Department of Pharmacology and Physiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Pathumwan, Bangkok, Thailand 10330.
Anticancer Res. 2013 Dec;33(12):5433-44.
The effect of extended exposure of cancer cells to nitric oxide (NO), an endogenous mediator frequently found increased in tumors, is largely unknown. In the present study, the effect of long-term NO exposure on chemotherapeutic resistance was investigated in lung cancer cells.
The effect of long-term exposure of human lung cancer cells to NO on susceptibility to chemotherapeutic agents-induced apoptosis was analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, co-staining of Hoechst 33342 and propidium iodide (PI), and Annexin V detection. The expression of survival-related proteins was analyzed by western blot. Gene manipulation was used for evaluation of the effect of expression proteins on susceptibility of the cells to chemotherapeutic agent-mediated death.
Long-term NO exposure for 7-14 days rendered the lung cancer cells resistant to cisplatin, doxorubicin, and etoposide dose- and time-dependently. The underlying mechanism was found to involve the adaptive responses of the cells, by increasing survival due to increase in the level of caveolin-1 (CAV1) and anti-apoptotic B-cell lymphoma-2 (BCL2), and up-regulation of activated protein kinase B (AKT). The gene manipulation study revealed that the increase of activated AKT and BCL2 was responsible for the resistance to all tested drugs, while the up-regulation of CAV1 only attenuated cell death mediated by doxorubicin and etoposide. Interestingly, NO-mediated drug resistance was found to be reversible when cells were further cultured in the absence of NO for five days.
These findings reveal the novel role of NO in the tumor environment, in attenuating chemotherapeutic susceptibility and this could be beneficial in contriving strategies to treat the disease.
癌细胞暴露于一氧化氮（NO）中时间延长，NO 是肿瘤中经常发现的内源性介质，其对癌细胞的影响目前还知之甚少。本研究旨在探讨长期 NO 暴露对肺癌细胞化疗耐药性的影响。
通过 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）测定、Hoechst 33342 和碘化丙啶（PI）共染色以及 Annexin V 检测分析长期暴露于 NO 对人肺癌细胞对化疗药物诱导的细胞凋亡敏感性的影响。通过 Western blot 分析生存相关蛋白的表达。通过基因操作评估表达蛋白对细胞对化疗药物介导的死亡敏感性的影响。
长期（7-14 天）NO 暴露使肺癌细胞对顺铂、阿霉素和依托泊苷的剂量和时间依赖性耐药。研究发现，其潜在机制涉及细胞的适应性反应，通过增加 caveolin-1（CAV1）和抗凋亡 B 细胞淋巴瘤-2（BCL2）的水平来增加细胞的存活，以及激活蛋白激酶 B（AKT）的上调。基因操作研究表明，激活 AKT 和 BCL2 的增加是导致所有测试药物耐药的原因，而 CAV1 的上调仅减弱了阿霉素和依托泊苷介导的细胞死亡。有趣的是，当细胞在没有 NO 的情况下进一步培养五天时，NO 介导的耐药性被发现是可逆的。
这些发现揭示了 NO 在肿瘤微环境中减弱化疗敏感性的新作用，这可能有助于制定治疗该疾病的策略。
