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结肠癌是一种常见的消化道恶性肿瘤,死亡率仅次于肺癌、肝癌及胃癌,居第4位,对人类的生命健康造成极大危害[1]。随着医疗水平的不断提升,结肠癌在诊断及治疗方面已取得明显进步,但是药物治疗不仅疗效不理想,而且还带来明显的不良反应及结肠癌细胞的转移[2]。因此,开发更有效的化疗药物已成为抗肿瘤治疗研究的热点。萝卜硫素是一种从十字花科植物中提取的异硫氰酸盐,研究[3-6]发现其具有抗肿瘤、抗氧化、抗炎、免疫调节等药理活性,然而萝卜硫素抗结肠癌的作用机制报道甚少。转化生长因子β1(TGF-β1)信号通路是细胞内重要的信号传导途径,可介导结肠癌细胞的增殖、分化、凋亡及迁移等生物学过程,抑制TGF -β1/Smad信号通路活化可以降低结肠癌细胞增殖、并诱导其凋亡[7-10]。本实验探讨萝卜硫素对结肠癌HT-29细胞增殖的抑制作用及其对细胞中TGF-β1/Smad信号通路的影响,以期为其开发应用提供理论依据。
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10、20、40 μmol/L萝卜硫素组细胞OD值相对于对照组明显降低(P < 0.01);同一浓度萝卜硫素处理48 h和72 h,细胞OD值相比于24 h也降低(P < 0.05~P < 0.01)。40 μmol/L萝卜硫素干预细胞72 h后,细胞抑制率高达76.25%,而低浓度萝卜硫素对HT-29细胞增殖抑制率表现出时间依赖性(P < 0.05)(见表 1)。
分组 n 24 h OD值 48 h OD值 72 h OD值 F P MS组内 对照组 3 1.4251±0.018 4 1.635 1±0.021 5 1.485 7±0.030 5 60.74 < 0.01 0.001 10 μmol/L萝卜硫素 3 1.014 3±0.016 5++ 0.951 7±0.021 6++* 0.627 1±0.020 4++**# 336.64 < 0.01 0.000 20 μmol/L萝卜硫素 3 0.540 6±0.012 6++▲▲ 0.485 1±0.013 5++*▲▲ 0.395 1±0.016 2++**#▲▲ 80.53 < 0.01 0.000 40 μmol/L萝卜硫素 3 0.385 1±0.009 4++▲▲■ 0.410 5±0.011 8++*▲▲■ 0.352 8±0.012 7++**#▲▲ 19.91 0.01 0.000 F — 3 120.49 3 043.56 1 894.27 — — — P — < 0.01 < 0.01 < 0.01 — — — MS组内 — 0.000 0.000 0.000 — — — q检验:与24 h比较*P < 0.05,** P < 0.01;与48 h比较##P < 0.01;与对照组比较++P < 0.01;与萝卜硫素10 μmol/L比较▲▲P < 0.01;与萝卜硫素20 μmol/L比较■■P < 0.01 表 1 各组间HT-29细胞OD值比较(x±s)
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10、20、40 μmol/L萝卜硫素干预HT-29细胞24 h,细胞凋亡率均高于对照组(P < 0.05~P < 0.01)(见图 1、表 2)。
分组 n 细胞凋亡率/% 对照组 3 3.85±0.61 10 μmol/L萝卜硫素 3 7.35±0.84* 20 μmol/L萝卜硫素 3 10.57±1.16**# 40 μmol/L萝卜硫素 3 19.53±1.84**##▲ F — 93.35 P — < 0.01 MS组内 — 1.452 q检验:与对照组比较*P < 0.05,** P < 0.01;与10 μmol/L萝卜硫素比较#P < 0.05,##P < 0.01;与20 μmol/L萝卜硫素比较▲P < 0.05 表 2 各组细胞凋亡率比较(x±s)
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ELISA结果表明,药物处理HT-29细胞24 h,10、20、40 μmol/L萝卜硫素组细胞培养液中TGF-β1水平均低于对照组(P < 0.05~P < 0.01)(见表 3)。
分组 n TGF β1/β-actin 对照组 3 1 315.26±40.17 10 μmol/L萝卜硫素 3 1 141.39±26.37* 20 μmol/L萝卜硫素 3 892.54±20.38**# 30 μmol/L萝卜硫素 3 627.43±15.27**##▲ F — 364.63 P — < 0.01 MS组内 — 739.381 q检验:与对照组比较*P < 0.05,** P < 0.01;与10 μmol/L萝卜硫素比较#P < 0.05,##P < 0.01;与20 μmol/L萝卜硫素比较▲P < 0.05 表 3 各组细胞TGF β1水平比较(x±s)
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与对照组相比,10、20、40 μmol/L萝卜硫素均能抑制p-Smad3、Smad4、Cyclin D1及c-Myc蛋白表达(P < 0.05~P < 0.01)(见图 2、表 4)。
分组 n p-Smad3/β-actin Smad4/β-actin Cyclin D1/β-actin c-Myc/β-actin 对照组 3 0.41±0.05 0.57±0.04 0.36±0.05 0.63±0.04 10 μmol/L萝卜硫素 3 0.24±0.03* 0.34±0.05* 0.22±0.04* 0.31±0.05* 20 μmol/L萝卜硫素 3 0.15±0.03** 0.21±0.03** 0.14±0.03** 0.16±0.03** 30 μmol/L萝卜硫素 3 0.07±0.02**##▲ 0.17±0.03**#▲ 0.06±0.03**#▲ 0.13±0.02**#▲ F — 54.36 66.08 33.29 116.50 P — < 0.01 < 0.01 < 0.01 < 0.01 MS组内 — 0.001 0.001 0.001 0.001 q检验:与对照组比较*P < 0.05,** P < 0.01;与10 μmol/L萝卜硫素比较#P < 0.05,##P < 0.01;与20 μmol/L萝卜硫素比较▲P < 0.05 表 4 各组p-Smad3、Smad4、Cyclin D1及c-Myc蛋白表达比较(x±s)
萝卜硫素调控TGF-β1/Smad信号通路抑制结肠癌HT-29细胞增殖的机制研究
Sulforaphane inhibits proliferation of colon cancer HT-29 cells via regulating TGF β1/Smad signaling pathway
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摘要:
目的研究萝卜硫素对结肠癌HT-29细胞增殖的影响及其可能的作用机制。 方法采用CCK8分析萝卜硫素对HT-29细胞增殖的影响,流式细胞术检测HT-29细胞凋亡,ELISA检测细胞培养液中转化生长因子β1(TGF-β1)的水平,Western blotting检测细胞中p-Smad3、Smad4、Cyclin D1及c-Myc蛋白表达。 结果CCK8结果显示,随着药物浓度和作用时间的增加,萝卜硫素对HT-29细胞的增殖抑制作用增强(P < 0.05~P < 0.01)。10、20、40 μmol/L萝卜硫素干预HT-29细胞24 h,细胞凋亡率均高于对照组(P < 0.05~P < 0.01)。ELISA结果表明,药物处理HT-29细胞24 h,10、20、40 μmol/L萝卜硫素组细胞培养液中TGF β1水平均低于对照组(P < 0.05~P < 0.01)。与对照组相比,10、20、40 μmol/L萝卜硫素均能抑制p-Smad3、Smad4、Cyclin D1及c-Myc蛋白表达(P < 0.05~P < 0.01)。 结论萝卜硫素可抑制结肠癌HT-29细胞的增殖,其作用机制可能与调控TGF-β1/Smad信号通路有关。 -
关键词:
- 结肠肿瘤 /
- 萝卜硫素 /
- 增殖 /
- TGF β1/Smad信号通路
Abstract:ObjectiveTo study the effect of sulforaphane on the proliferation of colon cancer HT-29 cells and its potential mechanism. MethodsCCK8 was used to detect the effect of sulforaphane on the proliferation of HT-29 cells.Flow cytometry was applied to determine the apoptosis of HT-29 cells.ELISA was performed to measure the levels of transforming growth factor β1 (TGF-β1).Western blotting was carried out to analyze the expression of p-Smad3, Smad4, Cyclin D1 and c-Myc protein. ResultsCCK8 results showed that the inhibitory effect of sulforaphane on the proliferation of HT-29 cells increased with the increase of drug concentration and time (P < 0.05 to P < 0.01).The apoptosis rate of HT-29 cells treated with 10, 20, 40 μmol/L sulforaphane was higher than that in control group (P < 0.05 to P < 0.01).The results of ELISA showed that the level of TGF-β1 in cell culture medium in 10, 20, 40 μmol/L sulforaphane group was lower than that in control group (P < 0.05 to P < 0.01).The expression of p-Smad3, Smad4, Cyclin D1 and c-Myc protein in HT-29 cells after treatment with 10, 20, 40 μmol/L sulforaphane was decreased compared with control group (P < 0.05 to P < 0.01). ConclusionsSulforaphane can inhibit the proliferation of colon cancer HT-29 cells, the mechanism of which might be related to the regulation of TGF-β1/Smad signaling pathway. -
Key words:
- colon neoplasms /
- sulforaphane /
- proliferation /
- TGF-β1/Smad signaling pathway
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表 1 各组间HT-29细胞OD值比较(x±s)
分组 n 24 h OD值 48 h OD值 72 h OD值 F P MS组内 对照组 3 1.4251±0.018 4 1.635 1±0.021 5 1.485 7±0.030 5 60.74 < 0.01 0.001 10 μmol/L萝卜硫素 3 1.014 3±0.016 5++ 0.951 7±0.021 6++* 0.627 1±0.020 4++**# 336.64 < 0.01 0.000 20 μmol/L萝卜硫素 3 0.540 6±0.012 6++▲▲ 0.485 1±0.013 5++*▲▲ 0.395 1±0.016 2++**#▲▲ 80.53 < 0.01 0.000 40 μmol/L萝卜硫素 3 0.385 1±0.009 4++▲▲■ 0.410 5±0.011 8++*▲▲■ 0.352 8±0.012 7++**#▲▲ 19.91 0.01 0.000 F — 3 120.49 3 043.56 1 894.27 — — — P — < 0.01 < 0.01 < 0.01 — — — MS组内 — 0.000 0.000 0.000 — — — q检验:与24 h比较*P < 0.05,** P < 0.01;与48 h比较##P < 0.01;与对照组比较++P < 0.01;与萝卜硫素10 μmol/L比较▲▲P < 0.01;与萝卜硫素20 μmol/L比较■■P < 0.01 表 2 各组细胞凋亡率比较(x±s)
分组 n 细胞凋亡率/% 对照组 3 3.85±0.61 10 μmol/L萝卜硫素 3 7.35±0.84* 20 μmol/L萝卜硫素 3 10.57±1.16**# 40 μmol/L萝卜硫素 3 19.53±1.84**##▲ F — 93.35 P — < 0.01 MS组内 — 1.452 q检验:与对照组比较*P < 0.05,** P < 0.01;与10 μmol/L萝卜硫素比较#P < 0.05,##P < 0.01;与20 μmol/L萝卜硫素比较▲P < 0.05 表 3 各组细胞TGF β1水平比较(x±s)
分组 n TGF β1/β-actin 对照组 3 1 315.26±40.17 10 μmol/L萝卜硫素 3 1 141.39±26.37* 20 μmol/L萝卜硫素 3 892.54±20.38**# 30 μmol/L萝卜硫素 3 627.43±15.27**##▲ F — 364.63 P — < 0.01 MS组内 — 739.381 q检验:与对照组比较*P < 0.05,** P < 0.01;与10 μmol/L萝卜硫素比较#P < 0.05,##P < 0.01;与20 μmol/L萝卜硫素比较▲P < 0.05 表 4 各组p-Smad3、Smad4、Cyclin D1及c-Myc蛋白表达比较(x±s)
分组 n p-Smad3/β-actin Smad4/β-actin Cyclin D1/β-actin c-Myc/β-actin 对照组 3 0.41±0.05 0.57±0.04 0.36±0.05 0.63±0.04 10 μmol/L萝卜硫素 3 0.24±0.03* 0.34±0.05* 0.22±0.04* 0.31±0.05* 20 μmol/L萝卜硫素 3 0.15±0.03** 0.21±0.03** 0.14±0.03** 0.16±0.03** 30 μmol/L萝卜硫素 3 0.07±0.02**##▲ 0.17±0.03**#▲ 0.06±0.03**#▲ 0.13±0.02**#▲ F — 54.36 66.08 33.29 116.50 P — < 0.01 < 0.01 < 0.01 < 0.01 MS组内 — 0.001 0.001 0.001 0.001 q检验:与对照组比较*P < 0.05,** P < 0.01;与10 μmol/L萝卜硫素比较#P < 0.05,##P < 0.01;与20 μmol/L萝卜硫素比较▲P < 0.05 -
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