Gilardini Montani Maria Saveria, Granato Marisa, Santoni Claudio, Del Porto Paola, Merendino Nicolò, D'Orazi Gabriella, Faggioni Alberto, Cirone Mara
Department of Experimental Medicine, La Sapienza University of Rome, V.le Regina Elena 324, 00161, Rome, Italy.
Department of Ecological and Biological Sciences (DEB), Tuscia University, Viterbo, Italy.
Cell Oncol (Dordr). 2017 Apr;40(2):167-180. doi: 10.1007/s13402-017-0314-z. Epub 2017 Feb 3.
Histone deacetylase inhibitors (HDACi) are anti-neoplastic agents that are known to affect the growth of different cancer types, but their underlying mechanisms are still incompletely understood. Here, we compared the effects of two HDACi, i.e., Trichostatin A (TSA) and Valproic Acid (VPA), on the induction of cell death and autophagy in pancreatic cancer-derived cells that exhibit a high metastatic capacity and carry KRAS/p53 double mutations.
Cell viability and proliferation tests were carried out using Trypan blue dye exclusion, MTT and BrdU assays. FACS analyses were carried out to assess cell cycle progression, apoptosis, reactive oxygen species (ROS) production and mitochondrial depolarization, while Western blot and immunoprecipitation analyses were employed to detect proteins involved in apoptosis and autophagy.
We found that both VPA and TSA can induce apoptosis in Panc1 and PaCa44 pancreatic cancer-derived cells by triggering mitochondrial membrane depolarization, Cytochrome c release and Caspase 3 activation, although VPA was more effective than TSA, especially in Panc1 cells. As underlying molecular events, we found that ERK1/2 was de-phosphorylated and that the c-Myc and mutant p53 protein levels were reduced after VPA and, to a lesser extent, after TSA treatment. Up-regulation of p21 and Puma was also observed, concomitantly with mutant p53 degradation. In addition, we found that in both cell lines VPA increased the pro-apoptotic Bim level, reduced the anti-apoptotic Mcl-1 level and increased ROS production and autophagy, while TSA was able to induce these effects only in PaCA44 cells.
From our results we conclude that both VPA and TSA can induce pancreatic cancer cell apoptosis and autophagy. VPA appears have a stronger and broader cytotoxic effect than TSA and, thus, may represent a better choice for anti-pancreatic cancer therapy.
组蛋白去乙酰化酶抑制剂（HDACi）是一类抗肿瘤药物，已知其可影响不同类型癌症的生长，但其潜在机制仍未完全明确。在此，我们比较了两种HDACi，即曲古抑菌素A（TSA）和丙戊酸（VPA），对具有高转移能力且携带KRAS/p53双突变的胰腺癌衍生细胞中细胞死亡和自噬诱导的影响。
使用台盼蓝拒染法、MTT和BrdU检测进行细胞活力和增殖测试。进行流式细胞术分析以评估细胞周期进程、凋亡、活性氧（ROS）产生和线粒体去极化，同时采用蛋白质印迹和免疫沉淀分析来检测参与凋亡和自噬的蛋白质。
我们发现VPA和TSA均可通过触发线粒体膜去极化、细胞色素c释放和半胱天冬酶3激活，诱导Panc1和PaCa44胰腺癌衍生细胞凋亡，尽管VPA比TSA更有效，尤其是在Panc1细胞中。作为潜在的分子事件，我们发现VPA处理后细胞外信号调节激酶1/2（ERK1/2）去磷酸化，c-Myc和突变型p53蛋白水平降低，TSA处理后程度较轻。同时观察到p21和Puma上调，伴随突变型p53降解。此外，我们发现两种细胞系中VPA均增加促凋亡蛋白Bim水平，降低抗凋亡蛋白Mcl-1水平，并增加ROS产生和自噬，而TSA仅能在PaCA44细胞中诱导这些效应。
根据我们的结果，我们得出结论，VPA和TSA均可诱导胰腺癌细胞凋亡和自噬。VPA似乎比TSA具有更强、更广泛的细胞毒性作用，因此可能是抗胰腺癌治疗的更好选择。
