Celecoxib-induced apoptosis is enhanced by ABT-737 and by inhibition of autophagy in human colorectal cancer cells.
Celecoxib-induced apoptosis is enhanced by ABT-737 and by inhibition of autophagy in human colorectal cancer cells.
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DOI：
10.4161/auto.6.2.11124
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发表时间：
2010-02
期刊：
影响因子：
13.3
通讯作者：
Sinicrope FA
中科院分区：
文献类型：
--
作者：
Huang S;Sinicrope FA
凋亡和自噬已被证明受促生存Bcl-2蛋白的负调控。我们确定了抗癌药物塞来昔布单独使用或与小分子Bcl-2/Bcl-xL拮抗剂（ABT-737）联合使用是否能诱导结肠癌细胞自噬。此外，我们还研究了抑制自噬是否会导致结肠癌细胞凋亡。塞来昔布可诱导细胞凋亡，但异位敲除Bcl-2或Bax可减弱细胞凋亡。ABT-737协同增强塞来昔布诱导的细胞毒性，主要是由于凋亡，如caspase切割和膜联蛋白V标记所示，被泛caspase抑制剂（z-VAD-fmk）减弱。赛来昔布触发自噬体相关蛋白轻链3 （LC3）从细胞质（LC3I）转化为膜结合（LC3II）形式，如免疫印迹和异位GFP-LC3蛋白的点状荧光模式所示。塞来昔布诱导LC3的转化是由于自噬诱导，正如溶酶体抑制剂ba_lomycin A1所支持的那样，它产生LC3II的积累。ABT-737增强塞来昔布诱导的LC3转化和p62/SQSTM1降解。然后研究了自噬的抑制作用，以推动细胞凋亡。3-甲基腺嘌呤（3-MA）阻断LC3转化，3-MA和wortmannin显著增强塞来昔布联合ABT-737处理的细胞的凋亡信号传导。此外，使用siRNA敲除Atg8/LC3B或Vps34可减弱p62降解并增强凋亡信号传导；Vps34 siRNA增强了塞来昔布+ ABT-737诱导的膜联蛋白V+、PI -标记细胞。综上所述，塞来昔布可诱导ABT-737增强细胞凋亡和自噬。抑制自噬可增强细胞凋亡，提示一种新的结肠癌治疗策略。
Apoptosis and autophagy have been shown to be negatively regulated by prosurvival Bcl-2 proteins. We determined whether the anticancer agent celecoxib, alone or combined with a small molecule Bcl-2/Bcl-xL antagonist (ABT-737), can induce autophagy in colon cancer cells. Furthermore, we determined whether inhibition of autophagy can drive colon cancer cells into apoptosis. Celecoxib was shown to induce apoptosis that was attenuated by ectopic Bcl-2 or Bax knockout. ABT-737 synergistically enhanced celecoxib-induced cytotoxicity that was primarily due to apoptosis as shown by caspase cleavage and Annexin V labeling that was attenuated by a pan caspase inhibitor (z-VAD-fmk). Celecoxib triggered conversion of the autophagosome-associated protein light chain 3 (LC3) from a cytosolic (LC3I) to a membrane-bound (LC3II) form, as shown by immunoblotting and a punctate _uorescence pattern of an ectopic GFP-LC3 protein. Celecoxib-induced conversion of LC3 was due to autophagy induction, as supported using the lysosome inhibitor, ba_lomycin A1, which produced an accumulation of LC3II. ABT-737 enhanced celecoxib-induced LC3 conversion and p62/SQSTM1 degradation. Inhibition of autophagy was then studied in an e_ort to drive cells into apoptosis. 3-methyladenine (3-MA) blocked LC3 conversion, and 3-MA and wortmannin signi_cantly enhanced apoptotic signaling in cells treated with celecoxib plus ABT-737. Furthermore, knockdown of Atg8/LC3B or Vps34 using siRNA attenuated p62 degradation and enhanced apoptotic signaling; Vps34 siRNA potentiated annexin V+, PI− labeled cells induced by celecoxib + ABT-737. In conclusion, celecoxib induces apoptosis and autophagy that can both be potentiated by ABT-737. Inhibition of autophagy was shown to enhance apoptosis, suggesting a novel therapeutic strategy against colon cancer.
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