P03.05 * THE HIV-DERIVED PROTEIN VPR(52-96) HAS PRO-APOPTOTIC AND ANTIPROLIFERATIVE ACTIVITY IN GLIOBLASTOMA CELL LINES IN VITRO

Abstract

BACKGROUND AND PURPOSE: Patients with actively replicating human immunodeficiency virus (HIV) are highly radiosensitive and exhibit adverse reactions even at low irradiation doses. One of the major underlying causes are high intra- and extracellular levels of a virus-encoded peptide termed viral protein R (Vpr). As Vpr efficiently crosses the blood-brain barrier and accumulates in astrocytes, we examined whether its active subunit (Vpr52-96) may be utilized as drug in the treatment of glioblastoma multiforme. MATERIAL AND METHODS: Four glioblastoma-derived cell lines with and without lentivirus-mediated MGMT-overexpression (U251, U87MG, U251-MGMT, U87-MGMT) were exposed to Vpr52-96, 100 µM of temozolomide (TMZ), conventional photon irradiation (2-6 Gy) or to combinations of the three. Acute toxicities and LD50 were examined using the MTT proliferation assay. Apoptosis was detected using a luminescent reaction measuring caspase-3 and -7 activities (Caspase Glo®). Clonogenic survival (survival fraction, SF) was determined using the colony-forming assay. RESULTS: Treatment of U251 cells with 1, 10 and 20 µM of medium-dissolved Vpr52-96 yielded cell viability rates of 82.4 ± 3.2%, 32.6 ± 9.1% and 10.5 ± 4.8% respectively, corresponding to a LD50 of 4.0 ± 1.1 µM. For U87MG, the corresponding viability rates were 89.3 ± 4.4%, 61.2 ± 12.4% and 43.6 ± 17.8%, resulting in an LD50 of 15.7 ± 7.5 µM. Combined treatment (Vpr + RT and/or TMZ) showed additive, but not synergistic effects in MTT assays. Vpr induced apoptosis in all cell lines within 6-12 hours in a dose-dependent fashion. Clonogenic assays also revealed an additive effect but no sensitization towards irradiation. Vpr alone efficiently inhibited clonogenic survival (SF at 10 µM Vpr: 0.24 ± 0.06 for U251 and 0.35 ± 0.09 for U87MG). In contrast to TMZ, it acts independently from the MGMT expression status (SF of U251-MGMT at 10 µM Vpr: 0.2 vs. 0.9 at 100 µM TMZ; SF of U87-MGMT at 10 µM Vpr: 0.3 vs. 0.7 at 100 µM TMZ). SUMMARY: Vpr52-96 exhibits all traits of a chemotherapeutic drug that may be employed for the treatment of glioblastoma multiforme. The protein acts additively to current standard therapies and shows undiminished antiproliferative activity in MGMT-overexpressing cells.