TRAIL诱导胃癌细胞凋亡的研究
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摘要
[目的] 研究TRAIL(tumor necrosis factor related apoptosis inducing ligand)对胃癌细胞AGS、MKN45、MKN28和SGC-7901的凋亡诱导作用，及凋亡调控相关蛋白NF-κB、survivin、Bcl-2和Caspase3在细胞中的表达，探讨胃癌细胞对TRAIL作用的耐受机制；观察硝普钠及α-生育酚单独及与TRAIL联合应用时细胞凋亡率，及用药前后细胞中上述蛋白的表达，阐明二者单独应用及与TRAIL联合应用时对胃癌细胞的作用。
[方法] 采用碘化丙啶(PI)染色及流式细胞术对TRAIL诱导的胃癌细胞凋亡做定量分析；应用western blot方法观察了NF-κB、survivin、Bcl-2和Caspase3在四种胃癌细胞中的表达；应用α-生育酚(α-TOS)、α-TOS+TRAIL分别作用于敏感和耐受细胞，观察单独应用α-TOS及与TRAIL联合用药时细胞凋亡率的不同；应用硝普钠(SNP)、SNP+TRAIL分别作用于敏感和耐受细胞，观察单独应用SNP及SNP与TRAIL联合用药时细胞凋亡率的不同，western blot方法观察用药前后细胞中NF-κB、survivin和Bcl-2的表达。
[结果] (1)在TRAIL浓度为300ng／ml作用24小时情况下，胃癌细胞株MKN28的凋亡率最高为36.05％，MKN45和AGS次之，分别为20.37％和16.50％，SGC-7901对TRAIL敏感性最低，仅为11.80％。统计学分析表明，MKN28细胞对TRAIL的敏感性远高于其余三种细胞(p＜0.05)，而其他三种细胞对TRAIL的敏感性无显著差别(p＞0.05)。(2)western blot结果显示，MKN28细胞中NF-κB、survivin的表达明显低于SGC-7901细胞，而Caspase3的表达明显高于SGC-7901细胞，四种细胞中Bcl-2的表达无明显差异。(3)单用α-TOS 60μmol／L，作用24小时，MKN28和SGC-7901细胞的凋亡率分别为9.0％和8.5％，单用TRAIL300ng／ml诱导两种胃癌细胞的凋亡率分别为36.05％和11.80％，而α-TOS 60μmol／L和TRAIL 300ng／ml联合应用，可使胃癌MKN28细胞和SGC-7901细胞的凋亡率分别提高至63.7％和48.1％，显著高于单独用药对细胞的凋亡诱导作用(p＜0.05)。联合用药对胃癌细胞的凋亡诱导作用呈时效、量效关系。(4)单独应用SNP对胃癌细胞无凋亡诱导作用，但联合应用TRAIL300ng／ml及SNP2.0mM，可使MKN28和SGC-7901细胞凋亡率分别提高至63.77％和41.92％，远高于单独应用TRAIL对细胞的作用(p＜0.05)，且联合用药的凋亡诱导作用呈时效、量效关系。(5)α-生育酚可使细胞中survivin和Bcl-2的表达下降，且α-生育酚联合
第三军医大学硕士学位论文
TRAIL处理后，这种下降更明显。(6) SNP对细胞中Bd一2和NF- KB的表达无影响，
但可使survivin的表达下降，联合应用sNP+TRAIL，可使细胞中NF- KB和survivin
的表达明显下降，但对Bd一2的表达无影响。
【结论】(1) TRAIL在50ng/m卜一300n岁ml剂量范围内，对四种胃癌细胞均具
有凋亡诱导作用，其作用具有量效关系。MKN28细胞对TRAIL的敏感性最高，而
SGC一7901细胞的敏感性最低。(2)胃癌细胞对一TRAIL的敏感性与细胞内Bd一2的表
达无关，与凋亡抑制因子NF一‘B和survivin的低表达及凋亡促进因子Caspase3的高
表达有关。(3) Q.TOS对MKN28细胞和SGC一7901细胞具有凋亡诱导作用，且这种
作用呈现量效关系，Q .TOS与TRAIL联合应用对TRAIL具有增敏作用，且这种作用
呈现时效和量效关系。Q一Tos通过降低细胞中survivin和Bcl一2的表达来发挥对TRAIL
的增敏作用。(4) SNP单独应用不能诱导胃癌细胞凋亡。SNP与TRAIL联合应用对
TRAIL具有增敏作用，且这种作用呈现时效和量效关系。SNP通过降低细胞中survivin
的表达来发挥对TRAIL的增敏作用。
Objective: To study the apoptosis-inducing activity of TRAIL on gastric cancer cells MKN28, MKN45, AGS and SGC-7901, and to investigate the expressions of apoptosis-related regulator genes, including NF-кB, survivin, Bcl-2 and Caspase3 in these four cell lines.Meanwhile, we observed the effect of a combination of α-Tocopheryl succinate (α-TOS) and TRAIL, or sodium nitroprusside(SNP) and TRAIL on the apoptosis of gastric cancer cells and their effects on the expression of NF-к B,survivin and Bcl-2.
Methods: Apoptosis of gastric cancer cells induced by TRAIL was analysized with PI staining method and flow cytometry (FCM); the expressions of NF- кB,survivin, Bcl-2 and Caspase3 were observed Using Western blot method in the four gastric cancer cell lines.
Results: (1) After the gastric cancer cells were exposed to TRAIL 300ng/ml for 24 hours, the apoptosis rate was 36.05%, 20.37%, 16.50% and 11.80% in MKN28, MKN45, AGS and SGC-7901 cells respectively. The sensitivity to TRAIL in MKN28 cells was significantly higher than that in MKN45, AGS and SGG7901 cells(p<0.05). (2)Western blot revealed that the expressions of NF-к B and survivin were lower in MKN28 cells than in MKN45,AGS and SGC-7901 cells. In contrast, the expression of Caspase3 was higher in MKN28 cells than in MKN45, AGS and SGG7901 cells and the expression of Bcl-2 had no difference among the four cell lines. (3) When SGG7901 cells and MKN28 cells were exposed to α -TOS 60 mol/1 for 24 hours, the apoptosis rate was 9.0% in MKN28, 8.5% in SGC-7901, while treatment of SGC-7901 cells and MKN28 cells with a combination of u -TOS 60 mol/1 and TRAIL 300ng/ml for 24 hours , the apoptosis rate was 48.1% and 63.7% respectively.When the cells were exposed to α -TOS alone, the level of Bcl-2 and survivin
was decreased. Theα-TOS and TRAIL combination decreased the expression of NF- кB, Bcl-2 and survivin in gastric cancer cells.(4) Treatment of SGG7901 cells and MKN28 cells with a combination of SNP2.0mM and TRAIL 300ng/ml for 24 hours, the apoptosis rate was 41.92% and 63.77% respectively, but treatment with SNP alone can not induce SGC-7901 and MKN28 cells to undergo apoptosis. SNP administration could decrease the expression of survivin, but could not decrease the expression of NF-к B in gastric cancer cells. Moreover, SNP and TRAIL combination can decreased the expression of NF- кB and survivin in SGC-7901 cells and MKN28 cells
Conclusions: (1) There is a selectivity of TRAIL potency to induce apoptosis in gastric cancer cells of different cell lines. SGG7901 cells was most insensitive to TRAIL while MKN28 cells was most sensitive. (2)The anticancer potency of TRAIL is associated with the decreased expressions of NF- кB and survivin and the increased expression of Caspase3 of the gastric cancer cells.(3) alpha-TOS is a strong inducer of apoptosis and exerted a cooperative proapoptotic activity with TRAIL due to the inhibition of expression of NF- кB, Bcl-2 and survivin.(4) SNP alone could not induce apoptosis but it could enhance the activity of TRAIL through inhibition of the expression ofsurvivin in gastric cancer cells.
[方法] 采用碘化丙啶(PI)染色及流式细胞术对TRAIL诱导的胃癌细胞凋亡做定量分析；应用western blot方法观察了NF-κB、survivin、Bcl-2和Caspase3在四种胃癌细胞中的表达；应用α-生育酚(α-TOS)、α-TOS+TRAIL分别作用于敏感和耐受细胞，观察单独应用α-TOS及与TRAIL联合用药时细胞凋亡率的不同；应用硝普钠(SNP)、SNP+TRAIL分别作用于敏感和耐受细胞，观察单独应用SNP及SNP与TRAIL联合用药时细胞凋亡率的不同，western blot方法观察用药前后细胞中NF-κB、survivin和Bcl-2的表达。
[结果] (1)在TRAIL浓度为300ng／ml作用24小时情况下，胃癌细胞株MKN28的凋亡率最高为36.05％，MKN45和AGS次之，分别为20.37％和16.50％，SGC-7901对TRAIL敏感性最低，仅为11.80％。统计学分析表明，MKN28细胞对TRAIL的敏感性远高于其余三种细胞(p＜0.05)，而其他三种细胞对TRAIL的敏感性无显著差别(p＞0.05)。(2)western blot结果显示，MKN28细胞中NF-κB、survivin的表达明显低于SGC-7901细胞，而Caspase3的表达明显高于SGC-7901细胞，四种细胞中Bcl-2的表达无明显差异。(3)单用α-TOS 60μmol／L，作用24小时，MKN28和SGC-7901细胞的凋亡率分别为9.0％和8.5％，单用TRAIL300ng／ml诱导两种胃癌细胞的凋亡率分别为36.05％和11.80％，而α-TOS 60μmol／L和TRAIL 300ng／ml联合应用，可使胃癌MKN28细胞和SGC-7901细胞的凋亡率分别提高至63.7％和48.1％，显著高于单独用药对细胞的凋亡诱导作用(p＜0.05)。联合用药对胃癌细胞的凋亡诱导作用呈时效、量效关系。(4)单独应用SNP对胃癌细胞无凋亡诱导作用，但联合应用TRAIL300ng／ml及SNP2.0mM，可使MKN28和SGC-7901细胞凋亡率分别提高至63.77％和41.92％，远高于单独应用TRAIL对细胞的作用(p＜0.05)，且联合用药的凋亡诱导作用呈时效、量效关系。(5)α-生育酚可使细胞中survivin和Bcl-2的表达下降，且α-生育酚联合
第三军医大学硕士学位论文
TRAIL处理后，这种下降更明显。(6) SNP对细胞中Bd一2和NF- KB的表达无影响，
但可使survivin的表达下降，联合应用sNP+TRAIL，可使细胞中NF- KB和survivin
的表达明显下降，但对Bd一2的表达无影响。
【结论】(1) TRAIL在50ng/m卜一300n岁ml剂量范围内，对四种胃癌细胞均具
有凋亡诱导作用，其作用具有量效关系。MKN28细胞对TRAIL的敏感性最高，而
SGC一7901细胞的敏感性最低。(2)胃癌细胞对一TRAIL的敏感性与细胞内Bd一2的表
达无关，与凋亡抑制因子NF一‘B和survivin的低表达及凋亡促进因子Caspase3的高
表达有关。(3) Q.TOS对MKN28细胞和SGC一7901细胞具有凋亡诱导作用，且这种
作用呈现量效关系，Q .TOS与TRAIL联合应用对TRAIL具有增敏作用，且这种作用
呈现时效和量效关系。Q一Tos通过降低细胞中survivin和Bcl一2的表达来发挥对TRAIL
的增敏作用。(4) SNP单独应用不能诱导胃癌细胞凋亡。SNP与TRAIL联合应用对
TRAIL具有增敏作用，且这种作用呈现时效和量效关系。SNP通过降低细胞中survivin
的表达来发挥对TRAIL的增敏作用。
Objective: To study the apoptosis-inducing activity of TRAIL on gastric cancer cells MKN28, MKN45, AGS and SGC-7901, and to investigate the expressions of apoptosis-related regulator genes, including NF-кB, survivin, Bcl-2 and Caspase3 in these four cell lines.Meanwhile, we observed the effect of a combination of α-Tocopheryl succinate (α-TOS) and TRAIL, or sodium nitroprusside(SNP) and TRAIL on the apoptosis of gastric cancer cells and their effects on the expression of NF-к B,survivin and Bcl-2.
Methods: Apoptosis of gastric cancer cells induced by TRAIL was analysized with PI staining method and flow cytometry (FCM); the expressions of NF- кB,survivin, Bcl-2 and Caspase3 were observed Using Western blot method in the four gastric cancer cell lines.
Results: (1) After the gastric cancer cells were exposed to TRAIL 300ng/ml for 24 hours, the apoptosis rate was 36.05%, 20.37%, 16.50% and 11.80% in MKN28, MKN45, AGS and SGC-7901 cells respectively. The sensitivity to TRAIL in MKN28 cells was significantly higher than that in MKN45, AGS and SGG7901 cells(p<0.05). (2)Western blot revealed that the expressions of NF-к B and survivin were lower in MKN28 cells than in MKN45,AGS and SGC-7901 cells. In contrast, the expression of Caspase3 was higher in MKN28 cells than in MKN45, AGS and SGG7901 cells and the expression of Bcl-2 had no difference among the four cell lines. (3) When SGG7901 cells and MKN28 cells were exposed to α -TOS 60 mol/1 for 24 hours, the apoptosis rate was 9.0% in MKN28, 8.5% in SGC-7901, while treatment of SGC-7901 cells and MKN28 cells with a combination of u -TOS 60 mol/1 and TRAIL 300ng/ml for 24 hours , the apoptosis rate was 48.1% and 63.7% respectively.When the cells were exposed to α -TOS alone, the level of Bcl-2 and survivin
was decreased. Theα-TOS and TRAIL combination decreased the expression of NF- кB, Bcl-2 and survivin in gastric cancer cells.(4) Treatment of SGG7901 cells and MKN28 cells with a combination of SNP2.0mM and TRAIL 300ng/ml for 24 hours, the apoptosis rate was 41.92% and 63.77% respectively, but treatment with SNP alone can not induce SGC-7901 and MKN28 cells to undergo apoptosis. SNP administration could decrease the expression of survivin, but could not decrease the expression of NF-к B in gastric cancer cells. Moreover, SNP and TRAIL combination can decreased the expression of NF- кB and survivin in SGC-7901 cells and MKN28 cells
Conclusions: (1) There is a selectivity of TRAIL potency to induce apoptosis in gastric cancer cells of different cell lines. SGG7901 cells was most insensitive to TRAIL while MKN28 cells was most sensitive. (2)The anticancer potency of TRAIL is associated with the decreased expressions of NF- кB and survivin and the increased expression of Caspase3 of the gastric cancer cells.(3) alpha-TOS is a strong inducer of apoptosis and exerted a cooperative proapoptotic activity with TRAIL due to the inhibition of expression of NF- кB, Bcl-2 and survivin.(4) SNP alone could not induce apoptosis but it could enhance the activity of TRAIL through inhibition of the expression ofsurvivin in gastric cancer cells.
引文
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71．涂水平等，细胞凋亡与消化道肿瘤，消化内镜。1997，3(1)：60-65．
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68. MaeDonale G, Shi L, Vandeovelde C, et al.Induction of apoptosis by TRAIL.J Exp Med.1999,189:133-144.
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71．涂水平等，细胞凋亡与消化道肿瘤，消化内镜。1997，3(1)：60-65．