Effect of Gossypol Acetate Acid on Invasion of Human Tongue Squamous Cell Carcinoma Cal-27 Cells in vitro
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摘要:
目的 研究醋酸棉酚（gossypol acetic acid，GAA）对体外培养的人舌鳞癌Cal-27细胞侵袭影响及其作用机制。 方法 制备0 μmol/L、2.5 μmol/L、5.0 μmol/L、10.0 μmol/L的GAA作用于人舌鳞癌Cal-27 细胞，通过Transwell实验检测GAA对人舌鳞癌Cal-27 细胞侵袭能力的影响；通过qRT-PCR及Western blot实验检测GAA对人舌鳞癌Cal-27 细胞基质金属蛋白酶-2（Matrix Metalloproteinase-2，MMP-2）、基质金属蛋白酶-9（Matrix Metalloproteinase-9，MMP-9）mRNA及蛋白的表达变化。 结果 Transwell实验显示：GAA对人舌鳞癌Cal-27细胞细胞侵袭产生抑制作用，实验组和对照组比较差异有统计学意义（P < 0.05）；qRT-PCR实验及Western blot显示，实验组人舌鳞癌Cal-27细胞中MMP-2、MMP-9 mRNA及蛋白的表达降低，与对照组相比差异有统计学意义（P < 0.05）。 结论 GAA能抑制人舌鳞癌Cal-27细胞的侵袭性，并降低人舌鳞癌Cal-27细胞MMP-2、MMP-9的mRNA及蛋白表达水平，这可能是GAA抑制肿瘤侵袭作用的机制之一。 Abstract:Objective To observe the effect of gossypol acetate acid（GAA）on the invasion of human tongue squamous cell carcinoma Cal-27 cells in vitro. Methods GAA of different concentrations（0 μmol/L, 2.5 μmol/L, 5.0 μmol/L, 10.0 μmol/L）was acted on Cal-27 cells, Transwell experiment was employed to detect the inhibitory effect of GAA on the invasion of Cal-27 cells. The effect of gossypol acetate on MMP-2 and MMP-9 mRNA expression was quantified by qRT-PCR. The effect of gossypol acetate acid on MMP-2 and MMP-9 protein expression was analyzed by Western-Blot experiment. Results Cell Transwell chamber experiment showed that GAA could inhibit the migration of human tongue squamous cell carcinoma Cal-27 cells, and the amount of invasion decreased with the increase in concentration. The difference between the control group and the experimental group was statistically significant（P < 0.05）. qRT-PCR experiment and Western-blot results showed that the expression of MMP-2 and MMP-9 mRNA and proteins was significantly reduced after Cal-27 cells of human tongue squamous cell carcinoma was treated with GAA, and the difference between the control group and the experimental group was statistically significant（P < 0.05）. Conclusions GAA can reduce the invasiveness of tongue squamous cell carcinoma Cal-27 cells. And GAA can reduce the mRNA and protein expression levels of MMP-2 and MMP-9 of human tongue squamous cell carcinoma Cal-27 cells. -
图 1 各组细胞Transwell迁移实验（×100）
Figure 1. The light microscope photos of the migration of Cal-27 cell（×100）
图 3 各组细胞Transwell侵袭实验（×100）
Figure 3. The light microscope photos of the invasion of Cal-27 cell（×100）
图 5 GAA对人舌鳞癌Cal-27细胞MMP-2、MMP-9 蛋白表达水平的影响
Figure 5. The protein expression level of MMP-2，MMP-9 in Cal- 27 cells by GAA
表 1 MMP-2、MMP-9、GAPDH的引物序列
Table 1. Primer sequences of MMP-2，MMP-9 and GAPDH
基因 引物序列 大小（bp） MMP-2（F） 5′-GAACCAGATCACATACAGGATC-3′ MMP-2（R） 5′-ATCCACTGTCTCTGGGTCCA-3′ 66 bp MMP-9（F） 5′-ACCTCAAGTGGCACCACCAC-3′ MMP-9（R） 5′-GCGGCAAGTCTTCCGAGTAG-3′ 63 bp GAPDH（F） 5′-GCCATCAATGACCCCTTCAT-3′ GAPDH（R） 5′-TTGGAACATGTAAACCATGT -3′ 51 bp 
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表 2 GAA对Cal-27细胞Transwell迁移个数对比（个）[（
$\bar{{{x}}}\pm {{s}}$ ，n = 3）]Table 2. Comparison of the migration of Cal-27 cell in different concentration of GAA（each）[（
$\bar{{{x}}}\pm {{s}}$ ，n = 3）]组别 0 h 6 h 12 h 18 h 24 h 对照组 14.00 ± 3.61 15.00 ± 3.00▽ 43.00 ± 2.65▽△ 75.33 ± 3.51▽△☆ 100.33 ± 4.93▽△☆◇ 2.5 μmol/L组 5.00 ± 1.00▲ 13.00 ± 2.65▲▽ 26.33 ± 3.51▲▽△ 43.00 ± 2.00▲▽△☆ 64.00 ± 3.00▲▽△☆◇ 5 μmol/L组 4.33 ± 2.08▲★ 10.33 ± 2.52▲★▽ 24.67 ± 1.53▲★▽△ 38.67 ± 2.52▲★▽△☆ 54.67 ± 3.51▲★▽△☆◇ 10 μmol/L组 2.33 ± 1.15▲★■ 7.00 ± 2.00▲★■▽ 8.67 ± 2.08▲★■▽△ 12.67 ± 2.08▲★■▽△☆ 21.33 ± 3.51▲★■▽△☆◇ 相同作用时间与对照组比较，▲P < 0.05；相同作用时间与2.5 μmol/L比较，★P < 0.05；相同作用时间与5 μmol/L比较，■P < 0.05；相同GAA浓度作用与0 h比较，▽P < 0.05；相同GAA浓度作用与6 h比较，△P < 0.05；相同GAA浓度作用与12 h比较，☆P < 0.05；相同GAA浓度作用与18 h比较，◇P < 0.05。
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表 3 GAA对Cal-27细胞Transwell侵袭个数对比（个）[（
$\bar{{{x}}}\pm {{s}}$ ），n = 3]Table 3. Comparison of the invasion of Cal-27 cell in different concentration of GAA（each） [（
$\bar{{{x}}}\pm {{s}}$ ），n = 3]组别 0 h 6 h 12 h 18 h 24 h 对照组 12.00 ± 2.65 13.33 ± 3.06▽ 39.00 ± 1.00▽△ 75.67 ± 5.69▽△☆ 100.67 ± 7.77▽△☆◇ 2.5 μmol/L组 6.00 ± 2.00▲ 12.33 ± 3.51▲▽ 28.33 ± 4.51▲▽△ 40.33 ± 1.53▲▽△☆ 62.67 ± 5.69▲▽△☆◇ 5 μmol/L组 5.33 ± 3.51▲★ 9.33 ± 4.73▲★▽ 24.33 ± 3.51▲★▽△ 32.33 ± 3.51▲★▽△☆ 52.33 ± 6.51▲★▽△☆◇ 10 μmol/L组 3.33 ± 1.53▲★■ 8.67 ± 3.06▲★■▽ 9.00 ± 1.00▲★■▽△ 10.67 ± 2.08▲★■▽△☆ 19.67 ± 1.53▲★■▽△☆◇ 相同作用时间与对照组比较，▲P < 0.05；相同作用时间与2.5 μmol/L比较，★P < 0.05；相同作用时间与5 μmol/L比较，■P < 0.05；相同GAA浓度作用与0 h比较，▽P < 0.05；相同GAA浓度作用与6 h比较，△P < 0.05；相同GAA浓度作用与12 h比较，☆p < 0.05；相同GAA浓度作用与18 h比较，◇P < 0.05。
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表 4 GAA对Cal-27细胞中MMP-2、MMP-9 mRNA表达的影响（
$\bar{{{x}}}\pm {{s}}$ ，n = 9）Table 4. The mRNA expression level of MMP-2，MMP-9 in Cal- 27 cells by GAA（
$\bar{{{x}}}\pm {{s}}$ ，n = 9）组别 MMP-2 MMP-9 对照组 1.02 ± 0.24 1.01 ± 0.15 2.5 μmol/L组 0.25 ± 0.04▲ 0.83 ± 0.10▲ 5 μmol/L组 0.16 ± 0.06▲ 0.69 ± 0.03▲★ 10 μmol/L组 0.10 ± 0.03▲★ 0.46 ± 0.18▲★■ 与对照组比较，▲P < 0.05；与2.5 μmol/L比较，★P < 0.05；与5 μmol/L比较，■P < 0.05。
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表 5 不同浓度GAA作用于人舌鳞癌Cal-27细胞MMP-2、MMP-9蛋白表达相对值（MMP-2、MMP-9/GAPDH）（
$\bar{{{x}}}\pm {{s}}$ ，n = 3）Table 5. The protein expression level of MMP-2、MMP-9 in cal- 27 cells by GAA（MMP-2、MMP-9/GAPDH） （
$\bar{{{x}}}\pm {{s}}$ ，n = 3）组别 MMP2/GAPDH MMP9/GAPDH 对照组 1.20 ± 0.02 1.20 ± 0.01 2.5 μmol/L组 0.98 ± 0.00▲ 0.91 ± 0.01▲ 5 μmol/L组 0.76 ± 0.00▲★ 0.75 ± 0.00▲★ 10 μmol/L组 0.61 ± 0.01▲★■ 0.53 ± 0.00▲★■ 与对照组比较，▲P < 0.05；与2.5 μmol/L比较，★P < 0.05；与5 μmol/L比较，■P < 0.05。
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