於CL1-5細胞中破壞β-tubulin/CCT-β蛋白質複合體引發細胞凋亡並藉抑制MMP-2及AKT/GSK-3β壓制其轉移和侵略

Abstract

之前實驗室的研究發現可藉由I-Trp的iodoacetyl基團來烷基化β-tubulin 的Cys354，而破壞β-tubulin及chaperonin-containing TCP-1β (CCT-β)蛋白-蛋白交互作用而成造成細胞凋亡[1]。在本論文中，我們發現在CL1-5細胞中，I-Trp是透過打斷β-tubulin/CCT-β複合體的結合，內質網壓力相關蛋白被活化後，誘發細胞蛋白酶體的活化來消彌在內質網內累積的過量蛋白質。並且由於內質網壓力所引起的細胞凋亡通常會伴隨細胞質內鈣離子濃度的上升和蛋白激酶 (MAPKs)的活化，我們也有觀察到鈣離子的濃度變化，MAPK蛋白的活性改變和粒線體上的細胞凋亡相關蛋白的表現量改變。 另外我們也有探討I-Trp是否也有抑制高轉移性肺癌細胞CL1-5的轉移能力。實驗中使用的I-Trp的最高濃度維持在細胞還有80%的存活率，以確保在不殺及過多細胞的情況下探討I-Trp抑制細胞遷移和侵入的能力。實驗結果顯示，當細胞受到I-Trp刺激之際，會隨著藥物濃度上升抑制細胞遷移和侵入的能力。機制是透過抑制磷酸化AKT，進而抑制下游基質金屬蛋白酶MMP-2的活性和表現量來減緩CL1-5細胞轉移的能力。 總括而論，本篇研究嘗試去釐清破壞β-tubulin/CCT-β 複合體所引發細胞凋亡及抗細胞轉移能力，作為未來研究肺癌療法的新策略。

We have previously demonstrated that I-Trp with an iodomethyl ketone warhead to alkylate Cys354 of β-tubulin, thereby disrupting the protein-protein interaction (PPI) of -tubulin with chaperonin-containing TCP-1β (CCT-β), causes cancer cell apoptosis [1]. In this study, we found that in CL1-5 cells, I-Trp activates both ER stress related proteins and proteasome activity to eliminate the imbalance proteins loading in ER, thereby mitigating ER stress, at the onset of β-tubulin/CCT-β complexes disruption. In addition, ER stress-associated apoptotic signaling is usually accompanied with intracellular Ca2+ release and the activation of MAPKs. We also observed the elevated intracellular Ca2+ levels, activation of MAPKs and caspase over-activation. Since CL1-5 cells are a highly metastatic lung cancer cell line, we assayed for its migration/invasion in the presence of I-Trp, where there were 80% survived cells to ensure most of the cells were not killed. The experimental results demonstrate the dose-dependent inhibition of CL1-5 cell migration and invasion. Furthermore, the mechanistic studies revealed that I-Trp inhibited phosphorylation AKT, and GSK-3β of CL1-5 cells, thereby downregulating MMP-2 expression and activation. In conclusion, this study reveals a novel therapeutic strategy potential for evoking apoptotic signaling by targeting β-tubulin/CCT-β complexes, and its anti-migration/invasion activity against human lung adenocarcinoma.