Physiologisches Institut, Universität Würzburg, Röntgenring 9, D-97070 Würzburg, Germany
1 To whom correspondence should be addressed. Fax: +49 931 312741. E-mail: gerald.schwerdt{at}mail.uni-wuerzburg.de.
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ABSTRACT |
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Key Words: cisplatin; hypoxia; collecting duct; apoptosis; mitochondria.
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INTRODUCTION |
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Apoptosis is a well-described sort of cell death induced by a variety of substances. The processes in an apoptotic cell are well characterized and several reports describe that mitochondria play a crucial role (Kroemer et al., 1997; Kroemer and Reed, 2000
). Release of cytochrome C and other proteins from mitochondria (Liu et al., 1996
; Patterson et al., 2000
) often induces a series of events which finally leads to activation of caspase-3, followed by DNA ladder formation and cell death. Besides different substances, inhibition of mitochondrial respiration was shown to lead to apoptosis induction (Wolvetang et al., 1994
). Additionally, inhibition of mitochondrial respiration forces the cell to increase the anaerobic glycolysis pathway to guarantee the ATP supply of the cell. In this case, the cell produces increased amounts of lactic acid which acidify either the cell interior and/or the surrounding media.
The collecting duct cell can experience an acidic microenvironment both physiologically and pathophysiologically: physiologically, in the collecting duct the pH underlies considerable variations from alkaline conditions down to pH 4.5 (Hamm and Alpern, 1992; Sabatini and Kurtzman, 1989
) and pathopysiologically hypoxic conditions with lowered extracellular pH (down to pH 6.0; Vaupel et al., 1989
) at the basolateral side of the cells may occur. Furthermore, pH maintenance plays an important role in tumor genesis. It is well known that cancer cells produce acidification of the extracellular compartment. This is due to increased use of anaerobic, lactic acid producing glycolysis, which is the main source of energy production of cancerous cells. At the same time the mitochondria-mediated oxidative phosphorylation is diminished (Gatenby and Gawlinski, 2003
; Warburg, 1956
). These observations underline the importance of mitochondria and extracellular pH on cell survival. Additionally, in collecting duct cells acidic apical pH leads to increased cellular apical uptake, increased transepithelial reabsorptive transport, and increased apoptosis rates induced by other nephrotoxic substances, e.g., ochratoxin A (Dahlmann et al., 1998
; Schwerdt et al., 1997
, 2004
; Zingerle et al., 1997
).
Intact mitochondria are thus a prerequisite for a cell to fulfill its responsibilities in an organism. Therefore, to investigate the role of mitochondria in cisplatin-induced apoptosis, we studied the effects of inhibition of mitochondria or of hypoxia on cisplatin-induced apoptosis in epithelial collecting duct cells. Additionally, the effects of extracellular pH and the role of the intracellular pH on cell survival after cisplatin administration of MDCK-C7 cells were studied.
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MATERIALS AND METHODS |
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Caspase-3 activity assay.
Before incubation with cisplatin or other substances, cells were incubated in serum-free medium for 24 h in Petri dishes (9.62 cm2 culture area, 104 µl media/cm2). Caspase-3 activity was measured according to the manufacturer's instructions (Clontech Laboratories GmbH, Heidelberg, Germany) with slight modifications: cells were washed once with PBS buffer (4°C) and incubated with 150 µl cell lysis buffer (Clontech) for 10 min on ice, harvested, and centrifuged at 16,000 x g for 10 min at 4°C. Sixty µl of the supernatant was incubated with 50 µM DEVD-AFC (end-concentration) for 60 min at 37°C, and fluorescence of the cleaved product, 7-amino-4-trifluoromethylcoumarin (AFC), was measured at 400 nm excitation and 505 nm emission wavelength using a multiwell-multilabel counter (Victor2, Wallac, Turku, Finland). Cleaved AFC was quantified by a calibration curve using known AFC concentrations. As control, cell extracts were incubated as described above but in the presence of caspase-3 inhibitor zDEVD-CHO. No activity could be found under these conditions. Protein content was determined with bicinchoninic acid assay (Pierce) using bovine serum albumin as standard.
LDH activity assay.
Activity of LDH in media and cell lysates was determined in an automatic analyzer (Cobas-Mira, Roche Diagnostics, Mannheim, Germany), using standard protocol (Bergmeyer and Bernt, 1974).
DNA ladder formation assay.
DNA ladder was visualized as described previously (Schwerdt et al., 1999). Briefly, cells in culture medium were collected by short centrifugation. Adherent cells on the Petri dish were harvested in cell lysis buffer (5 mM Tris, 20 mM EDTA pH 8.0, 0.5% Triton X-100), briefly incubated on ice and together with the previous cell pellet centrifuged (20 min, 16,000 x g, 4°C). 50 µg/ml proteinase K and 40 µg/ml RNase A were added to the supernatant and incubated for 60 min at 37°C. DNA was extracted by adding the same volume phenol/chloroform/isoamylalcohol (25:24:1 in TE buffer [10 mM Tris, 1 mM EDTA, pH 8.0]). After shaking and centrifugation (30 min, 3420 x g) the upper phase was collected. One tenth volume 3 M sodium acetate pH 5.2 and two volumes ice cold (20°C) ethanol were added and samples were left overnight at 20°C. After centrifugation (30 min, 16,000 x g, 4°C), pellet was washed with 70% ice cold ethanol and dried. DNA was solved in water. DNA concentration was measured at 260 nm in a photometer. DNA ladder was visualized in 1.5% agarose gel.
Measurement of lactic acid in the medium.
Lactic acid concentration in the media was measured using the lactic acid determination kit from Sigma (Deisenhofen, Germany) following their instructions with slight modifications. In brief, samples were incubated in a 96-well plate for 30 min at 37°C in 200 µl of 200 mM glycine/hydrazine buffer, pH 9.2, containing 1.2 mM NAD+ and 8.3 U/ml lactate-dehydrogenase and absorbance was measured in a multilabel counter at 334 nm. Lactic acid concentrations in the samples were calculated from calibration curve of lactic acid standard solutions.
Determination of intracellular pH.
Intracellular pH of MDCK-C7 cells was measured using the pH sensitive fluorescent dye 2'-7'bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) according to Weiner and Hamm (1989). Cells were incubated on coverslips with the media of interest at 37°C and 5% CO2. After the desired time 2 µM BCECF (final concentration) was added. After 15 min the medium on the coverslips was exchanged against BCECF free medium from cells grown in parallel under the same conditions. Coverslips were transferred to the stage of an inverted Axiovert 100 TV microscope (Zeiss, Oberkochen, Germany) and kept in an aerated and heated chamber under 5% CO2 and 37°C. Excitation wavelengths were 460 and 488 nm. The emitted light was filtered through a bandpass filter (515565 nm). The data acquisition rate was one fluorescence intensity ratio every 10 s using an ICCD camera (Hamamatsu Deutschland, Herrsching, Germany). Images were digitized on-line using video-imaging software (Aquacosmos image acquisition and analysis system, Hamamatsu). After background subtraction fluorescence intensity ratios were calculated. pH calibration was performed after each determination by the nigericin technique, using two calibration solutions (132 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES) adjusted to pH 6.8 and 7.6.
Statistics.
Experiments were done in triplicate with three Petri dishes used each time if not indicated otherwise in the text or figure legends. All data are given as mean values ± SEM. Columns represent the means and error bars the SEM. The significance of difference was determined by the unpaired Student's t-test. p < 0.05 was considered to be statistically significant. To compare the dependent data an analysis of variance with appropriate post-hoc analysis was made (ANOVA-test, Prophet 5.0 software). Expected mean results for the effect of two substances were calculated as follows:
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RESULTS |
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Therefore, no correlation between the intracellular pH and caspase-3 activity could be detected, neither in the presence nor in the absence of cisplatin. This shows that the increased activation of caspase-3 after inhibition of mitochondria is independent of the intracellular pH.
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DISCUSSION |
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Because mitochondria often play a major role in apoptotic processes and mitochondria seem to be one target of cisplatin (Liang and Ullyatt, 1998; Melendez-Zajgla et al., 1999
; Murata et al., 1990
) we investigated the effects of inhibition of mitochondrial function on caspase-3 activity in the presence of cisplatin. We observed that blocking of the respiratory chain by rotenone or the F1Fo-ATP synthase was not enough to increase caspase-3 activity or to induce DNA ladder formation. Cisplatin significantly increased the effect of inhibition of the respiratory chain or blockade of F1Fo-ATP synthase cisplatin on caspase-3 activity. Therefore, functional mitochondria seem not to be necessary for apoptosis or rather caspase-3 activation as reported by others (Li et al., 2003
; Wolvetang et al., 1994
). Only after treatment of cells with CCCP caspase-3 activity and DNA ladder formation were not significantly increased by cisplatin due to the already high caspase-3 activity after treatment of the cells with CCCP. This difference in the effect which the inhibitors of mitochondrial function exert are explainable by the observation that (1) CCCP alone already can induce apoptosis (de Graaf et al., 2004
) and (2) the action of CCCP is not only restricted to mitochondria but also to other cellular targets such as endosomes or the cell membrane (Zhang et al., 2000
). Necrotic cell death induced by cisplatin and inhibition of mitochondria showed the same pattern as apoptotic cell death. These observations demonstrate that a disturbance of the mitochondria caused by a single substance, which itself may lead to only mild perturbations can be potentiated when cisplatin is present. Notably, this occurs already at cisplatin concentrations which itself do not lead to increased cell death. Specific inhibition of mitochondria therefore shifts the threshold concentration of cisplatin-induced cell death down to lower cisplatin concentrations.
Under ischemic conditions or oxygen deprivation the oxygen-dependent mitochondria do not participate in energy supply. As the cisplatin-induced caspase-3 activity is increased after artificial blockage of mitochondria we tested the effect of a turn off of mitochondria by pronounced hypoxic conditions. Similar to the artificial blockage of mitochondria by rotenone or other inhibitors, the cisplatin-induced caspase-3 activity was increased under hypoxic conditions. This demonstrates that respiring mitochondria are necessary to avoid cisplatin-induced apoptotic events in MDCK-C7 cells and that an increased glycolysis rate leads to increased cisplatin-induced apoptosis.
Cellular ATP levels may be diminished after blockade of mitochondria although increased glycolysis may compensate partially the impending ATP fall. Under these conditions, ATP-fueled export of cisplatin by transporters such as p-gp (multidrug resistence protein, MDR1) or MRP2 (multidrug resistence associated protein) may be limited. This would lead to increased intracellular cisplatin levels which could explain the observed toxic effects. However, there is no evidence of p-gp expression in MDCK cells and in MDCK cells transfected with p-gp no transport of cisplatin was observed (Pastan et al., 1988). Additionally, we were not able to measure a MRP2 transport activity in MDCK-C7 cells (not shown). This shows that limited export of cisplatin cannot explain the effects of blocked mitochondria on cisplatin-induced cell death (at least for these two transporters).
Concomitant with an increase in caspase-3 activity, an acidification of the extracellular medium was observed when mitochondria were blocked or after hypoxic conditions. This acidification was due to enhanced production of lactic acid under conditions of blocked mitochondria. Mitochondrial inhibitors when administered alone led to increased lactate formation, but in the presence of cisplatin this lactate formation was significantly increased. This demonstrates an increased use of anaerobic gylcolysis to fulfill the cellular energy demand when cells are exposed to cisplatin and mitochondrial inhibitors.
Because the extracellular media were acidified during simultaneous inhibition of mitochondria and cisplatin exposure, we investigated the effect of extracellular pH on caspase-3 activity. A slight acidification or alkalization led to no significant potentiation or antagonisation of the effect which cisplatin at low dose (20 µM) had on caspase-3 activity. Only the effect of high cisplatin concentration (100 µM) was reduced by extracellular alkalization. At more acidic conditions (pH 6.8) the effect of cisplatin (100 µM) on caspase-3 activity was potentiated significantly. Thus, the results presented here show that the extracellular pH plays an important role on cell survival in the presence of cisplatin. It is important to remember that acidic pH occurs daily in the collecting duct under physiologic conditions. But also under pathophysiologic conditions acidic pH occurs. Hypoxic conditions leading to acidic cell surroundings are not unusual in tumor tissues and an aggravation of cell death may be desirable in that case.
We also found that the intracellular pH is not a decisive factor for apoptosis induction in the presence of cisplatin. Even though many studies have demonstrated that apoptosis is associated with a decrease in cytosolic pH (Matsuyama et al., 2000; Segal and Beem, 2001
), no correlation between caspase-3 activity and intracellular pH was observed. For example, 24 h exposure to rotenone led to a pHin of 6.94 and caspase-3 activity was close to the activity of control cells (126% compared to control). Under more alkaline conditions, with pHin of 7.435, the caspase-3 activity was nearly the same as with rotenone (121% compared to control).
In conclusion, we show that inhibition of mitochondria or severe hypoxic conditions can significantly increase the damaging effect of cisplatin in collecting duct-derived cells. This effect is not dependent on the intracellular pH but dependent on extracellular pH and on functional respiring mitochondria. Blocked or because of hypoxia not-used mitochondria increase the apoptosis-inducing effect of cisplatin. This suggests avoiding hypoxia or acidic conditions in the collecting duct when cisplatin is administered.
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ACKNOWLEDGMENTS |
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