RESUMEN
Increasing studies have suggested that some cardiac glycosides, such as conventional digoxin (DIG) and digitoxin, can induce immunogenic cell death (ICD) in various tumors. We previously found that 3'-epi-12ß-hydroxyfroside (HyFS), a novel cardenolide compound isolated by our group, could induce cytoprotective autophagy through inactivation of the Akt/mTOR pathway. However, whether HyFS can induce ICD remains unknown. In this study, we extend our work to further investigate whether HyFS could induce both autophagy and ICD, and we investigated the relationship between autophagy and ICD in three TNBC cell lines. Unexpectedly, compared to DIG, we found that HyFS could induce complete autophagy flux but not ICD in three human triple-negative breast cancer (TNBC) cell lines and one murine TNBC model. Inhibition of HyFS-induced autophagy resulted in the production of ICD in TNBC MDA-MB-231, MDA-MB-436, and HCC38 cells. A further mechanism study showed that formation of RIPK1/RIPK3 necrosomes was necessary for ICD induction in DIG-treated TNBC cells, while HyFS treatment led to receptor-interacting serine-threonine kinase (RIPK)1/3 necrosome degradation via an autophagy process. Additionally, inhibition of HyFS-induced autophagy by the autophagy inhibitor chloroquine resulted in the reoccurrence of ICD and reversion of the tumor microenvironment, leading to more significant antitumor effects in immunocompetent mice than in immunodeficient mice. These findings indicate that HyFS-mediated autophagic degradation of RIPK1/RIPK3 necrosomes leads to inactivation of ICD in TNBC cells. Moreover, combined treatment with HyFS and an autophagy inhibitor may enhance the antitumor activities, suggesting an alternative therapeutic for TNBC treatment.
Asunto(s)
Neoplasias de la Mama Triple Negativas , Animales , Humanos , Ratones , Apoptosis , Autofagia , Línea Celular Tumoral , Muerte Celular Inmunogénica , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Microambiente TumoralRESUMEN
Toxicarioside O (TCO), a natural product derived from Antiaris toxicaria, has been identified to be a promising anticancer agent. In this study, we aimed to investigate the effect of TCO on the proliferation and epithelial-mesenchymal transition (EMT) of lung cancer cells and its molecular mechanisms. Here, we indicated that TCO inhibits the proliferation of lung cancer cells both in vitro and in vivo. Our results demonstrated that TCO induces apoptosis in lung cancer cells. Moreover, we found that TCO suppresses EMT program and inhibits cell migration in vitro. Mechanistically, TCO decreases the expression of trophoblast cell surface antigen 2 (Trop2), resulting in inhibition of the PI3K/Akt pathway and EMT program. Overexpression of Trop2 rescues TCO-induced inhibition of cell proliferation and EMT. Our findings demonstrate that TCO markedly inhibits cell proliferation and EMT in lung cancer cells and provides guidance for its drug development.
RESUMEN
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.
Asunto(s)
Antineoplásicos/farmacología , Autofagia/efectos de los fármacos , Cardenólidos/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Animales , Antineoplásicos/administración & dosificación , Western Blotting , Cardenólidos/administración & dosificación , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Formazáns/análisis , Perfilación de la Expresión Génica , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Ratones Endogámicos BALB C , Ratones Desnudos , Microscopía Electrónica de Transmisión , Trasplante de Neoplasias , Proteína Oncogénica v-akt/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Coloración y Etiquetado , Serina-Treonina Quinasas TOR/metabolismo , Sales de Tetrazolio/análisis , Resultado del TratamientoRESUMEN
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.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Productos Biológicos/farmacología , Cardenólidos/farmacología , Proliferación Celular/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Animales , Calotropis/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Cloroquina/farmacología , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 4 Dependiente de la Ciclina/genética , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quimioterapia Combinada , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proteínas Asociadas a Microtúbulos/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismoRESUMEN
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.
RESUMEN
Two azaphilonidal derivatives [penicilazaphilones B (1) and C (2)], have been isolated from the fermented products of marine fungus strain Penicillium sclerotiorum M-22, penicilazaphilones C was a new compound. The compound's structures were identified by the analysis of spectroscopic data including 1D and 2D NMR techniques (1H-NMR, 13C-NMR, COSY, HMQC, and HMBC). Biological evaluation revealed that penicilazaphilones B and C showed selective cytotoxicity against melanoma cells B-16 and human gastric cancer cells SGC-7901 with IC50 values of 0.291, 0.449 and 0.065, 0.720 mM, respectively, while exhibiting no significant toxicity to normal mammary epithelial cells M10 at the same concentration. Moreover, penicilazaphilones C also exhibited strong antibacterial activity against Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumonia and Escherichia coli with MIC values 0.037-0.150 mM, while penicilazaphilones B's bacteriostatic action was weaker.
Asunto(s)
Antibacterianos/farmacología , Antineoplásicos/farmacología , Benzopiranos/farmacología , Penicillium , Pigmentos Biológicos/farmacología , Animales , Antibacterianos/química , Antibacterianos/aislamiento & purificación , Antineoplásicos/química , Antineoplásicos/aislamiento & purificación , Benzopiranos/química , Benzopiranos/aislamiento & purificación , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Relación Dosis-Respuesta a Droga , Hongos , Humanos , Melanoma Experimental , Ratones , Pruebas de Sensibilidad Microbiana/métodos , Pigmentos Biológicos/química , Pigmentos Biológicos/aislamiento & purificaciónRESUMEN
The combination of several potential strategies so as to develop new tumor vaccines is an attractive field of translational medicine. Pulsing tumor lysates with dendritic cells (DCs), in-vivo attraction of DCs by macrophage inflammatory protein 3α (MIP-3α), and reversion of the tumor suppressive microenvironment have been tested as strategies to develop tumor vaccines. In this study, we generated an alginate microsphere (named PaLtTcAdMIP3α) that encapsulated tumor lysates, live tumor cells engineering with a recombinant MIP-3α adenovirus and BCG. We used PaLtTcAdMIP3α as a model vaccine to test its antitumor activities. Our results showed that PaLtTcAdMIP3α expressed and excreted MIP-3α, which effectively attracted DCs ex vivo and in vivo. Injection of PaLtTcAdMIP3α into tumor-bearing mice effectively induced both therapeutic and prophylactic antitumor immunities in CT26, Meth A, B16-F10 and H22 models, but without any ensuing increase in adverse effects. Both tumor-specific cellular and humoral immune responses, especially the CD8(+) T cell-dependent cytotoxic T immunity, were found in the mice injected with PaLtTcAdMIP3α. The anti-tumor activity was abrogated completely by depletion of CD8(+) and partially by CD4(+) T lymphocytes. In addition, the number of IFN-γ-producing CD8(+) T cells in spleen and tumor tissues was significantly increased; but the number of CD4(+)CD25(+)FOXP3(+) regulatory T cells (Treg) in tumor tissues was decreased. These data strongly suggest that a combination of multi-current-using strategies such as the novel approach of using our PaLtTcAdMIP3α microspheres could be an effective tumor model vaccine.
Asunto(s)
Vacunas contra el Cáncer/administración & dosificación , Quimiocina CCL20/inmunología , Composición de Medicamentos , Animales , Línea Celular Tumoral , Ratones , Microambiente TumoralRESUMEN
The IgE Fcε3 domain is an active immunotherapeutic target for asthma and other allergic diseases. However, previous methods for preparing IgE fusion protein vaccines are complex. Antigen 43 (Ag43) is a surface protein found in Escherichia coli that contains α and ß subunits (the α subunit contains multiple T epitopes). Here we constructed a novel Ag43 surface display system (Ag43 system) to express Ag43 chimeric proteins to disrupt immune tolerance against IgE. The Ag43 system was constructed from the E. coli strain Tan109, in which the Ag43 gene was deleted and a recombinant plasmid (pETAg43) expressing a partial Ag43 gene was introduced. The Fcε3 domain of the IgE gene was then subcloned into plasmid pETAg43, resulting in a recombinant plasmid pETAg43/Fcε3, which was used to transform Tan109 for Ag43/Fcε3 surface expression. Thereafter, Ag43/Fcε3 was investigated as an asthma vaccine in a mouse model. Ag43/Fcε3 was expressed on and could be separated from the bacterial surface by heating to 60° while retaining activity. Ag43/Fcε3, as a protein vaccine, produced neutralizing autoantibodies to murine IgE, induced significant anti-asthma effects, and regulated IgE and T helper cytokines in a murine asthma model. Data show that Ag43/Fcε3 chimeric protein is a potential model vaccine for asthma treatment, and that the Ag43 system may be an effective tool for novel vaccine preparation to break immune tolerance to other self-molecules.
Asunto(s)
Adhesinas de Escherichia coli/inmunología , Asma/prevención & control , Hiperreactividad Bronquial/prevención & control , Receptores de IgE/inmunología , Vacunas Sintéticas/inmunología , Adhesinas de Escherichia coli/biosíntesis , Adhesinas de Escherichia coli/genética , Traslado Adoptivo , Animales , Anticuerpos Neutralizantes/sangre , Asma/sangre , Asma/inmunología , Asma/fisiopatología , Autoanticuerpos/sangre , Hiperreactividad Bronquial/sangre , Hiperreactividad Bronquial/inmunología , Hiperreactividad Bronquial/fisiopatología , Broncoconstricción , Células Cultivadas , Clonación Molecular , Citocinas/metabolismo , Modelos Animales de Enfermedad , Histamina/metabolismo , Tolerancia Inmunológica , Inmunoglobulina E/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Ovalbúmina/inmunología , Receptores de IgE/biosíntesis , Receptores de IgE/genética , Proteínas Recombinantes de Fusión/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Factores de Tiempo , Vacunas Sintéticas/biosíntesis , Vacunas Sintéticas/genéticaRESUMEN
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.
Asunto(s)
Adhesinas de Escherichia coli/inmunología , Vacunas contra el Cáncer/inmunología , Carcinoma Pulmonar de Lewis/terapia , Tolerancia Inmunológica/inmunología , Péptidos y Proteínas de Señalización Intracelular/inmunología , Neovascularización Patológica/terapia , Adhesinas de Escherichia coli/genética , Animales , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Antígenos de Superficie/genética , Antígenos de Superficie/inmunología , Carcinoma Pulmonar de Lewis/inmunología , Endoglina , Epítopos de Linfocito T , Escherichia coli/genética , Escherichia coli/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Neovascularización Patológica/inmunología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunologíaRESUMEN
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.
Asunto(s)
Citocalasina D/farmacología , Neoplasias Pulmonares/secundario , Melanoma Experimental/metabolismo , Tromboplastina/metabolismo , Animales , Línea Celular Tumoral , Factor VIIa/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Melanoma Experimental/genética , Ratones , Ratones Endogámicos C57BL , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Fosforilación , Interferencia de ARN , ARN Interferente Pequeño , Tromboplastina/biosíntesis , Tromboplastina/genética , Regulación hacia Arriba , Proteínas Quinasas p38 Activadas por Mitógenos/biosíntesis , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Novel antibacterial agents against Mycobacterium tuberculosis (MTB) are crucial due to the high infection and mortality rates associated with the disease. Our previous study confirmed that aptamers from a whole bacterium obtained by the Systematic Evolution of Ligands by Exponential Enrichment method specifically bound to the MTB virulent strain (H37Rv). In the present study, the function of aptamers against MTB in a mouse model was further determined. It was demonstrated that the NK2 aptamer has marked inhibitory effects on the adhesion/invasion of H37Rv to macrophages in vitro and stimulates intracellular IFN-γ production in CD4+ T-cells. The aptamer pool exhibited the strongest inhibitory effect on H37Rv adhesion/invasion to CD8+ T-cells in vitro compared with all aptamers-treated and control groups. Histopathological examination of lung biopsy specimens revealed a correlation between aptamer presence and lower pulmonary alveoli fusion, swelling and more prominent air spaces. Acid-fast staining of biopsy specimens from the lungs of the mice demonstrated parallel treatment effects. Results of the present study indicate that the 10th pool aptamers and NK2 may play an active role against H37Rv, however, the effect was different in vivo and in vitro. The treatment effect of 10th pool aptamers was found to be better in comparison to NK2 in vivo. Additional target sites involved in pathogenicity of H37Rv were also revealed and the NK2 binding site and aptamers, including the 10th pool aptamers, may antagonize these sites. Further studies are required to screen for other valuable aptamers which may be used as therapeutic drugs in combination with NK2.
Asunto(s)
Aptámeros de Nucleótidos/farmacología , ADN de Cadena Simple/química , Mycobacterium tuberculosis/efectos de los fármacos , Animales , Aptámeros de Nucleótidos/química , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/microbiología , Modelos Animales de Enfermedad , Interferón gamma/metabolismo , Pulmón/microbiología , Pulmón/patología , Macrófagos/metabolismo , Macrófagos/microbiología , Ratones , Ratones Endogámicos C57BL , Mycobacterium tuberculosis/patogenicidad , Técnica SELEX de Producción de AptámerosRESUMEN
OBJECTIVE: To observe the immune response induced by complex gene vaccine pcSAG1-ROP5 of Toxoplasma gondii in mice. METHODS: The recombinant eukaryotic expression plasmids pcSAG1, pcROP5 and pcSAG1-ROP5 were constructed and identified by PCR, restriction enzyme digestion, and sequencing. The three recombinant plasmids were transfected into HeLa cells to express in vitro and identified by Western blotting analysis. Seventy Kunming mice were randomly divided into 5 groups with 14 each, i.e. pcSAG1 group, pcROP5 group, pcSAG1-ROP5 group, blank plasmid group and PBS control group. The mice were immunized intramuscularly with pcSAG1, pcROP5, pcSAG1-ROP5, pcDNA3.1, and PBS, respectively, every two weeks for three times. Sera were collected before each injection and 2 weeks after the last immunization. The titer of mice serum in pcSAG1-ROP5 group combined with recombinant protein SAG1, ROP5 and SAG1-ROP5 and the level of IgG against T. gondii in 5 groups were determined by ELISA. Three weeks after the last immunization, ten mice of each group were challenged with 10(3) tachyzoites of the virulent T. gondii RH strain to observe the survival time. One week later, the rest four mice in each group were sacrificed and the supernatant of cultured splenocytes was collected for the detection of IFN-gamma and IL-4. RESULTS: Western blotting showed that the recombinant plasmids pcSAG1, pcROP5 and pcSAG1-ROP5 were expressed in HeLa cells with M(r) 31 000, 57 000, and 88 000, respectively. The serum titer in pcSAG1-ROP5 group combined with SAG1, ROP5 and SAG1-ROP5 was 1:320, 1:160, and 1:2560, respectively. The IgG level kept rising in pcSAG1, pcROP5 and pcSAG1-ROP5 groups. Two weeks after the last immunization, the IgG level in pcSAG1-ROP5 group was higher than those in other groups (P<0.05). After a lethal challenge of T. gondii RH strain, the survival time of the mice in pcSAG1-ROP5 group was (288 +/- 7) h, which was 48 h and 96h longer than the groups of pcSAG1 and pcROP5, respectively (P< 0.05). Four weeks after the last immunization, IFN-gamma in splenocyte culture of pcSAG1-ROP5 group [(908.52 +/- 6.31) pg/ml] was higher than other groups (P<0.05), with no significant difference in IL-4 (P>0.05). CONCLUSION: Compared with the single gene vaccines pcSAG1 and pcROP5, higher levels of IgG and IFN-gamma and longer survival time are observed in mice immunized with pcSAG1-ROP5.
Asunto(s)
Vacunas Antiprotozoos/inmunología , Toxoplasma/inmunología , Toxoplasmosis Animal/inmunología , Vacunas de ADN/inmunología , Animales , Anticuerpos Antiprotozoarios/sangre , Antígenos de Protozoos/inmunología , Femenino , Células HeLa , Humanos , Inmunización , Inmunoglobulina G/sangre , Interferón gamma/inmunología , Ratones , Ratones Endogámicos , Plásmidos , Proteínas Protozoarias/inmunología , Proteínas Recombinantes/genética , Toxoplasma/genética , Toxoplasmosis Animal/prevención & control , TransfecciónRESUMEN
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.
Asunto(s)
Glicósidos Cardíacos/farmacología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neovascularización Patológica , Factor de Crecimiento Transformador beta/metabolismo , Alginatos/química , Animales , Apoptosis , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Endoglina , Células Endoteliales/citología , Femenino , Ácido Glucurónico/química , Ácidos Hexurónicos/química , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Microcirculación , Modelos Químicos , Invasividad Neoplásica , Trasplante de Neoplasias , Neoplasias/patología , Fosforilación , ARN Interferente Pequeño/metabolismo , Transducción de SeñalRESUMEN
OBJECTIVE: To study the antitumor efficacy of an immunoconjugate composed of adriamycin (ADM) and downsized Fab fragment of mouse anti-Endoglin monoclonal antibody. METHODS: The Fab fragment of mouse anti-Endoglin monoclonal antibody was downsized and conjugated with ADM by m-Maleimidobenzoyl-N-hydroxysuccinimide ester (MBS). The antitumor effect of the conjugate was tested in mice bearing subcutaneous injection of H22 tumor in vivo. RESULTS: The molecular ratio of Fab:ADM in conjugate was approximately 1:2. The Fab-ADM conjugate inhibited the growth of H22 by 91.94% on day 14 after injection at the dose of 0.4 mg/ kg, much higher the inhibition rate of 25.00% by the equivalent dose of free ADM. The median survival time of the mice treated with the conjugate was longer than those treated with free ADM. The Fab-ADM conjugate was significantly more effective than free ADM in tumor suppression and life span prolongation. CONCLUSION: Fab -ADM displayed more significant antitumor efficacy than free ADM in vivo and might be a novel candidate for cancer treatment.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Doxorrubicina/farmacología , Inmunotoxinas/farmacología , Péptidos y Proteínas de Señalización Intracelular/inmunología , Animales , Antibióticos Antineoplásicos/farmacología , Endoglina , Masculino , Ratones , Neoplasias Experimentales/tratamiento farmacológicoRESUMEN
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.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma Hepatocelular/tratamiento farmacológico , Neoplasias Colorrectales/tratamiento farmacológico , Citocalasina D/farmacología , Neoplasias Hepáticas Experimentales/tratamiento farmacológico , Melanoma Experimental/tratamiento farmacológico , Polietilenglicoles/farmacología , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacocinética , Antineoplásicos/toxicidad , Apoptosis/efectos de los fármacos , Disponibilidad Biológica , Carcinoma Hepatocelular/irrigación sanguínea , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Química Farmacéutica , Neoplasias Colorrectales/irrigación sanguínea , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Citocalasina D/administración & dosificación , Citocalasina D/análogos & derivados , Citocalasina D/farmacocinética , Citocalasina D/toxicidad , Relación Dosis-Respuesta a Droga , Semivida , Inyecciones Intravenosas , Liposomas , Neoplasias Hepáticas Experimentales/irrigación sanguínea , Neoplasias Hepáticas Experimentales/metabolismo , Neoplasias Hepáticas Experimentales/patología , Masculino , Melanoma Experimental/irrigación sanguínea , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Neovascularización Patológica , Polietilenglicoles/administración & dosificación , Polietilenglicoles/farmacocinética , Polietilenglicoles/toxicidad , Solubilidad , Distribución Tisular , Carga Tumoral/efectos de los fármacosRESUMEN
To investigate the association between apolipo-protein E (APOE) polymorphisms and insulin resistance and Traditional Chinese Medicine (TCM) syndromes in type 2 diabetes mellitus (T2DM) with macroangiopathy, 60 patients with T2DM macroangiopathy were enrolled and divided into three groups: dryness-heat due to deficiency of yin, Qi-Yin deficiency, and Yin-Yang deficiency, according to the TCM syndromes, with a control group of 20 healthy individuals. APOE genotype analysis was performed with polymerase chain reaction amplification and restriction fragment length polymorphism, and the results showed that the proportion of the ε4/4 and ε3/4 genotypes and frequencies of the ε4 and ε3 alleles were higher in the Qi-Yin deficiency group (P<0.05). Among the T2DM macroangiopathy patients, the E4 group had the largest number of cases, as well as a significantly longer disease course compared to the E2 group (P<0.05). The insulin resistance index (IRI), insulin action index and body mass index (BMI) of patients in the Yin-Yang deficiency group were significantly different from those of patients with dryness-heat due to deficiency of yin and Qi-Yin deficiency. Furthermore, correlation analysis of the BMI and IRI of patients in the Yin-Yang deficiency group revealed a correlation coefficient r=0.696 (P<0.01) and a typical correlation between them. In conclusion, the Qi-Yin deficiency in T2DM patients with macroangiopathy is associated with the APOE E4 and E3 genotypes. Thus, the APOE gene polymorphism can, to some degree, reflect the TCM syndrome types of T2DM patients with macroangiopathy. Insulin resistance plays an important role in the occurrence of T2DM macroangiopathy and is closely associated with the Yin-Yang deficiency according to the TCM differentiating types.
Asunto(s)
Apolipoproteínas E/genética , Diabetes Mellitus Tipo 2/genética , Angiopatías Diabéticas/genética , Resistencia a la Insulina , Polimorfismo Genético , Anciano , Índice de Masa Corporal , Diabetes Mellitus Tipo 2/complicaciones , Angiopatías Diabéticas/complicaciones , Femenino , Frecuencia de los Genes , Genotipo , Humanos , Masculino , Medicina Tradicional China , Persona de Mediana Edad , Isoformas de Proteínas/genética , Síndrome , Deficiencia Yang/clasificación , Deficiencia Yang/complicaciones , Deficiencia Yang/genética , Deficiencia Yin/clasificación , Deficiencia Yin/complicaciones , Deficiencia Yin/genéticaRESUMEN
Interleukin-5 (IL-5) involves in the development of airway inflammation and hyperresponsiveness through activation of eosinophils. Thus, inhibition of IL-5 expression seems to be an attractive approach for asthma therapy. In this study, an antisense IL-5 gene transferred by recombinant adeno-associated virus (asIL-5) was constructed to transfect murine allergic asthma model. Our results showed that asIL-5 efficiently inhibited the IL-5 mRNA expression and significantly attenuated the inflammation in lung tissues. Significant decreasing of eosinophils and inflammatory cells were found in peripheral blood and bronchoalveolar lavage fluid (BALF). In addition, significant inhibition of airway hyperresponsiveness (AHR) was also found in the mice treated with asIL-5. These observations demonstrate that antisense oligonucleotid against IL-5 delivered by adeno-associated virus system is possibly an efficacious therapeutic strategy for allergic asthma and other eosinophil-related disorders.
Asunto(s)
Asma/terapia , Movimiento Celular/inmunología , Eosinófilos/inmunología , Interleucina-5/antagonistas & inhibidores , Oligodesoxirribonucleótidos Antisentido , Animales , Asma/genética , Asma/inmunología , Dependovirus , Modelos Animales de Enfermedad , Humanos , Interleucina-5/genética , Interleucina-5/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Transducción GenéticaRESUMEN
Interleukin-5 (IL-5) accompanies the development of airway inflammation and hyperresponsiveness through the activation of eosinophils. Therefore, interference of IL-5 expression in lung tissue seems to be an accepted approach in asthma therapy. In this study, we designed a small interfering RNA (siRNA) to inhibit the expression of IL-5. The siRNAs against IL-5 were constructed in a lentivirus expressing system, and 1.5x10(6) IFU (inclusion-forming unit) lentiviruses were administered intratracheally to ovalbumin (OVA)-sensitized murine asthmatic models. Our results show that lentivirus-delivered siRNA against IL-5 efficiently inhibited the IL-5 messenger ribonucleic acid (mRNA) expression and significantly attenuated the inflammation in lung tissue. Significant decrease of eosinophils and inflammatory cells were found in peripheral blood, bronchoalveolar lavage fluid (BALF), and lung tissue. In addition, significant inhibition of airway hyperresponsiveness (AHR) was found in the mice treated with siRNA against IL-5. These observations demonstrate that siRNA delivered by means of the lentivirus system is possibly an efficacious therapeutic approach for asthma.
Asunto(s)
Asma/inmunología , Asma/prevención & control , Hiperreactividad Bronquial/inmunología , Terapia Genética/métodos , Interleucina-5/inmunología , Neumonía/inmunología , Neumonía/prevención & control , ARN/uso terapéutico , Animales , Hiperreactividad Bronquial/prevención & control , Modelos Animales de Enfermedad , Humanos , Interleucina-5/genética , Masculino , Ratones , Ratones Endogámicos BALB C , Resultado del TratamientoRESUMEN
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.
Asunto(s)
Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/patología , Proteínas Recombinantes de Fusión/uso terapéutico , Tromboplastina/uso terapéutico , Trombosis/tratamiento farmacológico , Factor A de Crecimiento Endotelial Vascular/uso terapéutico , Animales , Línea Celular Tumoral , Clonación Molecular , Neoplasias del Colon/irrigación sanguínea , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Expresión Génica , Ratones , Ratones Endogámicos BALB C , Trasplante de Neoplasias , Plásmidos/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Tromboplastina/genética , Tromboplastina/aislamiento & purificación , Tromboplastina/metabolismo , Trombosis/patología , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/aislamiento & purificación , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
BACKGROUND: Eosinophils play a pivotal role in the generation of asthma inflammation. Interleukin (IL)-5 is the major activator of eosinophils. We hypothesize that modulating IL-5 activity could be an effective strategy for asthma therapy. In this study, we tested whether the plasmid encoding human IL-5 as a xenogeneic DNA vaccine could induce the production of autoantibodies, and be used for asthma treatment. METHODS: A eukaryotic plasmid encoding the human IL-5 was constructed, and used as a DNA vaccine. A mouse model of asthma was established to observe its antiasthma activities. Eosinophils in tissue, blood and the bronchoalveolar lavage were stained and counted. Airway hyperresponsiveness (AHR) was determined by whole body plethysmography. Antibody characters and cytokines were detected with immunological methods. RESULTS: Immunization with a plasmid encoding the human IL-5 as DNA vaccine reduced airway inflammation, reversed Th2 cytokines, and decreased AHR in mice. In addition, this immunization induced the production of polyclonal antibodies that were cross-reactive with native murine IL-5, and IgG1 and IgG2a were the major subclasses. Adoptive transfer of the purified antibodies from the sera of mice immunized with the plasmid encoding the human IL-5 resulted in similar antiasthma effects. CONCLUSIONS: Our results suggest that active vaccination against IL-5 may be a rational therapeutic approach for the treatment of asthma and potentially other eosinophilic disorders.