| Objective:In this in vitro study, we took A549 cell line as working model and evaluated the changes of MMP-9mRNA and proteins expression, adhesion ability, migration ability and invasiveness ability of A549 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.Methods:(1)The effect of matrine on proliferation of A549 cells measured by MTT assay Growing cells were treated with matrine at a final concentration of 0.0625g/L,0.1250g/L,0.2500g/L,0.5000g/L, respectively. Culture medium was added in control group.Fresh culture medium with different concentration of matrine or control medium was changed every two days.After exposure to various matrine for 1-6d, respectively. MTT was added and the plates were incubated for an additional 4h before absorbance at 570nm was measured. The inhibitory rate was calculated according to the formula. (2)Cell Matrivgel adhesion assay Each well of 96-well plates was coated with Matrivgel 40μL(2g/L) and 2% BSA 20μL.A549 cells treated with matrine for 48h were collected and resuspended at 1×109 cells/L in RPMI1640,still treated with matrine at a final concentration of 0.0625g/L,0.1250g/L,0.2500g/L or culture medium, each hole by adding 100μL.Those cells that could not adhere to Matrivgel were washed by PBS after incubated for 1h at 37℃.MTT was added and the plates were incubated for another 4h before absorbance at 570nm was measured. The inhibitory rate of cellular adhesion was calculated according to the formula.(3)Cell migration assay Cancer cell migration was measured by examining cell traverse through fibronectin-coated polycarbonate filters, using modified transwell chambers.A549 cells treated with matrine for 48h were collected and resuspended at 1×109cells/L in RPM1640.150μL of suspension was added into each chamber and treated with matrine at a final concentration of 0.0625g/L,0.1250g/L,0.2500g/L.Control group was performed by adding culture medium. After incubated for addtional 10h,the cells on the upper side of polyethylene membrane were wiped off with a cotton swab. The remaining cells that traversed the polyethylene membrane were fixed, stained and six random fields of vision were counted under light microscope. The number of migration cells was equal to the number of cells on the lower surface of the membrane. The inhibitory rate of cellular migration was calculated according to the formula.(4)Cell Matrivgel invasiveness assay 50μLof Matrivgel(0.5g/L) was added to each transwell's botton, relocated in 24-well plates and dried for 4h. A549 cells treated with matrine for 48h were collected and resuspended at 1×109 cells/L in RPM1640.150μL of suspension was added into each chamber and treated with matrine at a final concentration of 0.0625g/L,0.1250g/L,0.2500g/L.Control group was performed by adding culture medium.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 Matrivgel 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.the inhibitory rate of cellular invasiveness was calculated according to the formula.(5)The MMP-9mRNA expression levels performed by semi-quantitative RT-PCR A549 cells were collected at after treated with 0.0625g/L,0.1250g/L,0.2500g/L matrine or culture medium for 48h. The total RNAs were extracted from the cells and then semi-quantiative RT-PCR was performed to evaluate the MMP-9mRNA expression.40 cycles of MMP-9 were performed and the length of amplification was about 196 base pairs.The internal control was made by PCR with Primers specific forβ-actin,40 cycles of P-actin were Performed and the length of PCR product was about 285 base pairs.(6)The MMP-9 proteins expression levels performed by Immunocytochemistry Logarithmic growth phase of the A549cells were collected, and resuspended at 5×107cells/L with complete culture medium, suspension was added into 24-well plate, each hole was 1mL,attachment after 24h and treated with matrine at a final concentration of 0.0625g/L, 0.1250g/L,0.2500g/L matrine or culture medium for 48h. Fixed with 4% formaldehyde, A549 cells were incubated at 4℃over night with MMP-9 polyclonal rabbit antibodies(1:100).Then, the steps were in accordance with the immunohistochemistry kit and five random fields of vision were counted under light microscope, the antigen visualized with the immunoperoxidase-based system.Results:(1)The inhibitory effect of matrine on proliferation of A549 cells Matrine can remarkably inhibit the proliferation of A549 cells with a dose dependent manner within the concentration of 0.1250g/L,0.2500g/L, 0.5000g/L(P<0.05).However, the inhibitory effect of matrine at low concentration of 0.0625g/L was weak.(2)The inhibitory effect of matrine on adhesion of A549 cells After treated with culture medium or various concentration matrine, the absorbance at 570nm was 0.561±0.040,0.542±0.043,0.337±0.053, 0.203±0.054,respectively. The absorbance at 570nm of A549 cells treated with matrine of group 0.1250g/L and 0.2500g/L were decreased significantly compared to that of control group(P<0.05).However, the difference in the group of 0.0625g/L did not exist. The inhibitory effect was significantly different between treatment groups(P<0.05).Besides, absorbance at 570nm decreased gradually when the concentration of matrine increased. The inhibitory effect of matrine had the obvious dosage-dependence.(3)The inhibitory effect of matrine on migration of A549 cells After treated with culture medium or various concentration matrine,the number of invasive cells of treated groups was 188.133±11.521, 153.833±10.926,136.367±10.008,102.367±10.762,respectively. Compared to control group, the number of migrative cells of treated groups was remarkably decreased(P<0.05).Besides,the inhibitory effect was significantly different between treatment groups(P<0.05).(4)The inhibitory effect of matrine on invasiveness of A549 cells After treated with culture medium or various concentration matrine, the number of invasive cells of treated groups was 155.267±8.221, 133.200±8.036,106.533±9.273,78.967±7.189,respectively. Compared to control group, the number of invasive cells of treated groups was remarkably decreased(P<0.05).Besides,the inhibitory effect was significantly different between treatment groups(P<0.05). (5)The inhibition of matrine on MMP-9mRNA expression of A549 cells After treated with culture medium or various concentration matrine, the ratio of optical density of MMP-9mRNA to that ofβ-actin in A549 cells was 0.820±0.016,0.648±0.022,0.416±0.067,0.222±0.019,respectively. The MMP-9mRNA expression of A549 cells treated with matrine of different final concentration was significantly decreased compared to that of control group(P<0.05).The inhibitory effect was significantly different between treatment groups(P<0.05).The inhibitory effect of matrine had the obvious dosage-dependence.(6)The inhibition of matrine on MMP-9 proteins expression of A549 cells After treated with culture medium or various concentration matrine,the integrate optical density of MMP-9 proteins in A549 cells was 0.392±0.133,0.381±0.202,0.251±0.088,0.152±0.182,respectively. The MMP-9 proteins expression of A549 cells treated with matrine of group 0.1250g/L and 0.2500g/L were decreased significantly compared to that of control group(P<0.05).However, the difference in the group of 0.0625g/L did not exist. The inhibitory effect was significantly different between treatment groups(P<0.05).The inhibitory effect of matrine had the obvious dosage-dependence.Conclusions:1.Matrine can remarkably inhibit the proliferation of A549 cells in a dose-dependent manner. 2.Matrine can significantly inhibit the adhesion, migration and invasiveness of A549 cells in a dose-dependent manner.3.Matrine can down-regulate the expression of MMP-9mRNA and proteins of A549 cells in a dose-dependent manner.4.Matrine has a significant inhibitory effect on the adhesion, migration and invasiveness of A549 cells by down-regulating the expression of MMP-9mRNA and proteins.
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