Inhibitory effect of ginsenoside Rg3 on ovarian cancer metastasis

Xu T-m, Cui Man-hua, Xin Y, Gu L-p, et al. Chinese Medical Journal, 2008, Vol. 121 No. 15 : 1394-1397 Background Ginsenosides are main components extracted from ginseng, and ginsenoside Rg3 is one of the most important parts. Ginsenoside Rg3 has been found to inhibit several kinds of tumor growth and metastasis. The present study was undertaken to investigate the effect of ginsenoside Rg3 on human ovarian cancer metastasis and the possible mechanism. Methods?The experimental lung metastasis models of ovarian cancer SKOV-3 and the assay of tumor-induced angiogenesis were used to observe the inhibitory effects of Rg3 on tumor metastasis and angiogenesis. The effect of Rg3 on invasive ability of SKOV-3 cells in vitro was detected by Boyden chamber, and immunofluorescence staining was used to recognize the expression of matrix metalloproteinase 9 (MMP-9) in SKOV-3 cells. Results In the experimental lung metastasis models of ovarian cancer, the number of tumor colonies in the lung and vessels oriented toward the tumor mass in

each ginsenoside Rg3 group, was lower than that of control group. The invasive ability and MMP-9 expression of SKOV-3 cells decreased significantly after treatment with ginsenoside Rg3. Conclusions?Ginsenoside Rg3 can significantly inhibit the metastasis of ovarian cancer. The inhibitory effect is partially due to inhibition of tumor-induced angiogenesis and decrease of invasive ability and MMP-9 expression of SKOV-3 cells. Ginseng is a medicinal herb widely used in Asian countries for its wide spectrum of medicinal effects such as tonic, immunomodulatory, adaptogenic, and anti-aging activities.1 These effects are attributed to the triterpene glycosides known as ginsenosides and Rg3 is one kind of the ginsenosides. The molecular formula of ginsenoside Rg3 is C42H72O13 and its molecular weight is 784.30. Ginsenoside Rg3 can inhibit catecholamine secretion, protect cultured cortical cells from glutamate- induced neurodegeneration, and anti-contraction of vascular smooth muscle.2-4 Researchers found that ginsenoside Rg3 can also resist tumor. For example, it inhibited invasion and metastasis of B16 melenoma without impairing cell growth, and proliferation of tumor cells combined with cyclophosphamide.5-7 However, the effect of ginsenoside Rg3 on human ovarian cancer has not been identified and the mechanism of its anti-tumor is unknown. The present study was to assess the effects of ginsenoside Rg3 on angiogenesis and metastasis produced by human ovarian cancer and on the invasion and the matrix metalloproteinase 9 (MMP-9) expression of SKOV-3 cells in vitro. METHODS Drug and reagents Ginsenoside Rg3 was provided by Department of Organic Chemistry of Preclinical Medicine of Jilin University. Its purity is more than 99.5%. Athymic mice were purchased from the Department of Experimental Animals of Jilin University. Boyden chamber and matrigel were purchased from BD Company, USA. Cell culture Human ovarian cancer SKOV-3 cell line and mouse NIH3T3 fibroblast cell were obtained from the Tumor Research Department of Jilin Province and maintained in RPMI1640 supplemented with 10% fetal bovine serum (FBS). Assay for experimental lung metastasis In 40 mice, each was injected with SKOV-3 cells (2×105) into the lateral tail vein. The mice were randomly divided into 4 groups (n=10 for each group): Rg3 groups (0.3, 1.0 and 3.0 mg/kg) and control group. Ginsenoside Rg3 was injected intraperitoneally at daily doses (0.3, 1.0 and 3.0 mg/kg) to the tumor-bearing mice the next day after tumor inoculation. The mice were killed 20 days after the inoculation. The lungs were fixed in Bouin’s solution and the lung tumor colonies were counted under a dissecting microscope. Cell invasion assay The invasive activity of SKOV-3 cells was assayed in Boyden chambers. The upper surface of the filters in Boyden chambers was precoated with 60 µl of matrigel. The SKOV-3 cells were harvested, washed 3 times and re-suspended to a final concentration of 1×106/ml in RPMI1640. Then the cells were divided into 3 groups after pretreatment with ginsenoside Rg3 of different concentrations (0, 2.5 and 5.0 µg/ml) at 37°C for 30 minutes. 200 µl of each cell suspension was added to the upper compartment of the chamber and 200 µl of conditioned medium of NIH3T3 cells to the lower compartment. After 5-hour incubation, the cells on the upper surface of the filters were removed by wiping with cotton swabs and the filters were fixed with FAA solution and stained with hematoxylin and eosin. The cells that had invaded through the matrigel and filters into the lower surface were manually counted under a microscope at a magnification of ×400. Each assay was performed in triplicate. The data were expressed as the number of invaded cells/field. Assay for tumor-induced angiogenesis In 20 athymic mice, each was inoculated intradermally with SKOV-3 cells (5×105) on the back. They were randomly divided into 4 groups (n=5): Rg3 groups (0.3, 1.0 and 3.0 mg/kg) and control group. Ginsenoside Rg3 at various doses was injected intraperitoneally to the mice 1, 2, 3, 4 and 5 days after tumor inoculation. Two days later, the mice were sacrificed and the skin was separated from the underlying tissues. Angiogenesis was quantified by counting the number of vessels oriented toward the tumor mass under a dissecting microscope. MMP-9 immunofluorescence studies The SKOV-3 cells that were seeded onto 24-well plates and treated with 0, 2.5 and 5.0 µg/ml of ginsenoside Rg3 for 24 hours were used for immunofluoresecent staining. First, the cells were washed briefly with phosphate buffered saline three times and then fixed in 4% phosphate buffered paraformaldehyde for 30 minutes. Second, they underwent permabilization with the addition of phosphate-buffered 0.1% Triton X-100 for 10 minutes and then were incubated in 1% bovine serum albumin for 30 minutes all at room temperature. Third, the cells were incubated with mouse anti-human MMP-9 monoclonal antibody (1: 100 dilution) and then with goat anti-mouse fluorescein isothiocyanate (FITC) antibody respectively for 1 hour at 4°C. Finally, the coverslips were mounted and sealed for examination under a confocal microscope. Statistical analysis Measurement data were evaluated by one-way analysis of variance (ANOVA) for multiple group comparisons, and the LSD test for two-group comparisons. Ranked data were evaluated by the Kruskal-Wallis test for multiple group comparisons and the Nemenyi test for two-group comparisons. P Cell invasion assay After the SKOV-3 cells were treated with ginsenoside Rg3 at three concentrations of 0, 2.5 and 5.0 µg/ml, the number of the cells invading through matrigel and filters into the lower surface was 157.3±29.4, 110.8±25.6 and 92.5±18.4 respectively. Among the three groups, the group of 0 µg/ml ginsenoside Rg3 was control group. Statistical analysis illustrated that the number of cells invading filters of the ginsenoside groups was less than that of the control group (P