Department of Chemistry, Tsinghua University, Beijing 100084, P. R. China.
State Key Laboratory of Chemical Oncogenomics, Division of Life Science and Health, The Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, P. R. China.
Cell Mol Biol (Noisy-le-grand). 2020 Sep 30;66(6):65-70.
The purpose of this study was to investigate the effects of RAPA on the proliferation and the expression of p53, Bcl-2 and Bax proteins in cultured human small cell lung cancer (NCI-H446) cells, and to explore the possible mechanism of RAPA-treated NCI-H446 cells with different concentrations of RAPA-treated NCI-H446 cells. The proliferation of NCI-H446 cells in all groups was assayed by the CCK-8 method. FITC-Annexin V/PI double staining method was used to determine the apoptosis of NCI-H446 cells. The immunohistochemical SP method was used to detect the expression of p53, Bcl-2 and Bax. Expression of p53, Bcl-2 and Bax mRNA was detected by RT-PCR. The results showed that, after 48h treatment, the proliferation of NCI-H446 cells treated with 5ng/mL, 10ng/mL and 15ng/mL RAPA decreased significantly (P < 0.05) and the proliferation inhibition rate increased significantly (P < 0.05) compared with the control group, and the proliferation inhibition rate had a dose-dependent relationship with RAPA. Compared with the control group, the apoptosis rate of NCI-H446 cells treated with 5ng/mL, 10ng/mL and 15ng/mL RAPA increased significantly (P < 0.05), and there was a dose-dependent relationship between the apoptosis rate and RAPA. The expression of Bcl-2 protein and mRNA was higher in the control group, while the expression of p53 and Bax protein and mRNA was lower. The expression of Bcl-2 protein and mRNA decreased and the expression of p53 and Bax protein and mRNA increased gradually with the increase of concentration and the prolongation of action time in 5ng/mL, 10ng/mL and 15ng/mL RAPA groups. In the control group, the intracellular Ca2+ concentration was constant, and there was no significant change with time; while in the 5ng / mL, 10ng / mL, and 15ng / mL RAPA group, the intracellular Ca2+ concentration in the RAPA group increased significantly after 12 h of administration (P <0.05); After that, with the prolonged action time of the medicine, the intracellular Ca2+ concentration in the 5ng / mL, 10ng / mL, and 15ng / mL RAPA group decreased, but at 72h, the effect was 5ng / mL, 10ng / mL, and 15ng / mL RAPA. The intracellular Ca2+ fluorescence intensity in the group was still significantly higher than that in the control group (P <0.05). In conclusion, RAPA can induce apoptosis of NCI-H446 cells by down-regulating Bcl-2 gene expression, up-regulating P53 and Bax gene expression, and increasing intracellular Ca2+ concentration and its apoptosis induction effect have timeliness and dose-effect.
本研究旨在探讨 RAPA 对培养的人小细胞肺癌（NCI-H446）细胞增殖和 p53、Bcl-2 和 Bax 蛋白表达的影响，并探讨不同浓度 RAPA 处理的 NCI-H446 细胞的可能机制。用 CCK-8 法检测各组 NCI-H446 细胞的增殖情况。FITC-Annexin V/PI 双染法检测 NCI-H446 细胞的凋亡情况。免疫组化 SP 法检测 p53、Bcl-2 和 Bax 的表达。用 RT-PCR 检测 p53、Bcl-2 和 Bax mRNA 的表达。结果显示，48h 处理后，与对照组相比，5ng/mL、10ng/mL 和 15ng/mL RAPA 处理的 NCI-H446 细胞增殖明显减少（P < 0.05），增殖抑制率明显升高（P < 0.05），且增殖抑制率与 RAPA 呈剂量依赖性。与对照组相比，5ng/mL、10ng/mL 和 15ng/mL RAPA 处理的 NCI-H446 细胞凋亡率明显升高（P < 0.05），且凋亡率与 RAPA 呈剂量依赖性。对照组 Bcl-2 蛋白和 mRNA 表达较高，而 p53 和 Bax 蛋白和 mRNA 表达较低。随着 5ng/mL、10ng/mL 和 15ng/mL RAPA 组浓度的增加和作用时间的延长，Bcl-2 蛋白和 mRNA 表达逐渐降低，p53 和 Bax 蛋白和 mRNA 表达逐渐升高。对照组细胞内 Ca2+浓度保持不变，随时间无明显变化；而在 5ng/mL、10ng/mL 和 15ng/mL RAPA 组，给药 12h 后 RAPA 组细胞内 Ca2+浓度明显升高（P <0.05）；此后，随着药物作用时间的延长，5ng/mL、10ng/mL 和 15ng/mL RAPA 组细胞内 Ca2+浓度逐渐降低，但在 72h 时，5ng/mL、10ng/mL 和 15ng/mL RAPA 组的细胞内 Ca2+荧光强度仍明显高于对照组（P <0.05）。综上所述，RAPA 通过下调 Bcl-2 基因表达、上调 P53 和 Bax 基因表达、增加细胞内 Ca2+浓度诱导 NCI-H446 细胞凋亡，其诱导凋亡作用具有时效性和剂量效应。
