BACKGROUND: Increasing the efficacy of chemotherapeutics by reducing chemoresistance may be a useful strategy in cancer therapy. Constitutive activation of nuclear factor-kappa B (NF-kappaB) is a hallmark of various cancers, including melanoma, which is almost universally resistant to chemotherapy. NF-kappaB is regulated by inhibitory kappaB (IkappaB) proteins, which are in turn phosphorylated by the IkappaB kinase (IKK) complex. METHODS: The effect on NF-kappaB activity of a novel small-molecule inhibitor of the beta subunit of IKK (KINK-1; kinase inhibitor of nuclear factor-kappaB-1) was assessed by measuring phosphorylation of the alpha subunit of IkappaB by immunoblotting, DNA binding by electrophoretic mobility shift assays, and nuclear translocation of NF-kappaB using immunofluorescence. Regulation of NF-kappaB-dependent gene expression was determined by microarray analysis, real-time and semiquantitative reverse transcription polymerase chain reaction (RT-PCR), and Western blot analyses. The effects of KINK-1 (alone and in combination with cytostatic agents) on melanoma cells were characterized by assessing proliferation, soft agar colony formation, and markers of apoptosis. The antitumoral efficacy of KINK-1 in combination with the cytostatic agents doxorubicin or camptothecin (all injected intraperitoneally) was tested in vivo by measuring lung weight and counting metastases in C57BL6 mice (groups of six) bearing metastases of melanoma cells. All statistical tests were two-sided. Results KINK-1 strongly suppressed both constitutive and induced NF-kappaB activity in melanoma cells. It reduced the expression of NF-kappaB-dependent gene products that regulate proliferation, cytokine production, and antiapoptotic responses but exhibited little antiproliferative or proapoptotic activity at the cellular level. However, KINK-1 markedly increased the activities of some cytostatic agents in vitro and abrogated doxorubicin-induced NF-kappaB activation. Combined treatment of C57BL6 mice that had been injected with melanoma cells with KINK-1 and doxorubicin or camptothecin reduced metastases and pulmonary tumor mass compared with either treatment alone (mean lung weight 19 days after injection of melanoma cells of mice treated with 3 mg/kg KINK-1 alone, 1 mg/kg doxorubicin alone, and 1 mg/kg doxorubicin plus 3 mg/kg KINK-1 = 260 mg, 95% confidence interval (CI) = 216 to 305 mg; 268 mg, 95% CI = 224 to 313 mg; and 181 mg, 95% CI = 171 to 192 mg, respectively, P < .001 from t tests comparing mean lung weight of double-treated mice to that in mice treated with either compound alone). CONCLUSION: Inhibition of constitutive and induced IKKbeta-activity through treatment with KINK-1 might increase tumor susceptibility to chemotherapy.

中文翻译：

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KINK-1，一种新型的IKKbeta小分子抑制剂，以及黑素瘤细胞对抗肿瘤治疗的敏感性。

背景：通过降低化学抗性来提高化疗药物的疗效可能是癌症治疗中的一种有用策略。核因子-κB（NF-kappaB）的组成性激活是包括黑素瘤在内的各种癌症的标志，它几乎普遍对化学疗法具有抗性。NF-kappaB受抑制性kappaB（IkappaB）蛋白调节，而该蛋白又被IkappaB激酶（IKK）复合物磷酸化。方法：通过免疫印迹法测定IkappaB的α亚基的磷酸化，评估一种新型的IKKβ亚基小分子抑制剂（KINK-1；核因子kappaB-1的激酶抑制剂）对NF-κB活性的影响。 ，通过电泳迁移率迁移测定法进行的DNA结合以及使用免疫荧光的NF-κB的核易位。通过微阵列分析，实时和半定量逆转录聚合酶链反应（RT-PCR）和Western印迹分析确定了NF-κB依赖性基因表达的调控。通过评估增殖，软琼脂集落形成和凋亡标记物来表征KINK-1（单独或与细胞抑制剂联合使用）对黑素瘤细胞的作用。通过测量肺部重量并计算携带黑色素瘤细胞转移的C57BL6小鼠（六只一组）中的转移，在体内测试KINK-1与细胞抑制剂阿霉素或喜树碱（均腹膜内注射）联合的抗肿瘤功效。所有统计检验都是两面的。结果KINK-1强烈抑制黑色素瘤细胞的本构和诱导的NF-κB活性。它降低了调节增殖，细胞因子产生和抗凋亡反应的NF-κB依赖性基因产物的表达，但在细胞水平上却几乎没有抗增殖或促凋亡活性。但是，KINK-1显着增加了某些细胞抑制剂的体外活性，并废除了阿霉素诱导的NF-κB活化。与单独使用任一治疗相比，联合注射KINK-1和阿霉素或喜树碱的黑素瘤细胞对C57BL6小鼠的联合治疗可减少转移和肺肿瘤的发生（注射3 mg / kg的黑素瘤细胞后19天的平均肺重）单独KINK-1，单独1 mg / kg阿霉素和1 mg / kg阿霉素加3 mg / kg KINK-1 = 260 mg，95％置信区间（CI）= 216至305 mg; 268 mg，95％CI = 224至313毫克；和181毫克，比较两次治疗的小鼠的平均肺重量与仅用任一化合物治疗的小鼠的平均肺重量，t试验的95％CI分别为171至192 mg，P <.001 结论：通过KINK-1治疗抑制本构和诱导的IKKbeta活性可能会增加肿瘤对化学疗法的敏感性。