RESUMEN
We studied the effects of all-trans-retinoic acid (ATRA), C-phycocyanin (C-PC), or ATRA+C-PC on the growth of cervical cells (HeLa cells), cell cycle distribution, and apoptosis. The anticancer mechanism of the drug combination was revealed. MTT assay was adopted to determine the effects of C-PC and ATRA on the growth of HeLa cells. The expression quantities of cyclin-dependent kinase (CDK) 4, cyclin D1, Bcl-2, caspase-3, and CD59 were determined by in situ hybridization, immunofluorescence, immunohistochemistry staining, Western blot, and RT-PCR. TUNEL assay was adopted to determine the cellular apoptosis levels. Both C-PC and ATRA could inhibit the growth of HeLa cells, and the combination of ATRA+C-PC functioned cooperatively to induce apoptosis in HeLa cells. The dosage of ATRA was reduced when it cooperated with C-PC to reduce the toxicity. ATRA treated with C-PC could induce more cell cycle arrests than the single drug used by decrease in cyclin D1 and CDK4 expression. The combination of the two drugs could upregulate caspase-3 and downregulate the Bcl-2 gene and induce cell apoptosis. Moreover, the combination therapy has an important immunological significance in decreased expression of the CD59 protein. Singly, C-PC or ATRA could inhibit the growth of HeLa cells, and the effects of treatment were further enhanced in the combination group. In combination with C-PC, the dosage of ATRA was effectively reduced. The C-PC + ATRA combination might take effect by inhibiting the progress of the cell cycle, inducing cell apoptosis and promoting complement-mediated cytolysis.
Asunto(s)
Antineoplásicos/administración & dosificación , Ficocianina/administración & dosificación , Tretinoina/administración & dosificación , Apoptosis/efectos de los fármacos , Antígenos CD59/análisis , Caspasa 3/análisis , Proliferación Celular/efectos de los fármacos , Ciclina D1/análisis , Quinasa 4 Dependiente de la Ciclina/análisis , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Proteínas Proto-Oncogénicas c-bcl-2/análisisRESUMEN
In the present study, we investigated the effects of the combination of all-transretinoic acid (ATRA) and natural nontoxic C-phycocyanin (C-PC) on the growth of A549 lung cancer cells in vitro and in vivo. Furthermore, the anticancer mechanism of the drug combination was revealed. Results showed both C-PC and ATRA could inhibit the growth of A549 cells in vivo. The combination of ATRA+C-PC could yield a higher inhibition rate. C-PC exerted a major effect on the proliferation of human embryo lung cells, but ATRA at a high concentration exerted an inhibitory effect. In addition, ATRA+C-PC could decrease the CDK4 mRNA level, but upregulated caspase-3 protein expression and induced cell apoptosis. A mouse model with tumor was constructed by a subcutaneous injection to the left axilla of nu nude (NU/NU) mice. Compared with the control group, the tumor weight was decreased in the single-drug treatment group and was the lowest in the combination group. C-PC+ATRA could upregulate tumor necrosis factor levels and downregulate Bcl-2 expression and the cyclin D1 gene in the tumor. C-PC could promote T cells' activities and spleen weight, but a single use of ATRA exerted an opposite effect. The dosage of ATRA could be reduced when combined with C-PC to reduce the toxic side-effects. In summary, the antitumor effects of the C-PC+ATRA combination were more significant than a single drug in vivo and in vitro.
Asunto(s)
Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Sinergismo Farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Ficocianina/farmacología , Tretinoina/farmacología , Animales , Antineoplásicos/farmacología , Western Blotting , Quimioterapia Combinada , Humanos , Neoplasias Pulmonares/metabolismo , Ratones , Ratones Desnudos , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The anticancer effects and mechanism of all-trans retinoic acid (ATRA), C-phycocyanin (C-PC) or ATRA+C-PC on the growth of A549 cells were studied in in vitro and in vivo experiments. The effects of C-PC and ATRA on the growth of A549 cells were determined. The expression of CDK-4 and caspase-3, and the cellular apoptosis levels were detected. The tumor model was established by subcutaneous injection of A549 cells to the left axilla of the NU/NU mice. The weights of tumor and the spleen were tested. The viabilities of T-cells and spleen cells, TNF levels, the expression of Bcl-2 protein and Cyclin D1 gene were examined. Results showed both C-PC and ATRA could inhibit the growth of tumor cells in vivo and in vitro. ATRA+C-PC cooperatively showed a higher antitumor activity. The dosage of ATRA was reduced when it was administered with C-PC together, and the toxicity was reduced as well. ATRA+C-PC could decrease CDK-4 but increase caspase-3 protein expression level and induce cell apoptosis. ATRA alone could lower the activities of T lymphocytes and spleen weights, but the combination with C-PC could effectively promote viability of T cells and spleen. C-PC+ATRA could up-regulate TNF, and down-regulate Bcl-2 and Cyclin D1 gene. The combination might inhibit tumor growth by inhibiting the progress of cell cycle, inducing cell apoptosis and enhancing the body immunity.
Asunto(s)
Antineoplásicos , Protocolos de Quimioterapia Combinada Antineoplásica , Neoplasias Pulmonares/tratamiento farmacológico , Ficocianina , Tretinoina , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Caspasa 3/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Ciclina D1/genética , Quinasa 4 Dependiente de la Ciclina/genética , Sinergismo Farmacológico , Femenino , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Masculino , Ratones Desnudos , Ficocianina/administración & dosificación , Ficocianina/farmacología , Ficocianina/uso terapéutico , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Bazo/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Tretinoina/administración & dosificación , Tretinoina/farmacología , Tretinoina/uso terapéutico , Carga Tumoral/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The effects of C-phycocyanin (C-PC) on atherosclerosis and the regulatory effects of CD59 gene on anti-atherosclerotic roles of C-PC were investigated. Apolipoprotein E knockout (ApoE(-/-)) mice were randomly divided into four groups: control group, C-PC treatment group, CD59 transfection group and C-PC+CD59 synergy group. The mice were fed with high-fat-diet and treated with drug intervention at the same time. Results showed the atherosclerotic mouse model was successfully established. CD59 was over-expressed in blood and tissue cells. Single CD59 or C-PC could reduce blood lipid levels and promote the expression of anti-apoptotic Bcl-2 but inhibit pro-apoptotic Fas proteins in endothelial cells. The expression levels of cell cycle protein D1 (Cyclin D1) and mRNA levels of cyclin dependent protein kinase 4 (CDK4) in smooth muscle cells were restrained by CD59 and C-PC. CD59 or C-PC alone could inhibit the formation of atherosclerotic plaque by suppressing MMP-2 protein expression. In addition, C-PC could promote CD59 expression. So both CD59 and C-PC could inhibit the progress of atherosclerosis, and the anti-atherosclerotic effects of C-PC might be fulfilled by promoting CD59 expression, preventing smooth muscle cell proliferation and the apoptosis of endothelial cells, reducing blood fat levels, and at last inhibiting the development of atherosclerosis.
Asunto(s)
Aterosclerosis/mortalidad , Células Endoteliales/metabolismo , Miocitos del Músculo Liso/metabolismo , Ficocianina/farmacología , Placa Aterosclerótica/metabolismo , Animales , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Aterosclerosis/genética , Aterosclerosis/patología , Aterosclerosis/terapia , Antígenos CD59 , Proliferación Celular/efectos de los fármacos , Ciclina D1/biosíntesis , Ciclina D1/genética , Quinasa 4 Dependiente de la Ciclina/biosíntesis , Quinasa 4 Dependiente de la Ciclina/genética , Grasas de la Dieta/efectos adversos , Grasas de la Dieta/farmacología , Modelos Animales de Enfermedad , Células Endoteliales/patología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Ratones , Ratones Noqueados , Miocitos del Músculo Liso/patología , Placa Aterosclerótica/patología , Placa Aterosclerótica/terapia , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/genética , Receptor fas/biosíntesis , Receptor fas/genéticaRESUMEN
BACKGROUND: How to find an effective gene locus resistant to atherosclerosis has become a hotspot of today's medicine. Membrane attack complex (MAC) has proved to be related with the occurrence and development of atherosclerosis. Complement regulatory protein CD59 is a key regulator of complement MAC assembly. So this study aimed at discussing the effects of CD59 gene on occurrence and development of atherosclerosis and relative mechanism. METHODS: Apolipoprotein E knockout (ApoE (-/-)) mice were randomly divided into four groups: control group, empty plasmid-treated group, 0.5 ml CD59-treated group and 1.0 ml CD59-treated group. At the end of the 12th week, CD59 mRNA levels in whole blood were determined by RT-PCR and CD59 protein expressions were detected by western blot. The biochemical indexes in blood serum were detected. The paraffin sections of aortic root of mice were made and the degrees of atherosclerotic plaques formation were observed by hematoxylin/eosin (HE) staining. The expressions of cell apoptosis-related proteins (Bcl-2 and Fas) and plaque stability related protein (MMP-2) were detected by immunohistochemistry. Then the cell apoptosis levels were detected by TUNEL, the expression of Cyclin D1 and the mRNA level of cyclin dependent protein kinase 4 (CDK4) were detected by immunofluorescence and in situ hybridization, respectively. RESULTS: Atherosclerotic mouse model was successfully established. CD59 gene was overexpressed in blood cells and tissue cells after liposome transfection. CD59 could reduce blood lipid levels, promote the expression of anti-apoptotic Bcl-2 protein and inhibit pro-apoptotic Fas proteins, so finally lead to degradation of apoptosis levels of endothelial cells. In addition, Cyclin D1 protein and CDK4 mRNA levels were restrained by CD59 so as to inhibit the proliferation of smooth muscle cells. CD59 could inhibit the formation of atherosclerotic vulnerable plaque by suppressing the MMP-2 expression, which was further confirmed by HE staining. The anti-atherosclerotic effects were enhanced with the increase of CD59 gene dose. CONCLUSIONS: CD59 could lower blood lipid levels, positively regulate cell cycle, maintain the stability of cell proliferation and apoptosis of aorta cells, slow down the development of atherosclerotic vulnerable plaque, and finally inhibit the progress of atherosclerosis. So CD59 gene might be a new genetic locus for the therapy of atherosclerosis.