Apoptosis of leukemic cells accompanies reduction in intracellular pH after targeted inhibition of the Na+/H+exchanger

Abstract

The Na+/H+ exchanger isoform 1 (NHE1) is primarily responsible for the regulation of intracellular pH (pHi). It is a ubiquitous, amiloride-sensitive, growth factor–activatable exchanger whose role has been implicated in cell-cycle regulation, apoptosis, and neoplasia. Here we demonstrate that leukemic cell lines and peripheral blood from primary patient leukemic samples exhibit a constitutively and statistically higher pHi than normal hematopoietic tissue. We then show that a direct correlation exists between pHi and cell-cycle status of normal hematopoietic and leukemic cells. Advantage was taken of this relationship by treating leukemic cells with the Na+/H+ exchanger inhibitor, 5-(N, N-hexamethylene)-amiloride (HMA), which decreases the pHiand induces apoptosis. By incubating patient leukemic cells in vitro with pharmacologic doses of HMA for up to 5 hours, we show, using flow cytometry and fluorescent ratio imaging microscopy, that when the pHi decreases, apoptosis—measured by annexin-V and TUNEL methodologies—rapidly increases so that more than 90% of the leukemic cells are killed. The differential sensitivity exhibited between normal and leukemic cells allows consideration of NHE1 inhibitors as potential antileukemic agents.

Amiloride derivatives induce apoptosis by depleting ER Ca2+ stores in vascular endothelial cells

“…KS Park et al minimizes NHE activity (Slepkov et al, 2007). The apoptotic effects of amiloride analogues observed in this condition thus cannot be attributed to the intracellular acidification resulting from NHE inhibition, as previously suggested (Rich et al, 2000). In liver cells, apoptosis induced by NHE inhibitors is associated with activation of JNK kinase (Suzuki and Tsukamoto, 2004) or with the inhibition of ERK kinase (Konstantinidis et al, 2006).…”

“…In contrast to their protective effects, amiloride derivatives also elicit growth inhibition and apoptotic cell death in hepatocytes (Suzuki and Tsukamoto, 2004), leukaemic cells (Rich et al, 2000), smooth muscle (Chen and O'Brien, 2003) and neurons (Schneider et al, 2004). Based on these apoptotic effects, amiloride analogues were proposed as potential therapeutic agents against metastasis and multi-drug resistant cancers (Harguindey and Cragoe, 1992).…”

“…Inhibition of Na +coupled H + efflux decreases intracellular pH (Counillon and Pouyssegur, 2000), and the subsequent cytosolic acidification can trigger apoptosis by promoting mitochondrial cytochrome c release and caspase activation (Furlong et al, 1997;Park et al, 1999;Matsuyama et al, 2000;Kristian et al, 2001). Thus, acidification induced by NHE inhibition was suggested to be the apoptogenic mechanism of amiloride derivatives (Rich et al, 2000;Schneider et al, 2004). Conversely, the characteristic cytosolic alkalinization of malignant tumour cells is in part due to growth factors and oncogenes up-regulating NHE expression (Reshkin et al, 2000;Cardone et al, 2005).…”

Reduction of intracellular pH inhibits constitutive expression of Cyclooxygenase‐2 in human colon cancer cells

“…An increase in pH i was shown to be an essential prerequisite for DNA synthesis, cell proliferation, and activation of glycolysis (Grinstein et al, 1989). Recently, Rich et al (2000) described a pH i approximately 0.4 units higher in various leukemia cell lines as compared to peripheral blood mononuclear cells (PBMC) from healthy donors. Importantly, they demonstrated that leukemia cells from patients also possess a more alkaline pH i than do PBMC from healthy donors suggesting an essential function of elevated pH i in tumor cells (Rich et al, 2000).…”

“…Recently, Rich et al (2000) described a pH i approximately 0.4 units higher in various leukemia cell lines as compared to peripheral blood mononuclear cells (PBMC) from healthy donors. Importantly, they demonstrated that leukemia cells from patients also possess a more alkaline pH i than do PBMC from healthy donors suggesting an essential function of elevated pH i in tumor cells (Rich et al, 2000). Further support for this hypothesis was found after transformation of NIH-3T3 cells by the E7 oncogene from human papilloma virus-16.…”