Medicine Department, Harbin Medical University Cancer Hospital, Haping Road No.150, Harbin, 150081, Heilongjiang, China.
BMC Cancer. 2021 Feb 25;21(1):195. doi: 10.1186/s12885-021-07901-w.
Activation of autophagy flux contributed to resistance of breast cancer (BC) cells to current chemotherapeutic drugs, which seriously limited their therapeutic efficacy and facilitated BC recurrence in clinic. However, the detailed mechanisms are still not fully understood. In the present study, we identified that inactivation of AMPK-ULK1 signaling cascade mediated protective autophagy sensitized BC cells to doxorubicin in vitro.
Cell counting kit-8 (CCK-8) assay and colony formation assay were performed to evaluate cell proliferation abilities. Trypan blue staining assay was used to examine cell viability, and Annexin V-FITC/PI double staining method was conducted to determine cell apoptosis. The autophagosomes in BC cells were observed and photographed by electronic microscope (EM). Western Blot analysis was employed to examine genes expressions at protein levels.
The parental doxorubicin-sensitive BC (DS-BC) cells were exposed to increasing concentrations of doxorubicin to establish doxorubicin-resistant BC (DR-BC) cells, and the DR-BC cells were much more resistant to high-dose doxorubicin treatment compared to the DS-BC cells. Interestingly, high-dose doxorubicin specifically increased LC3B-II/I ratio, promoted autophagosomes formation and decreased p62 expression levels to facilitate autophagy in DR-BC cells, instead of DS-BC cells, and the autophagy inhibitor 3-methyladenine (3-MA) enhanced the cytotoxic effects of high-dose doxorubicin on DR-BC cells. In addition, we proved that high-dose doxorubicin triggered protective autophagy in DR-BC cells by activating AMPK-ULK1 pathway. Functionally, high-dose doxorubicin increased the expression levels of phosphorylated AMPK (p-AMPK) and ULK1 (p-ULK1) to activate AMPK-ULK1 pathway in DR-BC cells, and the inhibitors for AMPK (compound C) and ULK1 (SBI-0206965) blocked autophagy to promote cell death and slow down cell growth in DR-BC cells treated with high-dose doxorubicin.
Collectively, our in vitro data indicated that blockage of AMPK-ULK1 signaling cascade mediated protective autophagy might be a promising strategy to increase doxorubicin sensitivity for BC treatment.
自噬通量的激活有助于乳腺癌（BC）细胞对当前化疗药物产生耐药性，这严重限制了它们的治疗效果，并在临床上促进了 BC 的复发。然而，其详细的机制仍未完全阐明。在本研究中，我们发现 AMPK-ULK1 信号级联的失活介导了保护性自噬，使 BC 细胞对阿霉素在体外敏感。
使用细胞计数试剂盒-8（CCK-8）测定和集落形成测定来评估细胞增殖能力。台盼蓝染色测定用于检测细胞活力，并用 Annexin V-FITC/PI 双染色法检测细胞凋亡。通过电子显微镜（EM）观察和拍摄 BC 细胞中的自噬体。采用 Western Blot 分析检测基因在蛋白质水平上的表达。
将亲本阿霉素敏感的 BC（DS-BC）细胞暴露于递增浓度的阿霉素中，以建立阿霉素耐药的 BC（DR-BC）细胞，与 DS-BC 细胞相比，DR-BC 细胞对高剂量阿霉素处理的耐药性更强。有趣的是，高剂量阿霉素特异性地增加了 LC3B-II/I 比值，促进自噬体的形成，并降低 p62 的表达水平，从而促进 DR-BC 细胞中的自噬，而不是 DS-BC 细胞，并且自噬抑制剂 3-甲基腺嘌呤（3-MA）增强了高剂量阿霉素对 DR-BC 细胞的细胞毒性作用。此外，我们证明高剂量阿霉素通过激活 AMPK-ULK1 通路触发 DR-BC 细胞中的保护性自噬。功能上，高剂量阿霉素增加了磷酸化 AMPK（p-AMPK）和 ULK1（p-ULK1）的表达水平，从而激活 DR-BC 细胞中的 AMPK-ULK1 通路，并且 AMPK 的抑制剂（化合物 C）和 ULK1（SBI-0206965）阻断自噬，促进高剂量阿霉素处理的 DR-BC 细胞中的细胞死亡和减缓细胞生长。
总的来说，我们的体外数据表明，阻断 AMPK-ULK1 信号级联介导的保护性自噬可能是提高 BC 治疗中阿霉素敏感性的一种有前途的策略。
