Chemo-resistant protein expression pattern of glioblastoma cells (A172) to perillyl alcohol

Abstract

Glioblastoma multiform (GBM) is by far the most malignant glioma. We have introduced a new treatment for GBMs that comprises the inhalation of a naturally occurring terpene with chemotherapeutic properties known as perillyl alcohol (POH). Clinical trial results on recurrent GBM patients showed that POH extends the average life by more than eight months, temporarily slows tumor growth, and in some cases even decreases tumor size. After approximately seven months, the tumor continues to grow and leads to a dismal prognosis. To investigate how these tumors become resistant to POH, we generated an A172 human glioblastoma cell culture tolerant to 0.06 mM of POH (A172r). We used Multidimensional Protein Identification Technology (MudPIT) to compare the protein expression profile of A172r cells to the established glioblastoma A172 cell line. Our results include a list of identified proteins unique to either the resistant or the nonresistant cell line. These proteins are related to cellular growth, negative apoptosis regulation, Ras pathway, and other key cellular functions that could be connected to the underlying mechanisms of resistance.

Figure 1

Magnetic resonance imaging (MRI) follow-up on a patient with recurrent primary glioblastoma multiform (GBM) in the lobar location under treatment with perillyl alcohol (POH) by intranasal delivery. A: MRI before starting the treatment with POH. B: MRI after 5 months of treatment with POH. C: MRI after 7 months of treatment with POH.

Figure 2

A TFold comparative plot between the A172r and the A172 cells. Only proteins found in all 6 MudPIT assays were considered for this analysis. Each protein (represented as a dot) is mapped according to its log₂ (fold change) on the ordinate axis and its -log₂ (t-test p-value) on the abscissa axis. The latter indicates how likely the observed fold change is a result of chance. Fold changes refer to the ratios of the average relative quantitation values obtained for each state. Accordingly, blue-dot proteins have p-value < 0.01 and an absolute fold change greater than 2.5, the established fold-change cutoff. Orange-dot proteins did not meet the fold-change cutoff but were indicated as statistically different. Green-dot proteins met the fold-change cutoff but cannot be claimed to be statistically different. Red dots did not satisfy the fold-change or the statistical cutoffs. The BH theoretical false-positive estimator (q < 0.05) indicates that all proteins selected as differentially expressed are likely to be truly differentially expressed. There are 57, 60, 72, and 1284 blue, orange, green, and red dots, respectively.

Figure 3

Western blot of HSP70: Slots 1 and 2 correspond to A172 and A172r, respectively. The western blot shows HSP70 to be over-expressed in A172r. Beta-tubulin, a protein not indicated as differentially expressed by the TFold, was used as an internal standard.

Figure 4

Approximately proportional Venn diagram of the identified proteins presents in all three replicates of each state: The dark green represents the proteins unique of the A172 cells; the yellow represents the proteins unique of the resistant cells (A172r). The number in parentheses represents the unique identified proteins in each condition. Only proteins present in all three replicates of a state were considered.