Bicyclic triterpenoid Iripallidal induces apoptosis and inhibits Akt/mTOR pathway in glioma cells

Background The highly resistant nature of glioblastoma multiforme (GBM) to chemotherapy prompted us to evaluate the efficacy of bicyclic triterpenoid Iripallidal against GBM in vitro. Methods The effect of Iripallidal on proliferation and apoptosis in glioma cell lines was evaluated by MTS, colony formation and caspase-3 activity. The effect of iripallidal to regulate (i) Akt/mTOR and STAT3 signaling (ii) molecules associated with cell cycle and DNA damage was evaluated by Western blot analysis. The effect of Iripallidal on telomerase activity was also determined. Results Iripallidal (i) induced apoptosis, (ii) inhibited Akt/mTOR and STAT3 signaling, (iii) altered molecules associated with cell cycle and DNA damage, (iv) inhibited telomerase activity and colony forming efficiency of glioma cells. In addition, Iripallidal displayed anti-proliferative activity against non-glioma cancer cell lines of diverse origin. Conclusion The ability of Iripallidal to serve as a dual-inhibitor of Akt/mTOR and STAT3 signaling warrants further investigation into its role as a therapeutic strategy against GBM.

Despite recent advances in understanding molecular mechanisms involved in GBM progression, the prognosis of the most malignant brain tumor continues to be dismal. Ras activation occurs in GBMs [4] and this high level of active Ras has been a target for glioma therapy. RasGRP3-is an exchange factor that catalyzes the formation of the active GTP-bound form of Ras-like small GTPases [5]. Importantly, Ras activation stimulates its downstream effector Akt that plays a major role in glioblastoma development as~80% of GBM cases express high Akt levels [6]. Akt activates mammalian target of rapamycin (mTOR), which is deregulated in glioblastoma [7]. mTOR phosphorylates p70 ribosomal S6 kinase (p70S6 kinase) that regulates translation of proteins involved in cellular proliferation and formation. Moreover, blocking mTOR signaling reduces glioma cell proliferation [8]. Given the importance of Akt/mTOR signaling in glioma cell survival, significant efforts are being invested in identifying inhibitors that target this pathway [8][9][10]. In addition to aberrant PI3K/Akt signaling; heightened STAT3 activation plays a critical role in glioblastoma and STAT3 inhibitors have shown promise as therapeutics for GBM [11][12][13]. In addition to RasGRP3 Iripallidal also binds to PKCα [2] which is known to induce cells ectopically expressing hyperactive Ras to undergo apoptosis [14]. Not only is STAT3 essential for Ras transformation [15] but constitutively activated STAT3 is negatively regulated by PKC-activated tyrosine phosphatase(s) [16]. As Iridals interacts with PKCα and RasGRP3-molecules that regulate Akt and STAT3 signaling, and since inhibition of Akt/mTOR and STAT3 signaling are being targeted for GBM treatment we evaluated the effect of Iripallidal on glioma cell proliferation and these signaling pathways.

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 gradient 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 institutional 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.

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].
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.

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 different 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 activation 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 activation and cleaved-PARP level was indicative of apoptosis induction by Iripallidal. These results suggest that Iripallidal 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 directed towards targeting this pathway. Treatment with Iripallidal decreased Akt phosphorylation in glioma cells (Fig. 2). As inhibition of PI3 kinase p110α blocks Akt phosphorylation in glioma cells [23], we investigated  whether this decrease in pAkt was the consequence of reduced p110α levels. Iripallidal had no effect on p110α levels (Fig. 2). As Iripallidal inhibited pAkt, we investigated its effect on Akt downstream target mTOR. Iripallidal downregulated phospho-mTOR in glioma cells (Fig. 2). mTOR activation results in phosphorylation of effector molecule p70S6K and S6 ribosomal protein, which subsequently leads to mTOR-dependent gene transcription that regulates cell growth, protein synthesis, and metabolism. 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 Iripallidal treatment (Fig. 3). These results indicate that Iripallidal 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 molecules 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 treatment (Fig. 4a).
As maintained DNA breaks induce apoptosis [26] and since H2AX is phosphorylated at sites of DNA double-strand breaks [27], we determined the expression of γ-H2AX in Iripallidal treated cells. While an increased γ-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 anticancer 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 telomerase reverse transcriptase (hTERT) expression in human cancer and primary cells [31]. Also, we have shown that inhibition of telomerase activity is associated 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 anchorage-independent growth of transformed cells [33]. Since Iripallidal decreased glioma cell survival we determined the ability of Iripallidal to effect the anchorage 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 antiproliferative 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 compared to their respective controls (Fig. 6a). These findings indicate that Iripallidal not only inhibits proliferation of GBM, but also exhibits anti-proliferative activity against a wide variety of human cancers. To show the selectivity of Iripallidal for tumor cells, the effect of Iripallidal was investigated on normal human monocytes. Treatment of monocytes with Iripallidal induced~8-10% decrease in viability, suggesting that the anti-proliferative ability of Iripallidal is selective for transformed cells (Fig. 6b).

Discussion
In vitro screening of compounds with anticancer properties by NCI identified Iridals for their anti-proliferative activity. Besides its ability to bind PKCα and RasGRP3 [2], nothing is known regarding the mechanism of action or bioavailability of Iripallidal. Our studies suggest that Iripallidal induce apoptosis in glioma cells and inhibits the Akt/ mTOR pathway. The efficacy of mTOR inhibitors in glioblastoma 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 insulin receptor substrate (IRS) by mTOR effector molecule S6 kinase [35,36], it was therefore not surprising that Rapamycin treatment induced Akt activation in some GBM patients in a Phase I clinical trial [9]. Moreover, dual inhibition of Akt and mTOR has proven effective in pre-clinical 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 inhibitor attenuates Ras activation and this attenuation correlates with an inhibition of RasGRP3 phosphorylation [38]. Interestingly, PKCα 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 signaling 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 viability of several other cancer cell types although to different 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-responsiveness 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 sensitivity 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 pathways suggest that its potential as a chemotherapeutic agent against GBM should be further evaluated. Importantly, 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