Introduction 1 2 3 4 5 6 7 8 9 10 11 12 13 15 16 17 We therefore hypothesized that the combination of TRAIL and lovastatin, neither of which alone has noxious effects on healthy cells, could generate a regime that was effective in killing cancer cells but caused minimal insult to normal healthy cells. In this study we report the effects of TRAIL in combination with a non-chemotherapeutic drug, lovastatin, on glioblastoma cells. Materials and methods Reagents 18 Cell culture 2 Measurement of cell viability 4 Cell cycle analysis 6 Apoptosis assay Apoptotic cells were determined by two methods, Annexin-V and PI stained cells by flow cytometry and DNA fragmentation assay. During apoptosis, translocation of phosphatidylserine from inner membrane to outer membrane is a common phenomenon. Cells were stained with Annexin V for analysis of phosphoserine inversion, which was considered to be a sensitive marker of apoptosis. Using an Annexin V-FITC apoptosis detection kit (Molecular Probe Inc, Eugene, OR), the levels of binding of Annexin V and staining with PI were measured for the detection of early and late apoptosis respectively. All of the procedures were preformed under manufacturer’s guidelines. Cells were treated with lovastatin or/and TRAIL for 48 h and then stained with Annexin-V and PI. Viable cells were recognized as negative for both Annexin-V and PI; early apoptotic events were positive for Annexin-V but negative for PI staining. Late apoptotic events were positive to both Annexin V and PI. Necrotic cells were positive for PI staining only. 4 RT-PCR Total RNA was isolated using Qiagen RNeasy extraction kit and performed according to the manufacturer’s protocol. Total RNA (5 μg) was reversely transcribed using Promega RT-PCR kit and thermal program was set at 42°C for 15 min and 95°C for 5 min. PCR reaction was performed using the following primers, which have previously been tested successfully: TRAIL-R1, 5′-CTG AGC AAC GCA GAC TCG CTG TCC AC-3′ and 5′-TCC AAG GAC ACG GCA GAG CCT GTG CCA T-3′; TRAIL-R2, 5′-GCC TCA TGG ACA ATG AGA TAA AGG TGG CT-3′ and 5′-CCA AAT CTC AAA GTA CGC ACA AAC GG-3′; TRAIL-R3, 5′-GAA GAA TTT GGT GCC AAT GCC ACT G-3′ and 5′-CTC TTG GAC TTG GCT GGG AGA TGT G-3′; TRAIL-R4, 5′-CTT TTC CGG CGG CGT TCA TGT CCT TC-3′ and 5′-GTT TCT TCC AGG CTG CTT CCC TTT GTA G-3′. The thermal program was set up as one cycle at 94°C for 5 min, 30 cycles at 94°C for 1 min, 55°C for 1 min, 72°C for 2 min, and one cycle at 72°C for 5 min. PCR products were resolved and visualized on a 2% agarose gel stained with ethidium bromide. Western blot analysis 19 20 6 Statistics t P Results Cell viability measured by MTT assay 1 1 Fig. 1 a b  2 2 2 Fig. 2 a b c P P  Cell cycle determination by PI staining 3 Fig. 3 0 1 0 1 0 1 P P  Apoptosis is the major mode of cell death 4 4 P 4 P 4 21 Fig. 4 a b c P P + P ++ P  DNA fragmentation in glioblastoma cells 5 Fig. 5 P P + P ++ P  The expression of TRAIL receptors in glioblastoma cells 5 6 Fig. 6 a b  P P 7 Fig. 7 a b c d a c) b d P P  Discussion 2 22 23 24 27 5 0 1 28 0 1 29 30 31 32 32 0 1 5 33 34 30 32 17 33 5 33 35 11 12 19 20 This study demonstrated a synergistic interaction between lovastatin and TRAIL, but the mechanisms of action by which lovastatin sensitized glioblastoma cells remains unknown. Our results are in agreement with the concept of combined cancer therapeutic action via both intrinsic and extrinsic apoptotic cell death pathways. This combination of non-chemotherapeutic agents, TRAIL and lovastatin, may offer a potential regime for glioblastoma treatment.