Background and objective: Lung cancer is a common malignant tumor all over the world, and Ca(2+) is a critical regulator for apoptosis of cancer cells. The monitoring of cytoplastic Ca(2+) level in real-time will contribute to further investigate the molecular mechanisms of apoptosis mediated by Ca2+ in lung cancer cells. To evaluate the Ca(2+) indicator fluo-3 and fluo-4 in the process of H2O2 induced the apoptosis of lung adenocarcinoma A549 cells. The cytoplastic Ca(2+) concentration ([Ca(2+)]i) was determined in real-time, and the correlations between [Ca(2+)]i and cell apoptosis were investigated. The differences in fluorescence intensity and measured value were compared between the two Ca(2+) indicators.
Methods: Cells were loaded with the Ca(2+) indicator fluo-3 or fluo-4 for 1 h, and then stimulated with 50 mM H2O2. Laser scanning confocal microscope was applied to perform real-time monitoring on the variation of [Ca(2+)]i in selected cells. DAPI staining was used to observe apoptosis in H2O2 treated cells.
Results: Our results showed that the fluorescence intensity of fluo-4 was stronger than that of fluo-3 in the same condition of dye concentration, loading time and image acquisition parameters before or after H2O2 stimulation. The cytoplastic [Ca(2+)]i was rapidly elevated in H2O2 stimulated A549 cells. The range of [Ca(2+)]i in selected cells loaded with fluo-3 was 112.2 nM-1,069.6 nM, and that in selected cells loaded with fluo-4 was 7.6 nM-505.4 nM. Moreover, the apoptotic rate was significantly increased in H2O2 treated cells, compared with untreated ones (P<0.01).
Conclusion: In summary, H2O2 promoted Ca(2+) release in A549 cells, and induced cell apoptosis. Ca(2+) indicator fluo-4 was probably more applicable to measure [Ca(2+)]i in cells with less content of Ca(2+).
背景与目的 肺癌是世界范围内常见的恶性肿瘤，Ca²⁺对于肿瘤细胞凋亡有重要的调控作用。实时监测肺癌细胞内Ca²⁺水平，有助于深入研究Ca²⁺介导肺癌细胞凋亡的分子机制。本研究旨在观察Ca²⁺荧光探针fluo-3和fluo-4在H₂O₂诱导的A549细胞凋亡过程中的应用，实时测定胞浆Ca²⁺浓度（[Ca²⁺]i），探讨[Ca²⁺]i与细胞凋亡的关系，并比较两种Ca²⁺探针在荧光强度及[Ca²⁺]i测定值方面的差异。 方法 采用Ca²⁺荧光探针fluo-3和fluo-4负载细胞，1 h后用不同浓度的H₂O₂刺激细胞，激光扫描共聚焦显微镜实时测定选取细胞的[Ca²⁺]i变化。采用DAPI染色试剂盒观察H₂O₂刺激后细胞凋亡情况。结果 在相同的探针浓度、负载时间和相同的图像采集参数的条件下，选定细胞内fluo-4平均荧光强度高于fluo-3。50 mM H₂O₂刺激后，A549细胞胞浆内[Ca²⁺]i迅速升高，通过公式计算发现采用fluo-3探针负载的选定细胞中[Ca²⁺]i变化范围是112.2 nM-1,069.6 nM，采用fluo-4探针负载的选定细胞中[Ca²⁺]i变化范围是7.6 nM-505.4 nM。同时发现经H₂O₂刺激后，凋亡细胞百分比明显增加（P<0.01）。结论 H₂O₂促进A549细胞内Ca²⁺释放，诱导细胞凋亡。Ca²⁺探针fluo-4可能更适合于监测含量较低的细胞中[Ca²⁺]i变化。