Department of Clinical Chemistry and Molecular Diagnostics, Poznan University of Medical Sciences, Przybyszewskiego 49 St., 60-355, Poznan, Poland.
Biocenter, Division of Medical Biochemistry, Innsbruck Medical University, Innrain 80-82, A-6020, Innsbruck, Austria.
BMC Cancer. 2018 Feb 13;18(1):185. doi: 10.1186/s12885-018-4095-1.
Several efforts have been focused on identification of pathways involved in malignancy, progression, and response to treatment in Glioblastoma (GB). Overexpression of PKCε was detected in histological samples from GB, anaplastic astrocytoma, and gliosarcoma and is considered an important marker of negative disease outcome. In multiple studies on GB, autophagy has been shown as a survival mechanism during cellular stress, contributing to resistance against anti-cancer agents. The main object of this research was to determine the influence of PKCε downregulation on the expression of genes involved in autophagy pathways in glioblastoma cell lines U-138 MG and U-118 MG with high PKCε level.
We conducted siRNA-mediated knockdown of PKCε in glioblastoma cell lines and studied the effects of autophagy pathway. The expression of autophagy-related genes was analyzed using qPCR and Western blot analysis was carried out to assess protein levels. Immunostaining was used to detect functional autophagic maturation process.
We found that these cell lines exhibited a high basal expression of autophagy-related genes. Our results suggest that the loss of PKCε contributes to the downregulation of genes involved in autophagy pathways. Moreover, most of the changes we observed in Western blot analysis and endogenous immunofluorescence experiments confirmed dysfunction of autophagy programs. We found that knockdown of PKCε induced a decrease in the expression of Beclin1, Atg5, PI3K, whereas the expression of other autophagy-related proteins mTOR and Bcl2 was increased. Treatment of control siRNA glioma cells with rapamycin-induced autophagosome formation and increase in LC3-II level and caused a decrease in the expression of p62. Additionally, PKCε siRNA caused a diminution in the Akt phosphorylation at Ser473 and in the protein level in both cell lines. Moreover, we observed reduction in the adhesion of glioblastoma cells, accompanied by the decrease in total FAK protein level and phosphorylation.
Effects of down-regulation of PKCε in glioma cells raised the possibility that the expression of PKCε is essential for the autophagic signal transduction pathways in these cells. Thus, our results identify an important role of PKCε in autophagy and may, more importantly, identifyit as a novel therapeutic target.
多项研究致力于鉴定胶质母细胞瘤（GB）中的恶性肿瘤、进展和治疗反应相关通路。在 GB、间变性星形细胞瘤和胶质肉瘤的组织样本中检测到 PKCε 的过表达，其被认为是疾病预后不良的重要标志物。在对 GB 的多项研究中，自噬已被证明是细胞应激过程中的一种生存机制，有助于抵抗抗癌药物。本研究的主要目的是确定 PKCε 下调对高 PKCε 水平的 U-138 MG 和 U-118 MG 胶质母细胞瘤细胞系中自噬通路相关基因表达的影响。
我们采用 siRNA 介导的 PKCε 下调，研究自噬通路的影响。采用 qPCR 分析自噬相关基因的表达，采用 Western blot 分析评估蛋白水平。采用免疫组化检测功能自噬成熟过程。
我们发现这些细胞系表现出自噬相关基因的高基础表达。我们的结果表明，PKCε 的缺失导致自噬通路相关基因下调。此外，Western blot 分析和内源性免疫荧光实验观察到的大多数变化证实自噬程序功能障碍。我们发现，PKCε 下调诱导 Beclin1、Atg5、PI3K 表达降低，而 mTOR 和 Bcl2 等其他自噬相关蛋白的表达增加。雷帕霉素处理对照 siRNA 胶质母细胞瘤细胞诱导自噬体形成，LC3-II 水平增加，导致 p62 表达降低。此外，PKCε siRNA 导致两种细胞系中 Akt 的 Ser473 磷酸化和蛋白水平降低。此外，我们观察到胶质母细胞瘤细胞黏附减少，总 FAK 蛋白水平和磷酸化降低。
胶质母细胞瘤细胞中 PKCε 下调的影响表明 PKCε 的表达对于这些细胞中自噬信号转导通路是必需的。因此，我们的结果确定了 PKCε 在自噬中的重要作用，更重要的是，将其确定为一个新的治疗靶点。
