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目的探讨曲古抑菌素A(TSA)诱导胰腺癌细胞PANC-1细胞凋亡机制。方法 TSA 0.1～0.6μmol.L-1培养PANC-1细胞0～48 h,MTT法检测细胞存活率并计算IC50。TSA 0.4μmol.L-1PANC-1培养0～48 h,Hoechst 33258染色观察细胞核形态变化。TSA 0.4μmol.L-1PANC-1培养0～12 h,检测胱天蛋白酶3活性。TSA 0.4μmol.L-1与PANC-1培养24 h,实时定量PCR检测c-myc,p53,Bcl-2,Bax,存活素,基质金属蛋白酶1(MMP1)和基质金属蛋白酶1组织抑制剂(TIMP-1)和Notch-1基因的表达。TSA 0.4和0.6μmol.L-1与PANC-1培养24 h,细胞免疫化学方法检测Notch-1蛋白的胞内活性形式NICD表达。结果TSA可以明显抑制PANC-1细胞增殖,12,24和48 h的IC50值分别为0.42,0.32和0.19μmol.L-1,具有量效(r=0.640,P=0.01)和时效(r=0.768,P=0.002)关系。Hoechst 33258染色结果表明,TSA 0.4μmol.L-1增加PANC-1细胞核的蓝色荧光并出现凋亡特征。TSA 0.4μmol.L-1作用4,8和12 h后,胱天蛋白酶3的活性分别为正常对照组的1.62±0.12,2.68±0.17和(3.92±0.23)倍。PCR结果显示,TSA 0.4μmol.L-1作用24 h后,p53,c-myc和存活素mRNA表达下降,分别为对照组的(18.3±5.1)%,(24.2±0.9)%和(15.8±1.0)%,Bcl-2和Notch-1 mRNA未见明显变化,而Bax,MMP1和TIMP-1 mRNA升高(P<0.05),Bcl-2/Bax比值降低到正常对照组的(13.0±2.8)%。Notch-1活性分子NICD明显升高(P<0.05)。结论TSA可通过线粒体途径诱导胰腺癌PANC-1细胞凋亡,而且使Notch-1激活,促进转移相关基因表达。
Objective To investigate the mechanism of trichostatin A (TSA) -induced pancreatic cancer cell PANC-1 cell apoptosis. Methods PANC-1 cells were cultured in TSA 0.1-0.6 μmol·L-1 for 0-48 h. Cell viability was assayed by MTT assay and IC50 was calculated. TSA 0.4μmol.L-1PANC-1 cultured 0 ~ 48 h, Hoechst 33258 staining observed nuclear morphological changes. TSA 0.4μmol.L-1PANC-1 cultured 0-12 h, detecting caspase 3 activity. The expression of c-myc, p53, Bcl-2, Bax, survivin, matrix metalloproteinase 1 (MMP1) and tissue inhibitor of matrix metalloproteinase 1 (TIMP-1) were detected by real- TIMP-1) and Notch-1 gene expression. TSA 0.4 and 0.6 μmol.L-1 and PANC-1 were cultured for 24 h, and the intracellular active form NICD expression of Notch-1 protein was detected by immunocytochemistry. Results TSA could significantly inhibit the proliferation of PANC-1 cells. The IC50 values at 12, 24 and 48 h were 0.42, 0.32 and 0.19 μmol·L-1, respectively, with significant effects (r = 0.640, P = 0.01) 0.768, P = 0.002). Hoechst 33258 staining results showed that TSA 0.4μmol.L-1 increased the blue fluorescence of PANC-1 cells and showed apoptotic features. After 4, 8 and 12 h TSA 0.4 μmol·L -1, the activities of caspase 3 were 1.62 ± 0.12, 2.68 ± 0.17 and 3.92 ± 0.23 times of the normal control group, respectively. The results of PCR showed that the expression of p53, c-myc and survivin mRNA decreased after treated with TSA 0.4 μmol·L-1 for 24 h, which were (18.3 ± 5.1)%, (24.2 ± 0.9)% and (15.8 ± (P <0.05). The ratio of Bcl-2 / Bax decreased to (13.0 ± 2.8)% in normal control group, but no significant change in Bcl-2 and Notch- )%. The NICD of Notch-1 activity was significantly increased (P <0.05). Conclusion TSA can induce apoptosis of pancreatic cancer PANC-1 cells through mitochondrial pathway, and activate Notch-1 to promote metastasis-related gene expression.