Eppstein Andrew C, Sandoval John A, Klein Patrick J, Woodruff Heather A, Grosfeld Jay L, Hickey Robert J, Malkas Linda H, Schmidt C Max
Department of Surgery, Indiana University School of Medicine and Riley Children's Hospital, Indianapolis, IN 46202, USA.
J Pediatr Surg. 2006 Jan;41(1):252-9. doi: 10.1016/j.jpedsurg.2005.10.047.
Neuroblastoma tumors are comprised of neuroblastic (N), substrate-adherent (S), and intermediate (I) cells. Because cell growth and differentiation often involve p44/p42 mitogen-activated protein kinase (MAPK) pathway signaling, we explored MAPK signaling and growth response in three NB cell types after MAPK kinase (MEK) inhibition to evaluate the feasibility of MAPK-targeted treatment strategies.
Three human NB cell cultures, SH-SY5Y (N-type), BE(2)-C (I-type), and SK-N-AS (S-type), were treated in monolayer cultures with increasing concentrations of the MEK inhibitor U0126. MAPK pathway intermediates MEK and ERK, their activated (phosphorylated) forms p-MEK and p-ERK, and p53 expression were assessed by Western blot at 1 and 24 hours. At 72 hours, cell counts determined growth inhibition and DNA fragmentation ELISA assessed apoptosis.
Among all three lines, total ERK and MEK expression were unaffected by U0126. However, constitutive total ERK and p53 expression were significantly greater in BE(2)-C (I-type) cells than in SH-SY5Y (N-type) and SK-N-AS (S-type). Active ERK (p-ERK) levels decreased in dose response to U0126 at 1 and 24 hours in all lines. Conversely, p-MEK levels increased with increasing U0126 concentrations at 1 hour in SH-SY5Y (N-type) and at 24 hours in all lines. BE(2)-C (I-type) cell counts decreased in concentration-dependent fashion with U0126, whereas SH-SY5Y (N-type) and SK-N-AS (S-type) showed a biphasic response with increased cell counts at 1 micromol/L U0126 and slightly decreased cell counts at 10 mumol/L U0126.
This study demonstrates that BE(2)-C (I-type) cells exhibit greater constitutive total ERK and p53 expression than SH-SY5Y (N-type) and SK-N-AS (S-type). Although all three lines exhibit p-ERK decreases with MEK inhibition, only BE(2)-C (I-type) cells significantly decrease their proliferation with U0126 treatment. Although MEK inhibition holds promise in targeting I-type NB cells, successfully treating this heterogeneous tumor may require combining agents against N- and S-type cells.
神经母细胞瘤肿瘤由成神经细胞（N）、基质黏附细胞（S）和中间型细胞（I）组成。由于细胞生长和分化常涉及p44/p42丝裂原活化蛋白激酶（MAPK）信号通路，我们探究了MAPK激酶（MEK）抑制后三种神经母细胞瘤细胞类型中的MAPK信号传导及生长反应，以评估MAPK靶向治疗策略的可行性。
三种人神经母细胞瘤细胞培养物，即SH-SY5Y（N型）、BE(2)-C（I型）和SK-N-AS（S型），在单层培养中用浓度递增的MEK抑制剂U0126处理。在1小时和24小时时通过蛋白质免疫印迹法评估MAPK信号通路中间体MEK和ERK、它们的活化（磷酸化）形式p-MEK和p-ERK以及p53表达。在72小时时，细胞计数确定生长抑制情况，DNA片段化酶联免疫吸附测定评估细胞凋亡。
在所有三株细胞系中，总ERK和MEK表达不受U0126影响。然而，BE(2)-C（I型）细胞中组成型总ERK和p53表达显著高于SH-SY5Y（N型）和SK-N-AS（S型）细胞。在所有细胞系中，活性ERK（p-ERK）水平在1小时和24小时时随U0126剂量增加而降低。相反，在SH-SY5Y（N型）细胞的1小时以及所有细胞系的24小时时，p-MEK水平随U0126浓度增加而升高。BE(2)-C（I型）细胞计数随U0126呈浓度依赖性下降，而SH-SY5Y（N型）和SK-N-AS（S型）细胞呈现双相反应，在1 μmol/L U0126时细胞计数增加，在10 μmol/L U0126时细胞计数略有下降。
本研究表明，BE(2)-C（I型）细胞比SH-SY5Y（N型）和SK-N-AS（S型）细胞表现出更高的组成型总ERK和p53表达。尽管所有三株细胞系在MEK抑制时均表现出p-ERK下降，但只有BE(2)-C（I型）细胞在U0126处理后显著降低其增殖。虽然MEK抑制有望靶向I型神经母细胞瘤细胞，但成功治疗这种异质性肿瘤可能需要联合针对N型和S型细胞的药物。
