Department of Surgery, Royal College of Surgeons in Ireland, Dublin, Ireland.
Royal College of Surgeons in Ireland, RCSI Smurfit Building, Beaumont Hospital, Dublin 9, Ireland.
Cell Oncol (Dordr). 2018 Feb;41(1):35-46. doi: 10.1007/s13402-017-0357-1. Epub 2017 Nov 13.
Triple-negative breast cancers (TNBC) lack expression of three common cell surface receptors, i.e., estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER2). Accordingly, TNBCs are associated with fewer treatment options and a relatively poor prognosis. Having screened a National Cancer Institute natural compound library, the purpose of this study was to investigate the bioactivity of compound C4 (Crassin) in TNBC cells.
Cell viability assays were performed in two TNBC cell lines, MDA-MB-231 and 4T1, following C4 treatment in the presence or absence of the antioxidant N-acetyl-L-cysteine (NAC). Phosphorylation of Akt and ERK was assessed by Western blotting. Apoptosis, necrosis, autophagy, necroptosis, ferroptosis and cytostasis assays were performed to explain viability deficits resulting from C4 exposure.
We found that the viability of the TNBC cells tested decreased in a concentration- and time-dependent fashion following C4 treatment. This decrease coincided with an unexpected increase in the expression of the cell survival effectors pAkt and pERK. In addition, we found that both the decreased cell viability and the increased pAkt/pERK levels could be rescued by the antioxidant NAC, suggesting a central role for reactive oxygen species (ROS) in the mechanism of action of C4. Necrosis, apoptosis, necroptosis and ferroptosis could be ruled out as cell death mechanisms. Instead, we found that C4 induced cytostasis downstream of ROS activation. Finally, we observed a synergistic effect between C4 and the chemotherapeutic drug doxorubicin in TNBC cells.
From our in vitro data we conclude that C4 exerts cytostatic effects on triple-negative breast cancer cells via a pathway involving reactive oxygen species. Its potential value in combination with cytotoxic therapies merits deeper investigation in pre-clinical models.
三阴性乳腺癌（TNBC）缺乏三种常见细胞表面受体的表达，即雌激素受体（ER）、孕激素受体（PR）和人表皮生长因子受体 2（HER2）。因此，TNBC 的治疗选择较少，预后相对较差。本研究通过筛选美国国立癌症研究所天然化合物库，旨在研究化合物 C4（Crassin）在 TNBC 细胞中的生物活性。
在存在或不存在抗氧化剂 N-乙酰-L-半胱氨酸（NAC）的情况下，用 C4 处理两种 TNBC 细胞系 MDA-MB-231 和 4T1 后，进行细胞活力测定。通过 Western blot 检测 Akt 和 ERK 的磷酸化。进行凋亡、坏死、自噬、坏死性凋亡、铁死亡和细胞抑制测定，以解释 C4 暴露导致的活力缺陷。
我们发现，随着 C4 处理，所测试的 TNBC 细胞的活力呈浓度和时间依赖性下降。这种减少伴随着细胞存活效应物 pAkt 和 pERK 的意外增加。此外，我们发现，抗氧化剂 NAC 可以挽救细胞活力的降低和 pAkt/pERK 水平的升高，表明活性氧（ROS）在 C4 作用机制中起核心作用。可以排除坏死、凋亡、坏死性凋亡和铁死亡作为细胞死亡机制。相反，我们发现 C4 诱导 ROS 激活下游的细胞静止。最后，我们观察到 C4 与化疗药物多柔比星在 TNBC 细胞中的协同作用。
根据我们的体外数据，我们得出结论，C4 通过涉及活性氧的途径对三阴性乳腺癌细胞发挥细胞抑制作用。其与细胞毒性疗法联合应用的潜在价值值得在临床前模型中进行更深入的研究。
