, as a vaccine strategy. Our results showed that combination of endoglin DNA and protein vaccines could enhance both protective and therapeutic anti-tumor efficacy in both colon carcinoma and Lewis lung carcinoma models. Significant inhibition of tumor angiogenesis was found in the tumor tissues. The titers of autoantibodies against murine endoglin were significantly increased and the antibody levels lasted longer in the mice with combined endoglin DNA and recombinant protein vaccination. CTL response against endoglin-positive HUVECs, but not against endoglin-negative tumor cells was found in the mice combined DNA with protein vaccination. In addition, combination of endoglin DNA and recombinant protein vaccination significantly induced IFN-gamma secreting cells. These observations suggested that a combination of endoglin DNA and recombinant protein immunization as a vaccine strategy was superior to those using endoglin DNA or recombinant protein alone as vaccines.