Growth inhibition of malignant glioblastoma by DING protein

Abstract

Malignant gliomas are a highly aggressive type of brain tumor with extremely poor prognosis. These tumors are highly invasive and are often surgically incurable and resistant to chemotherapeutics and radiotherapy. Thus, novel therapies that target pathways involved in growth and survival of the tumor cells are required for the treatment of this class of brain tumors. Previous studies revealed that epidermal growth factor receptor and extracellular-signal-regulated kinases (ERKs), which are involved in the induction of cell proliferation, are activated in the most aggressive type of glioma, i.e. glioblastoma multiforme (GBM). In fact, GBMs with increased levels of ERK activity exhibit a more aggressive phenotype than the others with moderate ERK activity, pointing to the importance of ERK and its kinase activity in the development and progression of these tumors. In this study, we have evaluated the effect of p38SJ, a novel member of the DING family of proteins, derived from Hypericum perforatum calluses, on the growth of malignant glioma cell lines, T98G and U-87MG by focusing on cell cycle and signaling pathways controlled by phosphorylation of various regulatory proteins including ERK. p38SJ, which exhibits profound phosphatase activity, shows the capacity to affect the phosphorylation status of several important kinases modulating signaling pathways, and cell growth and proliferation. Our results demonstrate that p38SJ reduces glioma cell viability and arrests cell cycle progression at G0/G1. The observed growth inhibitory effect of p38SJ is likely mediated by the downregulation of several cell cycle gatekeeper proteins, including cyclin E, Cdc2, and E2F-1. These results suggest that p38SJ may serve as a potential candidate for development of a therapeutic agent for the direct treatment of malignant gliomas and/or as a potential radiosensitizer.

Fig. 1

p38SJ inhibits cell proliferation. a Cell viability assay using T98G glioma cells transfected with various plasmids expressing p38SJ or their control empty vectors. The quantitative representation of cell viability assay is displayed after 5 days. b Colony formation assay showing representative images of T98G glioma cells with and without p38SJ-expressing plasmids (top panel). The quantitative representation of colony assays for T98G and U-87MG glioma cells is displayed (bottom panel). The number of colonies that survived in the presence of blasticidin was determined after 3 weeks as described in materials and methods. The average number of colonies from three independent experiments were determined and arbitrarily set at 100% for the control

Fig. 2

N-terminal region of p38SJ is important for the inhibition of cell proliferation. a Schematic presentation of p38SJ protein and its N-terminal deletion mutants highlighting the various regions of the protein with potential phosphate binding sites spanning between amino acids 8–149 as depicted by P (9–11), P (34), P (64), and P (143,148,149). The position of DING spanning amino acids 1–8 is shown. b. Colony formation assay using U-87MG cells and various deletion mutants of p38SJ. The percent inhibition of colony formation caused upon expression of p38SJ or its mutant variants is shown

Fig. 3

Effect of exogenously added p38SJ on glioma cell viability. a Coomassie blue stained gel illustrating purified p38SJ before and after treatment with proteinase K (PK). M indicates size marker proteins. b MTT assay illustrating percent of affected U-87MG or T98G cells after treatment with p38SJ or p38SJ plus PK

Fig. 4

Effect of p38SJ on various proteins associated with the cell. Western blot analysis of extracts from T98G cells after treatment with p38SJ (Panels a and b) or U-87MG cells after transfection with YFP-p38SJ (Panel c)

Fig. 5

Effect of p38SJ on cell cycle progression. a FACS cell cycle analysis of NIH 3T3 cells treated with p38SJ extract demonstrates a delay in entry between G0/G1 and S phases. The labeled bars at the bases of the graphs indicate the relative stage of cell cycle arrest for p38SJ treated cells compared to control cells treated with p38SJ + PK. b FACS cell cycle analysis of U87MG cells treated with p38SJ extract demonstrates a slow progression of cells from G0/G1 to S phase

Fig. 6

Effect of p38SJ on MAPK signaling. a Phospho-MAPK arrays treated with transfected glioma cell lysates. The top panel displays results from protein extracts from T98G cells expressing only YFP, and the bottom panel displays results from T98G cells expressing YFP-p38SJ. b Quantitative representation of phospho-MAPK protein levels in p38SJ-expressing T98G cells as determined by densitometric analysis of each spot. The values were normalized to the levels seen in YFP expressing cells. c Western blot analysis of extracts from YFP-p38SJ-expressing T98G cells for NF-κB p65 and phospho-c-Jun, c-Jun, YFP-p38SJ and the housekeeping protein, Grb2. d Quantification of the differences in NF-κB levels as determined by densitometric analysis of the band corresponding to NF-κB p65 that was normalized to the level of Grb2 is shown. e Quantification of the differences in phospho-c-Jun after being normalized to the level of total c-Jun is shown. Grb2 served as a loading control