Koul et al. BMC Cancer 2010, 10:328 http://www.biomedcentral.com/1471-2407/10/328 whether this decrease in pAkt was the consequence of reduced p110a levels. Iripallidal had no effect on p110a levels (Fig. 2). As Iripallidal inhibited pAkt, we investi- gated its effect on Akt downstream target mTOR. Iripal- lidal downregulated phospho-mTOR in glioma cells (Fig. 2). mTOR activation results in phosphorylation of effector molecule p70S6K and S6 ribosomal protein, which sub- sequently leads to mTOR-dependent gene transcription that regulates cell growth, protein synthesis, and meta- bolism. We therefore determined the effect of Iripallidal on the status of p70S6K and pS6 kinase. Iripallidal inhibited phosphorylation of mTOR targets 70S6K and ribosomal protein S6 (Fig. 2). These results indicate that iripallidal acts as a dual inhibitor of Akt/mTOR pathway. Iripallidal downregulates STAT3 phosphorylation in glioma cells As mTOR inhibitor blocks STAT activation and glial differentiation [24] and since STAT3 inhibitors induce apoptosis in glioma cells [12], we determined the status of STAT3 activation in Iripallidal treated cells. A decrease in pSTAT3 Tyr705 was observed upon Iripalli- dal treatment (Fig. 3). These results indicate that Iripalli- dal inhibits STAT3 activation in glioma cells. Iripallidal affects expression of molecules involved in cell cycle regulation and DNA damage response Inhibition of PI3-K/Akt/mTOR signaling effects cell cycle progression [23,25]. mTOR inhibitors induce cell cycle arrest through down regulation of Cyclin D and upregulation of p27 [8]. Since Iripallidal inhibited glioma cell proliferation, we determined the expression of mole- cules associated with cell cycle progression. An increase in p21 and p27, and decrease in cyclin D1 and cMyc levels was observed in glioma cells upon Iripallidal treat- ment (Fig. 4a). As maintained DNA breaks induce apoptosis [26] and since H2AX is phosphorylated at sites of DNA double- Page 4 of 8 strand breaks [27], we determined the expression of g-H2AX in Iripallidal treated cells. While an increased g-H2AX expression was observed in Iripallidal treated cells (Fig. 4b), the levels of total H2AX was unaffected (Fig. 4b). Iripallidal suppresses telomerase activity in glioma cells Inhibition of telomerase activity is an important antic- ancer modality since its inhibition causes apoptosis in human cancers [28]. Telomerase activity is regulated by Ras/PI3K/Akt pathway [29] and mTOR inhibitor rapamycin inhibits telomerase activity in endometrial cancer cells [30]. Besides, STAT3 regulates human tel- omerase reverse transcriptase (hTERT) expression in human cancer and primary cells [31]. Also, we have shown that inhibition of telomerase activity is asso- ciated with decrease glioma cell proliferation [18,20]. Since Iripallidal inhibits mTOR and STAT3 activation in glioma cells we investigated its ability to regulate telomerase activity. An approximate 50% reduction in telomerase activity was observed in glioma cells upon treatment with 20 μM Iripallidal (Fig. 5a). Telomerase inhibitors are known to reduce colony formation in soft agar assays [32] and STAT3 is essential for ancho- rage-independent growth of transformed cells [33]. Since Iripallidal decreased glioma cell survival we determined the ability of Iripallidal to effect the ancho- rage independent growth of glioma cells. Treatment with 20 μM Iripallidal reduced colony forming ability of glioma cells in soft agar by ~40%, as compared to control (Fig. 5b). Iripallidal inhibits proliferation of non-glioma cancer cells of diverse origin in vitro We next evaluated whether Iripallidal also exhibits anti- proliferative property against other human malignancies, by testing its effects against a panel of non-glioma human cancer cell lines in vitro. Treatment with 20 μM Iripallidal reduced viability of MCF-7, HeLa, HepG2, THP1 and HT-29 cells lines by ~35% to 60%, as Figure 3 Treatment with Iripallidal decreases STAT3 phosphorylation in glioma cells. Western blotting analysis demonstrates a decrease in pSTAT3 levels in A172, LN229, T98G and U87MG glioma cells upon Iripallidal treatment. Representative blot is shown from three independent experiments with identical results. C and I denote control and Iripallidal, respectively. Blots were reprobed for b-actin to establish equivalent loading.