Koul et al. BMC Cancer 2010, 10:328 http://www.biomedcentral.com/1471-2407/10/328 Page 6 of 8 Figure 6 Effect of Iripallidal on the viability of non-glioma cancer cell lines and normal human monocytes. (a) Viability of Iripallidal treated non-glioma cancer cell lines and (b) normal human monocytes were determined by MTS assay. Graph represents the percentage viable MCF-7 (breast), HeLa (cervical), HepG2 (hepatocellular carcinoma), THP1 (acute myeloid leukemia), HT-29 (colon carcinoma) cells and monocytes treated with 10 and 20 μM concentration of Iripallidal for 24 hours, as determined by MTS assay. C denotes control. * Significant decrease from control (P < 0.05). Discussion In vitro screening of compounds with anticancer proper- ties by NCI identified Iridals for their anti-proliferative activity. Besides its ability to bind PKCa and RasGRP3 [2], nothing is known regarding the mechanism of action or bioavailability of Iripallidal. Our studies suggest that Iripal- lidal induce apoptosis in glioma cells and inhibits the Akt/ mTOR pathway. The efficacy of mTOR inhibitors in glio- blastoma cell lines [8,10] has prompted their clinical trials for GBM [9,34]. As rapamycin activates Akt pathway by a negative feedback loop involving phosphorylation of insu- lin receptor substrate (IRS) by mTOR effector molecule S6 kinase [35,36], it was therefore not surprising that Rapa- mycin treatment induced Akt activation in some GBM patients in a Phase I clinical trial [9]. Moreover, dual inhi- bition of Akt and mTOR has proven effective in pre-clini- cal model of GBM [23], suggesting that dual Akt/mTOR inhibitor can effectively overcome the effects of feeback loop efficiently than a single inhibitor selectively targeting mTOR. As mTOR blockade is a biomarker of therapeutic efficacy in glioma [37], the unique ability of Iripallidal to inhibit both Akt and mTOR can be exploited as novel anti-glioma therapy. In addition to inhibiting Akt/mTOR axis, Iripallidal also inhibited STAT3 signaling. PKC inhi- bitor attenuates Ras activation and this attenuation corre- lates with an inhibition of RasGRP3 phosphorylation [38]. Interestingly, PKCa regulates mTOR [37] as well as STAT3 activation [16]. It is possible that Iripallidal effects Akt/mTOR and STAT3 signaling pathways through its ability to bind PKC. Iripallidal mediated decrease in STAT3 activation was concurrent with decreased cyclin D1 and increased p21 expression. While cyclin D1 overexpression and STAT3 activation are mutually exclusive events [39], p21 inhibits STAT3 signaling [40]. Besides, inhibition of mTOR signal- ing induces cell cycle arrest through regulation of Cyclin D and p27 [8]. As telomerase inhibition is known to cause apoptosis in human cancers [28], the ability of Iripallidal to down-regulate telomerase activity may also represent a mechanism for its anti-proliferative effect on glioma cells. Besides glioma cell lines, Iripallidal also decreased the via- bility of several other cancer cell types although to differ- ent extents. It is known that cytotoxic responses is a reflection of an integrated readout of all targets and/or biochemical pathways affected upon drug exposure [41]. As strong co-relation exists between chemo-responsive- ness and gene expression [41], it is likely that differential expression of cellular pathways in cancer cell types of diverse origin could have resulted in differences in sensi- tivity to Iripallidal. Taken together our studies suggest that (i) Iripallidal induces glioma cell apoptosis and (ii) inhibits Akt/mTOR and STAT3 pathway. This ability of Iripallidal to act as a multi-inhibitor that blocks Akt/mTOR and STAT3 path- ways suggest that its potential as a chemotherapeutic agent against GBM should be further evaluated. Impor- tantly, Iripallidal is not only a promising candidate for the treatment of GBM but a wide variety of malignancies, since it elicits cell death in many tumor cell types. Conflict of Interest “Bicyclic triterpenoid Iripallidal as a novel anti-glioma and anti-neoplastic therapy in vitro” has been filed for Indian patent (#2915/DEL/2008) and International