ABSTRACT
Title
Type-3 metabotropic glutamate receptor blockade enables the cytotoxic activity of temozolomide on human glioma stem cells
Authors
C. Ciceroni,1M. Bonelli,2 E. Mastrantoni,2 C. Niccolini,2 M. Laurenza,2 A. Traficante,3 P. Spinanti, 2 L. Ricci-Vitiani,4 A. Arcella,3 R. De Maria,4 F. Stocchi,1 F. Nicoletti,2,3 G. Battaglia,3 D. Melchiorri1,2
1I.R.C.C.S. San Raffaele Pisana, 00163 Roma, Italy; 2Department of Physiology an Pharmacology, University “Sapienza”, 00185 Roma, Italy; 3I.N.M. Neuromed, 86077 Pozzilli, Italy; 4Department of Hematology, Istituto Superiore di Sanità, 00161 Rome, Italy.
1I.R.C.C.S. San Raffaele Pisana, 00163 Roma, Italy; 2Department of Physiology an Pharmacology, University “Sapienza”, 00185 Roma, Italy; 3I.N.M. Neuromed, 86077 Pozzilli, Italy; 4Department of Hematology, Istituto Superiore di Sanità, 00161 Rome, Italy.
Abstract
Drug treatment of malignant gliomas is limited by mechanisms of chemoresistance that are intrinsic to cancer stem cells present in the tumor mass. Cancer stem cells isolated from surgical specimens of human glioblastoma (glioma stem cells or GSCs) expressed type-3 metabotropic glutamate (mGlu3) receptors coupled to multiple transduction pathways including the phosphatidylinositol-3-kinase (PtdIns-3-K) pathway. Exposure of cultured GSCs to a combination of Temozolomide (TMZ, 250 µM), the gold standard in GM therapy, and the selective type-3 mGluR antagonist, LY341495 (100 nM), for either 24h or 48h, induced a time-dependent cell death, as assessed by MTT assay. No effect on cell viability was observed following exposure of GSCs to either TMZ or LY341495 by themselves. Similar results were obtained by knocking down mGluR3 receptors with siRNA. To investigate the molecular mechanisms underlying the permissive effect of LY341495 on TMZ-induced toxicity, we focused on the phosphatidylinositol-3-kinase (PI-3K) pathway which is coupled to the activation of mGluR3, and is classically involved in the regulation of cell proliferation and survival. Exposure of cells to TMZ and the selective inhibitor of the PI-3K, LY290042 (100nM), which was inactive by itself, induced cell death, reproducing the effect observed with the co-administration of LY341495 and TMZ.
We extended the study to signalling pathways that lie downstream of Akt and regulate survival of glioma cells. Akt activates nuclear factor kB (NFkB) by phosphorylating the IKB kinase. NFkB blockade enhances the pro-apoptotic activity of DNA alkylating agents in glioma cells. The NFkB inhibitor, JSH-2326, enabled temozolomide toxicity and occluded the permissive action of LY341495 in GSCs. Similar effects were obtained with salicylic acid, which also inhibits NFkB. As opposed to LY341495, JSH-23 could still enhance temozolomide toxicity in GSC expressing the constitutively active form of Akt indicating that NFkB lies downstream of Akt in the pathway that restrains temozolomide toxicity.
Temozolomide resistance of cancer cells is thought to be mediated by the expression of high levels of the DNA repairing enzyme O6-methylguanine-DNA methyltransferase (MGMT). The MGMT gene promoter contains two NFkB binding sites, and NFkB enhances MGMT expression, thus inducing resistance to DNA alkylating agents.
The association between temozolomide and mGlu3 receptor blockade caused a dramatic reduction in the expression of MGMT. Overexpression of MGMT abolished the synergism between mGlu3 receptor blockade and temozolomide. Again, the action of LY341495 was mimicked by a siRNA-induced knockdown of mGlu3 receptors, by the PtdIns-3-K inhibitor, LY294002, and by the NFkB inhibitor, JSH-23. The permissive action of LY341495, LY294002, or JSH-23 on temozolomide toxicity was not seen in GSCs overexpressing MGMT.
Stereotaxic implantation of GFP+-GSCs was performed into the left caudate nucleus of CD1 nude mice. After 15 days from cell implantation, mice were subcutaneously implanted with osmotic minipumps releasing LY341495 (1mg/kg/die) or saline, and treated with TMZ (60 mg/kg/die) every other day for one week. In mice killed 45 days after cell implantation, tumor cells were confined to the medial portion of the caudate nucleus close to the wall of the lateral ventricle. In mice killed at 75 days, tumour cells formed an infiltrating mass in the ipsilateral caudate nucleus and, in most of the animals, they spread to the ipsilateral septum and to the ipsi-and contralateral portion of the corpus callosum. In both sets of experiments, treatments with temozolomide or LY341495 alone had small, if any, effect on tumour growth. In contrast, a combined treatment with temozolomide and LY341495 significantly reduced tumour growth in nice agreement with in vitro data.
We extended the study to signalling pathways that lie downstream of Akt and regulate survival of glioma cells. Akt activates nuclear factor kB (NFkB) by phosphorylating the IKB kinase. NFkB blockade enhances the pro-apoptotic activity of DNA alkylating agents in glioma cells. The NFkB inhibitor, JSH-2326, enabled temozolomide toxicity and occluded the permissive action of LY341495 in GSCs. Similar effects were obtained with salicylic acid, which also inhibits NFkB. As opposed to LY341495, JSH-23 could still enhance temozolomide toxicity in GSC expressing the constitutively active form of Akt indicating that NFkB lies downstream of Akt in the pathway that restrains temozolomide toxicity.
Temozolomide resistance of cancer cells is thought to be mediated by the expression of high levels of the DNA repairing enzyme O6-methylguanine-DNA methyltransferase (MGMT). The MGMT gene promoter contains two NFkB binding sites, and NFkB enhances MGMT expression, thus inducing resistance to DNA alkylating agents.
The association between temozolomide and mGlu3 receptor blockade caused a dramatic reduction in the expression of MGMT. Overexpression of MGMT abolished the synergism between mGlu3 receptor blockade and temozolomide. Again, the action of LY341495 was mimicked by a siRNA-induced knockdown of mGlu3 receptors, by the PtdIns-3-K inhibitor, LY294002, and by the NFkB inhibitor, JSH-23. The permissive action of LY341495, LY294002, or JSH-23 on temozolomide toxicity was not seen in GSCs overexpressing MGMT.
Stereotaxic implantation of GFP+-GSCs was performed into the left caudate nucleus of CD1 nude mice. After 15 days from cell implantation, mice were subcutaneously implanted with osmotic minipumps releasing LY341495 (1mg/kg/die) or saline, and treated with TMZ (60 mg/kg/die) every other day for one week. In mice killed 45 days after cell implantation, tumor cells were confined to the medial portion of the caudate nucleus close to the wall of the lateral ventricle. In mice killed at 75 days, tumour cells formed an infiltrating mass in the ipsilateral caudate nucleus and, in most of the animals, they spread to the ipsilateral septum and to the ipsi-and contralateral portion of the corpus callosum. In both sets of experiments, treatments with temozolomide or LY341495 alone had small, if any, effect on tumour growth. In contrast, a combined treatment with temozolomide and LY341495 significantly reduced tumour growth in nice agreement with in vitro data.