Inhibitory Effects Of Matrine On Invasiveness And Metastasis Of Human Malignant Melanoma Cell Line A375 In Vitro

Section 1:The effects of matrine on proliferation and apoptotis of human malignant melanoma cell line A375Malignant melanoma (MM) is one of the most aggressive malignancies. The mortality of MM is so high that the vast majority of patients die within the first year after diagnosis due to lymph or blood widespread. However, mechanisms responsible for the progression of MM remain largely unknown and no effective therapy is available to the date. It becomes urgent to search for effective drugs and therapeutic modalities against MM. As one kind of Chinese traditional medicine, matrine, which being the most important component of Sophora Flavesces, is found to possess various kinds of pharmacological activities, including anti-tumor activity. The distinctive anti-tumor activity of matrine has aroused people's interest day by day, and related experiments and clinical researches are on the rise. However, to our knowledge, few investigations were reported about the effects of matrine on proliferation and apoptosis of MM cell line. Therefore, in this study, we took the A375 cell line cultured in vitro as research object and observed the influences of matrine on the proliferation and apoptosis of A375 cells.methods1 , The effect of matrine on proliferation of A375 cells measured by MTT assay Growing cells were treated with matrine at a final concentration of 0.25mg/mL, 0.5mg/mL,1.0mg/mLor 2.0mg/mL respectively. Culture medium was added in controlgroup. Each concentration treatment was done in quadruplicate wells. Fresh culture medium with different concentration of matrine or control medium was changed every two days. After exposure to various matrine for l⁶d respectively, MTT was added and the plates were incubated for an additional 4h before absorbance at 570nm was measured. All experiments were repeated for three times. The inhibitory rate was calculated according to the formula of [1-A₅₇₀test/A₅₇₀control] 100%.2 , The effect of matrine on apoptosis of A375 cells assessed by the Annexin-V-FITC/PI affinity assayA375 cells were treated with matrine at a final concentration of 0.125mg/mL, 0.25mg/mL, 0.5mg/mL or 1 .Omg/mL.Control group was performed by adding culture medium. Each concentration treatment was done in triplicate. After being treated for 48h, cells were collected and the supernatant (100uL/tube) were incubated with Annexin-V-FITC(5uL) and PI(5uL) for 15 minutes at room temperature in the dark. Cells were then analyzed by flow-cytometry. All experiments were repeated for three times.3, Morphologic observation of A375 cells by light and electron microscopyTreated with matrine at a final concentration of 0.25mg/mL, 0.5mg/mL, l.Omg/mL or culture medium for 48h, A375 cells were fixed with 10% formaldehyde, then were stained with HE method and observed under inverted light microscopy. At the same time,the samples were prefixed in 2.5% glutaraldehyde, then in 1% OsO4, dehydrated in ethanol series, and replaced in propene oxide. The ultrastructures of A3 75 cells were examined under transmission electron microscope.4, Statistic analysisThe results were expressed as mean ± SD. Data were analyzed using SPSS 10.0. A P-value of <0.05 was considered statistically significant.Results1 -. The inhibitory effect of matrine on proliferation of A375 cellsMatrine could remarkably inhibit the proliferation of A375 cells with a dose and time dependent manner within the range of certain concentration (0.5².0mg/mL).However, the inhibitory effect of matrine at low concentration (<0.25mg/mL) was still weak ,even if the incubation time lengthened.2x The outcome of flow-cytometryMatrine at a final concentration ranged from 0.5 to 1.0 mg/mL could induce more Annexin-V stained cells in a dose-dependent manner.3> The morphological changes of A375 cells observed by light and electron microscopyMorphologic features of apoptosis of A375 cells treated with matrine were observed under both light and electron microscopy. Cytoplasmic vacuoles were observed when the final concentration of matrine was l.Omg/mL.Conclusions1-. Matrine plays an inhibitory role in proliferation of tumor cells with a dose and time dependent manner in malignant melanoma cells in vitro.2, Matrine induces the cellular apoptosis in a dose-dependent manner in malignant melanoma cells in vitro.Section 2:Inhibition of invasiveness and expression of heparanase-mRNA in human malignant melanoma cell line A375 by matrineThe recent researches have showed that matrine can inhibit proliferation of tumor cells, induce celluar differentiation and apoptosis, and suppress neovascularization, and so on. However, there are still few reports about the effects of matrine on tumor invasiveness and metastasis. Heparanase is the only endo-p-glucoronidase which is essential for degradation of the extracellular matrix and basement membrane therefore facilitate metastasis of tumor cells. In this in vitro study, we took A375 cell line as working model and evaluated the changes of heparanse-mRNA expression, adhesion ability and invasiveness ability of A375 cells after treatment with various concentrations of matrine, in order to further clarify the possible mechanism of inhibitory effects of matrine on invasiveness and metastasis of tumor cells.MethodsK The heparanase-mRNA expression levels performed by semi-quantitative RT-PCRA375 cells were collected after treated with 0.125mg/mL, 0.25mg/mL, 0.5mg/mL matrine or culture medium for 48h . The total RNAs were extracted from the cells and then semi-quantitative RT-PCR was performed to evaluate the heparanase-mRNA expression. 35 cycles of heparanase were performed and the length of amplification was about 584 base pairs. The internal control was made by PCR with primers specific for (3-actin. 30 cycles of 6-actin were performed and the length of PCR product was about 225 base pairs. All experiments were repeated for four times.2> Cell Matrigel adhesion assayEach well of 96-well plates was coated with Matrigel gel 10ug(30uL) and2%BSA 20uL. A375 cells treated with matrine for 48h were collected , and still treated with matrine at a final concentration of 0.125mg/mL, 0.25mg/mL , 0.5mg/mL or culture medium. Each concentration treatment was done in quadruplicate. Those cells that couldn't adhere to Matrigel gel were washed by PBS after incubated for lh at 37°C . MTT was added and the plates were incubated for another 4h before absorbance at 570nm was measured. All experiments were performed in triplicate .The inhibitory rate of cellular adhesion was calculated according to the formula of [1-A57otest/As7ocontrol] 100%.3 % Matrigel invasiveness assaylOOuL of Matrigel gel (lmg/mL) was added to each Boyden chamber's botton, relocated in 24-well plates and dried for 4h. A375 cells were collected and resuspended at 2xl05cells/mL in RPMI1640. lOOuL of suspension was added into each chamber and treated with matrine at a final concentration of 0.125mg/mL, 0.25mg/mL or 0.5mg/mL.Control group was performed by adding culture medium. Each concentration treatment was done in quadruplicate. After incubated for additional 48h, the cells on the upper side of polyethylene membrane were wiped off with a cotton swab. The remaining cells that traversed the Matrigel gel were fixed , stained and six random fields of vision were counted under light microscope . The number of invasive cells was equal to the number of cells on the lower surface of the membrane plus the number of adhesive cells in corresponding well;the inhibitory rate of cellular invasiveness was calculated according to the formula of [1-the number of test /the number of control] 100%.4^ Statistic analysisThe results were expressed as mean ± SD. Data were analyzed using SPSS 10.0. A-P-value of <0.05 was considered statistically significant.Results1-. The inhibition of matrine on heparanase-mRNA expression of A375 cells After treated with culture medium or various concentration matrine, the ratio of optical density of heparanase-mRNA to that of P-actin in A375 cells was 1.45±0.12,1.01±0.13, 0.65±0.01, 0.32±0.08, respectively. The heparanase-mRNA expression of A375 cells treated with matrine of different final concentration was significantly decreased compared to that of control group.Besides, the heparanase-mRNA expression decreased gradually when the concentration of matrine increased. The inhibitory effect of matrine had the obvious dosage-dependence. 2^ The inhibitory effect of matrine on adhesion of A375 cells Compared to control group, the adhesion to Matrigel gel in treated groups was significantly suppressed. The inhibitory rate was 26.17±1.77%, 50.01±5.68%, 73.07±3.24%, respectively. The inhibitory effect of matrine on the adhesion of A375 cells was in a dose-dependent manner.3n The inhibitory effect of matrine on invasiveness of A375 cells The inhibitory rate of invasiveness of A3 75 cells treated with matrine of 0.125mg/mL, 0.25mg/mL or 0.5mg/mL was 27.47±2.55%, 53.76±2.18%, 87.09±1.00%, respectively. Compared to control group, the number of invasive cells of treated groups was remarkably decreased. Besides.the inhibitory effect was significantly different between treatment groups ?Conclusions1 ^ Matrine can down-regulate the heparanase-mRNA expression of A375 cells in a dose-dependent manner.2> Matrine can significantly inhibit the adhesion and invasiveness of A3 75 cells in a dose-dependent manner in vitro .3^ By down-regulating the expression of heparanase-mRNA, matrine has a significant inhibitory effect on the adhesion and invasiveness of A375 cells.