Schön Margarete, Wienrich B Gregor, Kneitz Susanne, Sennefelder Helga, Amschler Katharina, Vöhringer Verena, Weber Olaf, Stiewe Thorsten, Ziegelbauer Karl, Schön Michael P
Department of Dermatology and Venereology, University Medical Center Göttingen, von-Siebold-Str 3, 37075 Göttingen, Germany.
J Natl Cancer Inst. 2008 Jun 18;100(12):862-75. doi: 10.1093/jnci/djn174. Epub 2008 Jun 10.
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.
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).
Inhibition of constitutive and induced IKKbeta-activity through treatment with KINK-1 might increase tumor susceptibility to chemotherapy.
通过降低化疗耐药性来提高化疗药物的疗效可能是癌症治疗中的一种有效策略。核因子-κB（NF-κB）的组成性激活是包括黑色素瘤在内的多种癌症的一个标志，黑色素瘤几乎对化疗普遍耐药。NF-κB由抑制性κB（IkappaB）蛋白调节，而IkappaB蛋白又被IkappaB激酶（IKK）复合物磷酸化。
通过免疫印迹法测量IkappaBα亚基的磷酸化、电泳迁移率变动分析检测DNA结合以及利用免疫荧光检测NF-κB的核转位，评估一种新型IKKβ亚基小分子抑制剂（KINK-1；核因子-κB-1激酶抑制剂）对NF-κB活性的影响。通过微阵列分析、实时和半定量逆转录聚合酶链反应（RT-PCR）以及蛋白质印迹分析确定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志物来表征KINK-1（单独以及与细胞生长抑制剂联合使用时）对黑色素瘤细胞的影响。通过测量C57BL6小鼠（每组6只）肺部重量并计数携带黑色素瘤细胞转移灶的小鼠体内转移灶数量，在体内测试KINK-1与细胞生长抑制剂阿霉素或喜树碱联合使用（均经腹腔注射）的抗肿瘤疗效。所有统计检验均为双侧检验。结果KINK-1强烈抑制黑色素瘤细胞中组成性和诱导性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，但在细胞水平上几乎没有抗增殖或促凋亡活性。然而，KINK-1在体外显著增强了一些细胞生长抑制剂的活性，并消除了阿霉素诱导的NF-κB激活。与单独使用任何一种治疗相比，用KINK-1和阿霉素或喜树碱联合治疗已注射黑色素瘤细胞的C57BL6小鼠可减少转移灶和肺部肿瘤肿块（单独用3mg/kg KINK-1、1mg/kg阿霉素以及1mg/kg阿霉素加3mg/kg KINK-1治疗的小鼠在注射黑色素瘤细胞19天后的平均肺重分别为260mg，95%置信区间（CI）=216至305mg；268mg，95%CI=224至313mg；以及181mg，95%CI=171至192mg，通过t检验比较联合治疗小鼠与单独用任一化合物治疗小鼠的平均肺重，P<.001）。
通过用KINK-1治疗抑制组成性和诱导性IKKβ活性可能会增加肿瘤对化疗的敏感性。
