[Effect of Indirubin and Sheng-Xue-Xiao-Ban Capsule (SXXBC) on Promoting Peripheral Platelet in ITP Model Mice].

OBJECTIVE: To compare the effect of Sheng-Xue-Xiao-Ban Capsule (SXXBC) and indirubin to the peripheral platelets of the Idiopathic thrombocytopenic purpura (ITP) model mouse. METHODS: The ITP mouse model was established by the method of passive immunization. SXXBC and indirubin were used for intervention treatment. Then the hemorrhagic phenomena of ITP mice were observed and the numbers of peripheral platelets, hemoglobin and white blood cells, bone marrow megakaryocytes and their classification and coagulation function were detected and compared. RESULTS: The improvement rate of hemorrhage in SXXBC group was 40% for small dose, 60% for medium dose and 80% for high dose, while the improvement rate of hemorrhage in indirubin group was 30% for small dose, 50% for medium dose and 60% for high dose. There was no statistically significant difference in the improvement rate of hemorrhage between the two groups (P＞0.05). Compared with the model control group, PLT and Hb increased in different doses of SXXBC and indirubin group 4th-8th day after drug intervention (P＜0.05, 0.01). However, there was no significant difference between the different doses of SXXBC group and indirubin group (P＞0.05). Compared with the model control group, the WBC in each group was significantly lower (P＜0.05, 0.01) on the 4th-8th day after drug intervention; However, there was no statistical significance between the two groups of SXXBC and indirubin (P＞0.05). Compared with the model control group, the total number of megakaryocytes in each treatment group were decreased (P＜0.05, P＜0.01), in which the number of primary megakaryocytes in the large and medium dose groups of SXXBC and indirubin were decreased (P＜0.05, 0.01), and the number of juvenile megakaryocytes in the large dose group of SXXBC and indirubin were also decreased (P＜0.05). The number of granular megakaryocytes were decreased in each intervention groups (P＜0.05, 0.01), and the number of thromocytogenic megakaryocyte was increased in the high and medium dose groups of SXXBC and indirubin (P＜0.01). The time of prothrombin was shortened in the high and medium dose groups of SXXBC and indirubin (P＜0.05), and the fibrinogen (FIB) content in the high and medium dose groups of SXXBC was close to that of the normal control group. CONCLUSION: Both of the SXXBC and the indirubin standard all show good hemostatic effects. Indirubin shows a positive effect on increasing the peripheral platelet and hemoglobin in ITP model mice, regulating the immune response, reducing the total number of bone marrow megakaryocytes, increasing the thromocytogenic megakaryocyte, and increasing coagulation function.

Ectopic expression of the WWOX gene suppresses stemness of human ovarian cancer stem cells.

The present study aimed to investigate the effects of the WW domain-containing oxidoreductase (WWOX) gene on the stem cell properties of human ovarian cancer stem cells. A eukaryotic expression vector containing the WWOX gene was transfected into human ovarian cancer stem cells and Western blotting was used to assess the expression of WWOX protein in the transfected cells compared with the control cells (untransfected cells and cells transfected with the empty vector). The self-renewal abilities of these three types of stem cells was also assessed in vitro. To monitor changes in their differentiation potential, cells were cultured in medium supplemented with serum, and the expression of specific stem cell markers was determined. Drug-sensitivity tests were used to measure the sensitivity of the stem cells to cisplatin, doxorubicin, and mitoxantrone. The cells were also transplanted into non-obese diabetic (NOD)/severe combined immunodeficiency (SCID) mice to determine the changes in their tumorigenicity in vivo. Cells transfected with the WWOX-expressing plasmid stably expressed WWOX protein, while no WWOX protein was detected in control cells. Compared with the two types of control cells, WWOX-expressing stem cells manifested significantly reduced self-renewal ability. Compared with control cells, the expression levels of stem cell markers, including CD133, CD117, ATP-binding cassette sub-family G member 2, Nanog, octamer-binding transcription factor 4 and breast cancer resistance protein, were significantly lower in WWOX-expressing cells, while the level of the differentiation marker E-cadherin was significantly higher in WWOX-expressing cells. Furthermore, WWOX-expressing cells were more sensitive to treatment with cisplatin, doxorubicin and mitoxantrone. In NOD/SCID mice, the tumorigenicity of WWOX-expressing cells was significantly lower compared with that of control cells. The results indicate that the tumor suppressor WWOX suppresses stem cell properties in cancer stem cells, including self-renewal ability, differentiation potential, in vivo tumorigenic capability, high-level expression of stem cell genes and multidrug resistance.

Effects of sodium valproate on the growth of human ovarian cancer cell line HO8910.

To explore a possible new treatment for human ovarian cancer, we studied the effects of sodium valproate on the growth of the HO8910 human cell line. HO8910 cells were cultured in vitro and treated with different concentrations of sodium valproate. Cell proliferation, cell cycling, and apoptosis were measured by flow cytometry, cell morphology under a microscope, and expression levels of WWOX and P27 by Western blotting and RT-PCR. Tumor xenografts were established to determine in vivo effects of sodium valproate. Our results showed that cell proliferation was decreased with increasing concentration of sodium valproate, with features of cytoplasmic retraction and floating cells. Moreover, cell cycle analysis revealed a higher apoptosis rate and G0/ G1 phase in the sodium valproate experimental group than in the control group. In addition, protein expression levels of WWOX and P27 were elevated. Importantly, sodium valproate decreased in vivo xenograft tumor burden and up-regulated WWOX and P27 expression in nude mice. In conclusion, sodium valproate might play a role in inhibition and control of ovarian cancer cell line HO8910 by inhibiting cell proliferation, interfering with the cell cycle and promoting apoptosis, so that it may be effective in the clinical treatment of ovarian cancer.

[Effects of WWOX gene transfection on cell growth of epithelial ovarian cancer].

OBJECTIVE: To study the effects of anti-oncogene WWOX on cell growth of epithelial ovarian cancer,in order to find a new approach of gene therapy for ovarian cancer. METHODS: A eukaryotic expression vector containing WWOX was transfected into ovarian cancer cell line HO8910 in vitro (recombinant plasmid group), and positive cell clones were selected and amplified. Expression of WWOX protein was detected by western blot. Untransfected cell (blank contrast group) and transfected empty plasmid cell (empty plasmid group) were served as control groups. In vitro, the biology effect of WWOX on HO8910 cell was analyzed through the methyl thiazolyl tetrazolium test, transwell chamber cell invasion assay in vitro, agarose clony-formation and flow cytometry. In vivo, the cell of transfection was transplanted intraperitoneally in to BALB/c nude mice. The survival time and growth ability of nude mice were observed. RESULTS: (1) Recombinant plasmid group cell could steadily express WWOX protein, while in empty plasmid group and blank control group the expression of WWOX protein were not detected. (2) The growth rate of recombinant plasmid group cell was inhibited. (3) The agarose clony-formation rate of recombinant plasmid group (19.8%) was significantly lower than that of the empty plasmid group (54.5%) and blank control group (56.0%, P < 0.05). (4) Flow cytometry showed that (72.08 +/- 0. 39)% of cells was arrested at G0/G1 stage in recombinant plasmid group, while in empty plasmid group and blank control group G0/G1 stage cells were at (41.02 +/- 1.08)% and (39.31 +/- 0.67)% (P < 0.05). (5) In vitro invasion assay showed that invasion cell number in recombinant plasmid group (89.7 +/- 3.1) was not significantly different from that of empty plasmid group (91.2 +/- 1.3) and blank control group (91.4 +/- 1.3, P > 0.05). (6) In vivo test in nude mice showed that WWOX gene could inhibit tumor growth of the HO8910 cells. CONCLUSIONS: Tumor suppressor gene WWOX could interfere with the cell cycles of ovarian cancer cell and inhibit cell proliferation. As a new valuable tool, it promises to have application in the gene therapy of ovarian cancer.