Dynamic proteomic overview of glioblastoma cells (A172) exposed to perillyl alcohol

Abstract

Perillyl alcohol (POH) is a naturally occurring terpene and a promising chemotherapeutic agent for glioblastoma multiform; yet, little is known about its molecular effects. Here we present results of a semi-quantitative proteomic analysis of A172 cells exposed to POH for different time-periods (1', 10', 30', 60', 4h, and 24h). The analysis identified more than 4000 proteins; which were clustered using PatternLab for proteomics and then linked to Ras signaling, tissue homeostasis, induction of apoptosis, metallopeptidase activity, and ubiquitin-protein ligase activity. Our results make available one of the most complete protein repositories for the A172. Moreover, we detected the phosphorylation of GSK3beta (Glycogen synthase kinase) and the inhibition of ERK's (extracellular signal regulated kinase) phosphorylation after 10', which suggests a new mechanism of POH's activation for apoptosis.

Figure 1

Graphical user interfaces for the new PatternLab modules A) The TrendQuest graphical user interface provides several quality filter and normalization options. The clustering result is displayed in the right panel. B) The XFold graphical user interface. On the left, the user can specify several parameters such as the normalization strategy and stringency parameters (e.g., minimum fold change and p-value). On the right side are the results in the form of a table showing groups of proteins (rows) that were differentially expressed for roughly the same set of different conditions. C) The AAPVD module’s graphical user interface. The number of unique proteins for each MudPIT run is shown for three biological states in an AAPVD. Clicking on the diagram’s annotations within the circles lists the proteins and further details such as the quantitation information for each experiment.

Figure 2

AAPVD of the identified proteins present in at least two replicates for each state. The labels T0, T1, and T24 stand for A172 cells not exposed to POH, exposed to 1.8 mM of POH for 1 hour, exposed to 1.8 mM of POH for 24 hours, respectively.

Figure 3

The three major trends obtained from clustering the normalized expression profiles of A172 membrane-enriched fractions exposed to 1.8 mM of POH are displayed in Panels A, B, and C.

Figure 4

The upper panel shows the nitrocellulose membrane stained with Ponceau red. Lanes 1–7 represent the exposure times of 0′, 1′, 10′, 30′, 60′, 4h, and 24h, respectively. The lower panel shows the western blot for the HSP70 antibody.

Figure 5

A172 enriched-membrane fraction were incubated with 1.8 mM of POH during 1′, 10′, 30′, 60′, 4h, and 24h, resolved by SDS-PAGE, and transferred to a nitrocellulose membrane. The membrane was probed with p-ERK, ERK, β-catenin, p-GSK-3β, and GSK-3β as described in Materials and Methods. This procedure was repeated twice.