Department of Pharmacology, Piramal Life Sciences, Piramal Enterprises Limited, 1 Nirlon Complex, Goregaon (East), Mumbai, Maharashtra 400 063, India.
Lung Cancer. 2013 Nov;82(2):214-21. doi: 10.1016/j.lungcan.2013.08.010. Epub 2013 Sep 3.
P276-00 is a novel cyclin-dependent kinase (CDK) inhibitor is in Phase II clinical trials. Valproic acid (VPA), an antiepileptic agent has been associated with anticancer activity, through the inhibition of histone deacetylase I. Here we investigate the effect of the combination of VPA and P276-00, in non-small-cell lung cancer (NSCLC) cell lines.
Cell growth inhibition was studied using the Propidium iodide (PI) assay. Cell cycle analysis and recovery were detected by flow cytometry. The expression levels of various proteins were detected by western blot. Inhibition of colony formation in H460 was checked in vitro. In vivo efficacy was studied in H460 xenograft model.
The combination of P276-00 and VPA showed synergistic effect on p53+ and p53- NSCLC cell lines in antiproliferative assay at both constant and non-constant ratio with marked decrease in colony forming potential. Flow cytometric analysis confirmed a significant time dependent increase in apoptosis with 64% apoptotic population at 96 h compared to VPA (1%) and P276-00 (28%) alone (p < 0.0001). Incubation of the cells after treatment, in fresh medium without drugs, led to the recovery of cells treated with P276-00 alone but not the cells treated with the combination of both the drugs. The combination treatment up-regulated tumor suppressor proteins like p53, p21 and p27 along with down-regulation of proliferation and survival proteins viz. cyclin D1 and Bcl-2. This was also associated with the upregulation of the pro-apoptotic protein Bax and significant accumulation of hyperacetylated histones in the combination treatment. Interestingly, VPA in combination with P276-00 was much more effective as an antitumor agent than alone, in the H460 xenograft tumor model in SCID mice.
This study indicates that the combination of HDAC inhibitor VPA with CDK inhibitor P276-00 is promising novel molecularly targeted therapeutic approach for NSCLC treatment.
P276-00 是一种新型细胞周期蛋白依赖性激酶（CDK）抑制剂，目前正在进行 II 期临床试验。丙戊酸（VPA）是一种抗癫痫药物，通过抑制组蛋白去乙酰化酶 I 具有抗癌活性。在这里，我们研究了 VPA 和 P276-00 联合应用于非小细胞肺癌（NSCLC）细胞系的效果。
使用碘化丙啶（PI）检测法研究细胞生长抑制。通过流式细胞术检测细胞周期分析和恢复。通过 Western blot 检测各种蛋白质的表达水平。在体外检查 H460 中的集落形成抑制。在 H460 异种移植模型中研究体内疗效。
在增殖测定中，P276-00 与 VPA 联合应用对 p53+和 p53- NSCLC 细胞系表现出协同作用，在固定和非固定比例下均显著降低集落形成潜力。流式细胞术分析证实，与 VPA（1%）和 P276-00（28%）单独处理相比，在 96 小时时，凋亡的细胞明显增加，凋亡细胞比例为 64%（p < 0.0001）。在用药物处理后，将细胞孵育在不含药物的新鲜培养基中，导致仅用 P276-00 处理的细胞恢复，但用两种药物联合处理的细胞未恢复。联合治疗上调了肿瘤抑制蛋白，如 p53、p21 和 p27，同时下调了增殖和存活蛋白，如细胞周期蛋白 D1 和 Bcl-2。这也与促凋亡蛋白 Bax 的上调和联合治疗中组蛋白乙酰化的显著积累有关。有趣的是，与单独使用相比，VPA 与 P276-00 联合使用在 SCID 小鼠的 H460 异种移植肿瘤模型中作为抗肿瘤药物更为有效。
本研究表明，HDAC 抑制剂 VPA 与 CDK 抑制剂 P276-00 的联合应用是治疗 NSCLC 的有前途的新型分子靶向治疗方法。
