Intracerebral C6 glioma xenografts spontaneously develop centrally located necrotic regions that are bordered by densely packed neoplastic cells. Proliferative and metabolic heterogeneity in these perinecrotic regions were assessed by bromodeoxyuridine (BrdU) incorporation, by immunocytological and by histochemical analyses. The borders of necrotic regions are comprised of glioma cells that express increased levels of the type 1 glucose transporter (GLUT-1) with rare cells having incorporated BrdU. By contrast, BrdU-positive glioma cells are located immediately adjacent to GLUT-1-positive cells bordering areas of necrosis. BrdU-positive glioma cells are also scattered throughout poorly vascularized, central regions of the tumor and are present at the highly vascularized tumor periphery. GLUT-1 expression increased considerably when C6 glioma cells were grown for 48 h under either the acidotic conditions of pH 6.8 or under hypoxic conditions. The perinecrotic GLUT-1-positive glioma cells in poorly vascularized, centrally located tumor regions demonstrated a 75% reduction in glycogen content and negligible glycogenolytic capacity, when compared with normal brain white matter. Cytochrome c oxidase (COX) and lactate dehydrogenase (LDH) maintained 50% enzymatic activity compared to controls, while succinate dehydrogenase (SDH) activity was 25% of control values. Based upon these findings, a metabolic model is proposed in which GLUT-1-positive perinecrotic cells are growth arrested and predominantly rely upon non-oxidative glycolysis. It is further postulated that BrdU-positive, GLUT-1-negative glioma cells within the poorly vascularized, central tumor region convert glucose-6-phosphate to nucleotide precursors for DNA replication.

Malignant glioma cells maintain an elevated intracellular pH (pH i ) within hypoxic-ischemic tumor microenvironments through persistent activation of sodium-proton transport (McLean et al., 2000). Amiloride has been reported to selectively kill human malignant glioma cell lines but not primary astrocytes (Hegde et al., 2004). While amiloride reduces pH i of malignant gliomas by inhibiting isoform 1 of sodium-proton exchange (NHE1), direct acidification was shown to be cytostatic rather than cytotoxic. At cytotoxic concentrations, amiloride has multiple drug targets including inhibition of NHE1 and sodium calcium exchange. Amiloride's glioma cytotoxicity can be explained, at least in part, by dual inhibition of NHE1 and of Na + -dependent calcium efflux by isoform 1.1 of the sodium calcium exchanger (NCX1.1) , which increases [Ca 2+ ] i and initiates glioma cell demise. As a result of persistent NHE1 activity, cytosolic free levels of sodium ([Na + ] i ) in U87 and C6 glioma cells are elevated 3-fold, as compared with normal astrocytes. Basal cytosolic free calcium levels ([Ca 2+ ] i ) also are increased 5-fold. 2′, 4′-dichlorobenzamil (DCB) inhibits the sodium-dependent calcium transporter (NCX1.1) much more potently than NHE1. DCB was employed in a concentration-dependent fashion in glioma cells to selectively inhibit the forward mode of NCX1.1 at ≤1uM, while dually inhibiting both NHE1 and NCX1.1 at ≥20uM. DCB (1uM) was not cytotoxic to glioma cells, while DCB (20μM) further increased basal elevated levels of [Ca 2+ ] i in glioma cells that was followed by cell demise. Cariporide and © 2010 Elsevier B.V. All rights reserved. * To whom correspondence should be sent (fagorin@ucdavis.edu).Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Conflict of Interest:Fredric Gorin, Michael Nantz, and the University of California have filed patents pertaining to the use of amiloride and novel derivatives as potential anti-cancer agents. Fredric Gorin is CEO of D3G, which contracts with publically owned and private labs to perform pre-clinical drug discovery, as it pertains to advance the treatment of primary and metastatic cancers affecting the central nervous system (brain and spinal cord). SEA0400 are more specific inhibitors of NHE1 and NCX1.1 than amiloride or DCB, respectively. Individually, Cariporide and SEA0400 are not cytotoxic, but in combination induced glioma cell death. Like amiloride, the combination of Cariporide and SEA0400 produced glioma cell death in the absence of demonstrable caspase-activation. NIH Public Access

The relative non-toxicity of the diuretic amiloride, coupled with its selective inhibition of the protease urokinase plasminogen activator (uPA), makes this compound class attractive for structure-activity studies. Herein we substituted the C(2)-acylguanidine of C(5)-glycyl-amiloride with amidine and amidoxime groups. The data show the importance of maintaining C(5)-hydrophobicity. The C(5)-benzylglycine analogs containing either C(2)-acylguanidine or amidine inhibited uPA with an IC50 ranging from 3 to 7μM and were cytotoxic to human U87 malignant glioma cells.

Inhibition of the sodium-proton exchanger (NHE) plays an important role in reducing tissue damage during ischemic reperfusion injury; however, pharmacological inhibitors of NHE have restricted access to acutely ischemic tissues because of severely compromised tissue perfusion. We describe the syntheses, characterization, and NHE inhibitory activities of a novel class of amiloride derivatives where peptides are conjugated to the amiloride C(5) amino group. These new peptide-C(5)-amiloride conjugates are inactive; however, peptide residues were chosen such that selective cleavage by neutral endopeptidase 24.11 (enkephalinase) liberates an amino acid-C(5)-amiloride conjugate that inhibits NHE in a glial cell line. These results confirm the feasibility of using peptide-amiloride conjugates as NHE inhibitor prodrugs. We envision the design of analogous peptide-amiloride prodrugs that can be administered prior to ischemic events and subsequently activated by endopeptidases selectively expressed by ischemic tissues.The sodium-proton exchanger (NHE) represents a family of sodium-dependent transport proteins that participate in various cellular functions (Orlowski and Grinstein, 1997). Seven isoforms (NHE1-7) have been identified (Numata and Orlowski, 2001;Brett et al., 2002;Slepkov and Fliegel, 2002). NHE1 and NHE5 to 7 are particularly important in maintaining the intracellular pH (pH i ) in human heart and brain. Additionally, increased NHE1 activity has been shown to maintain an alkalotic pH i in several human cancer cell types, including transformed astrocytes, i.e., malignant glioma cells (McLean et al., 2000;Hegde et al., 2004). Inhibition of NHE1 in glioma cells causes a reduction of intracellular sodium and associated cytotoxic edema (F. A. Gorin, R. Sriram, W. Harley, C. Floyd, A. Marshall, B. Lyeth, and T. Jue, unpublished data).There is a shift from oxidative to nonoxidative glycolysis during ischemia with increased intracellular acidosis. This reduction in pH i activates NHE, which increases intracellular Na ϩ ([Na ϩ ] i ) levels (step 2, Fig. 1) (Orlowski and Grinstein, 1997). The specifics of normalizing increased [Na ϩ ] i remain unclear but include regulation by Na ϩ /K ϩ ATPase and sodium-dependent calcium influx (reverse mode) by the sodium-calcium exchanger (NCX) (Satoh et al., 2003). Persistent activation of the reverse mode of NCX during vascular perfusion further increases intracellular Ca 2ϩ ([Ca 2ϩ ] i ) (step 4, Fig. 1), which is believed to initiate the irreversible cellular damage observed during ischemia-reperfusion (I/R) (Piper et al., 1996). This sequence of physiological events leading to I/R injury is initiated by NHE activation; consequently, the controlled inhibition of NHE is an area of intense research (Masereel et al., 2003) (Fig. 1).The significance of NHE participation in I/R injury in animals has been shown by demonstrating that NHE inhibitors, such as cariporide (Fig. 2), are effective in preventing cellular damage resulting from cerebral and myocardial ischemia when ad...

The human glioma cell lines, U87 and T98G, were evaluated for their ability to survive and form colonies in an acidic environment of pH ext 6.0. In contrast to U87, which showed an 80-90% survival rate, only 40% of T98G cells survived 6 days at pH ext 6.0 and lost their colony forming ability when returned to a normocidic environment. Although both U87 and T98G cells maintain an intracellular pH (pH i ) of 7.0 at pH ext 6.0 and arrest mostly in G1 phase of the cell cycle, only T98G demonstrated a major loss of cyclin D1 that was prevented by the proteasome inhibitor MG132. Colony forming ability was restored by stably transfecting T98G cells with a cyclin D1-expressing plasmid. Both U87 and T98G cells demonstrated increased cytoplasmic localization of cyclin D1 during exposure at pH ext 6.0. Upon prolonged (24 h) incubation at pH ext 6.0, nuclear cyclin D1 was nearly absent in T98G in contrast to U87 cells. Thus, an acidic environment triggers cytoplasmic localization and proteasomal degradation of cyclin D1.