Inhibition of constitutively activated nuclear factor-kappaB radiosensitizes human melanoma cells.
Inhibition of constitutively activated nuclear factor-kappaB radiosensitizes human melanoma cells.
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DOI：
10.1158/1535-7163.985.3.8
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发表时间：
2004-08
影响因子：
5.7
通讯作者：
A. Munshi;J. Kurland;T. Nishikawa;P. Chiao;M. Andreeff;R. Meyn
中科院分区：
文献类型：
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作者：
A. Munshi;J. Kurland;T. Nishikawa;P. Chiao;M. Andreeff;R. Meyn
黑色素瘤肿瘤和培养的细胞系对电离辐射的细胞毒性作用具有相对的抵抗力，从而限制了放射治疗在黑色素瘤临床治疗中的应用。因此，增敏黑色素瘤细胞的新策略值得研究。为了识别和靶向参与辐射抗性的信号通路，我们研究了核因子-kappaB(NF-kappaB)的作用，它是一种已知的转录因子，可以抑制多种刺激诱导的细胞凋亡并促进辐射抗性。用两种人转移性黑色素瘤细胞株A375和MeWo检测了核因子-kappaB途径抑制剂的放射增敏作用。用凝胶迁移率改变分析法检测这些细胞系的核提取液中的活性核因子-kappaB。两种黑色素瘤细胞系均可通过凝胶迁移率改变实验观察到核因子-kappaB的组成性激活。为了逆转NF-kappaB的活性，细胞被单独用赋形剂或蛋白酶体抑制剂Z-Leu-H(MG132；照射前2小时10微克/L)处理，后者同时抑制原生性和辐射诱导的NF-kappaB的活性。克隆形成细胞存活实验显示，MG132可增强肿瘤细胞的放射敏感性，2Gy时的存活系数由载体剂组的48+/-0.8%和48+/-1.6%降至MG132处理的MeWo和A375细胞的27.7+/-0.32%和34.3+/-0.7%。为了更直接地测试核因子-kappaB在辐射抗性中的作用，将显性-负性突变体IkappaBalpha结构稳定地导入MeWo细胞，导致结构性和辐射诱导的核因子-kappaB活性的抑制。稳定表达的MeWo细胞的放射敏感性也有一定程度的恢复，2Gy值的存活因子从亲代MeWo细胞的47+/-0.8%降低到稳定转染组的32.9+/-0.7%。由于结构性激活的丝裂原活化蛋白激酶(MEK)途径已被证明导致激活的NF-kappaB，我们想要确定激活的MEK在人黑色素瘤细胞中的相对贡献。为了测试这一点，将MeWo和A375黑色素瘤细胞暴露于MEK抑制剂PD184352。PD184352处理部分逆转了核因子-kappaB的活性，但不增加对辐射的敏感性。我们的结果表明，激活的核因子-kappaB可能是导致黑色素瘤细胞辐射抵抗的途径之一，抑制其影响的策略可能有助于恢复黑色素瘤的辐射反应。
Melanoma tumors and cultured cell lines are relatively resistant to the cytotoxic effects of ionizing radiation, thereby limiting the use of radiotherapy for the clinical treatment of melanoma. New strategies for sensitizing melanoma cells therefore deserve examination. In an attempt to identify and target signaling pathways that contribute to radioresistance, we investigated the role of nuclear factor-kappaB (NF-kappaB), a transcription factor known to inhibit apoptosis induced by a variety of stimuli and promote radioresistance. Two human metastatic melanoma cell lines, A375 and MeWo, were used to examine the radiosensitizing effects of inhibitors of the NF-kappaB pathway. Nuclear extracts from these cell lines were tested for active NF-kappaB using the electrophoretic mobility shift assay. Both melanoma cell lines had constitutively activated NF-kappaB as observed by electrophoretic mobility shift assay. In an attempt to reverse NF-kappaB activity, cells were treated either with vehicle alone (DMSO) or with a proteasome inhibitor Z-Leu-Leu-Leu-H (MG132; 10 micromol/L for 2 hours prior to irradiation) that inhibited both constitutive and radiation-induced NF-kappaB activity. The clonogenic cell survival assay showed that pretreatment with MG132 enhanced tumor cell radiosensitivity with the survival factor at 2 Gy being reduced from 48 +/- 0.8% and 48 +/- 1.6% in vehicle-treated cells to 27.7 +/- 0.32% and 34.3 +/- 0.7% in MG132-treated MeWo and A375 cells, respectively. To test the role of NF-kappaB in radioresistance more directly, MeWo cells were stably transfected with a dominant-negative mutant IkappaBalpha construct, which led to the inhibition of both constitutive and radiation-induced NF-kappaB activity. A modest restoration of radiosensitivity was also observed in the stably transfected MeWo cells with survival factor at 2 Gy values being reduced from 47 +/- 0.8% in parental MeWo cells to 32.9 +/- 0.7% in stable transfectants. Because constitutively activated mitogen-activated protein kinase kinase (MEK) pathway has been shown to lead to activated NF-kappaB, we wanted to determine the relative contribution of activated MEK in the human melanoma cells. To test this, MeWo and A375 melanoma cells were exposed to the MEK inhibitor PD184352. Treatment with PD184352 partially reversed NF-kappaB activity but did not impart radiation sensitivity to these cells. Our results indicate that activated NF-kappaB may be one of the pathways responsible for the radioresistance of melanoma cells and that strategies for inhibiting its influence may be useful in restoring the radioresponse of melanomas.