Rapid Tumor Cell Swelling and Bursting: Beware of Collateral Damage

To the editor: We read with interest the article recently published in Molecular Therapy by Tannous and colleagues entitled “Mutant Sodium Channel for Tumor Therapy.”1Tannous BA Christensen AP Pike L Wurdinger T Perry KF Saydam O et al.Mutant sodium channel for tumor therapy.Mol Ther. 2009; 17: 810-819Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar Their aim was to determine whether expression of a constitutively active sodium (Na+) channel in the membrane of glial tumor cells would cause efficient killing not only of the infected cells but also of the neighboring tumor cells. Indeed, this approach would take advantage of the fact that glial cells possess gap junctions: infecting one cell in one area of the tumor should result in near-instantaneous changes in all cells connected to it. The investigators used a well-described viral vector—herpes simplex virus/Epstein–Barr virus hybrid amplicon vector2Wang S Vos JM A hybrid herpesvirus infectious vector based on Epstein-Barr virus and herpes simplex virus type 1 for gene transfer into human cells in vitro and in vivo.J Virol. 1996; 70: 8422-8430PubMed Google Scholar—that was engineered to encode a constitutively active mutant brain Na+ channel, ASIC2a (acid-sensing ion channel 2a).3Jasti J Furukawa H Gonzales EB Gouaux E Structure of acid-sensing ion channel 1 at 1.9 Å resolution and low pH.Nature. 2007; 449: 316-323Crossref PubMed Scopus (868) Google Scholar Control experiments were performed with the same vector, encoding a wild-type ASIC2a. Expression of the proteins of interest was confirmed in vitro with immunohistochemistry and western blotting. In vivo experiments were also performed by injecting the engineered vector directly into glial tumors growing on the flanks of nude mice. The experimental system was elegant in that the expression of the heterologous proteins was under the strict control of a doxycycline-dependent promoter. Ultimately the authors provided convincing evidence that the in vitro and in vivo data supported their working hypothesis: expression of the mutant Na+ channel resulted in “robust killing” of tumor cells infected by the viral vector and their noninjected neighbors, but not normal cells in the brain. They observed that cells infected with the mutant channels swelled and burst within hours after exposure to doxycycline, an effect not seen with the wild-type channel. Importantly, they also demonstrated that this effect was due to Na+ influx using direct Na+ current measurements (with or without amiloride) as well as monitoring changes in the intensity of an Na+-sensitive intracellular dye (sodium-binding benzofuran isophthalate–acetoxymethyl ester). The authors concluded that these changes must have been caused by an “inflow of water with sodium” into infected cells. We wish to raise a few issues that relate to the potential clinical application of this antitumor strategy, but first we provide a succinct review of the mechanisms that may be responsible for rapid cellular swelling in this setting. The simplest and most intuitive way to examine the mechanisms involved in this unique experimental setting is to perform a sequential analysis of the events leading to cellular bursting. The authors report that induction of expression of the constitutively active ASCI2a resulted in a rapid change in cellular shape “from flat to round” within 3 hours of exposure to doxycycline; after 12 hours, all infected cells had burst. 1. Exposure to doxycyline rapidly triggers transcription of ASIC2a genes (both wild-type and mutant), followed within hours by translocation of the translated protein to its target subcellular domain (in this case, the cell membrane). Insertion of wild-type Na+ channels should be harmless, in that the extracellular pH is not expected to be below 6.9 (ASIC2a is “normally activated by low pH”).4Kellenberger S Schild L Epithelial sodium channel/degenerin family of ion channels: a variety of functions for a shared structure.Physiol Rev. 2002; 82: 735-767Crossref PubMed Scopus (864) Google Scholar However, constitutively active Na+ channels would be expected to trigger an immediate influx of Na+, because its activity is no longer modulated by pH changes. 2. Extrusion of this new intracellular Na+ via the Na+−K+-ATPase is required to preserve functional integrity of the cell because it is critically dependent on a specific value for the negative intracellular voltage (K+ refers to potassium). This marked increase in “local work” would rapidly cause a “fuel crisis” if local ATP demands outstrip the vascular supply of oxygen. Exceeding aerobic metabolic capacity in this way would cause increased rates of anaerobic ATP regeneration (via glycolysis) until cellular demands for glucose again surpass endogenous and vascular supply.5Lowell BB Spiegelman BM Towards a molecular understanding of adaptive thermogenesis.Nature. 2000; 404: 652-660Crossref PubMed Scopus (1322) Google Scholar A large rise in [H+] would be expected in the vicinity of affected cells. In addition, this could lead to a reduced supply of glucose for normal oxidation of neighboring, unaffected cells. 3. Once all these compensatory mechanisms are overwhelmed, extra Na+ ions entering via constitutively active ASIC2a would now stay “trapped” intracellularly. At this point, overall electroneutrality may be preserved only by the exit of an abundant intracellular cation from these cells (K+), by the influx of an abundant extracellular anion (chloride; Cl–), or by both mechanisms. Because the overall cell volume increased in the process, as was convincingly documented by the authors, electroneutrality was probably achieved in large part by entry of extracellular Cl– via transmembrane Cl– channels. This massive influx of Cl– would be responsible (along with Na+) for the robust cell swelling observed. Based on the above discussion, the following questions could be addressed experimentally. 1. The glucose concentration and the pH of the culture medium of cells infected with the mutant ASCI2a should be lower than those of the wild type after its induction with doxycycline; the degree of change in these parameters will be affected by the cell mass, the quantity of glucose, and the buffer capacity of the incubation medium. 2. If low glucose and a high [H+] are indeed documented, it may be possible to examine the contribution of these factors in parallel to Na+ and Cl– uptake on cell swelling and bursting. 3. Reduction of Cl– influx should slow the process of cellular swelling and rupture; this could in theory be tested experimentally by either reducing the concentration of Cl– in the medium or using a Cl– channel inhibitor (see the article by Meng and Reeves for an example6Meng X Reeves WB Effects of chloride channel inhibitors on H2O2-induced renal epithelial cell injury.Am J Physiol Renal Physiol. 2000; 278: 83-97PubMed Google Scholar). First and foremost, if one of the goals of this therapeutic approach were to target intracranial tumors of neuronal and/or glial origin, it would be critical to monitor very closely the changes in intracranial pressure. Indeed, shortly after induction of the mutant Na+ channel expression via exposure to doxycycline, one can expect rapid and considerable swelling of the tumor as a whole as its constituent cells are bursting. In the worst-case scenario, this process may cause acute cerebral herniation. It is noteworthy that this problem should be specific to intracranial tumors, in that this is the only body compartment with a fixed surrounding volume. However, even if treating extracranial tumors in this way, clinicians may need to consider documenting conclusively the absence of another intracranial tumor (for example, a benign meningioma). Indeed, if a fraction of the engineered vectors “escaped” from the target tumor and successfully infected an occult one, the same deleterious effects could in theory be observed. Second, “robust killing” of tumor cells via a “rapid, explosive mechanism” may at first sound like a panacea. On the other hand, chilling considerations soon emerge as one considers the biochemical imbalances that may result from this rapid process. Massive cellular bursting from any mechanism is expected to cause the release of a large amount of K+ into venous blood, potentially leading to severe arterial hyperkalemia and cardiac arrhythmias. It is important to recognize that there is minimal opportunity for this intravenous “K+ bolus” to mix with the entire extracellular fluid before it reaches the heart. This is akin to the larger fall in arterial versus brachial venous Na+ concentration following an acute, large, oral water load.7Shafiee MA Charest AF Cheema-Dhadli S Glick DN Napolova O Roozbeh J et al.Defining conditions that lead to the retention of water: the importance of the arterial sodium concentration.Kidney Int. 2005; 67: 613-621Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar Cell lysis would also result in acute hyperphosphatemia, because phosphate is the major intracellular anion. A sudden rise in venous inorganic phosphate concentration may cause acute hypocalcemia due to precipitation of calcium phosphate. Calcium carbonate precipitation may also occur simultaneously, as we recently described.8Brown K Halperin LF Malhotra A Tsang J Grynpas M Halperin ML Hypocalcemia and a low cardiac output after intravenous codeine phosphate injection: need for an additional mechanism to remove ionized calcium.Nephro Dial Transplant. 2005; (in press)Google Scholar It is interesting to note that incubation of cells with the mushroom toxin phallolysin induces cellular changes that are in many ways analogous to that presented by Tannous and colleagues: rapid cell swelling (within minutes)9Seitz J Adler G Stofft E Faulstich H The mechanism of cytolysis of erythrocytes by the mushroom toxin phallolysin. Morphological and biochemical evidence for sodium influx and swelling.Eur J Cell Biol. 1981; 25: 46-53PubMed Google Scholar that is due to massive Na+ (and Cl–) influx via transmembrane ion channels formed by the toxin,10Wilmsen HU Faulstich H Eibl H Boheim G Phallolysin. A mushroom toxin, forms proton and voltage gated membrane channels.Eur Biophys J. 1985; 12: 199-209Crossref PubMed Scopus (13) Google Scholar resulting in patchy cellular bursting.11Petzinger E Seeger R Scanning electron microscopic studies on the cytolytic effect of phallolysin on isolated rat hepatocytes and AS-30 D hepatoma cells.Naunyn Schmiedebergs Arch Pharmacol. 1976; 295: 211-213Crossref PubMed Scopus (26) Google Scholar Considered a potential antitumor agent in the early 1970s,12Seeger R Lehmann D [Antitumor activity of phallolysin from Amanita phalloides].Naunyn Schmiedebergs Arch Pharmacol. 1973; 279: 235-242Crossref PubMed Scopus (10) Google Scholar its development as a therapeutic agent was hampered by its lethality in most species; it triggered severe hyperkalemia caused by extreme intravascular hemolysis and cytolysis of other cell types.13Odenthal KP Seeger R Vogel G Toxic effects of phallolysin from Amanita phalloides.Naunyn Schmiedebergs Arch Pharmacol. 1975; 290: 133-143Crossref PubMed Scopus (15) Google Scholar Although the therapeutic regimen proposed by Tannous and colleagues is obviously much more sophisticated (and specific) than that proposed by the proponents of phallolysin in the 1970s, this historical vignette provides a cautionary reminder that rapid lysis of a large number of cells is associated with significant intracellular content spillage. In conclusion, to reduce the potential risks associated with the therapeutic approach proposed by Tannous et al., we suggest that additional strategies to slow the rate of cell swelling and/or Na+ (and its anion) entry merit consideration. Perhaps inducing a slower entry of Cl– and/or having fewer active mutant Na+ channels could minimize the “collateral damage.”

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