John C. Lyons

Acidic environment causes apoptosis by increasing caspase activity.

SummaryAn exposure of HL-60 human promyelocytic leukaemia cells to acidic media with pH 6.2–6.6 caused an up-regulation of Bax protein expression within 2 h, which lasted for longer than 6 h. On the other hand, the apoptosis, as judged from PARP cleavage, DNA fragmentation and flow cytometric determination of cell population with sub-G1 DNA content, occurred after the cells were incubated in the acidic media for longer than 4 h. The PARP cleavage and DNA fragmentation in the cells exposed to an acidic environment could be effectively suppressed by inhibitors specific for ICE or CPP32, indicating that activation of these caspases is an essential step in acidic stress-induced apoptosis. It has been known that Bax is involved in the activation of caspases. Taken together, it appears that acidic stress first up-regulates Bax protein thereby activating caspases followed by PARP cleavage and DNA fragmentation. The observation that inhibition of either ICE or CPP32 could suppress acidic stress-induced apoptosis suggested that ICE activates pro-CPP32, which then cleaves PARP. Flow cytometric analysis indicated that acidic stress-induced apoptosis occurs mainly in G1 cells. The finding in the present study demonstrated that acidic intra-tumour environment may markedly perturb the tumour cell proliferation and tumour growth.

Modification of Intracellular pH and Thermosensitivity

The effects of amiloride (an inhibitor of Na+/H+ antiport), DIDS (an inhibitor of Na(+)-coupled and Na(+)-independent HCO3-/Cl- exchange) and nigericin (K+/H+ ionophore) alone and in various combinations on the intracellular pH (pHi) and thermosensitivity of SCK tumor cells were studied. Hyperthermia alone at 43 degrees C for 2 h decreased pHi of SCK cells by 0.15-0.20 pH units, as measured fluorometrically using the pH-sensitive dye BCECF. When the cells were treated with 0.5 mM amiloride at 37 degrees C, the pHi declined by 0.10-0.15 pH units at an extracellular pH (pHe) of both 7.2 and 6.6. Amiloride at 0.5 mM enhanced the thermal damage to SCK cells at pHe 6.6 but not at pHe 7.2. DIDS alone at 0.1 mM exerted no effect on pHi or cellular thermosensitivity. DIDS, however, enhanced the effects of amiloride in decreasing pHi and in increasing the thermoresponse of SCK cells, particularly at pHe 6.6. Treatment of the cells with nigericin at 0.1-1.0 micrograms/ml lowered the pHi and enhanced the thermosensitivity of the cells in a dose-dependent manner. Reductions in pHi and increases in thermosensitivity by nigericin at the lower concentration at pHe 6.6 were far greater than at pHe 7.2. When a mixture of 1.0 micrograms/ml nigericin, 0.5 mM amiloride, and 0.1 mM DIDS was present in the medium, the pHi rapidly decreased by about 0.3 and 0.4 pH units at pHe 7.2 and 6.6, respectively. This drug combination was also extremely effective in sensitizing SCK cells to heat, particularly at pHe 6.6. The fact that the thermosensitization by these drugs at pHe 6.6 is more pronounced than at pHe 7.2 and that intratumor environments are known to be acidic strongly suggested that it may then be possible to enhance the thermal damage with such drugs preferentially in tumors relative to normal tissues.

Increase in Tumor Oxygenation and Radiosensitivity Caused by Pentoxifylline

The effects of pentoxifylline (PTX), a drug commonly used for vascular disorders in humans, on the pO2 in SCK tumors of A/J mice and FSa-II tumors of C3Heb/FeJ mice as well as on the radioresponse of SCK tumors were investigated. When the host mice were injected intraperitoneally (ip) with 5 mg/kg PTX, the tumor pO2 increased slowly, peaked 20-50 min postinjection, and returned to its original level in 70-90 min. The magnitude of the increase in tumor pO2 varied markedly depending on the site and tumors. The magnitude of the changes in tumor pO2 after an ip injection of 25 or 50 mg/kg PTX was similar to that caused by 5 mg/kg PTX, but the pO2 tended to remain elevated longer with the higher dose of PTX. When the A/J mice bearing SCK tumors in the legs were injected ip with 50 mg/kg PTX and the tumors were X-irradiated 20 min later, the radiation-induced growth delay of the tumors was greater than that caused by X irradiation alone. The present study demonstrated that PTX is potentially useful for increasing the pO2 and the radioresponse of human tumors.

Radiation-induced apoptosis in different pH environments in vitro

Abstract Purpose: The effect of environmental pH on the radiation-induced apoptosis in tumor cells in vitro was investigated. Methods and Materials: Mammary adenocarcinoma cells of A/J mice (SCK cells) were irradiated with γ-rays using a 137 Cs irradiator and incubated in media of different pHs. After incubation at 37°C for 24–120 h the extent of apoptosis was determined using agarose gel electrophoresis, TdT-mediated dUTP -biotin nick end labeling (TUNEL) staining, flow cytometry, and release of 3 H from 3 H-thymidine labeled cells. The clonogenicity of the cells irradiated in different pH medium was determined, and the progression of cells through the cell cycle after irradiation in different pHs was also determined with flow cytometry. Results: Irradiation with 2–12 Gy of γ-rats induced apoptosis in SCK cells in pH 7.5 medium within 48 h as judged from the results of four different assays mentioned. Radiation-induced apoptosis declined as the medium pH was lowered from 7.5 to 6.4. Specifically, the radiation-induced degradation of DNA including the early DNA breaks, as determined with the TUNEL method, progressively declined as the medium pH was lowered so that little DNA fragmentation occurred 48 h after irradiation with 12 Gy in pH 6.6 medium. When the cells were irradiated and incubated for 48 h in pH 6.6 medium and the medium was replaced with pH 7.5 medium, DNA fragmentation promptly occurred. DNA fragmentation also occurred even in pH 6.6 medium when the cells were irradiated and maintained in pH 7.5 medium for 8 or longer post-irradiation before incubation in pH 6.6 medium. The radiation-induced G 2 arrest in pH 6.6 medium lasted markedly longer than that in pH 7.5 medium. Conclusion: Radiation-induced apoptosis in SCK cells in vitro is reversibly suppressed in an acidic environment. Taking the results of four different assays together, it was concluded that early step(s) in the apoptotic pathway, probably the DNA break or upstream of DNA break, is reversibly halted by an acidic environment in irradiated cells. Radiation-induced G 2 arrest is prolonged in an acidic environment indicating that the suppression of radiation-induced apoptosis and prolongation of radiation-induced G 2 arrest in an acidic environment are related.

Mechanism of action of lonidamine in the 9L brain tumor model involves inhibition of lactate efflux and intracellular acidification

Malignant gliomas have been associated with a high rate of glycolytic activity which is believed necessary to sustain cellular function and integrity. Since lonidamine (LND) is believed to reduce tumor glucose utilization by inhibition of the mitochondrially-bound glycolytic enzyme hexokinase (HK), 31P magnetic resonance spectroscopy (MRS) was used to noninvasively follow the effects of LND on both tumor pH and the high-energy phosphate metabolites; ATP, phosphocreatine (PCr) and inorganic phosphate (Pi) in subcutaneous rat 9L gliosarcomas. 31P tumor spectra acquired in 5 min intervals pre- and post LND administration of 50 and 100 mg/kg, i.p. revealed an acidotic pH shift of − 0.25 and − 0.45 pH units, respectively within 30 min post administration. The ATP/Pi ratio of 9L tumors decreased to 40% of control and Pi levels increased to 280% of control over a 3 hr period. LND exerted no effect on tumor blood flow and mean arterial blood pressure. Brain and muscle metabolite levels and pH were also unaffected by LND. In vitro measurements of cultured 9L tumor cell intra- and extracellular lactate, pentose phosphate pathway (PPP) and hexokinase (HK) activities suggest that the mode of action of LND involves inhibition of lactate efflux and intracellular acidification. The selective reduction of tumor energy metabolites and pH by LND may be exploitable for sensitizing gliomas to radiation, chemotherapy or hyperthermia.

Cell cycle progression and apoptosis after irradiation in an acidic environment

We investigated the effect of an acidic environment on the radiation-induced G2/M arrest and apoptosis using RKO·C human colorectal cancer cells expressing wild-type p53 and RC10·1 cells, a subline of RKO·C cells deficient in p53 as well as p53+/+ MEFs and p53−/− MEFs (mouse embryonic fibroblasts). The cells were irradiated with 4 Gy or 12 Gy of γ-rays in pH 7.5 medium or pH 6.6 medium. p53 accentuated the progression of cells from radiation-induced G2/M arrest to apoptosis and the pH 6.6 environment suppressed the progression of cells through G2/M-phase to apoptosis after irradiation. Further analysis indicated that the radiation-induced G2/M arrest was due mainly to G2 arrest in both pH 7.5 and pH 6.6. Therefore, it was concluded that p53 enhances, and an acidic environment suppresses, the exit of cells from radiation-induced G2 arrest by altering cyclin B1-Cdc2 kinase activity. Cell Death and Differentiation (2000) 7, 729–738

Effects of amiloride on intracellular pH and thermosensitivity.

Amiloride, a diuretic drug, is a potent inhibitor of Na+/H+ exchange through the plasma membrane, and has been reported to enhance thermal damage in tumor cells in vitro. We investigated the possible relationship between changes in the thermal response of SCK mouse mammary tumor cells in vitro and changes in intracellular pH (pHi) due to amiloride in the present study. At a concentration of 0.5 mM, amiloride reduced the shoulder (Dq) without causing significant change in the slope (Do) of the survival curve of SCK cells heated once at 43 degrees C. On the other hand, 0.5 mM of amiloride sensitized thermotolerant cells to heat as shown by a reduction in Do. The presence of amiloride during the interval between the first and second heatings slightly reduced or inhibited the development of thermotolerance. The pHi was measured with the BCECF fluorescence method. The presence of 0.5 mM amiloride significantly reduced pHi in both pH 7.2 and 6.6 medium. Heating the SCK cells at 43 degrees C in pH 7.2 or 6.6 medium also reduced the pHi. The combined effect of heat and amiloride in reducing the pHi of SCK cells was additive. These results suggest that the effects of amiloride on the thermal response of SCK cells might be mediated in part by a decrease in pHi. The possibility that the effects of amiloride on the thermal response of tumor cells are mediated by other biochemical changes, such as inhibition of protein synthesis, however, could not be ruled out.

Apoptosis and Cell Cycle Progression in an Acidic Environment after Irradiation

Abstract Park, H.J., Lyons, J.C., Griffin, R.J., Limm, B.U. and Song, C.W. Apoptosis and Cell Cycle Progression in an Acidic Environment after Irradiation. Apoptosis and cell cycle progression in HL60 cells irradiated in an acidic environment were investigated. Apoptosis was determined by TUNEL staining, PARP cleavage, DNA fragmentation, and flow cytometry. The majority of the apoptosis that occurred in HL60 cells after 4 Gy irradiation took place after G2/M-phase arrest. When irradiated with 12 Gy, a fraction of the cells underwent apoptosis in G1 and S phases while the rest of the cells underwent apoptosis in G2/M phase. The apoptosis caused by 4 and 12 Gy irradiation was transiently suppressed in medium at pH 7.1 or lower. An acidic environment was found to perturb progression of irradiated cells through the cell cycle, including progression through G2/M phase. Thus it was concluded that the suppression of apoptosis in the cells after 4–12 Gy irradiation in acidic medium was due at least in part to a delay in cell cycle progression, particularly the prolongation of G2/M-phase arrest. Irradiation with 20 Gy indiscriminately caused apoptosis in all cell cycle phases, i.e. G1, S and G2/M phases, rapidly in neutral pH medium and relatively slowly in acidic pH medium. The delay in apoptosis in acidic medium after 20 Gy irradiation appeared to result from mechanisms other than prolonged G2/M-phase arrest.

Effect of acidic environment and p53 on apoptosis induction by hyperthermia

The effect of environmental acidity on the induction of apoptosis by heat was investigated. Human colorectal tumour RKO.C cells, carrying wild-type p53 and isogenic RC10.1 cells deficient in p53, were heated at 42.0 C for 1h in pH7.5 or pH6.6 medium and the apoptosis was assessed based on the flow cytometic determination of DNA content, DNA fragmentation, and PARP cleavage. The degree of apoptosis after heating in pH6.6 medium was greater than that in pH7.5 medium in both RKO.C cells and RC10.1 cells. When heated in the same pH medium, more apoptosis occurred in the RC10.1 cells than in the RKO.C cells. Heating increased the expression of p53 protein and p21 protein markedly in RKO.C cells and slightly in RC10.1 cells. Expression of these proteins was slightly greater in pH7.5 medium than in pH6.6 medium. The expressions of Bax protein and Bcl-2 protein, which are known to control apoptosis, were not altered by heating. It was concluded that an acidic environment enhances heat-induced apoptosis. It was also concluded that heat-induced apoptosis is lessened by p53 and that Bcl-2 and Bax are not involved in the induction of apoptosis by hyperthermia.

Killing of Hypoxic Cells by Lowering the Intracellular pH in Combination with Hyperthermia

The acidic intracellular environment or low intracellular pH (pHi) increases the thermosensitivity of mammalian cells. The cells in a hypoxic environment produce a greater amount of acidic metabolites than those in an oxygenated environment. However, the hypoxic cells are not more thermosensitive than oxygenated cells since the decrease in pHi is minimized by the mechanisms that regulate the pHi such as Na+/H+ antiport. We hypothesized, therefore, that blocking the regulation of pHi might greatly reduce the pHi and increase the thermosensitivity of hypoxic cells. We tested our hypothesis by heating SCK tumor cells under oxygenated and hypoxic conditions in pH 7.5 or 6.6 medium with or without amiloride, an inhibitor of Na+/H+ antiport. We observed that amiloride increased the thermosensitivity of hypoxic cells markedly. Such a thermosensitization of hypoxic cells by amiloride was more pronounced in an acidic environment, with an enhancement ratio of 1.5, than in a neutral environment, with an enhancement ratio of 1.5. We concluded that lowering the pHi by blocking the regulation of pHi in combination with hyperthermia may be a useful way to eliminate the radioresistant hypoxic cells.