RESUMEN
OBJECTIVE: To investigate the effects of matrine on the anti-tumor efficiency of TIM2 gene-modified murine hepatocarcinoma H22 cells. METHODS: A combined eukaryotic expression vector pIRES2-EGFP-TIM2 was constructed and transfected into H22 cells by lipofectamin. The monoclone of positive H22-TIM2 cells and negative control H22-EGFP cells transfected with pIRES2-EGFP vector were selected by G418 pressure and limited dilution method in turn and were inoculated to establish the tumor-bearing mouse model. Next, matrine was administered to the tumor-bearing mice and the inhibitory effect of matrine was determined. RESULTS: The co-expression of EGFP protein and TIM2 gene was detected in H22 cells selected after TIM2 gene transfecion. After subcutaneous injection of H22-TIM2 cells, the rate of tumor formation (41%) was lower than that of H22 cells and H22-EGFP cells injection (92%) in mice. The tumor growth was significantly inhibited in mice vaccinated with H22-TIM2 cells. After the experiment was completed, the volume of tumors in mice of H22-TIM2 group was 31.34 +/- 9.21 mm3, smaller than those in H22-EGFP group (98.25 +/- 25.23)mm3 and H22 cells group (114.08 +/- 36.45)mm3 (P < 0.01). Matrine dramatically enhanced the anti-tumor efficiency of TIM2 gene-modified H22 cells, with the highest tumor inhibitory rate (IR) 90.6% among the H22-TIM2 group, matrine treatment group and H22-EGFP cells combined with matrine treatment group (69.2%, 67.5% and 70.8%, respectively) in the experimental mice. CONCLUSION: The tumorigenesity of H22 cells has been markedly impaired after modification by TIM2 gene. Matrine can enhance its inhibitory effect on tumors of H22-TIM2 cells in vivo. These data indicate importance to further study on the biological role of TIM2 gene in tumor immunity and explore the molecular mechanism of matrine in suppressing of tumor growth.
Asunto(s)
Alcaloides/farmacología , Antineoplásicos Fitogénicos/farmacología , Neoplasias Hepáticas Experimentales/patología , Proteínas de la Membrana/genética , Quinolizinas/farmacología , Carga Tumoral/efectos de los fármacos , Animales , Línea Celular Tumoral , Vectores Genéticos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Neoplasias Hepáticas Experimentales/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Trasplante de Neoplasias , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfección , MatrinasRESUMEN
OBJECTIVE: To investigate the effects of matrine on the anti-tumor efficiency of H22 murine hepatocarcinoma cell-based vaccine modified by TIM2 gene in vivo. METHOD: The combinant eukaryotic expression vector pIRES2-EGFP-TIM2 was constructed and transfected into H22 cells by lipofectamin. The monoclone of the positive H22-TIM2 cells and negative control H22-EGFP cells were selected by G418 pressure and limited dilution method in turn. The H22 whole-cell-based vaccine were inoculated to establish the tumor-bearing mouse model, and its oncogenicity and immunogenicity were observed in vivo. Then the matrine was administered to the tumor-bearing mice inoculated by H22-TIM2 cells, H22-EGFP cells and H22 cells, and the inhibitory effect of matrine on tumor was studied. RESULT: The co-expression of EGFP protein and TIM2 mRNA were detected in H22-TIM2 cells. The rate of tumor formation in mice injected of H22-TIM2 cells was 41%, lower than that of H22 cells and H22-EGFP cells injection (92%) in mice. The growth of tumor were significantly inhibited vaccinated with H22-TIM2 cells in mice. The inhibitory rate of tumor (IR) was 69.2% in mice of H22-TIM2 group, higher than that of mice treated with matrine and H22 cells injection, the later was 67.5%. Matrine could dramatically strengthen the anti-tumor efficiency of H22 cells modified by TIM2 gene, with the highest tumor inhibitory rate (IR) (90.6%) in all the experimental mice. The spleen index, populations of CD4-positive lymphocytes and the ratio of CD4-positive to CD8-positive lymphocytes of spleen in mice vaccinated of H22-TIM2 cells were obviously higher than those in the other groups. CONCLUSION: The oncogenicity of H22 cells is markedly impaired after modified by TIM2 gene. Matrine can strengthen the inhibitory effect of H22-TIM2 cells on tumor in mice. These data give us important clues to further study the biological role of TIM2 gene in tumor immunity and explore the molecular mechanism of matrine in suppressing tumor.
Asunto(s)
Alcaloides/farmacología , Antineoplásicos Fitogénicos/farmacología , Carcinoma Hepatocelular/tratamiento farmacológico , Expresión Génica/efectos de los fármacos , Proteínas de la Membrana/genética , Quinolizinas/farmacología , Alcaloides/administración & dosificación , Animales , Antineoplásicos Fitogénicos/administración & dosificación , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/inmunología , Carcinoma Hepatocelular/metabolismo , Línea Celular Tumoral , Femenino , Humanos , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Neoplasias Experimentales , Quinolizinas/administración & dosificación , Bazo/efectos de los fármacos , Bazo/inmunología , MatrinasRESUMEN
OBJECTIVE: To construct an eukaryotic expression vector of open reading frame of unknown KH gene (KH-ORF), and investigate its effect on cell proliferation. METHODS: The pCI-neo-KH-ORF expression vector was constructed by DNA recombinant technique and was introduced into COS-7 cells and K562 cells by lipofectactin-mediated DNA transfection. Expression of KH-ORF mRNA was detected by RT-PCR. The effect of KH-ORF on cell cycle of COS-7 cells and K562 cells was evaluated by flow cytometry (FCM). Effect on cell proliferation of COS-7 cells was tested by MTT assay and that on K562 cells was analyzed by growth curves and LDH activity measurement. RESULTS: (1) KH-ORF mRNA was expressed both in COS-7 cells and K562 cells. (2) The cell cycle and cell proliferation of COS-7 cells were unaffected significantly. (3) The proportion of cells in S phase was increased in pCI-neo-KH-ORF-transfected K562 cells; and growth curves and LDH activity indicated enhanced cell proliferation. CONCLUSION: KH gene may be a leukemia gene related to proliferation of K562 cells.
Asunto(s)
Proliferación Celular , Genes Relacionados con las Neoplasias/genética , Vectores Genéticos , Sistemas de Lectura Abierta/genética , Animales , Células COS , Chlorocebus aethiops , Regulación Neoplásica de la Expresión Génica , Genes Relacionados con las Neoplasias/fisiología , Humanos , Células K562 , L-Lactato Deshidrogenasa/metabolismo , Sistemas de Lectura Abierta/fisiología , Plásmidos , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Fase S , TransfecciónRESUMEN
OBJECTIVE: To investigate the inhibitory effect of matrine on tumor growth in tumor-bearing mice and explore its possible mechanisms of anti-tumor action in vivo. METHODS: Hepatocellular carcinoma cells H(22) were subcutaneously injected into BALB/c mice and matrine was administered to the tumor-bearing mice. The kinetics of tumor formation and tumor growth were measured, tumor growth inhibition rate (IR) was calculated, and tumor tissue samples were taken and examined by light and electron microscopy to assess the inhibitory effects of matrine on tumor growth in the mice. RESULTS: Marked inhibitory effect of matrine on the transplanted hepatocellular carcinoma H(22) was observed in the tumor-bearing mice. The inhibitory rates were 62.5% and 60.7% in the groups treated with high and low dosage of matrine, respectively (P < 0.01 vs. control group). The tumor formation was significantly retarded and tumor growth was inhibited in matrine-treated groups compared with those in control mice. Histopathological examination revealed widespread necrosis with massive accumulation of infiltrating lymphocytes and plasmacytes in the tumors. Numerous apoptotic cells and apoptotic bodies were observed in the tumors under the electron microscope. CONCLUSION: Matrine has marked inhibitory effects on tumor growth in vivo, which is probably related to inhibition of cell division and tumor cell proliferation, directly killing of tumor cells and/or induction of apoptosis and modulation of anti-tumor immune responses.
Asunto(s)
Alcaloides/uso terapéutico , Antineoplásicos Fitogénicos/uso terapéutico , Neoplasias Hepáticas Experimentales/tratamiento farmacológico , Quinolizinas/uso terapéutico , Animales , Apoptosis/efectos de los fármacos , Neoplasias Hepáticas Experimentales/patología , Ratones , Ratones Endogámicos BALB C , Trasplante de Neoplasias , MatrinasRESUMEN
BACKGROUND & OBJECTIVE: The differentiation of K562 cells can be induced by a given concentration of matrine. This study was designed to investigate the mechanism of signal transduction in induced differentiation of K562 cells. METHODS: ELISA coupled with streptavidin-biotin system was used to dynamically detect the activity of protein tyrosine kinase and phosphatase in the cytoplasm and the membrane of K562 cells treated by matrine. RESULTS: Matrine inhibited the activity of protein tyrosine kinase in K562 cells, and the inhibitive effect depended on matrine of the concentration within 0.1 mg/ml. The effects of different concentration of matrine on the protein tyrosine phosphatase activity had no significant difference. Activity of the protein tyrosine phosphatase decreased transiently accompanied with the activity of the protein tyrosine kinase in K562 cells treated with 0.1 mg/ml matrine. CONCLUSIONS: The change of protein tyrosine kinase activity is involved in the differentiation of K562 cells induced by matrine. The tyrosine kinase activity in cell membrane decreased more rapidly than that in cell cytoplasm, suggesting there be a transmembrane signal transduction. The change of tyrosine phosphatase activity following the kinase indicates the real time regulation of phosphorylation and dephosphorylation. Moreover, the inhibitory effect of matrine on the protein tyrosine kinase shows the characters of specificity and saturation, suggesting the exist of matrine-associated receptor in K562 cells.
Asunto(s)
Alcaloides/farmacología , Antineoplásicos Fitogénicos/farmacología , Diferenciación Celular/efectos de los fármacos , Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Células K562 , Quinolizinas , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , MatrinasRESUMEN
OBJECTIVE: To compare gene expression profile in K562 cells induced by matrine, so as to screen for differentiation related genes. METHODS: K562 cells were exposed to 0.1 mg/ml matrine for 3 hours, and gene expression profiles in matrine-treated and non-treated cells were studied by cDNA microarray. RESULTS: From 8465 screened genes, 30 differentially expressed genes were found. Among them 18 showed decreased expression including oncogene and proto-oncogene, signal transduction gene, DNA binding and transcription gene etc, 12 showed increased expression including cell receptor gene, immune-associated gene, metabolism-associated gene etc in matrine-treated K562 cells as compared with that in non-treated cell. CONCLUSION: The genes, that are closely associated with differentiation of can-cer cell, could be the potential targets for cancer treatment.
Asunto(s)
Alcaloides/farmacología , Diferenciación Celular/efectos de los fármacos , Células K562/metabolismo , Quinolizinas/farmacología , Diferenciación Celular/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Células K562/citología , Células K562/efectos de los fármacos , Análisis de Secuencia por Matrices de Oligonucleótidos , Proto-Oncogenes Mas , MatrinasRESUMEN
BACKGROUND & OBJECTIVE: It has been reported that matrine had the anti-tumor activity, and our previous study have provided that matrine had the effects of inhibiting proliferation and inducing differentiation in K562 cell line, but its molecular mechanism is unknown yet. METHOD: The expression of several proto-oncogenes(c-myc, c-jun, H-ras, p21, and HNF-1 alpha) in K562 cell line treated by 0.2 mg/ml matrine were measured by RT-PCR. RESULT: The c-myc, c-jun, and HNF-1 alpha mRNA of K562 cells treated by 0.2 mg/ml matrine were dramatically decreased at the early stage (3 h), while the H-ras and p21 mRNA were increased obviously at the same time. CONCLUSION: The changes of several proto-oncogenes in K562 cells treated by 0.2 mg/ml matrine may be related to inhibiting proliferation and inducing differentiation.