Department of Medicine, Section of Hematology and Medical Oncology, Tulane University Health Sciences Center, 1430 Tulane Ave, New Orleans, LA 70112, USA.
Breast Cancer Res. 2012 May 21;14(3):R79. doi: 10.1186/bcr3192.
Of the more than one million global cases of breast cancer diagnosed each year, approximately fifteen percent are characterized as triple-negative, lacking the estrogen, progesterone, and Her2/neu receptors. Lack of effective therapies, younger age at onset, and early metastatic spread have contributed to the poor prognoses and outcomes associated with these malignancies. Here, we investigate the ability of the histone deacetylase inhibitor panobinostat (LBH589) to selectively target triple-negative breast cancer (TNBC) cell proliferation and survival in vitro and tumorigenesis in vivo.
TNBC cell lines MDA-MB-157, MDA-MB-231, MDA-MB-468, and BT-549 were treated with nanomolar (nM) quantities of panobinostat. Relevant histone acetylation was verified by flow cytometry and immunofluorescent imaging. Assays for trypan blue viability, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) proliferation, and DNA fragmentation were used to evaluate overall cellular toxicity. Changes in cell cycle progression were assessed with propidium iodide flow cytometry. Additionally, qPCR arrays were used to probe MDA-MB-231 cells for panobinostat-induced changes in cancer biomarkers and signaling pathways. Orthotopic MDA-MB-231 and BT-549 mouse xenograft models were used to assess the effects of panobinostat on tumorigenesis. Lastly, flow cytometry, ELISA, and immunohistochemical staining were applied to detect changes in cadherin-1, E-cadherin (CDH1) protein expression and the results paired with confocal microscopy in order to examine changes in cell morphology.
Panobinostat treatment increased histone acetylation, decreased cell proliferation and survival, and blocked cell cycle progression at G2/M with a concurrent decrease in S phase in all TNBC cell lines. Treatment also resulted in apoptosis induction at 24 hours in all lines except the MDA-MB-468 cell line. MDA-MB-231 and BT-549 tumor formation was significantly inhibited by panobinostat (10 mg/kg/day) in mice. Additionally, panobinostat up-regulated CDH1 protein in vitro and in vivo and induced cell morphology changes in MDA-MB-231 cells consistent with reversal of the mesenchymal phenotype.
This study revealed that panobinostat is overtly toxic to TNBC cells in vitro and decreases tumorigenesis in vivo. Additionally, treatment up-regulated anti-proliferative, tumor suppressor, and epithelial marker genes in MDA-MB-231 cells and initiated a partial reversal of the epithelial-to-mesenchymal transition. Our results demonstrate a potential therapeutic role of panobinostat in targeting aggressive triple-negative breast cancer cell types.
在每年诊断出的超过 100 万例全球乳腺癌病例中，约有 15%的病例表现为三阴性，缺乏雌激素、孕激素和 Her2/neu 受体。缺乏有效的治疗方法、发病年龄较早以及早期转移扩散导致了这些恶性肿瘤相关的不良预后和结局。在这里，我们研究了组蛋白去乙酰化酶抑制剂帕比司他（LBH589）在体外选择性靶向三阴性乳腺癌（TNBC）细胞增殖和存活以及体内肿瘤发生的能力。
用纳摩尔（nM）数量的帕比司他处理 TNBC 细胞系 MDA-MB-157、MDA-MB-231、MDA-MB-468 和 BT-549。通过流式细胞术和免疫荧光成像验证相关组蛋白乙酰化。使用台盼蓝活力测定、3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）增殖和 DNA 片段化测定来评估整体细胞毒性。用碘化丙啶流式细胞术评估细胞周期进展的变化。此外，还使用 qPCR 阵列探测 MDA-MB-231 细胞中帕比司他诱导的癌症生物标志物和信号通路的变化。使用 MDA-MB-231 和 BT-549 原位异种移植小鼠模型来评估帕比司他对肿瘤发生的影响。最后，应用流式细胞术、ELISA 和免疫组织化学染色检测钙粘蛋白-1（Cadherin-1）和上皮钙粘蛋白（E-cadherin，CDH1）蛋白表达的变化，并与共聚焦显微镜相结合，以检查细胞形态的变化。
帕比司他处理增加了组蛋白乙酰化，降低了所有 TNBC 细胞系的细胞增殖和存活，并在 G2/M 期阻断细胞周期进展，同时 S 期减少。除 MDA-MB-468 细胞系外，所有细胞系在 24 小时时还诱导了细胞凋亡。在小鼠中，帕比司他（10mg/kg/天）显著抑制了 MDA-MB-231 和 BT-549 肿瘤的形成。此外，帕比司他在体外和体内均上调了 CDH1 蛋白，并诱导 MDA-MB-231 细胞的细胞形态发生变化，与上皮表型的逆转一致。
本研究表明，帕比司他在体外对 TNBC 细胞具有明显的毒性，并在体内降低肿瘤发生。此外，治疗还上调了 MDA-MB-231 细胞中的增殖抑制、肿瘤抑制和上皮标记基因，并启动了上皮-间充质转化的部分逆转。我们的结果表明帕比司他在靶向侵袭性三阴性乳腺癌细胞类型方面具有潜在的治疗作用。
