Biochemistry Department, Alexandria University, Moharam, Beck, Alexandria, Egypt.
Anticancer Res. 2011 Apr;31(4):1345-57.
We previously reported novel quinuclidinone analogs that showed both additive and synergistic cytotoxicity in lung cancer cells. We aimed at understanding the mechanism of these analogs and also their cytotoxic effect on normal cells. The effects of these analogs were studied in response to gamma radiation in H1299 human large cell lung carcinoma cells that are null for p53, normal lung epithelial cell line (NL-20) and H1299 cells stably transfected with p53.
The effects of the analogs were investigated by MTT assay, clonogenic survival assay, sphingomylinase activity, Cox-2 activity, ELISA-based apoptotic assay, terminal deoxynucleotidyl transferase dUTP nick end labeling assay, immunofluoresence staining, flow cytometry, real-time reverse transcription polymerase chain reaction and Western blot analysis.
Our data indicated that 8a and 8b reduced cell proliferation and induced apoptosis in H1299 cells more than H1299-wt p53 cells and NL-20 cells, they also radiosensitize H1299 cells to gamma radiation more than NL-20 cells. 8a and 8b decreased cells in G(2) phase in H1299 cells more than NL-20 cells, which is confirmed by increased expression of cyclin B in H1299 cells, with no significant increase in H1299-wtp53. 8a increased sphingomylinase activity and ceramide level in H1299 more than the rest of cells, it also reduced expression level and activity of COX-2 while it increased caspase-3 activity and induced PARP-1 cleavage. Both derivatives increased expression of p53 in H1299-wt p53 level, while they did not show significant increase in NL-20 cells. Interestingly, these analogs induced apoptosis in H1299 and p53 stably transfected H1299 cells, but they had less effect on normal lung epithelial cells (NL-20).
All these results confirm that our quinuclidinone derivatives provoke cytotoxicity in lung cancer cells more than normal cells, which is a feature not present in most chemotherapeutic drugs.
我们之前报道了新型奎宁环酮类似物，它们在肺癌细胞中表现出相加和协同细胞毒性。我们旨在了解这些类似物的作用机制及其对正常细胞的细胞毒性作用。我们研究了这些类似物在人非小细胞肺癌 H1299 细胞（p53 缺失）、正常肺上皮细胞系（NL-20）和 H1299 细胞中稳定转染 p53 后的γ辐射反应中的作用。
通过 MTT 测定、集落形成存活测定、神经鞘氨醇酶活性、Cox-2 活性、ELISA 法凋亡测定、末端脱氧核苷酸转移酶 dUTP 缺口末端标记测定、免疫荧光染色、流式细胞术、实时逆转录聚合酶链反应和 Western blot 分析研究类似物的作用。
我们的数据表明，8a 和 8b 比 H1299-wt p53 细胞和 NL-20 细胞更能降低 H1299 细胞的增殖并诱导其凋亡，它们也比 NL-20 细胞更能增敏 H1299 细胞对γ辐射。8a 和 8b 比 NL-20 细胞使 H1299 细胞更多地进入 G2 期，这一点通过 H1299 细胞中环素 B 的表达增加得到证实，而在 H1299-wtp53 中则没有明显增加。8a 增加了 H1299 中的神经鞘氨醇酶活性和神经酰胺水平，比其余细胞减少 COX-2 的表达水平和活性，同时增加 caspase-3 活性并诱导 PARP-1 裂解。两种衍生物均增加了 H1299-wt p53 水平的 p53 表达，而在 NL-20 细胞中则没有明显增加。有趣的是，这些类似物在 H1299 和稳定转染 p53 的 H1299 细胞中诱导凋亡，但对正常肺上皮细胞（NL-20）的影响较小。
所有这些结果证实，我们的奎宁环酮衍生物在肺癌细胞中的细胞毒性比正常细胞更强，这是大多数化疗药物所不具备的特征。
