National Institute of Health and Medical Research, Medical Research Unit S-1165/Paris Diderot University, University Institute of Hematology, Saint-Louis Hospital, 75010 Paris, France.
National Institute of Health and Medical Research, Unit 1234/Rouen University, Faculty of Medicine and Pharmacy, 76183 Rouen, France.
Int J Oncol. 2018 Apr;52(4):1246-1254. doi: 10.3892/ijo.2018.4272. Epub 2018 Feb 9.
Pentoxifylline (PTX), a xanthine family molecule and simvastatin (SIM), an anti-hypercholesterolemic agent, have recently been considered as sensitizers to chemotherapy and radiotherapy. The present in vitro study evaluated their antitumor synergistic effects on MDA‑MB‑231 breast cancer cells characterized by the triple‑negative phenotype (TNP). The anti-proliferative effects of these two agents were evaluated by MTT and clonogenic assays. Cell cycle progression was examined using propidium iodide staining. Apoptosis was investigated by Annexin V labeling, and by examining caspase 3 activity and DNA fragmentation. Autophagic vesicles and reactive oxygen species (ROS) levels were monitored by flow cytometry. Western blot analysis was performed to evaluate molecular targets. Our results revealed that when used alone, PTX and SIM exerted antitumor effects. Nevertheless, used in combination, the inhibition of cell proliferation was synergistically superior (80% vs 42%) than that observed following treatment with each agent alone after 48 h. PTX alone (0.5 mM) induced both apoptosis (25%) and autophagy (25%); however, when used in combination with SIM (0.5 µM), the balance between these processes was disrupted and the cells underwent apoptosis (>65%) as opposed to autophagy (<13%). This imbalance was associated with an increase in ERK1/2 and AKT activation, but not with an increase in mTOR phosphorylation, and with the suppression of the NF-κB pathway. In addition, in the cells treated with both agents, almost 78% of the cells were arrested at the G0/G1 phase and lost their colony-forming ability (38±5%) compared to the cells treated with PTX alone (115±5%). On the whole, these results suggest that the induction of autophagy may be a protective mechanism preventing MDA‑MB‑231 cancer cell death. The combined use of PTX and SIM may drive dormant autophagic cancer cells to undergo apoptosis and thus this may be a novel treatment strategy for breast cancer characterized by the TNP.
己酮可可碱（PTX）是黄嘌呤家族的一种分子，辛伐他汀（SIM）是一种抗高胆固醇药物，最近被认为是化疗和放疗的增敏剂。本体外研究评估了它们对三阴性表型（TNP）特征的 MDA-MB-231 乳腺癌细胞的抗肿瘤协同作用。通过 MTT 和集落形成试验评估这两种药物的抗增殖作用。通过碘化丙啶染色检查细胞周期进程。通过 Annexin V 标记和检查 caspase 3 活性和 DNA 片段化来研究细胞凋亡。通过流式细胞术监测自噬小泡和活性氧（ROS）水平。通过 Western blot 分析评估分子靶标。我们的结果表明，PTX 和 SIM 单独使用时均具有抗肿瘤作用。然而，联合使用时，抑制细胞增殖的协同作用明显优于单独使用时（48 小时后分别为 80%和 42%）。PTX 单独使用（0.5 mM）诱导凋亡（25%）和自噬（25%）；然而，当与 SIM（0.5 μM）联合使用时，这些过程之间的平衡被打破，细胞发生凋亡（>65%）而不是自噬（<13%）。这种不平衡与 ERK1/2 和 AKT 激活增加有关，但与 mTOR 磷酸化增加无关，与 NF-κB 途径抑制有关。此外，在用两种药物处理的细胞中，与单独用 PTX 处理的细胞（115±5%）相比，约 78%的细胞停滞在 G0/G1 期，丧失其集落形成能力（38±5%）。总的来说，这些结果表明，自噬的诱导可能是一种保护机制，防止 MDA-MB-231 癌细胞死亡。PTX 和 SIM 的联合使用可能会促使休眠的自噬癌细胞发生凋亡，因此这可能是 TNP 特征的乳腺癌的一种新的治疗策略。
