Leal Letícia F, Bueno Ana Carolina, Gomes Débora C, Abduch Rafael, de Castro Margaret, Antonini Sonir R
Department of Pediatrics, Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto, Sao Paulo, Brazil.
Department of Pediatrics, School of Medicine, Federal University of Uberlandia, Uberlândia, Minas Gerais, Brazil.
Oncotarget. 2015 Dec 15;6(40):43016-32. doi: 10.18632/oncotarget.5513.
To date, there is no effective therapy for patients with advanced/metastatic adrenocortical cancer (ACC). The activation of the Wnt/beta-catenin signaling is frequent in ACC and this pathway is a promising therapeutic target.
To investigate the effects of the inhibition of the Wnt/beta-catenin in ACC cells.
Adrenal (NCI-H295 and Y1) and non-adrenal (HeLa) cell lines were treated with PNU-74654 (5-200 μM) for 24-96 h to assess cell viability (MTS-based assay), apoptosis (Annexin V), expression/localization of beta-catenin (qPCR, immunofluorescence, immunocytochemistry and western blot), expression of beta-catenin target genes (qPCR and western blot), and adrenal steroidogenesis (radioimmunoassay, qPCR and western blot).
In NCI-H295 cells, PNU-74654 significantly decreased cell proliferation 96 h after treatment, increased early and late apoptosis, decreased nuclear beta-catenin accumulation, impaired CTNNB1/beta-catenin expression and increased beta-catenin target genes 48 h after treatment. No effects were observed on HeLa cells. In NCI-H295 cells, PNU-74654 decreased cortisol, testosterone and androstenedione secretion 24 and 48 h after treatment. Additionally, in NCI-H295 cells, PNU-74654 decreased SF1 and CYP21A2 mRNA expression as well as the protein levels of STAR and aldosterone synthase 48 h after treatment. In Y1 cells, PNU-74654 impaired corticosterone secretion 24 h after treatment but did not decrease cell viability.
Blocking the Tcf/beta-catenin complex inhibits the Wnt/beta-catenin signaling in adrenocortical tumor cells triggering increased apoptosis, decreased cell viability and impairment of adrenal steroidogenesis. These promising findings pave the way for further experiments inhibiting the Wnt/beta-catenin pathway in pre-clinical models of ACC. The inhibition of this pathway may become a promising adjuvant therapy for patients with ACC.
迄今为止，对于晚期/转移性肾上腺皮质癌（ACC）患者尚无有效的治疗方法。Wnt/β-连环蛋白信号通路在ACC中频繁激活，该通路是一个有前景的治疗靶点。
研究抑制Wnt/β-连环蛋白对ACC细胞的影响。
用PNU-74654（5-200μM）处理肾上腺（NCI-H295和Y1）及非肾上腺（HeLa）细胞系24-96小时，以评估细胞活力（基于MTS的检测法）、细胞凋亡（膜联蛋白V）、β-连环蛋白的表达/定位（定量PCR、免疫荧光、免疫细胞化学和蛋白质印迹法）、β-连环蛋白靶基因的表达（定量PCR和蛋白质印迹法）以及肾上腺类固醇生成（放射免疫测定、定量PCR和蛋白质印迹法）。
在NCI-H295细胞中，PNU-74654在处理96小时后显著降低细胞增殖，增加早期和晚期细胞凋亡，减少细胞核β-连环蛋白积累，在处理48小时后损害CTNNB1/β-连环蛋白表达并增加β-连环蛋白靶基因。在HeLa细胞中未观察到影响。在NCI-H295细胞中，PNU-74654在处理24和48小时后降低皮质醇、睾酮和雄烯二酮分泌。此外，在NCI-H295细胞中，PNU-74654在处理48小时后降低SF1和CYP21A2 mRNA表达以及STAR和醛固酮合酶的蛋白水平。在Y1细胞中，PNU-74654在处理24小时后损害皮质酮分泌，但未降低细胞活力。
阻断Tcf/β-连环蛋白复合物可抑制肾上腺皮质肿瘤细胞中的Wnt/β-连环蛋白信号通路，引发细胞凋亡增加、细胞活力降低和肾上腺类固醇生成受损。这些有前景的发现为在ACC临床前模型中进一步抑制Wnt/β-连环蛋白通路的实验铺平了道路。抑制该通路可能成为ACC患者有前景的辅助治疗方法。
