Yang Tsung-Ming, Barbone Dario, Fennell Dean A, Broaddus V Courtney
Division of Pulmonary and Critical Care Medicine, Chang Gung Memorial Hospital, Chiayi, Taiwan.
Am J Respir Cell Mol Biol. 2009 Jul;41(1):14-23. doi: 10.1165/rcmb.2008-0320OC. Epub 2008 Dec 18.
Combinatorial therapies using the proteasome inhibitor, bortezomib, have been found to induce synergistic apoptosis in cancer cells grown as monolayers; however, three-dimensional spheroid culture may be a better model for the multicellular resistance found in solid tumors, such as lung cancer. We tested the combinatorial apoptotic strategy of using bortezomib together with TNF-related apoptosis-inducing ligand (TRAIL), both in monolayers and in spheroids of A549 lung cancer cells. Indeed, bortezomib plus TRAIL induced synergistic apoptosis in A549 cells grown as monolayers, but had little effect on A549 cells grown as three-dimensional multicellular spheroids. The acquired resistance of spheroids was not due to a limitation of diffusion, to survival pathways, such as NF-kappaB or PI3K/Akt/mTOR, or to the up-regulation of FLIP(S) (Fas-associated death domain-like IL-1 beta-converting enzyme inhibitory protein, short). We then investigated a role for the Bcl-2 family of anti- and proapoptotic proteins. When cells formed spheroids, antiapoptotic Bcl-2 increased, whereas antiapoptotic Mcl-1 decreased. ABT-737, a small molecule that inhibits Bcl-2, but not Mcl-1, abolished the multicellular resistance of A549 spheroids to bortezomib plus TRAIL. In another lung cancer cell line, H1299, acquisition of multicellular resistance in spheroids was also accompanied by an increase in Bcl-2 and decrease in Mcl-1. In H1299 spheroids compared with those of A549, however, Mcl-1 remained higher, and Mcl-1 knockdown was more effective than ABT-737 in removing multicellular resistance. Our study suggests that the balance of Bcl-2 family proteins contributes to the acquired multicellular resistance of spheroids, and suggests a possible target for improving the response of lung cancer to bortezomib therapies.
已发现使用蛋白酶体抑制剂硼替佐米的联合疗法可诱导单层培养的癌细胞发生协同凋亡；然而，三维球体培养对于实体瘤（如肺癌）中发现的多细胞耐药性而言可能是一个更好的模型。我们在A549肺癌细胞的单层培养物和球体中测试了硼替佐米与肿瘤坏死因子相关凋亡诱导配体（TRAIL）联合使用的凋亡策略。实际上，硼替佐米加TRAIL可诱导单层培养的A549细胞发生协同凋亡，但对三维多细胞球体培养的A549细胞几乎没有影响。球体获得的耐药性并非由于扩散受限、生存途径（如核因子κB或磷脂酰肌醇-3激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白），也不是由于FLIP(S)（Fas相关死亡结构域样白细胞介素-1β转换酶抑制蛋白，短型）上调所致。然后，我们研究了抗凋亡和促凋亡Bcl-2家族蛋白的作用。当细胞形成球体时，抗凋亡的Bcl-2增加，而抗凋亡的Mcl-1减少。ABT-737是一种抑制Bcl-2但不抑制Mcl-1的小分子，它消除了A549球体对硼替佐米加TRAIL的多细胞耐药性。在另一种肺癌细胞系H1299中，球体中多细胞耐药性的获得也伴随着Bcl-2的增加和Mcl-1的减少。然而，与A549球体相比，H1299球体中的Mcl-1仍然较高，并且Mcl-1基因敲低在消除多细胞耐药性方面比ABT-737更有效。我们的研究表明，Bcl-2家族蛋白的平衡有助于球体获得多细胞耐药性，并提示了一个可能改善肺癌对硼替佐米治疗反应的靶点。
