Anticancer effects of crude extract from Melia toosendan Sieb. et Zucc on hepatocellular carcinoma in vitro and in vivo / 中国结合医学杂志

<p><b>OBJECTIVE</b>To investigate the anti-cancer effects of crude extract from Melia toosendan Sieb. et Zucc and its possible molecular mechanisms in vitro and in vivo.</p><p><b>METHODS</b>Transonic alcohol-chloroform extraction method was used to extract toosendanin from the bark of Melia toosendan Sieb. et Zucc, and the content of toosendanin in the crude extract was measured by high performance liquid chromatography (HPLC). Anti-cancer effects of crude extract from Melia toosendan Sieb. et Zucc were investigated in in vivo and in vitro studies. In the in vitro experiment, human hepatocellular carcinoma cell lines SMMC-7721 and Hep3B were co-incubated with toosendanin crude extract of different concentrations, respectively. In the in vivo experiment, BALB/c mice were subcutaneously inoculated with mouse hepatocellular carcinoma H22 cells and treated with crude extract.</p><p><b>RESULTS</b>HPLC revealed the content of toosendanin was about 15%. Crude extract from Melia toosendan Sieb. et Zucc inhibited cancer cells growth in a dose- and time-dependent manner. The 50% inhibitory concentration (IC50, 72 h) was 0.6 mg/L for SMMC-7721 cells and 0.8 mg/L for Hep3B cells. Both high-dose [0.69 mg/(kg d)] and low-dose [0.138 mg/(kg d)] crude extract could markedly suppress cancer growth, and the inhibition rate was greater than 50%. Hematoxylin and eosin staining showed necrotic area in cancers and transmission electron microscopy displayed necrotic and apoptotic cancer cells with apoptotic bodies. Immunohistochemistry showed that the expression of Bax and Fas increased and the expression of Bcl-2 reduced.</p><p><b>CONCLUSIONS</b>Toosendanin extract has potent anti-cancer effects via suppressing proliferation and inducing apoptosis of cancer cells in vivo and in vitro. The mechanism of apoptosis involves in mitochondrial pathway and death receptor pathway.</p>

Investigation of the sensitivities of two different serotypes of Streptococcus pneumoniae in C57BL/6 mice with acute otitis media / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To compare the sensitivities of two different serotypes of Streptococcus pneumoniae in acute otitis media of C57BL/6 mice.</p><p><b>METHODS</b>The middle ear cavity of C57BL/6 mice were inoculated via intratympanic injection of Streptococcus pneumoniae TIGR4 or 6B 1×10(8) CFU, and the control group were inoculated equivalent phosphate buffered solution (PBS). The incidence of mice, development of otitis media and the middle ear lavage fluid pathological changes by HE staining, as well as cell counts and cytokine levels were investigated.</p><p><b>RESULTS</b>After inoculated with Streptococcus pneumoniae TIGR4, 6B and PBS, the survival rate of 6B group was significantly less than TIGR4 group and PBS control group (P < 0.01). Inflammatory cells in the middle ear cavity were mainly neutrophils, and the inflammatory cells recruitment in TIGR4 group were more than 6B group; The levels of IL-6 and TNF-α in the middle ear lavage fluid in TIGR4 group and 6B group were significantly increased compared with PBS control group, while the TIGR4 group were significantly increased compared with 6B group;6B group had delayed bacterial clearance in the middle ear.</p><p><b>CONCLUSION</b>The study implied that Streptococcus pneumoniae 6B had higher pathogenicity for acute otitis media in C57BL/6 mice than TIGR4.</p>

Enhancing effect of matrine on the tumor-inhibition by TIM2 gene-modified hepatocarcinoma H22 cells in mice / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the effects of matrine on the anti-tumor efficiency of TIM2 gene-modified murine hepatocarcinoma H22 cells.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Effects of matrine on oncogenicity of H22 cells modified by TIM2 gene in vivo / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the effects of matrine on the anti-tumor efficiency of H22 murine hepatocarcinoma cell-based vaccine modified by TIM2 gene in vivo.</p><p><b>METHOD</b>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.</p><p><b>RESULT</b>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.</p><p><b>CONCLUSION</b>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.</p>

Inhibition of tumor growth in tumor-bearing mice treated with matrine / 中华肿瘤杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Construction of eukaryotic expression vector of unknown KH gene and its effect on cell proliferation / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To construct an eukaryotic expression vector of open reading frame of unknown KH gene (KH-ORF), and investigate its effect on cell proliferation.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>(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.</p><p><b>CONCLUSION</b>KH gene may be a leukemia gene related to proliferation of K562 cells.</p>

Gene expression profile in early differentiation of K562 cells induced by matrine / 中华血液学杂志

<p><b>OBJECTIVE</b>To compare gene expression profile in K562 cells induced by matrine, so as to screen for differentiation related genes.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>The genes, that are closely associated with differentiation of can-cer cell, could be the potential targets for cancer treatment.</p>