Akt参与热化疗对人小细胞肺癌H446细胞凋亡的协同作用。

Involvement of Akt in synergistic effects of thermo-chemotherapy on human small cell lung cancer H446 cell apoptosis.

作者信息

Wang Lin, Liu Xin-Kui, Shi Xiu-Qin

机构信息

Department of Radiotherapy, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052 PR China.

出版信息

Chin J Cancer. 2010 Apr;29(4):391-5. doi: 10.5732/cjc.009.10585.

DOI:10.5732/cjc.009.10585

PMID:20346214

Abstract

BACKGROUND AND OBJECTIVE

Akt pathway plays an important role in cell growth and apoptosis. This study was to characterize the role of Akt in the synergistic effects of thermo-chemotherapy on lung cancer cell growth and its underlying mechanisms.

METHODS

H446 cells were subjected to different thermo-chemotherapy schemes: 43centigrade + paclitaxel (120 microg/L) (thermo-chemotherapy group), 43centigrade + paclitaxel (120 microg/L) + Wortmannin (1 micromol/L, PI3K/Akt pathway inhibitor) (Wortmannin group), 43centigrade + paclitaxel (120 microg/L) + N-acety-L-cysteine (NAC) (30 micromol/L, reactive oxygen species, ROS inhibitor) (NAC group), and paclitaxel (120 microg/L) group. The cells without any treatment were used as the control. MTT assay was conducted to measure the cell proliferation rate. Cell apoptosis was analyzed by flow cytometry (FCM). ROS was detected with fluorescence. Phosphorylation of Akt and the expressions of Caspase-3 were determined by Western blot.

RESULTS

The cell proliferation rate was significantly lower in the thermo-chemotherapy group than in the control and the chemotherapy groups ((59.83 +/- 3.36)% vs. (100.00 +/- 0.00)% and (69.16 +/- 2.95)%, P < 0.05). The rate of cell apoptosis was the highest in the thermo-chemotherapy group (27.59 +/- 5.47)% (P < 0.05). The ROS expression level was higher in the cells of thermo-chemotherapy group (102.14 +/- 18.34) than in the other groups (P < 0.05), which could be inhibited by NAC(28.01 +/- 1.19), but not by the PI3K inhibitor Wortmannin (99.87 +/- 8.35). Phosphorylation of Akt significantly decreased in the thermo-chemotherapy group (0.69+/-0.03) (P < 0.05), which could be blocked by Wortmannin (0.00 +/- 0.00), but increased by NAC (1.05 +/- 0.29) (P < 0.05). The expression level of Caspase-3 was higher in the thermo-chemotherapy group (1.07 +/- 0.08) than in other groups (P < 0.05).

CONCLUSION

Thermo-chemotherapy has a stronger inhibitory effect than chemotherapy alone in lung tumor cell growth, probably through induction of ROS production and subsequent inhibition of Akt pathway activation and Caspase pathway-induced cancer cell apoptosis.

摘要

背景与目的

Akt信号通路在细胞生长和凋亡中起重要作用。本研究旨在探讨Akt在热化疗协同抑制肺癌细胞生长中的作用及其潜在机制。

方法

将H446细胞分为不同热化疗方案组：43℃+紫杉醇（120μg/L）（热化疗组）、43℃+紫杉醇（120μg/L）+渥曼青霉素（1μmol/L，PI3K/Akt信号通路抑制剂）（渥曼青霉素组）、43℃+紫杉醇（120μg/L）+N-乙酰半胱氨酸（NAC）（30μmol/L，活性氧，ROS抑制剂）（NAC组）以及紫杉醇（120μg/L）组。未作任何处理的细胞作为对照组。采用MTT法检测细胞增殖率。通过流式细胞术（FCM）分析细胞凋亡情况。用荧光法检测ROS。采用蛋白质免疫印迹法检测Akt的磷酸化水平及Caspase-3的表达。

结果

热化疗组细胞增殖率显著低于对照组和化疗组（（59.83±3.36）% 对（100.00±0.00）% 和（69.16±-2.95）%，P<0.05）。热化疗组细胞凋亡率最高（27.59±5.47）%（P<0.05）。热化疗组细胞中ROS表达水平高于其他组（102.14±18.34）（P<0.05），NAC（28.01±1.19）可抑制其表达，但PI3K抑制剂渥曼青霉素（99.87±8.35）不能抑制。热化疗组Akt磷酸化水平显著降低（0.69±0.03）（P<0.05），渥曼青霉素（0.00±0.00）可阻断其降低，而NAC（1.05±0.29）可使其升高（P<0.05）。热化疗组Caspase-3表达水平高于其他组（1.07±0.08）（P<0.05）。