Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, VA, USA.
Cancer Biol Ther. 2009 Nov;8(21):2084-96. doi: 10.4161/cbt.8.21.9895. Epub 2009 Nov 21.
Prior studies demonstrated that resistance to the ERBB1/2 inhibitor Lapatinib in HCT116 cells was mediated by increased MCL-1 expression. We examined whether inhibition of BCL-2 family function could restore Lapatinib toxicity in Lapatinib adapted tumor cells and enhance Lapatinib toxicity in naive cells. The BCL-2 family antagonist Obatoclax (GX15-070), that inhibits BCL-2/BCL-X(L)/MCL-1 function, enhanced Lapatinib toxicity in parental HCT116 and Lapatinib adapted HCT116 cells. In breast cancer lines, regardless of elevated ERBB1/2 expression, GX15-070 enhanced Lapatinib toxicity within 3-12 h. The promotion of Lapatinib toxicity neither correlated with cleavage of caspase 3 nor was blocked by inhibition caspases; and was not associated with changes in the activities of ERK1/2, JNK1/2 or p38 MAPK but with reduced AKT, mTOR and S6K1 phosphorylation. The promotion of Lapatinib toxicity by GX15-070 correlated with increased cytosolic levels of apoptosis inducing factor (AIF) and expression of ATG8 (LC3), and the formation of large vesicles that intensely stained for a transfected LC3-GFP construct. Knock down of the autophagy regulatory proteins ATG5 or Beclin1 suppressed the induction of LC3-GFP vesicularization and significantly reduced cell killing, whereas knock down of MCL-1 and BCL-X(L) enhanced the induction of LC3-GFP vesicularization and significantly enhanced cell killing. Knockdown of Beclin1 and AIF abolished cell killing. Collectively, our data demonstrate that Obatoclax mediated inhibition of MCL-1 rapidly enhances Lapatinib toxicity in tumor cells via a toxic form of autophagy and via AIF release from the mitochondrion.
先前的研究表明，HCT116 细胞对 ERBB1/2 抑制剂拉帕替尼的耐药性是由 MCL-1 表达增加介导的。我们研究了抑制 BCL-2 家族功能是否可以恢复拉帕替尼适应肿瘤细胞中的拉帕替尼毒性，并增强拉帕替尼在未适应细胞中的毒性。BCL-2 家族拮抗剂 Obatoclax（GX15-070）可抑制 BCL-2/BCL-X(L)/MCL-1 功能，增强亲本 HCT116 和拉帕替尼适应 HCT116 细胞中的拉帕替尼毒性。在乳腺癌系中，无论 ERBB1/2 表达升高，GX15-070 均可在 3-12 小时内增强拉帕替尼的毒性。促进拉帕替尼的毒性既与 caspase 3 的裂解无关，也不受 caspase 抑制剂的阻断，也与 ERK1/2、JNK1/2 或 p38 MAPK 的活性变化无关，而是与 AKT、mTOR 和 S6K1 磷酸化减少有关。GX15-070 通过增加细胞溶质中的凋亡诱导因子 (AIF) 和 ATG8 (LC3) 的表达，以及强烈染色转染的 LC3-GFP 构建体的大泡来促进拉帕替尼的毒性。自噬调节蛋白 ATG5 或 Beclin1 的敲低抑制了 LC3-GFP 囊泡化的诱导，显著降低了细胞杀伤率，而 MCL-1 和 BCL-X(L) 的敲低增强了 LC3-GFP 囊泡化的诱导，并显著增强了细胞杀伤率。Beclin1 和 AIF 的敲低消除了细胞杀伤。总的来说，我们的数据表明，Obatoclax 通过抑制 MCL-1 快速增强肿瘤细胞中的拉帕替尼毒性，通过一种有毒形式的自噬和通过 AIF 从线粒体释放。
