Department of Comparative Anatomy and Anthropology, Maria Curie-Sklodowska University, Lublin, Poland.
Department of Human Physiology, University of Rzeszow, Rzeszów, Poland.
Pharmacol Rep. 2017 Aug;69(4):779-787. doi: 10.1016/j.pharep.2017.03.008. Epub 2017 Mar 14.
Gliomas are aggressive brain tumors with very high resistance to chemotherapy. Therefore, the aim of the present study was to investigate the effectiveness of sorafenib and Temozolomide in elimination of human glioma cells through apoptosis and autophagy.
MOGGCCM (anaplastic astrocytoma) and T98G (glioblastoma multiforme) cell lines incubated with sorafenib and/or Temozolomide were used in the experiments. Cell morphology (ER stress, apoptosis, autophagy, and necrosis) was analyzed microscopically while apoptosis and mitochondrial membrane potential were assessed with flow cytometry. Beclin1, LC3, p62, Hsp27, and Hsp72 levels were analyzed by immunoblotting. The activity of caspase 3, 8, and 9 was evaluated fluorometrically. Expression of Hsps was blocked by transfection with specific siRNA.
In MOGGCCM cells, Temozolomide most frequently induced autophagy, which was accompanied by decreased p62 and increased beclin1 and LC3II levels. Sorafenib initiated mainly apoptosis. Additional incubation with Temozolomide, synergistically potentiated the pro-apoptotic properties of sorafenib, but it was mediated in a caspase-independent way. In T98G cells, the effect of the analyzed drugs on programmed cell death induction was different from that in MOGGCCM cells. Sorafenib induced autophagy, while Temozolomide initiated mainly apoptosis. After simultaneous drug application, apoptosis dominated, suggesting synergistic action of both drugs. Inhibition of Hsp27 and Hsp72 expression increased the sensitivity of both cell lines to ER stress and, to a lesser extent, to induction of apoptosis, but not autophagy.
Sorafenib and Temozolomide applied in combination are potent apoptosis inducers in T98G and MOGGCCM cells. ER stress precedes the elimination. Blocking of Hsp expression has a greater impact on ER stress rather than apoptosis induction.
神经胶质瘤是一种侵袭性脑肿瘤，对化疗具有很高的耐药性。因此，本研究旨在探讨索拉非尼和替莫唑胺通过凋亡和自噬消除人神经胶质瘤细胞的效果。
本实验使用索拉非尼和/或替莫唑胺孵育的 MOGGCCM（间变性星形细胞瘤）和 T98G（多形性胶质母细胞瘤）细胞系。通过显微镜分析细胞形态（内质网应激、凋亡、自噬和坏死），通过流式细胞术评估凋亡和线粒体膜电位。通过免疫印迹分析 Beclin1、LC3、p62、Hsp27 和 Hsp72 水平。通过荧光法评估 caspase 3、8 和 9 的活性。通过特异性 siRNA 转染阻断 HSP 的表达。
在 MOGGCCM 细胞中，替莫唑胺最常诱导自噬，这伴随着 p62 的减少和 Beclin1 和 LC3II 水平的增加。索拉非尼主要引发凋亡。与替莫唑胺联合孵育，协同增强了索拉非尼的促凋亡作用，但这种作用是 caspase 非依赖性的。在 T98G 细胞中，分析药物对程序性细胞死亡诱导的作用与 MOGGCCM 细胞不同。索拉非尼诱导自噬，而替莫唑胺主要诱导凋亡。在联合用药后，凋亡占主导地位，提示两种药物具有协同作用。抑制 Hsp27 和 Hsp72 的表达增加了两种细胞系对内质网应激的敏感性，并且在较小程度上增加了对凋亡的敏感性，但对自噬没有影响。
索拉非尼和替莫唑胺联合应用是 T98G 和 MOGGCCM 细胞中有效的凋亡诱导剂。内质网应激先于消除。抑制 HSP 表达对内质网应激的影响大于诱导凋亡。
