Koul et al. BMC Cancer 2010, 10:328 http://www.biomedcentral.com/1471-2407/10/328 Materials and methods Cell culture and treatment Glioblastoma cell lines A172, LN229, T98G and U87MG were obtained from American Type Culture Collection and cultured in DMEM supplemented with 10% fetal bovine serum. Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll/Histopaque density gra- dient centrifugation. Adherent monocytes were purified from PBMC following adherence on glass petri-dish for three hours after flushing the non-adherent cells by extensive washing with PBS. All experiments with human PBMC were conducted under an approved insti- tutional Human Ethics Committee protocol. On attaining semi-confluence, cells were switched to serum free media and after 6 hours, cells were treated with different concentration of Iripallidal (in Dimethyl sulphoxide, DMSO) in serum free media for 24 hours. DMSO treated cells were used as controls. Iripallidal was purchased from Calbiochem, USA. All reagents were purchased from Sigma unless otherwise stated. Colon cancer cell line HT29, breast cancer line MCF-7, cervical cancer cell line HeLa, hepatocellular carcinoma cell line HepG2, acute myeloid leukemic cell line THP1 and human monocytes were similarly treated with Iripallidal. Determination of cell viability Viability of Iripallidal treated monocytes and cancer cell lines was assessed using the [3-(4,5-dimethylthiazol-2- yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)- 2H- tetrazolium, inner salt] (MTS) (Promega) as described earlier [17]. Assay of Caspase 3 activity The Colorimetric Assay kits for caspase 3 (Sigma) were used to determine its enzymatic activity in Iripallidal treated glioma cells as described previously [18]. Western Blot Analysis Protein from whole cell lysates were isolated as described previously [19]. Protein (20-50 μg) isolated from control and Iripallidal treated cells was electrophoresed on 6% to 10% polyacrylamide gel and Western blotting performed as described [19]. Antibodies were purchased from Cell Signaling Technology (Danvers, MA) unless otherwise mentioned. The following antibodies were used: p21 (BD Biosciences), p27 (Abcam), pSTAT3 (Tyr705), pmTOR (Ser2448), mTOR, Akt, pAkt (Ser473), Cyclin D1 (Abcam), phospho-p70S6K (Thr389), cMyc (Santa Cruz), phospho-S6K (Ser235/236), pH2AX Ser139 (Upstate), cleaved-PARP and b actin. Secondary antibodies were purchased from Vector Laboratories. After addition of chemiluminescence reagent (Amersham), blots were exposed to Chemigenius, Bioimaging System (Syngene, Page 2 of 8 UK) for developing and images were captured using Gen- esnap software (Syngene). The blots were stripped and reprobed with anti-b-actin to determine equivalent load- ing as described [19]. TeloTAGGG Telomerase PCR ELISA PLUS Telomerase activity was determined using the Telo- TAGGG Telomerase PCR ELISA PLUS kit (Roche, Germany) as described previously [18]. Colony formation in soft agar The soft agar colony formation assay was performed using CytoSelect™ 96-Well Cell Transformation Assay kit (Cell Biolabs, Inc), as described previously [20]. Statistical Analysis All comparisons between groups were performed using two-tailed Paired student’s t-Test. All values of p less than 0.05 were taken as significant. Results Iripallidal decreases viability and induces apoptosis in glioma cells To determine whether Iripallidal affects viability of glioma cells, MTS assay was performed on A172, LN229, T98G and U87MG glioma cells treated with dif- ferent concentrations of Iripallidal for 24 hours. While no significant cell death was observed in cells treated with 10 μM Iripallidal, a ~50% decrease in cell viability was observed in all the glioma cell lines tested upon treatment with 20 μM Iripallidal (Fig. 1a). Since the acti- vation of caspase-3-like proteases is crucial in apoptotic cell death [21], we determined the caspase-3 activity in Iripallidal treated glioma cells. Decrease in viability was accompanied by a significant ~2.5 to 3-fold increase in caspase-3 activity in all the cell lines, as compared to control (Fig. 1b). As Caspase-3 activity was elevated in Iripallidal treated cells, we determined the expression of PARP in these cells. Treatment with Iripallidal elevated the level of cleaved PARP as compared to control, in all glioma cells tested (Fig. 1c). Increase in caspase-3 activa- tion and cleaved-PARP level was indicative of apoptosis induction by Iripallidal. These results suggest that Iripal- lidal induce apoptosis in glioma cells. Iripallidal inhibits Akt/mTOR signaling in glioblastoma cells As aberrant activation of the PI3K/Akt occurs frequently in glioblastomas [22], therapeutics approaches are direc- ted towards targeting this pathway. Treatment with Iri- pallidal decreased Akt phosphorylation in glioma cells (Fig. 2). As inhibition of PI3 kinase p110a blocks Akt phosphorylation in glioma cells [23], we investigated