Enhanced sensitivity of celecoxib in human glioblastoma cells: Induction of DNA damage leading to p53-dependent G1 cell cycle arrest and autophagy
Enhanced sensitivity of celecoxib in human glioblastoma cells: Induction of DNA damage leading to p53-dependent G1 cell cycle arrest and autophagy
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DOI：
10.1186/1476-4598-8-66
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发表时间：
2009-08-25
期刊：
影响因子：
37.3
通讯作者：
Wong, Meng Cheong
中科院分区：
文献类型：
--
作者：
Kang, Khong Bee;Zhu, Congju;Wong, Meng Cheong
背景：选择性环氧合酶(COX)-2抑制剂在多种肿瘤中引起抗增殖反应，但其潜在的抗肿瘤机制尚不清楚。肿瘤抑制基因p53突变失活在恶性胶质瘤中很常见。p53突变在人类胶质母细胞瘤细胞中选择性COX-2抑制剂塞来昔布的抗肿瘤反应中的作用尚不清楚。在这项研究中，我们使用了具有不同p53状态的人胶质母细胞瘤细胞；U87MG （p53功能水平高低）、LN229（功能性p53）和U373MG（突变型p53）细胞。用E6癌蛋白（U87MG-E6）转染U87MG细胞，并用可逆性p53抑制剂（U87MG- pft）处理后，实现了p53的抑制。我们研究了塞来昔布的抗胶质母细胞瘤反应是否依赖于p53，以及塞来昔布是否诱导DNA损伤导致p53依赖的G(1)细胞周期阻滞，随后发生自噬或凋亡。结果：我们的研究结果表明，在治疗24小时和72小时后，塞来昔布浓度依赖性地降低了胶质母细胞瘤细胞的活力。抑制胶质母细胞瘤细胞中的功能性p53可显著降低塞来昔布的抗增殖作用。在U87MG细胞中，塞来昔布（8 μ M和30 μ M）显著诱导DNA损伤，抑制DNA合成，与p53激活相对应。塞来昔布诱导U87MG细胞G(1)期细胞周期阻滞，并伴有p21活化。塞来昔布对U87MG-E6和U87MG-PFT细胞周期进程无影响。同时，塞来昔布在LN229细胞中诱导G(1)细胞周期阻滞，但在U373MG细胞中没有。塞来昔布在U87MG和LN229细胞中诱导自噬，结果显示，与未处理的对照组相比，吖啶橙染色细胞数量显著增加，LC3-II蛋白水平显著升高。塞来昔布在缺乏功能性p53的U87MG-PFT、U87MG-E6和U373MG细胞中未诱导显著的自噬。无论p53状态如何，塞来昔布对U87MG、U87MG- pft、U87MG- e6和U373MG细胞的凋亡水平均无显著影响。结论：我们的研究结果表明p53增加了人胶质母细胞瘤对塞来昔布的敏感性。塞来昔布通过诱导DNA损伤抑制胶质母细胞瘤细胞活力，导致p53依赖性G(1)细胞周期阻滞和p53依赖性自噬，但不导致细胞凋亡。
Background: Selective cyclooxygenase (COX)-2 inhibitors elicit anti-proliferative responses in various tumours, however the underlying anti-tumour mechanisms are unclear. Mutational inactivation of the tumour suppressor p53 gene is frequent in malignant gliomas. The role of p53 mutation in the anti-tumour responses of the selective COX-2 inhibitor celecoxib in human glioblastoma cells is unknown. In this study, we used human glioblastoma cells with various p53 status; U87MG (with high and low p53 functional levels), LN229 (functional p53) and U373MG (mutant p53) cells. Inhibition of p53 was achieved in U87MG cells transfected with E6 oncoprotein (U87MG-E6) and treated with pifithrin-alpha, a reversible inhibitor of p53 (U87MG-PFT). We investigated whether the anti-glioblastoma responses of celecoxib were p53-dependent, and whether celecoxib induced DNA damage leading to p53-dependent G(1) cell cycle arrest, followed by autophagy or apoptosis.Results: Our findings demonstrated that celecoxib concentration-dependently reduced glioblastoma cell viability, following 24 and 72 hours of treatment. Inhibition of functional p53 in glioblastoma cells significantly reduced the anti-proliferative effect of celecoxib. In U87MG cells, celecoxib (8 and 30 mu M) significantly induced DNA damage and inhibited DNA synthesis, corresponding with p53 activation. Celecoxib induced G(1)-phase cell cycle arrest, accompanied with p21 activation in U87MG cells. Cell cycle progression of U87MG-E6 and U87MG-PFT cells was not affected by celecoxib. In parallel, celecoxib induced G(1) cell cycle arrest in LN229 cells, but not in U373MG cells. Autophagy was induced by celecoxib in U87MG and LN229 cells, as shown by the significantly greater population of acridine orange-stained cells and increased levels of LC3-II protein (in comparison with non-treated controls). Celecoxib did not induce significant autophagy in U87MG-PFT, U87MG-E6 and U373MG cells, which lack functional p53. Regardless of p53 status, celecoxib caused no significant difference in apoptosis level of U87MG, U87MG-PFT, U87MG-E6 and U373MG cells.Conclusion: Our findings reveal that p53 increases human glioblastoma sensitivity to celecoxib. Celecoxib inhibits glioblastoma cell viability by induction of DNA damage, leading to p53-dependent G(1) cell cycle arrest and p53-dependent autophagy, but not apoptosis.