Daniel R. Premkumar

Coadministration of Sorafenib with Rottlerin Potently Inhibits Cell Proliferation and Migration in Human Malignant Glioma Cells

Mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) are activated in the majority of gliomas and contribute to tumor cell growth and survival. Sorafenib (Bay43-9006; Nexavar) is a dual-action Raf and vascular endothelial growth factor receptor inhibitor that blocks receptor phosphorylation and MAPK-mediated signaling and inhibits growth in a number of tumor types. Because our initial studies of this agent in a series of glioma cell lines showed only partial growth inhibition at clinically achievable concentrations, we questioned whether inhibition of PKC signaling using the PKC-δ inhibitor rottlerin might potentiate therapeutic efficacy. Proliferation assays, apoptosis induction studies, and Western immunoblot analysis were conducted in cells treated with sorafenib and rottlerin as single agents or in combination. Sorafenib and rottlerin reduced proliferation in all cell lines when used as single agents, and the combination produced marked potentiation of growth inhibition. Flow-cytometric measurements of cells stained with Annexin V-propidium iodide and immunocytochemical assessment of cytochrome c and apoptosis-inducing factor release demonstrated that addition of rottlerin resulted in significantly higher levels of apoptosis than sorafenib alone. In addition, the combination of sorafenib and rottlerin reduced or completely inhibited the phosphorylation of extracellular signal-regulated kinase and Akt and down-regulated cell cycle regulatory proteins such as cyclin-D1, cyclin-D3, cyclin-dependent kinase (cdk)4, and cdk6 in a dose- and time-dependent manner. Our results clearly indicate that inhibition of PKC-δ signaling enhances the antiproliferative effect of sorafenib in malignant human glioma cell lines and support the examination of combinations of signaling inhibitors in these tumors.

Bortezomib Sensitizes Malignant Human Glioma Cells to TRAIL, Mediated by Inhibition of the NF-κB Signaling Pathway

Previous studies have shown that the tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) has significant apoptosis-inducing activity in some glioma cell lines, although many lines are either moderately or completely resistant, which has limited the therapeutic applicability of this agent. Because our recent studies showed that inhibition of proteasomal function may be independently active as an apoptosis-inducing stimulus in these tumors, we investigated the sensitivity of a panel of glioma cell lines (U87, T98G, U373, A172, LN18, LN229, LNZ308, and LNZ428) to TRAIL alone and in combination with the proteasome inhibitor bortezomib. Analysis of these cell lines revealed marked differences in their sensitivity to these treatments, with two (LNZ308 and U373) of the eight cell lines revealing no significant induction of cell death in response to TRAIL alone. No correlation was found between sensitivity of cells to TRAIL and expression of TRAIL receptors DR4, DR5, and decoy receptor DcR1, caspase 8, apoptosis inhibitory proteins XIAP, survivin, Mcl-1, Bcl-2, Bcl-Xl, and cFLIP. However, TRAIL-resistant cell lines exhibited a high level of basal NF-κB activity. Bortezomib was capable of potentiating TRAIL-induced apoptosis in TRAIL-resistant cells in a caspase-dependent fashion. Bortezomib abolished p65/NF-κB DNA-binding activity, supporting the hypothesis that inhibition of the NF-κB pathway is critical for the enhancement of TRAIL sensitization in glioma cells. Moreover, knockdown of p65/NF-κB by shRNA also enhanced TRAIL-induced apoptosis, indicating that p65/NF-κB may be important in mediating TRAIL sensitivity and the effect of bortezomib in promoting TRAIL sensitization and apoptosis induction. Mol Cancer Ther; 10(1); 198–208. ©2011 AACR.

Synergistic interaction between 17-AAG and phosphatidylinositol 3-kinase inhibition in human malignant glioma cells

The phosphatidylinositol 3′‐kinase (PI3K)/Akt pathway is often constitutively activated in malignant glioma cells, in many cases as a result of mutation of phosphatase and tensin homologue deleted on chromosome ten (PTEN), an endogenous inhibitor of Akt, which renders tumor cells resistant to cytotoxic insults, including those related to anticancer drugs. Pharmacological inhibition of this pathway may potentially restore or augment the effectiveness of conventional chemotherapy or other signaling‐targeted agents. Because the heat shock protein (HSP) is involved in the conformational maturation of a number of signaling proteins critical to the proliferation of malignant glioma cells, we hypothesized that the combination of the PI3K inhibitor LY294002 and the HSP90 inhibitor 17‐allyl‐aminogeldanamycin (17‐AAG) would promote glioma cytotoxicity by decreasing both the activation status and levels of Akt, as well as downregulating the levels of other relevant signaling effectors. We, therefore, examined the effects of LY294002 and 17‐AAG, alone and in combination, on signal transduction and apoptosis in a series of malignant glioma cell lines. Simultaneous exposure to these inhibitors significantly induced cell death, and irreversibly inhibited proliferative activity and colony forming ability of the glioma cell lines. Quantitative analysis revealed that enhancement by LY294002 of 17‐AAG‐induced cytotoxicity was synergistic, leading to a pronounced increase in active caspase‐3 and poly (adenosine diphophate‐ribose) polymerase (PARP) cleavage together with the release of cytochrome c and apoptosis inducing factor (AIF). No significant growth inhibition or caspase activation was seen in control cells. The enhanced cytotoxicity of this combination was associated with diminished Akt activation and a significant downregulation of epidermal growth factor receptor (EGFR), Raf‐1, and mitogen activated protein kinase. Combination of 17‐AAG and LY294002 did not modify phospho‐JNK/SPK and phospho‐p38. Cells exposed to 17‐AAG and LY294002 displayed a significant reduction in cell‐cycle regulatory proteins, such as retinoblastoma (Rb), cyclin dependent kinase (CDK)4, CDK6, cyclin D1, and cyclin D3. Taken together, these findings suggest that the PI3K/Akt pathway plays a critical role in regulating the apoptotic response to 17‐AAG and that targeting this pathway could provide a potent strategy to treat patients with malignant gliomas.

Bortezomib-induced sensitization of malignant human glioma cells to vorinostat-induced apoptosis depends on reactive oxygen species production, mitochondrial dysfunction, Noxa upregulation, Mcl-1 cleavage, and DNA damage.

Glioblastomas are invasive tumors with poor prognosis despite current therapies. Histone deacetylase inhibitors (HDACIs) represent a class of agents that can modulate gene expression to reduce tumor growth, and we and others have noted some antiglioma activity from HDACIs, such as vorinostat, although insufficient to warrant use as monotherapy. We have recently demonstrated that proteasome inhibitors, such as bortezomib, dramatically sensitized highly resistant glioma cells to apoptosis induction, suggesting that proteasomal inhibition may be a promising combination strategy for glioma therapeutics. In this study, we examined whether bortezomib could enhance response to HDAC inhibition in glioma cells. Although primary cells from glioblastoma multiforme (GBM) patients and established glioma cell lines did not show significant induction of apoptosis with vorinostat treatment alone, the combination of vorinostat plus bortezomib significantly enhanced apoptosis. The enhanced efficacy was due to proapoptotic mitochondrial injury and increased generation of reactive oxygen species. Our results also revealed that combination of bortezomib with vorinostat enhanced apoptosis by increasing Mcl‐1 cleavage, Noxa upregulation, Bak and Bax activation, and cytochrome c release. Further downregulation of Mcl‐1 using shRNA enhanced cell killing by the bortezomib/vorinostat combination. Vorinostat induced a rapid and sustained phosphorylation of histone H2AX in primary GBM and T98G cells, and this effect was significantly enhanced by co‐administration of bortezomib. Vorinostat/bortezomib combination also induced Rad51 downregulation, which plays an important role in the synergistic enhancement of DNA damage and apoptosis. The significantly enhanced antitumor activity that results from the combination of bortezomib and HDACIs offers promise as a novel treatment for glioma patients.

ABT-737 synergizes with bortezomib to induce apoptosis, mediated by Bid cleavage, Bax activation and mitochondrial dysfunction in an Akt-dependent context in malignant human glioma cell lines

We observed that glioma cells are differentially sensitive to N-{4-[4-(4′-chloro-biphenyl-2-ylmethyl)-piperazin-1-yl]-benzoyl}-4-(3-dimethylamino-1-phenylsulfanylmethyl-propylamino)-3-nitro-benzenesulfonamide (ABT-737) and administration of ABT-737 at clinically achievable doses failed to induce apoptosis. Although elevated Bcl-2 levels directly correlated with sensitivity to ABT-737, overexpression of Bcl-2 did not influence sensitivity to ABT-737. To understand the molecular basis for variable and relatively modest sensitivity to the Bcl-2 homology domain 3 mimetic drug ABT-737, the abundance of Bcl-2 family members was assayed in a panel of glioma cell lines. Bcl-2 family member proteins, Bcl-xL, Bcl-w, Mcl-1, Bax, Bak, Bid, and Noxa, were found to be expressed ubiquitously at similar levels in all cell lines tested. We then examined the contribution of other apoptosis-resistance pathways to ABT-737 resistance. Bortezomib, an inhibitor of nuclear factor-kappaB (NF-κB), was found to enhance sensitivity of ABT-737 in phosphatase and tensin homolog on chromosome 10 (PTEN)-wild type, but not PTEN-mutated glioma cell lines. We therefore investigated the association between phosphatidylinositol 3-kinase (PI3K)/Akt activation and resistance to the combination of ABT-737 and bortezomib in PTEN-deficient glioma cells. Genetic and pharmacological inhibition of PI3K inhibition sensitized PTEN-deficient glioma cells to bortezomib- and ABT-737-induced apoptosis by increasing cleavage of Bid protein, activation and oligomerization of Bax, and loss of mitochondrial membrane potential. Our data further suggested that PI3K/Akt-dependent protection may occur upstream of the mitochondria. This study demonstrates that interference with multiple apoptosis-resistance signaling nodes, including NF-κB, Akt, and Bcl-2, may be required to induce apoptosis in highly resistant glioma cells, and therapeutic strategies that target the PI3K/Akt pathway may have a selective role for cancers lacking PTEN function.

Cooperative inhibitory effect of ZD1839 (Iressa) in combination with 17-AAG on glioma cell growth.

ZD1839 (“Iressa”) is an orally active, selective epidermal growth factor (EGF) receptor‐tyrosine kinase inhibitor. We evaluated the antitumor activity of ZD1839 in combination with HSP90 antagonist, 17‐AAG in malignant human glioma cell lines. ZD1839 independently produced a dose‐dependent inhibition of cellular proliferation in glioma cells grown in culture with time‐ and dose‐dependent accumulation of cells in G1 phase of the cell cycle on flow cytometric analysis, although the concentrations required for optimal efficacy were at or above the limits of clinically achievable levels. Because the heat shock protein (HSP) is involved in the conformational maturation of a number of signaling proteins critical to the proliferation of malignant glioma cells, we hypothesized that the HSP90 inhibitor 17‐AAG would potentiate ZD 1839‐mediated glioma cytotoxicity by decreasing the activation status of EGF receptor, as well as downregulating the levels of other relevant signaling effectors. We, therefore, examined the effects of ZD1839 and 17‐AAG, alone and in combination, on signal transduction and apoptosis in a series of malignant glioma cell lines. Simultaneous exposure to these inhibitors significantly induced cell death and quantitative analysis revealed that interaction between ZD1839 and 17‐AAG‐induced cytotoxicity was synergistic, leading to a pronounced increase in active caspase‐3 and PARP cleavage. No significant growth inhibition or caspase activation was seen in control cells. The enhanced cytotoxicity of this combination was associated with diminished Akt activation and a significant downregulation of EGFR receptor, Raf‐1 and mitogen activated protein kinase (MAPK). Cells exposed to 17‐AAG and ZD1839 displayed a significant reduction in cell cycle regulatory proteins, such as CDK4 and CDK6. Taken together, these findings suggest that ZD1839, an EGF receptor tyrosine kinase inhibitor, plays a critical role in regulating the apoptotic response to 17‐AAG and that multi‐site targeting of growth signaling and cell survival pathways could provide a potent strategy to treat patients with malignant gliomas.

YM-155 Potentiates the Effect of ABT-737 in Malignant Human Glioma Cells via Survivin and Mcl-1 Downregulation in an EGFR-Dependent Context

Antiapoptotic proteins are commonly overexpressed in gliomas, contributing to therapeutic resistance. We recently reported that clinically achievable concentrations of the Bcl-2/Bcl-xL inhibitor ABT-737 failed to induce apoptosis in glioma cells, with persistent expression of survivin and Mcl-1. To address the role of these mediators in glioma apoptosis resistance, we analyzed the effects of YM-155, a survivin suppressant, on survival on a panel of glioma cell lines. YM-155 inhibited cell growth and downregulated survivin and Mcl-1 in a dose- and cell line–dependent manner. While U373, LN18, LNZ428, T98G, LN229, and LNZ308 cells exhibited an IC50 of 10 to 75 nmol/L, A172 cells were resistant (IC50 ∼ 250 nmol/L). No correlation was found between sensitivity to YM-155 and baseline expression of survivin or cIAP-1/cIAP-2/XIAP. However, strong correlation was observed between EGF receptor (EGFR) activation levels and YM-155 response, which was confirmed using EGFR-transduced versus wild-type cells. Because we postulated that decreasing Mcl-1 expression may enhance glioma sensitivity to ABT-737, we examined whether cotreatment with YM-155 promoted ABT-737 efficacy. YM-155 synergistically enhanced ABT-737–induced cytotoxicity and caspase-dependent apoptosis. Downregulation of Mcl-1 using short hairpin RNA also enhanced ABT-737–inducing killing, confirming an important role for Mcl-1 in mediating synergism between ABT-737 and YM-155. As with YM-155 alone, sensitivity to YM-155 and ABT-737 inversely correlated with EGFR activation status. However, sensitivity could be restored in highly resistant U87-EGFRvIII cells by inhibition of EGFR or its downstream pathways, highlighting the impact of EGFR signaling on Mcl-1 expression and the relevance of combined targeted therapies to overcome the multiple resistance mechanisms of these aggressive tumors. Mol Cancer Ther; 12(3); 326–38. ©2013 AACR.

Inhibition of Phosphatidylinositol 3-Kinase/AKT Signaling by NVP-BKM120 Promotes ABT- 737-induced toxicity in a caspase-dependent manner through mitochondrial dysfunction and DNA damage response in established and primary cultured glioblastoma cells

Identification of therapeutic strategies that might enhance the efficacy of B-cell lymphoma-2 (Bcl-2) inhibitor ABT-737 [N-{4-[4-(4-chloro-biphenyl-2-ylmethyl)-piperazin-1-yl]-benzoyl}-4-(3-dimethylamino-1-phenylsulfanylmethyl-propylamino)-3-nitro-benzenesulfonamide] is of great interest in many cancers, including glioma. Our recent study suggested that Akt is a crucial mediator of apoptosis sensitivity in response to ABT-737 in glioma cell lines. Inhibitors of phosphatidylinositol 3-kinase (PI3K)/Akt are currently being assessed clinically in patients with glioma. Because PI3K/Akt inhibition would be expected to have many proapoptotic effects, we hypothesized that there may be unique synergy between PI3K inhibitors and Bcl-2 homology 3 mimetics. Toward this end, we assessed the combination of the PI3K/Akt inhibitor NVP-BKM120 [5-(2,6-dimorpholinopyrimidin-4-yl)-4-(trifluoromethyl)pyridin-2-amine] and the Bcl-2 family inhibitor ABT-737 in established and primary cultured glioma cells. We found that the combined treatment with these agents led to a significant activation of caspase-8 and -3, PARP, and cell death, irrespective of PTEN status. The enhanced lethality observed with this combination also appears dependent on the loss of mitochondrial membrane potential and release of cytochrome c, smac/DIABLO, and apoptosis-inducing factor to the cytosol. Further study revealed that the upregulation of Noxa, truncation of Bid, and activation of Bax and Bak caused by these inhibitors were the key factors for the synergy. In addition, we demonstrated the release of proapoptotic proteins Bim and Bak from Mcl-1. We found defects in chromosome segregation leading to multinuclear cells and loss of colony-forming ability, suggesting the potential use of NVP-BKM120 as a promising agent to improve the anticancer activities of ABT-737.

Abrogation of Mitogen-Activated Protein Kinase and Akt Signaling by Vandetanib Synergistically Potentiates Histone Deacetylase Inhibitor-Induced Apoptosis in Human Glioma Cells

Vandetanib is a multitargeted tyrosine kinase inhibitor. Our initial studies demonstrated that this agent blocks vascular endothelial growth factor receptor, epidermal growth factor receptor, and platelet-derived growth factor receptor phosphorylation and mitogen-activated protein kinase (MAPK)-mediated signaling in glioma cell lines in a dose-dependent manner. Despite these effects, we observed that vandetanib had little effect on apoptosis induction at clinically achievable concentrations. Because histone deacetylase inhibitors (HDACIs) have been suggested to regulate signaling protein transcription and downstream interactions via modulation of protein chaperone function through the 90-kDa heat shock protein, we investigated whether combining vandetanib with an HDACI could synergistically potentiate signaling pathway inhibition and apoptosis induction in a panel of malignant human glioma cell lines. Proliferation assays, apoptosis induction studies, and Western immunoblot analysis were conducted in cells treated with vandetanib and HDACIs as single agents or in combination. Vandetanib and suberoylanalide hydroxamic acid reduced proliferation in all cell lines when used as single agents, and the combination produced marked potentiation of growth inhibition as assessed by combinatorial methods. These effects were paralleled by potentiation of Akt signaling inhibition and apoptosis induction. Our results indicate that inhibition of histone deacetylation enhances the antiproliferative effect of vandetanib in malignant human glioma cell lines by enhancing inhibition of MAPK, Akt, and other downstream effectors that may have application in combinatorial therapeutics for these tumors.

Dasatinib synergizes with JSI-124 to inhibit growth and migration and induce apoptosis of malignant human glioma cells.

Background: Src family kinases (SFK) collectively regulate a variety of cellular functions in many cancer types, including proliferation, invasion, motility, survival, differentiation, and angiogenesis. Although Dasatinib (BMS-354825), an ATP-competitive, small molecule tyrosine kinase inhibitor, suppresses the activity of SFKs at nanomolar concentrations, IC50 values for antiproliferative effects in glioma cell lines were well above the clinically achievable range, suggesting the need to interfere with other components of receptor-induced downstream signaling in order to achieve an optimal therapeutic effect. Materials and Methods: The cytotoxic effects of combining Src and STAT3 inhibition on glioma cell lines were evaluated using assays to measure cell proliferation, apoptosis and migration. Western blotting and immunocytochemistry was used to monitor its effects on cell signaling and morphology. Results: Silencing Src and STAT3 expression each partially inhibited cell proliferation and migration. In addition, JSI-124 significantly enhanced the efficacy of dasatinib in vitro. Combination of dasatinib and JSI-124 achieved significant inhibition of migration in all cell lines, which correlated with the inhibition of Src and downstream mediators of adhesion (e.g. focal adhesion kinase). Cells exposed to dasatinib and JSI-124 exhibited morphological changes that were consistent with an upstream role for Src in regulating focal adhesion complexes. Conclusions: Targeting the Src and STAT pathways may contribute to the treatment of cancers that demonstrate increased levels of these signaling mediators, including malignant human glioma. Clinical studies in these tumor types are warranted.