Departments of Neurosurgery (G.B.S., H.A.H., N.C., L.B., S.T., J.S., P.D.) and Medicine (S.G., A.P.), Virginia Commonwealth University, Richmond, Virginia.
Mol Pharmacol. 2013 Oct;84(4):562-71. doi: 10.1124/mol.113.088005. Epub 2013 Jul 22.
The present studies were undertaken to determine whether the multikinase inhibitors sorafenib/regorafenib cooperated with clinically relevant , phosphatidyl inositol 3 kinase (PI3K)-thymoma viral proto-oncogene (AKT) inhibitors to kill tumor cells. In liver, colorectal, lung, breast, kidney, and brain cancer cells, at clinically achievable doses, sorafenib/regorafenib and the PI3K inhibitor acetic acid (1S,4E,10R,11R,13S,14R)-[4-diallylaminomethylene-6-hydroxy-1-methoxymethyl-10,13-dimethyl-3,7,17-trioxo-1,3,4,7,10,11,12,13,14,15,16,17-dodecahydro-2-oxa-cyclopenta[a]phenanthren-11-yl ester (PX-866) cooperated in a greater than additive fashion to kill tumor cells. Cells lacking phosphatase and tensin homolog were as sensitive to the drug combination as cells expressing the protein. Similar data were obtained using the AKT inhibitors perifosine and 8-[4-(1-aminocyclobutyl)phenyl]-9-phenyl-1,2,4-triazolo[3,4-f] [1,6]naphthyridin-3(2H)-one hydrochloride (MK2206). PX-866 treatment abolished AKT/glycogen synthase kinase 3 (GSK3) phosphorylation, and cell killing correlated with reduced activity of AKT and mammalian target of rapamycin (mTOR). Expression of activated AKT and to a lesser extent activated mTOR reduced drug combination lethality. Expression of B-cell lymphoma-extra large or dominant negative caspase 9, but not cellular FLICE (FADD-like IL-1b-converting enzyme)-inhibitory protein short, protected cells from the drug combination. Treatment of cells with PX-866 increased protein levels of p62, lysosome-associated membrane protein 2 (LAMP2), and microtubule-associated protein light chain (LC) 3 and LC3II that correlated with a large increase in LC3-green fluorescent protein (GFP) vesicle numbers. Exposure of PX-866 treated cells to sorafenib reduced p62 and LAMP2 levels, decreased the ratio of LC3 to LC3II, and reduced LC3-GFP vesicle levels. Knockdown of Beclin1 or autophagy-related 5 suppressed drug toxicity by ∼40%. In vivo, sorafenib and PX-866 or regorafenib and MK2206 cooperated to suppress the growth of established HuH7 and HCT116 tumors, respectively. Collectively our data demonstrate that the combination of sorafenib family kinase inhibitors with inhibitors of the PI3K/AKT pathway kills tumor cells in vitro and in vivo.
本研究旨在确定多激酶抑制剂索拉非尼/regorafenib 是否与临床相关的磷脂酰肌醇 3 激酶（PI3K）-胸腺瘤病毒原癌基因（AKT）抑制剂协同杀死肿瘤细胞。在肝癌、结直肠癌、肺癌、乳腺癌、肾癌和脑癌细胞中，在临床可达到的剂量下，索拉非尼/regorafenib 和 PI3K 抑制剂醋酸（1S，4E，10R，11R，13S，14R）-[4-二烯丙基氨基亚甲基-6-羟基-1-甲氧基甲基-10、13-二甲基-3、7、17-三氧代-1、3、4、7、10、11、12、13、14、15、16、17-十二氢-2-氧代环戊[a]菲-11-基酯（PX-866）与肿瘤细胞协同作用大于相加。磷酸酶和张力蛋白同源物缺失的细胞与表达该蛋白的细胞一样对药物组合敏感。使用 AKT 抑制剂 perifosine 和 8-[4-（1-氨基环丁基）苯基]-9-苯基-1,2,4-三唑[3,4-f][1,6]萘啶-3（2H）-酮盐酸盐（MK2206）也获得了类似的数据。PX-866 处理消除了 AKT/糖原合成酶激酶 3（GSK3）磷酸化，细胞杀伤与 AKT 和哺乳动物雷帕霉素靶蛋白（mTOR）活性降低相关。激活的 AKT 的表达以及在较小程度上激活的 mTOR 降低了药物组合的致死性。表达激活的 AKT 和在较小程度上表达激活的 mTOR 减少了药物组合的致死性。表达 B 细胞淋巴瘤-extra large 或显性负性半胱天冬酶 9，但不是细胞 FLICE（FADD 样 IL-1b 转化酶）-抑制蛋白短，可保护细胞免受药物组合的影响。用 PX-866 处理细胞会增加 p62、溶酶体相关膜蛋白 2（LAMP2）和微管相关蛋白轻链（LC）3 和 LC3II 的蛋白水平，这与 LC3-GFP 囊泡数量的大幅增加相关。用索拉非尼处理 PX-866 暴露的细胞会降低 p62 和 LAMP2 水平，降低 LC3 与 LC3II 的比值，并降低 LC3-GFP 囊泡水平。Beclin1 或自噬相关 5 的敲低抑制了约 40%的药物毒性。在体内，索拉非尼和 PX-866 或regorafenib 和 MK2206 分别协同抑制已建立的 HuH7 和 HCT116 肿瘤的生长。总的来说，我们的数据表明，索拉非尼家族激酶抑制剂与 PI3K/AKT 通路抑制剂的联合使用可在体外和体内杀死肿瘤细胞。
