![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, May 2006, p. 1664-1671, Vol. 50, No. 5
0066-4804/06/$08.00+0 doi:10.1128/AAC.50.5.1664-1671.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Kozie,
Krzysztof Zabocki,* and
Jerzy Duszy
ski
Nencki Institute of Experimental Biology, Pasteura 3, 02-093 Warsaw, Poland
Received 2 December 2005/ Returned for modification 10 January 2006/ Accepted 10 February 2006
 |
ABSTRACT |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
INTRODUCTION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
) across the inner membrane. Their de-energization due to inhibition of the respiratory chain or to uncoupling of oxidative phosphorylation may result in cellular energy deficiency and impairment of -dependent processes, among them intracellular calcium signaling (11, 15, 26, 27). In metazoa mitochondrial DNA (mtDNA) typically encodes 13 proteins involved in oxidative phosphorylation; two of them are elements of the Fo subunit of mitochondrial ATPase, while 11 proteins are components of the respiratory chain complexes I, III, and IV. In addition, mtDNA contains 22 genes encoding tRNAs and two genes for rRNA. Therefore, perturbations in mtDNA content and/or processing may cause severe impairments in the cellular energy metabolism. Ciprofloxacin is a 4-fluoroquinolone antibiotic commonly used in therapy of many microbial infections. Its antibacterial activity is based on the inhibition of the bacterial enzyme DNA gyrase. Unfortunately, this compound also inhibits mammalian topoisomerase II, especially its mitochondrial isoform. This side effect results in improper mtDNA replication and therefore causes mtDNA fragmentation and a gradual decrease in mtDNA content (4, 14). A prolonged exposure of murine leukemia cells to ciprofloxacin applied at concentrations ranging from 20 to 80 µg/ml results in a gradual depletion of mtDNA with a concomitant reduction of the respiration rate and stimulation of glycolysis (14).
Disturbance of the mitochondrial energy metabolism has been proposed to explain the cytotoxic effect of ciprofloxacin. The sensitivities of various cell lines to ciprofloxacin may differ significantly (13, 14). Some of them, for example, bladder cancer cells, exposed to low concentrations of this compound may undergo irreversible changes leading to apoptotic death (2, 3). Ciprofloxacin applied at a concentration of 80 µg/ml was found to both stimulate and inhibit transcription of many genes in human lymphocytes (6).
There is a growing body of evidence that ciprofloxacin, especially at higher concentrations, in the range from 50 µg/ml to 400 µg/ml, interferes with the cell cycle at the S/G2 checkpoint and commits cells to the apoptotic pathway. It also induces apoptosis in activated Jurkat cells (12). Recently, numerous reports have been published concerning the antitumor activity of ciprofloxacin based on stimulation of apoptosis in various malignant cells. This makes ciprofloxacin a candidate anticancer drug (3, 10).
Capacitative calcium entry, the most common pathway of Ca2+ influx in electrically nonexcitable cells, is preceded by calcium release from the lumen of the endoplasmic reticulum (ER). In other words, opening of the plasma membrane Ca2+ channels, the so-called store-operated calcium channels (SOCs), depends on the filling state of intracellular calcium stores (17, 19, 20). In many electrically nonexcitable cell types, including Jurkat cells, energized mitochondria are necessary for intensive calcium influx through SOCs to occur. It is well established that respiring, well-coupled mitochondria buffer the excess of calcium accumulated in the vicinity of calcium channels and thereby limit local Ca2+ concentration. This reduces the feedback inhibition of calcium channels and promotes their open state (7, 8, 11, 15, 16).
The main purpose of this study was to correlate the effects of long-term exposure to a relatively low concentration (25 µg/ml) of ciprofloxacin on the mtDNA content and mitochondrial energy state (expressed as the respiration rate and ) with those on capacitative calcium influx and viability of human lymphoidal cells (Jurkat). We show that ciprofloxacin induces a decrease in both the mitochondrial respiration rate and in parallel with a reduction of the mtDNA content. More importantly, the changes in the mitochondrial energy metabolism correlate with decreased calcium entry into these cells. Because the capacitative mode of Ca2+ influx is the major route of calcium entry into lymphocytes, it seems possible that ciprofloxacin-evoked inhibition of cell proliferation may, at least partially, result from aberrant calcium signals.
|
MATERIALS AND METHODS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-32P]dCTP, the Megaprime DNA labeling system, and salmon sperm DNA were from Amersham Biosciences (United Kingdom). Pronase was purchased from Roche Diagnostics GmbH (Mannheim, Germany). The Gene Ruler 1-kb DNA ladder and HindIII were from Fermentas MBI (Hanover, MD). Gel blot paper GB 003 and nylon transfer membrane Nytran SuPerCharge were from Schleicher and Schuell GmbH (Dassel, Germany). The QIAquick gel extraction kit and Taq PCR core kit (250) were from QIAGEN GmbH (Hilden, Germany). Other chemicals were of analytical grade. Cell culture. Human lymphoblastoid T cells (Jurkat) were grown in RPMI 1640 medium supplemented with 2 mM glutamine (Gibco BRL), 50 µM uridine, 10% fetal bovine serum (Gibco BRL), penicillin (100 U/ml), and streptomycin (50 µg/ml) in a humidified atmosphere of 5% CO2-95% air at 37°C. In cases indicated in the figures, the culture medium was supplemented with ciprofloxacin (25 µg/ml). The cells were seeded every 2 days, and all experiments were conducted 1 day after seeding.
Cytosolic Ca2+ concentration ([Ca2]c) measurement. The buffered saline solution (BSS) consisted of 132 mM NaCl, 5 mM KCl, 25 mM HEPES, 1 mM MgCl2, 0.5 mM NaH2PO4, 1 mM pyruvate, and 5 mM glucose. The pH was adjusted to 7.2 at 30°C. As indicated in Fig. 6, 0.12 µM oligomycin or 100 nM thapsigargin (both dissolved in dimethyl sulfoxide) was added.
|
Cellular ATP content and oxygen uptake. ATP was measured spectrofluorimetrically in neutralized cellular acidic extracts as an increase in NADPH fluorescence (340-nm and 465-nm excitation and emission wavelengths, respectively) using a standard enzymatic assay with hexokinase and glucose-6-phosphate dehydrogenase (25). Mitochondrial respiration was measured in digitonin-permeabilized cells with a Clark-type oxygen electrode (YSI, Yellow Springs, OH) equipped with a homemade electronic device that enabled recording of the first derivative of the O2 concentration trace (equivalent to the O2 uptake rate, d[O2]/dt) (24).
Mitochondrial membrane potential ().
The electric potential of the inner mitochondrial membrane was measured in situ using flow cytometry in intact cells stained with the JC-1 fluorescent probe (5). The cells (5 x 106) were suspended in 2 ml of the culture medium supplemented with 2 µl of JC-1 (stock 2 mg/ml in dimethyl sulfoxide). The cells were incubated for 10 min at 37°C and washed twice with BSS containing 1 mM pyruvate and 5 mM glucose. Finally, the cells were suspended in BSS and, as indicated in Fig. 3, treated with 0.12 µM oligomycin 15 min prior to measurements. JC-1 fluorescence was measured using a flow cytometer (Becton Dickinson) (excitation with an argon laser at 488 nm and emission wavelength of 530/590 nm) in 104 cells.
|
generated due to oxidation of succinate as a sole substrate was measured in digitonin-permeabilized cells and evaluated from the uptake of safranine O (1). This was determined from fluorescence quenching monitored at the wavelengths of 495 nm and 586 nm for excitation and emission, respectively, with a Shimadzu RF 5000 spectrofluorimeter. Isolation of cellular DNA. Cells (12 x 107) were collected by centrifugation at 200 x g for 5 min at room temperature. The supernatant was discarded, and the pellet was rinsed with phosphate-buffered saline (PBS) and centrifuged as above. Then, the pellet was suspended in 7.5 ml of a solution containing 75 mM NaCl and 0.25 mM EDTA, pH 8.0. After the addition of pronase (1 mg/ml) and sodium dodecyl sulfate (1%), the suspension was incubated for 16 h at 37°C. Incubation was terminated by the addition of 3 ml of 6 M NaCl (1.7 M final concentration). This suspension was centrifuged at 18,000 x g at 18°C. The supernatant was collected and centrifuged again under the same conditions. The final supernatant was mixed with a double volume of ice-cold ethanol (96%) and incubated at –20°C for 30 min. After centrifugation (18,000 x g for 10 min at 4°C) the supernatant was discarded, and the pellet was suspended in 1 ml of 70% cold ethanol, incubated at 4°C for 10 min, and centrifuged at 4°C for 10 min at 18,000 x g. This step was performed in duplicate. The final pellet was air dried. The dry DNA pellet was dissolved in 100 µl of 10 mM Tris-HCl and 1 mM EDTA, pH 7.4, and the DNA concentration was determined spectrophotometrically (21).
Preparation of DNA probes.
For determination of the proportion of mtDNA to total isolated DNA, two 32P-labeled DNA probes homologous to HindIII-digested DNA fragments were prepared. One of them (3,013 bp) was specific for a 5,477-bp mtDNA fragment, and the second one (1,185 bp) was for a nucleotide sequence of about 9,500 bp within the nuclear gene encoding 18S rRNA. Purified probes obtained by PCR were labeled with [-32P]dCTP using a Megaprime DNA labeling system (Amersham Biosciences).
Gel electrophoresis, Southern hybridization, and autoradiography. Isolated total cellular DNA (10 µg per sample) was incubated with HindIII endonuclease (7 U per µg of DNA; Fermentas) for 18 h at 37°C in R+ buffer (10 mM Tris-HCl, pH 8.5, 10 mM MgCl2, 100 mM KCl, 0.1 mg/ml bovine serum albumin) in a total volume of 20 µl. The digestion was terminated by the addition of 2 µl of sample buffer containing 200 mM EDTA and 40% Ficoll 400 (wt/vol).
Digested DNA was subjected to electrophoresis in 0.7% horizontal agarose gel (20 cm long) in 0.5x TBE buffer (45 mM Tris-borate, 2 mM EDTA). Following the electrophoresis at 30 V for 56 h, the gel was stained with ethidium bromide, and DNA was visualized in UV light at 254 nm. DNA was transferred onto a nitrocellulose filter (NYTRAN SuPerCharge; Schleicher and Schuell) and hybridized to previously prepared 32P-labeled DNA probes (21).
Expression of results. Calculated values are means ± standard errors for the number of independent experiments indicated in the figure legends. The statistical evaluation of differences was calculated by Student's t test.
|
RESULTS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Effect of ciprofloxacin on mtDNA content.
Figure 1A (a typical Southern blot) and B (collective data from three experiments, reflecting changes in mtDNA content in relation to nuclear DNA) show that exposure of Jurkat cells to ciprofloxacin for several days resulted in a substantial reduction in mtDNA content. The levels of mtDNA found on days 4, 7, and 11 of ciprofloxacin treatment were similar and amounted to around 30% of the control value. Presumably, this ciprofloxacin-induced reduction in mtDNA content was secondary to the inhibition of mitochondrial topoisomerase, which in turn caused disturbances in mtDNA replication/maintenance. This effect, also described by other authors (13), was the basis for our further studies. The decrease in mtDNA level was expected to perturb the mitochondrial energy state expressed by a lowered mitochondrial respiration rate and reduced and ATP formation.
|
.
As shown in Fig. 2, treatment of Jurkat cells with ciprofloxacin reduced the rate of oxygen consumption (measured polarographically with a Clark electrode) by digitonin-permeabilized cells incubated in the presence of malate plus glutamate. The inhibition of respiration started to be visible after 7 days of incubation with ciprofloxacin. A similar pattern was also observed with succinate (in the presence of rotenone), a respiratory substrate entering the respiratory chain via complex II. Interestingly, in cells treated with ciprofloxacin for 11 days, the rate of oxygen consumption with succinate plus rotenone (but not with malate plus glutamate) returned to the control value, which suggests that Jurkat cells may partially adapt to ciprofloxacin. In other words, oxidation of succinate (occurring via complexes II, III, and IV) becomes efficient again whereas oxidation of malate plus glutamate (occurring via complexes I, III, and IV) is blocked. This observation indicates a progressive and permanent inhibition of respiratory complex I, strongly limiting the electron flow through the respiratory chain, with the activities of complexes III and IV being apparently impaired to a lower extent, allowing respiration from succinate. Ciprofloxacin added directly to the assay using cells grown in the absence of the drug did not inhibit oxygen consumption (data not shown).
|
as measured by flow cytometry in intact cells stained with the JC-1 fluorescent probe and incubated in the presence of 1 mM pyruvate and 5 mM glucose. This effect was clearly visible 7 days after the drug was added to the culture medium (Fig. 3B, left column). Moreover, addition of 0.12 µM oligomycin 2 min before measurements to inhibit mitochondrial ATPase and thereby prevent formation at the expense of hydrolyzed ATP resulted in a decrease of in cells exposed to ciprofloxacin for 4 days and enhanced the effect of ciprofloxacin in cells incubated with the drug for longer periods (Fig. 3B, right column). Oligomycin did not affect in control cells (Fig. 3A). Because the cells were incubated in a glucose-containing medium, the effect of oligomycin strongly suggests that Jurkat cells with impaired respiratory function were able to restore (at least to some extent) at the expense of ATP synthesized glycolytically. When the mitochondrial ATPase was inhibited (e.g., by oligomycin), this route of generation was blocked.
Figure 4 shows the inhibitory effect of ciprofloxacin on measured with safranine O as a fluorescent probe in digitonin-permeabilized cells incubated with succinate (plus rotenone) as a sole substrate. It is noteworthy that the recovery of respiration with succinate observed in cells growing for 11 days with ciprofloxacin (Fig. 2) was not accompanied by a parallel recovery of (Fig. 4).
|
|
(Fig. 3). |
DISCUSSION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
in ciprofloxacin-treated cells indicates that may be sustained at the expense of glycolytically generated ATP. It seems that impairment of respiratory chain-related generation of precedes the inhibition of ATPase (which catalyzes the ATP-dependent restoration of ). This in turn suggests a longer lifetime of FoF1-ATPase (encoded partially by mtDNA) in comparison to mitochondrion-encoded respiratory chain subunits. Alternatively, it may indicate that the total activity of mitochondrial ATPase significantly exceeds the maximal activity of the respiratory chain.
An incomplete collapse of as observed in intact cells through day 7 after ciprofloxacin was added to the culture medium and its only partial sensitivity to oligomycin indicate that a large portion of mitochondria are still energetically efficient. In addition, despite the attenuation of mitochondrial ATP synthesis triggered by ciprofloxacin, the availability of ATP is not limiting for the generation of , since may be even further abolished by oligomycin. These facts as well as a full recovery of the cellular ability to restore intracellular ATP content in the presence of glucose after 11 days of cell exposure to ciprofloxacin are most likely due to upregulation of glycolysis and explain why 25 µg/ml ciprofloxacin does not cause cellular necrosis. This is additionally supported by a low proportion of ciprofloxacin-treated cells stained with trypan blue, which was not significantly higher than that observed in control cells (data not shown).
The recovery of respiration in the presence of succinate (plus rotenone) as a sole substrate (Fig. 2) (but not with NADH-delivering substrates) indicates that complexes III and IV are not responsible for the almost complete inhibition of oxygen consumption observed in the presence of glutamate plus malate after 11 days of incubation with ciprofloxacin. On the other hand, no recovery of under such conditions may result from either the uncoupling of oxidative phosphorylation due to an enhanced proton leak through the inner mitochondrial membrane (i.e., through impaired FoF1-ATPase or directly across the lipid bilayer) or from the affected vectorial proton transport by complexes III and IV (despite uninhibited electron flow through the respiratory chain). The latter possibility resembles the so called "slip" phenomenon described previously (18). In that case, ciprofloxacin (most probably acting via partial depletion and/or damage of mtDNA and therefore downregulation of certain elements of the respiratory chain or components of FoF1-ATPase) could alter the stoichiometry of the mitochondrial proton pump(s) in respiratory complexes III and/or IV. Such an effect has been described for bupivacaine-treated mitochondria (22). Oxygen consumption in the presence of malate and glutamate is permanently inhibited most probably because of a serious functional damage of complex I. Interestingly, rotenone exhibits only a partial inhibitory effect on oxygen consumption in cells grown in a ciprofloxacin-containing medium, incubated in the presence of malate plus glutamate, while in the control cells it fully blocks cellular respiration (Fig. 2). In addition, the inhibitory effect of rotenone disappears gradually during the course of ciprofloxacin treatment. This finding indicates that cells exposed to ciprofloxacin may intensively utilize substrates entering the respiratory chain downstream complex I. Most likely, the endogenous levels of such substrates are increased in cells exposed to ciprofloxacin. Taken together, the decrease in mtDNA content observed in Jurkat cells treated with ciprofloxacin (Fig. 1) leads to the complete inactivation of complex I, while the activities of complexes III and IV, although reduced, are still sufficient for electron transport from complex II (succinate dehydrogenase) and from other NAD+-independent substrates. However, it must be emphasized that mitochondrial Ca2+ uptake and therefore the rate of Ca2+ entry into the cell through SOCs directly depend on the but not on the rate of respiration per se (see below). Thus, the decrease in the mitochondrial potential is sufficient to permanently inhibit Ca2+ influx. Explanation of the mechanism responsible for the restoration of oxygen consumption without concomitant energization of the mitochondrial membrane in ciprofloxacin-treated cells incubated with succinate as a sole substrate, although tempting, is beyond the scope of this paper.
The full recovery of the cellular ability to synthesize ATP in the presence of glucose (Fig. 5) as well as the increase in the respiration rate in the presence of succinate observed on day 11 of the experiment (Fig. 2), preceded by a significant reduction of both parameters detected on day 7, may indicate adaptation of Jurkat cells to ciprofloxacin. On the other hand, the persistently abolished (Fig. 3 and 4), irreversibly inhibited respiration in the presence of NAD+-utilizing substrates (Fig. 2), and progressive inhibition of capacitative calcium entry (Fig. 6) point to partial adaptation to ciprofloxacin rather than to selection of ciprofloxacin-resistant cells.
The transient inhibition of glycolytic ATP synthesis on day 7 of incubation with ciprofloxacin indicates that this antibiotic not only acts in an mtDNA-dependent manner but also influences metabolic processes independent of mitochondrial gene expression.
Ca2+ entry into the mitochondrial matrix is driven by . Because the mitochondrial Ca2+ uniporter has a relatively low affinity for calcium ions, only mitochondria exposed to high [Ca2+]c (which happens in the neighborhood of Ca2+ channels) are able to accumulate calcium efficiently. As has been documented by several authors (7, 11, 15, 16), mitochondria located in close vicinity to SOCs accumulate Ca2+ and therefore protect these channels against feedback inhibition. A decrease in prevents mitochondrial Ca2+ uptake and consequently may lead to an inhibition of Ca2+ entry into the cell. The effect of ciprofloxacin on calcium influx requires several days to occur, which coincides with a decrease in . Thus, impairment in the mitochondrial energy state explains the ciprofloxacin-evoked inhibition of capacitative calcium entry into Jurkat cells. These effects may seriously affect calcium signaling and cause improper regulation of many calcium-dependent cellular processes, including cell division, gene expression, and exocytosis. Presumably, the decreased rate of proliferation observed in cells grown in the presence of ciprofloxacin is connected with a depression of capacitative calcium entry. It also seems possible that prolonged treatment with this antibiotic may influence the response of lymphocytes to antigens. However, these conjectures need to be confirmed. Therefore, understanding the precise mechanisms of ciprofloxacin action in human cells seems to be very important.
It has been suggested that ciprofloxacin induces apoptosis in some cell lines. Therefore, this compound could be considered as a potential anticancer drug. However, for such a purpose ciprofloxacin has to be applied at much higher concentrations, and its effects require only a few hours to develop. Such ciprofloxacin-induced cell death is attributed to stimulation of the mitochondrial permeability transition pore and to activation of the mitochondrion-dependent apoptotic pathway (2). The reduction of mtDNA content due to an inhibition of mitochondrial topoisomerase needs several cell cycles to occur. Therefore, the short-term proapoptotic effects of ciprofloxacin cannot be attributed to any long-term decrease in mtDNA content. The data presented in this paper concern the side effects of antimicrobial therapy rather than those related to antitumor treatment and might be important when long-term administration of 4-fluoroquinolones is undertaken.
|
ACKNOWLEDGMENTS |
|---|
This work was partially supported by the State Committee for Scientific Research under grant 3 P04A 041 23.
|
FOOTNOTES |
|---|
|
REFERENCES |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
ski. 2000. The role of mitochondria in the regulation of calcium influx into Jurkat cells. Eur. J. Biochem. 267:877-884.[Medline]
ckowski, D. Dymkowska, and L. Wojtczak. 2004. Effects of N-acylethanolamines on mitochondrial energetics and permeability transition. Biochim. Biophys. Acta 1657:151-163.[Medline]ski. 2003. pH-dependent effect of mitochondria on calcium influx into Jurkat cells: a novel mechanism of cell protection against calcium entry during energy stress. Cell Calcium 33:91-99.[CrossRef][Medline]ski. 2005. Extracellular pH modifies mitochondrial control of capacitative calcium entry in Jurkat cells. J. Biol. Chem. 280:3516-3521.This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Clin. Vaccine Immunol. | Clin. Microbiol. Rev. |
|---|---|
| J. Clin. Microbiol. | ALL ASM JOURNALS |
