EFFECTS OF VIETNAMESE SOPHORA ROOT ON GROWTH, ADHESION, INVASION AND MOTILITY OF MELANOMA CELLS

Background: Vietnamese Sophora Root mainly contains active constituents such as alkaloids, and it has anti-tumour, antibacterial, and anti-inflammatory effects. The objective of the paper was to study the effects of Vietnamese Sophora Root on growth, adhesion, invasion and motility of mouse melanoma B 16 BL 6 cells, and to preliminarily explore its mechanism of action. Materials and Methods: MTT assay was used to detect the effect of Vietnamese Sophora Root aqueous extract on B 16 BL 6 cell proliferation. Cell adhesion assay, reconstituted basement membrane invasion assay and chemotactic motility assay were used to observe the effects of Vietnamese Sophora Root aqueous extract on adhesion, invasion and motility of B 16 BL 6 cells. Results: Different concentrations of Vietnamese Sophora Root aqueous extracts had different degrees of inhibitory effects on B 16 BL 6 proliferation. With the decrease of concentration, the proliferation inhibitory effect decreased and even turned to promoting effect. The extract significantly inhibited the adhesion of B 16 BL 6 cells to the basement membrane component LN, and had a significant effect on both the invasive and migratory capacities of B 16 BL 6 cells through the basement membrane. Conclusion: We concluded that the aqueous extract of Vietnamese Sophora Root can inhibit the proliferation of melanoma cells, as well as their adhesion and movement.


Medicinal materials
Chinese medicinal herb Vietnamese Sophora Root, was purchased from Baidu Medicine Company, Shenyang，China.The herb was identified by Professor Kang Yangguo, and was placed in the pharmacy centre.ID number is 2011-3-311.

Cell lines
Melanoma B 16 BL 6 cells were provided by the Affiliated Hospital of China Medical University.

Preparation of Vietnamese Sophora Root aqueous extract
In reference to the method in Xiao et al. (2000), Vietnamese Sophora Root was pulverised, added with distilled water and kept overnight, followed by centrifugation at 3500 rpm for 20 min.The supernatant was concentrated to 1/10 of its original volume and centrifuged at 20000 rpm for 30 min.The supernatant was then freeze-dried to obtain Vietnamese Sophora Root extract.

Cell cultivation
Cells were cultured in RPMI 1640 medium added with 10% fetal bovine serum under conditions of 37 ℃ and 5% CO 2 .The culture medium was replaced every 2 ~3 d.

B 16 BL 6 cell proliferation inhibition assay
With reference to Carmichael's MTT method in Carmichael et al. (1987), B 16 BL 6 cells in the logarithmic growth phase were collected and digested with 0.25% trypsin.The cell concentration was adjusted to 5 ×10 4 /ml, and the cells were seeded in 96 well plates at 100 μL per well.6 duplicate wells were set up.After culturing for 24 h, 100 μL of drug-containing culture media were added.Final concentrations of Vietnamese Sophora Root extracts were 400, 500, 600, 700 and 800 μg/ml respectively.The negative control group was added with equal volume of culture medium.After incubation at 37 ℃ for 48 h, each well was added with 20 μl of MTT (2 mg/ml), and the incubation was continued for another 4 h.Then, the supernatant was discarded, and each well was added with 100 μl of DMSO to dissolve crystals.Afterwards, OD values were measured at wavelength of 570 nm using microplate reader.The inhibition rate (IR) of the drug on cell growth was calculated according to the following formula: IR (%) = (1 -average OD value of experimental group / average OD value of control group) × 100%.

B 16 BL 6 cell adhesion assay (Trapp et al., 2010)
With reference to the literature, 96-well plates were coated with Matrigel at 2 μg/well, placed on a super clean bench and air-dried for later use.The plates were blocked with 1% BSA at 40 μL per well, and incubated at 37℃ for 1 h.They were then washed 3 times with PBS for later use.B16BL6 melanoma cells in the logarithmic growth phase were collected and the cell concentration was adjusted to 1×10 6 /mL.The cells were added to the above 96-well plates at 90 μL per well.Afterwards,10 μL of DMEM media containing different concentrations of Vietnamese Sophora Root extracts were added to each well, and the plates were incubated at 37℃ for 1 h.The plates were then washed twice in PBS, added with 20 μL of MTT solution (2 mg/ml) and cultured for an additional 4 h in the incubator.The supernatant was discarded and 100 μL of DMSO solution was added to each well.After mixing well by shaking, OD value was measured at 570 nm using microplate reader, and inhibition rate (IR) of the drug on cell adhesion was calculated according to the following formula: IR (%) = (1 -average OD value of experimental group / average OD value of control group) × 100%

B 16 BL 6 cell reconstituted basement membrane invasion assay (Gu et al., 2007)
PVPF filter membrane was attached to the Transwell chamber.The outer surface of the membrane was coated with 5 μg of FN, and inner surface with 10 μg of Matrigel.The membrane was dried overnight at room temperature to form a matrix barrier layer.B 16 BL 6 cells, which were pre-treated with drug for 24 h, were digested with 0.25% trypsin, and re-suspended in serum-free DMEM medium containing 0.1% BSA at a density of 2×10 6 /ml. 100 μL of the above cell suspension was added to the Transwell chamber.The chamber was placed in a 24-well plate, and the plate was added with 600 μl of serum-free DMEM medium containing 0.1% BSA, and incubated at 37℃ for 4 h.The membrane was fixed in methanol for 1 min, stained with haematoxylin and eosin.Cells that did not permeate the membrane on one side of Matrigel were wiped off, and the filter membrane was sealed on the slide with Eukitt.5 different fields of the membrane, namely up, down, left, right and centre, were selected under a 400x optical microscope.The number of invasive cells in each field was counted, and the average value was calculated.The inhibition rate (IR) of the drug on cell invasion was calculated according to the following formula: IR (%) = (1 -average number of invasive cells of experimental group / average number of invasive cells of control group) × 100%

B 16 BL 6 chemotactic motility assay (Zheng et al., 2003)
The steps were the same as the invasion assay except for not coating Matrigel on the inner surface of PVPF membrane.The inhibition rate (IR) of the drug on cell motility was calculated according to the following formula: IR (%) = (1 -average number of motile cells of experimental group / average number of motile cells of control group) × 100%

Statistical analysis
The experimental results were analysed using SPSS 10.0 statistical software.The count data were compared with the χ 2 test, and measurement data were compared using the t test.P<0.05 indicated statistically significant difference.

Effect of Vietnamese Sophora Root aqueous extract on proliferative capacity of B 16 BL 6 cells
With the decreasing concentration of Vietnamese Sophora Root aqueous extract, inhibition rate of B16BL6 cell proliferation gradually decreased.The proliferation was even promoted at lower concentrations.When the concentration of Vietnamese Sophora Root extract was 800 μg/ml, the inhibition rate was 27.09%, which showed a significant difference.When the concentration was decreased to 500 μg/ml, the proliferation promoting effect was present.The results are shown in Table 1. between tumour cells and matrix Matrigel.When the concentration was 800 μg/ml, the inhibition rate reached 20.93%, and when the concentration was 400 μg/ml, the inhibition rate also reached 3.82%.The results are shown in Table 2.

Effect of Vietnamese Sophora Root on reconstituted basement membrane invasion of B 16 BL 6 cells
The results showed that compared with the control group each Vietnamese Sophora Root concentration group can significantly inhibit the number of melanoma cells and basement membrane-invasive cells, and the difference was significant.Moreover, the inhibitory effect increased with increasing drug concentration.The results are shown in Table 3.

Table 1 :
Determination of the effect of Vietnamese Sophora Root on B 16 BL 6 cell proliferation by MTT assay (n=6,

Effect of Vietnamese Sophora Root on adhesion capacity of B 16 BL 6 cells to the basement membrane component
Experimental results showed that when the tumour cells had been adherent to the matrix, that is, when the tumour cells have completed binding adhesion to the matrix, Vietnamese Sophora Root was added, and it was found that the Vietnamese Sophora Root inhibited the adhesion

Table 2 :
Effect of Vietnamese Sophora Root on adhesion capacity of B 16 BL 6 cells to LN (n=6,

Table 3 :
Effect of Vietnamese Sophora Root on reconstituted basement membrane invasive capacity of B 16 BL 6 cells (n=6,

Effect of Vietnamese Sophora Root on chemotactic motility of B 16 BL 6 cells
Vietnamese Sophora Root aqueous extract can significantly inhibit the motility of B16BL6 cells through the basement membrane at high concentrations.The results are shown in Table4.

Table 4 :
Effect of Vietnamese Sophora Root on chemotactic motility of B 16 BL 6 cells (n=6,