This study aims to establish the S_(180) tumor-bearing mice model, and to investigate the influence of Shenqi Erpi Granules(SQEPG) on immune function, as well as the drug's tumor-suppressive effect and mechanism. SPF grade KM mice(half male and half female) were randomly divided into 6 groups: a control group, a model group, a cyclophosphamide group(50 mg·kg~(-1)), as well as SQEPG groups in low-, medium-, and high-dose(5.25, 10.5, 21 g·kg~(-1)). The control group and the model group were given distilled water, and the other 4 groups were given the corresponding drugs by gavage. The administration continued for 10 days before the mice were sacrificed. The antitumor and immune regulation effects of SQEPG were evaluated. The effect of SQEPG on delayed type hypersensitivity reaction(DTH), carbon clearance index, and serum hemolysin antibody level was observed to reflect the effect on the immune function of tumor-bearing mice. Tumor weight was recorded to calculate the tumor suppression rate and the immune organ index. Hematoxylin-eosin(HE) staining was used to detect morphological changes in tumor tissues. Flow cytometry was employed to detect the percentage of CD4~+ and CD8~+ T-cells in the spleen tissues and the tumor tissue apoptosis levels. Immunohistochemistry was conducted to detect the KI67 protein expression level of tumor tissues. ELISA resorted to the detection of the following expression levels in tumor tissues: tumor necrosis factor-α(TNF-α), interleukin-2(IL-2), interferon-γ(IFN-γ). Western blot was performed to detect the expression levels of caspase-3, B-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax), cyclin-dependent kinases 4(CDK4), G_1/S-specific cyclin D1(cyclin D1), and vascular endothelial growth factor A(VEGFA). The results showed that, compared with the model group, the SQEPG could increase the swelling of the auricle of the tumor-bearing mice; significantly increase the phagocytic index of carbon granule contour(P<0.05 or P<0.01), and the middle dose of SQEPG could significantly increase the antibody level of hemolysin(P<0.05); different doses of SQEPG significantly inhibit the growth of the tumor, and decrease the mass of the tumor tissues(P<0.05 or P<0.01); the low dose of SQEPG significantly decreased spleen index(P<0.05), low and high doses of SQEPG increased thymus index, while medium doses of SQEPG decreased thymus index. High doses of SQEPG significantly elevated the levels of CD4~+ and CD8~+ T-cells in the spleens of the homozygous mice(P<0.01 or P<0.001), and increased the apoptosis rate of the cells of the tumor tissues(P<0.05); Meanwhile, high-dose SQEPG elevated the levels of immunity factors such as IL-2, IFN-γ and TNF-α in the serum of tumor-bearing mice(P<0.01); medium-and high-dose SQEPG significantly lowered the rate of positive expression of KI67 protein in tumor tissues(P<0.01). Compared with the model group, high-dose SQEPG significantly up-regulated the expression of caspase-3 and Bax proteins in tumor tissues(P<0.05), and significantly down-regulated the expression of CDK4, cyclin D1, and VEGFA proteins(P<0.05 or P<0.01). In conclusion, SQEPG has the effect of improving immune function and inhibiting tumor growth in tumor-bearing mice. Its mechanism of tumor-suppressive effects may be related to apoptosis promotion, cell cycle progression block, and tumor cell proliferation inhibition.
Animals ; Mice ; Drugs, Chinese Herbal/pharmacology* ; Male ; Female ; Apoptosis/drug effects* ; Sarcoma 180/genetics* ; Humans

Mouse hepatoma H22 cells and sarcoma cells (H22 and S180) were infected with EGFP-N1 by electroblot, and the acquired gfp-H22 and gfp-S180 cells expressing strong green fluorescence protein (GFP) fluorescence were supplemented with medium G418 Sigma (800 mg/ml). Meanwhile, the models bearing cancer (gfp H22 and gfp S180) subcutaneously and with abdominal cavity were established. There were no statistically significant differences by comparison on the cell phenotype, ultramicrostructure, growth curve and bearing cancer time between the H22 cells and S180 cells (P>0.05). The GFP fluorescence was detected with whole body GFP imaging system in vivo and with fluorescence microscope. According to the results of in vitro and in vivo assay, it was shown that, by application of fluorescence technology, the GFP-expressing H22 cells and S180 cells could be used in further studies on the tumor biological behavior.
Animals ; Electroporation ; Green Fluorescent Proteins ; genetics ; metabolism ; Liver Neoplasms, Experimental ; genetics ; metabolism ; Mice ; Microscopy, Fluorescence ; Sarcoma 180 ; genetics ; metabolism ; Transfection ; methods ; Tumor Cells, Cultured

OBJECTIVETo investigate effects of Sargassum confusum polysaccharide (SP) on the expressions of p53 and Rb genes.

METHODSThe mice bearing S180 sarcoma were treated with abdominal SP injection at the daily dose of 50, 100 or 200 mg/kg for 10 consecutive days, The mice injected with 0.85% saline served as the negative control group and those with 5-fluorouracil (5-FU) injection as the positive control group. The tumor weight was measured to calculate the tumor inhibition rate. RT-PCR and Western blotting were used to detect the expression levels of p53 and Rb genes at the mRNA and protein levels, respectively.

RESULTSThe 10-day SP treatment at 50, 100 and 200 mg/kg resulted in tumor growth inhibition rates of 30.5%, 47.6% and 63.5%, respectively. SP treatment upregulated the mRNA expressions and increased the protein levels of p53 and Rb in the tumors.

CONCLUSIONSP has inhibitory effect against S180 sarcoma in vivo and can increase the expression levels of p53 and Rb genes at both the mRNA and protein levels.

Animals ; Blotting, Western ; Cell Line, Tumor ; Female ; Gene Expression Regulation, Neoplastic ; drug effects ; Male ; Mice ; Polysaccharides ; isolation & purification ; pharmacology ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation ; Retinoblastoma Protein ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Sarcoma 180 ; genetics ; metabolism ; pathology ; Sargassum ; chemistry ; Tumor Suppressor Protein p53 ; genetics ; metabolism

OBJECTIVETo investigate the effect of Ganoderma lucidum polysaccharides (GLP) on the nucleotide contents and cell cycle distribution of the tumor cells in S180 ascitic tumor-bearing mice and explore the possible mechanism of the antitumor effect of GLP.

METHODSMice bearing S180 ascitic tumor were subjected to intragastric administration of GLP (100, 200, and 400 mg/kg), normal saline or subcutaneous injection of cyclophosphamide (CTX) at 25 mg/kg, respectively. The treatment was given once daily for 9 consecutive days, after which the ascitic tumor cells were harvested for determination of the RNA and DNA contents and their ratio as well as the cell cycle alterations. Laser scanning confocal microscopy and acridine orange staining was performed to evaluate the DNA and RNA fluorescence intensity, and flow cytometry with propidium iodide (PI) staining was utilized for cell cycle analysis of the tumor cells.

RESULTSCompared with normal saline group, the tumor cells in the 3 GLP groups all showed reduced RNA and DNA contents, and this reduction was statistically significant in 200 mg/kg GLP group (P=0.000). Significantly reduced RNA/DNA ratio was noted in all the 3 GLP groups (P=0.003, 0.000, 0.008 corresponding to 400, 200, and 100 mg/kg groups), suggesting that ganoderma polysaccharides more effectively reduced RNA content than DNA content. CTX also resulted in reduced RNA and DNA contents but not the RNA/DNA ratio. At the doses of 400, 200, and 100 mg/kg, GLP increased the percentage of G2/G2 phase cells (P=0.003, 0.000, and 0.000) whereas CTX showed the contrary effect (P=0.000). GLP produced no obvious effect on S-phage cells but CTX significantly reduced their percentage (P=0.000). GLP at the 3 doses all decreased the percentage of G2/M phase tumor cells (P=0.014, 0.049, 0.016) and CTX again induced contrary effect (P=0.000).

CONCLUSIONWith different effects from CTX on DNA and RNA contents and cell cycle, GLP inhibits DNA and RNA synthesis in the tumor cells by mobilizing the host immune function to interfere with the normal cell cycles, which might be one of the mechanisms for the antitumor effect of GLP.

Animals ; Antineoplastic Agents ; pharmacology ; Ascitic Fluid ; Cell Cycle ; drug effects ; Cell Line, Tumor ; DNA ; drug effects ; metabolism ; Dose-Response Relationship, Drug ; Immunohistochemistry ; Male ; Mice ; Polysaccharides ; pharmacology ; RNA ; drug effects ; metabolism ; Reishi ; chemistry ; Sarcoma 180 ; genetics ; pathology ; Xenograft Model Antitumor Assays

OBJECTIVETo study the anti-tumor angiogenesis effect of soluble VEGF receptor fragment by blocking the combination of VEGF and its receptor in vivo and in vitro.

METHODSRT-PCR technique was used to amplify Flk-1/KDR fragment from embryo mouse liver, which was recombinated to expression vector pET-28b(+) and retrovirus vector PLXSN, which was induced to be expressed, purified and identified with EcoR I and Hind III. Mouse endothelial cells were separated, cultured and identified by immunocytochemistrical staining using VIII factor-related antigen antibody. The expressed product was analyzed about its effect on endothelial cell's growth in vitro with MTT method. The retrovirus vector was transfected to tumor cell lines S180 and B16 by liposome method to observe the biological specificity in vitro after gene transfection.

RESULTS1000 bp size sVEGFR fragment was amplify from E9, E11 embryo mouse liver tissues, which was recombinated to TA clone vector and identified by sequence analysis. This fragment was cloned to expression vector pET-28b(+), the expressed product was purified and identified correctly. The in vitro study showed this expressed product can effectively inhibit endothelial cell(s), growth and proliferation. The fragment was then cloned to retrovirus vector PLXSN and transfected to tumor cell lines S180 and B16 successfully with RT-PCR and SDS-PAGE. The experiments in vivo showed that the weight of tumor smaller, the size decreased significantly, the microvessel density was fewer and Flk1 protein expression were higher in the group of gene transfection than that of control.

CONCLUSIONSoluble VEGFR fragment is a kind of effective gene engineer product for anti-tumor angiogenesis gene therapy and the development of anti-tumor drug.

Animals ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cloning, Molecular ; Endothelial Cells ; cytology ; Genetic Vectors ; Melanoma, Experimental ; metabolism ; pathology ; Mice ; Mice, Inbred BALB C ; Neoplasm Transplantation ; Neovascularization, Pathologic ; pathology ; RNA, Messenger ; biosynthesis ; genetics ; Retroviridae ; genetics ; Sarcoma 180 ; metabolism ; pathology ; Transfection ; Vascular Endothelial Growth Factor Receptor-2 ; biosynthesis ; genetics ; physiology