Neuroblastoma is a common childhood cancer with high mortality. We evaluated the capacity of the flavonoid, isoliquiritigenin (4,2’,4’-trihydroxychalcone; ISL) to inhibit cellular proliferation and migration in the human neuroblastoma cell line SH-SY5Y. Incubation of cultured SH-SY5Y cells with 20–100 μM ISL decreased cell confluency (15–70%) after 24 h incubation, while 10–100 μM ISL (24 h) depleted intracellular ATP stores (15–90% vs vehicle-treated control) after 24 h incubation. ISL-mediated cell toxicity did not involve intracellular caspase 3/7 activation, externalization of phosphatidylserine on the cell membrane or stimulation of TNF and IL-1β release, all indicating that the flavonoid did not induce apoptosis. Pre-treatment of cells with necrostatin-1, a necroptosis inhibitor, significantly restored ATP levels (ATP levels increased 12–42%) in ISL-treated neuroblastoma cells indicative of enhanced viability. By contrast, RIP1 phosphorylation status remained unchanged in cells treated with ISL although the intracellular ratio of phosphorylated/total parental RIP1 increased after ISL treatment on SH-SY5Y cells indicating that ISL decreased levels of native RIP1. In addition, ISL treatment inhibited SH-SY5Y cell migration/proliferation in a scratch assay and arrested cell cycle transition by significantly decreasing the number of cells in G0/G1 phase and increasing populations by ~10% in S (primarily) and G2/M (lesser extent) phases. The intracellular ratio of phosphorylated/total ERK 1/2 and p38 remained unchanged after ISL treatment (up to 40 μM); ERK activation was only determined at ISL dose well above the experimental IC₅₀ value as judged by ELISA analyses and this did not correlate with ISL cytotoxicity at lower dose <40 μM; Western blot assay confirmed the detection of phosphorylated (p-)ERK1/2 and (p-)p38 in ISL treated cells. Together the results suggest that ISL exerts anti-proliferative and cytotoxic activity on SHSY5Y cells through the loss of ATP, induction of cell cycle arrest, and cell death largely via a necroptotic mechanism in the absence of apoptotic activity.

神经母细胞瘤是一种常见的儿童癌症，死亡率高。我们评估了类黄酮，异寡糖原蛋白（4,2'，4'-三羟基查耳酮； ISL）抑制人类神经母细胞瘤细胞系SH-SY5Y中细胞增殖和迁移的能力。培养的SH-SY5Y细胞与20–100μMISL一起孵育24小时后会降低细胞融合度（15–70％），而10–100μMISL（24 h）会耗尽细胞内ATP储存（与媒介物处理相比为15–90％）对照）孵育24小时后。ISL介导的细胞毒性不涉及细胞内caspase 3/7活化，磷脂酰丝氨酸在细胞膜上的外在化或TNF和IL-1β释放的刺激，均表明类黄酮不诱导细胞凋亡。用坏死抑制剂necrostatin-1预处理细胞，ISL处理的神经母细胞瘤细胞可显着恢复ATP水平（ATP水平增加12–42％），表明生存能力增强。相比之下，ISL处理后的细胞中RIP1磷酸化状态保持不变，尽管在SH-SY5Y细胞上进行ISL处理后磷酸化/总亲本RIP1的细胞内比率增加，这表明ISL降低了天然RIP1的水平。此外，ISL处理在刮擦试验中抑制了SH-SY5Y细胞迁移/增殖，并通过显着减少G0 / G1期的细胞数量并使S（主要是）和G2 / M的种群增加了约10％，从而阻止了细胞周期的转变。 （程度较小）阶段。ISL处理后（高达40μM）磷酸化/总ERK 1/2和p38的细胞内比率保持不变；通过ELISA分析判断为₅₀，与较低剂量<40μM的ISL细胞毒性无关。Western印迹测定法证实在ISL处理的细胞中检测到磷酸化的（p-）ERK1 / 2和（p-）p38。在一起的结果表明，ISL通过ATP的丧失，诱导细胞周期停滞和细胞死亡，主要是通过在没有凋亡活性的情况下的坏死性机制，对SHSY5Y细胞发挥抗增殖和细胞毒性的作用。