* Department of Cell and Developmental Biology and
Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7090
Received September 9, 2002; accepted January 21, 2003
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ABSTRACT |
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Key Words: salicylate; mitochondrial permeability transition; t-butylhydroperoxide; A23187; tumor necrosis factor; necrosis; apoptosis.
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INTRODUCTION |
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Salicylate, the active metabolite of aspirin, decreases the threshold for onset of the MPT in isolated rat liver and kidney mitochondria (Al Nasser, 1999; Trost and Lemasters, 1996
). Other nonsteroidal anti-inflammatory drugs (NSAIDS), such as diclofenac and mefenamic acid, also promote the MPT (Uyemura et al., 1997
). In cultured hepatocytes, salicylate alone can cause the MPT and cell death at high concentrations (> 3 mM), and salicylate-dependent onset of the MPT may be responsible for Reyes syndrome in children receiving aspirin and some other therapeutic agents, such as valproic acid (Trost and Lemasters, 1996
, 1997
).
Aspirin and other NSAIDS appear to be chemopreventive against colon, lung, and breast cancer, possibly by promoting apoptosis of transformed cells (Shiff and Rigas, 1997). In cancer chemotherapy, the goal is to cause selective killing of tumor cells. Chemotherapeutic agents induce apoptosis by a variety of mechanisms, including induction of the MPT, as shown for the chemotherapeutic agent VP-16 (Custodio et al., 2001
). However, cancer cells frequently develop resistance to chemotherapy, and treatments that sensitize cells to onset of the MPT might reverse resistance to chemotherapeutic agents.
Accordingly, our goal was to test the hypothesis that the primary metabolite of aspirin, salicylate, potentiates necrotic and apoptotic cell killing mediated by onset of the MPT. We show here that salicylate at a nontoxic concentration makes cultured rat hepatocytes more vulnerable to necrotic cell killing from oxidant stress and calcium ionophore treatment and to apoptotic killing after TNF-, an effect that is prevented or delayed by the MPT blocker, CsA. Moreover, salicylate enhances tumor cell apoptosis by both intrinsic and extrinsic pathways. These findings suggest that salicylate has the potential to promote MPT-dependent apoptosis in cancer chemoprevention and chemotherapy.
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MATERIALS AND METHODS |
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Tumor cell lines.
MCF-7 human breast tumor cells with caspase 3 were the gift of Dr. Anna-Liisa Nieminen and were cultured in RPMI 1640 medium with 10% fetal calf serum in 24-well microtiter plates. Experiments were performed after the cells had grown to approximately 90% confluency. PLC/PRF/5 human hepatoma cells were purchased from the American Type Culture Collection (Rockville, MD) and grown to 90% confluency in RPMI 1640 medium with 10% fetal calf serum in 24-well microtiter plates.
Cell viability assay.
Viability of hepatocytes cultured on microtiter plates was monitored by propidium iodide fluorometry using a multi-well fluorescence scanner (FLUOstar 403, BMG LabTechnologies GmbH, Offenburg, Germany), as described (Nieminen et al., 1992). Briefly, hepatocytes in 24-well microtiter plates were incubated in KRH containing 30 µM propidium iodide. Fluorescence from each well was measured using excitation and emission wavelengths of 544 nm (25 nm band pass) and 590 nm (35 nm band pass), respectively. For each experiment, an initial fluorescence measurement (A) was made 20 min after addition of propidium iodide and then at intervals thereafter. Individual experiments were terminated with 375 µM digitonin to permeabilize all cells, and a final fluorescence measurement (B) was obtained 20 min later. The percentage of cell death (D) was calculated as D = 100 - 100(B - X)/(B - A), where X is fluorescence at any given time. As the plasma membrane permeability barrier fails at onset of necrotic cell death, propidium iodide enters cells, binds to DNA, and increases its fluorescence (Nieminen et al., 1992
). Loss of cell viability assessed by propidium iodide fluorometry correlates closely with trypan blue uptake and enzyme release (Gores et al., 1988
) and is indicative of the secondary necrosis of late apoptosis.
Induction of apoptosis.
To sensitize hepatocytes and PLC/PRF/5 human hepatoma cells to apoptosis induced by TNF- and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), respectively, cultured cells were infected with an Ad5I
B adenovirus that expresses an I
B superrepressor that blocks the anti-apoptotic NF
B signaling pathway, as described previously (Bradham et al., 1998
; Kim et al., 2002
). Briefly, in 90% confluent hepatoma cells or 2 h after plating of hepatocytes, the culture medium was changed to hormonally defined medium (HDM) containing 30 plaque forming units/cell of Ad5I
B for 2 h at 37°C (Bradham et al., 1998
). After two more hours, the cultures were washed three times with phosphate-buffered saline, and then the cells were treated with TNF-
/TRAIL and/or salicylate in HDM. After exposure, cell viability was determined by propidium iodide fluorometry.
Caspase-3 measurement.
Caspase-3 activity was performed using a commercial kit (Bio-Rad Laboratories, Hercules, CA) based on 7-amido-4-trifluoromethyl-coumarin (AFC) release from a tetrapeptide fluorogenic substrate following the manufacturers instructions. Briefly, whole cell lysates were incubated 2 h at 37°C. Fluorescence (excitation at 370 nm and emission at 490 nm) was monitored at 1 h intervals using a fluorescence plate reader and normalized to Lowry protein concentration. Caspase 3 activity was expressed as the percentage increase over the basal activity of untreated Ad5IB-infected hepatocytes.
Confocal microscopy.
For confocal microscopy, TNF--treated hepatocytes were incubated in HDM supplemented with 25 mM Na-HEPES buffer, pH 7.4 to stabilize pH during confocal microscopic observations. Approximately 30 min before imaging, hepatocytes were loaded with 250 nM tetramethylrhodamine methylester and 1 µM calcein-AM (TMRM, Molecular Probes, Eugene, OR) in TNF-
-free HDM for 15 min. After loading, the hepatocytes were washed, and the original TNF-
-containing HDM was added back. The coverslips were then mounted in a Focht Chamber (Bioptechs, Butler, PA) on the stage of a Zeiss LSM-410 laser scanning confocal microscope. Images of green calcein fluorescence and red TMRM fluorescence were collected, as described previously (Nieminen et al., 1995
). Briefly, green and red fluorescence was excited simultaneously with the 488 and 568 nm lines of an argon-krypton laser. Emitted fluorescence was then divided by a 568 nm emission dichroic reflector and detected through a 525545 nm (green) bandpass filter and 590 nm (red) long pass filter. All imaging was performed with a Zeiss 63X (N.A. 1.4) planapochromat oil immersion objective lens. Pinholes were set to 0.9 airy units in both red and green channels to maximize z-axis resolution.
Statistics.
Differences between means were compared by the Students t-test using a level of significance of p < 0.05. Data are shown as means ± SEM.
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RESULTS |
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Enhancement by Salicylate of TNF--Induced Apoptosis and Protection by CsA
To render normally unresponsive hepatocytes sensitive to TNF-, we blocked NF-
B with an I
B-superrepressor expressing adenovirus (Ad5I
B). After treatment with 30 ng/ml TNF-
, we showed previously that about 85% of I
B superrepressor-expressing cultured rat hepatocytes lost viability after 24 h, as measured by trypan blue uptake (Bradham et al., 1998
). This cell killing was apoptosis, as documented by cell shrinkage, chromatin condensation, nuclear fragmentation, internucleosomal DNA fragmentation, and caspase activation. In the current study, 1 mM salicylate caused very little cell killing assessed by propidium iodide fluorometry in I
B superrepressor expressing hepatocytes and in hepatocytes not treated with adenovirus (Fig. 3
; some data not shown). At a 30-fold lower concentration than used previously, TNF-
(1 ng/ml) caused only modest apoptotic cell death 3.3, 8.8, and 35.5% after 8, 12, and 24 h, respectively (Fig. 3
). By contrast, when cells were exposed to TNF-
plus salicylate, cell killing doubled or more to 22.9, 32.1, and 70.1%, respectively, after 12, 16, and 24 h (Fig. 3
). After longer times, virtually all cells lost viability (data not shown). CsA nearly completely prevented this salicylate-enhanced apoptosis after low-dose TNF-
(Fig. 3
).
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DISCUSSION |
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In previous work from our laboratory, salicylate was shown to lower the threshold for onset of the MPT in isolated rat liver mitochondria, although salicylate by itself did not cause mitochondrial inner membrane permeabilization (Trost and Lemasters, 1997). In intact hepatocytes, high concentrations of salicylate (
3 mM) promoted onset of the MPT and subsequent cell killing, especially when the hepatocytes were loaded with Ca2+ (Trost and Lemasters, 1997
). However, at low dose (
1 mM), salicylate did not cause cell killing, although in isolated mitochondria such salicylate concentrations still decreased the threshold for onset of the MPT. Since previous work showed that necrotic cell death caused by t-BuOOH and Br-23187 and apoptotic cell death induced by TNF-
, TRAIL, and etoposide involve the MPT and are blocked by CsA, the aim of current study was to test the hypothesis that salicylate lowers the threshold for apoptotic and necrotic cell killing in these models.
Here, we confirm that 1 mM salicylate alone caused little necrotic or apoptotic killing of hepatocytes. Nonetheless, this nontoxic salicylate concentration enhanced the acute cytotoxicity to hepatocytes of Br-A23187 (Fig. 1) and t-BuOOH (Fig. 2
) and the apoptotic response to low-dose TNF-
(Fig. 3
). CsA inhibited salicylate-sensitized cell killing with all three treatments, although least with t-BuOOH. These findings suggest that this sensitization by salicylate is mediated by the MPT.
CsA also inhibits calcineurin, a protein phosphatase (Liu et al., 1991). Calcineurin inhibition is the basis for the immunosuppression by CsA, tacrolimus, and other drugs. However, tacrolimus unlike CsA does not inhibit the MPT (Griffiths and Halestrap, 1991
), and tacrolimus does not protect against TNF-
-induced apoptosis in hepatocytes (Qian and Lemasters, unpublished observations). Furthermore, CsA analogs that do not inhibit calcineurin, such as 4-methylvaline cyclosporin and NIM811, nonetheless protect against both apoptosis and necrosis in hepatocyte models of cell killing (Trost and Lemasters, 1997
; Waldmeier et al., 2002
). Similar results have been found in other cell types (Friberg et al., 1998
; Karpinich et al., 2002
; Zamzami et al., 1996
). Furthermore, confocal microscopy shows directly that CsA blocks mitochondrial depolarization and inner membrane permeabilization induced in these models (Bradham et al., 1998
; Nieminen et al., 1995
; Qian et al., 1999
; Waldmeier et al., 2002
). In the present work, we used confocal microscopy to show that CsA specifically inhibits mitochondrial depolarization and inner membrane permeabilization during salicylate-enhanced apoptosis induced by TNF-
(Fig. 6
). Thus, the protective action of CsA is most likely mediated by inhibition of the MPT.
Br-A23187 and related Ca2+ ionophores are widely used to study calcium-dependent cell injury. Ca2+ ionophores like Br-A23187 increase intracellular and intramitochondrial calcium and induce onset of the MPT with subsequent acute necrotic cell death (Qian et al., 1999). Lower doses also induce apoptosis (Zhu and Loh, 1995
). The minimal dose of Br-A23187 causing a maximal rate of necrotic cell killing is about 10 µM (Qian et al., 1999
). Here, we employed 1 µM Br-A23187, which causes a slower rate of cell killing. At this lower dose, about 15% of hepatocytes remained viable even after 3 h of exposure to the toxicant. Salicylate (1 mM) accelerated cell killing after 1 µM Br-A23187, and the time to half maximal cell killing decreased from 60 min to 30 min. Moreover, in the presence of salicylate virtually no cells remained viable after 2.5 h (Fig. 1
). CsA, a specific inhibitor of the MPT, blocked Br-A23187 plus salicylate-induced cell killing substantially. These observations are consistent with the conclusion that killing of hepatocytes induced by salicylate plus Br-A23187 is mediated by onset of the MPT, as concluded previously for Br-A23187 toxicity alone (Qian et al., 1999
).
Similarly, the oxidant chemical, t-BuOOH, induces the MPT in hepatocytes, leading to necrotic cell killing. Previously, 100 µM t-BuOOH was shown to be the minimal concentration causing a maximal rate of cell killing in rat hepatocytes (Nieminen et al., 1995). In the present study, 20 µM t-BuOOH was employed, which caused a slower rate of necrotic cell killing. Supplementation of 20 µM t-BuOOH with 1 mM salicylate restored the maximum rate and extent of cell killing described previously. CsA delayed but did not prevent cell killing after exposure of hepatocytes to 20 µM t-BuOOH plus salicylate. This inhibition was less than that which occurred with Br-A23187 but is consistent with effects of CsA reported previously in isolated mitochondria and intact hepatocytes where CsA delays rather than prevents the MPT induced by t-BuOOH (Broekemeier and Pfeiffer, 1995
; Imberti et al., 1993
). Overall, our observations are consistent with the conclusion that salicylate enhances the cytotoxicity of 20 µM t-BuOOH by lowering the threshold for onset of the MPT.
The MPT is implicated in the mitochondrial pathway to apoptosis (Zamzami and Kroemer, 2001). After onset of the MPT in hepatocytes exposed to TNF-
, mitochondria undergo large amplitude swelling, which then leads to cytochrome c release and subsequent caspase 3 activation (Bradham et al., 1998
). In the current study, salicylate enhanced apoptosis by a low concentration of TNF-
(1 ng/ml) in Ad5I
B-sensitized hepatocytes. In the absence of salicylate, low dose TNF-
induced less than 40% apoptosis after 24 h. In this model, salicylate elicited a faster and stronger apoptotic response (Fig. 3
) accompanied by greater caspase 3 activation (Fig. 4
). We also directly visualized onset of the MPT by confocal imaging after exposure of hepatocytes to low-dose TNF-
in the presence of salicylate (Fig. 5
). Mitochondrial depolarization and inner membrane permeabilization occurred within 10 h, changes that were blocked by CsA (Fig. 6
). These findings again support the conclusion that salicylate enhances cell killing by decreasing the threshold to onset of the MPT.
ATP declines precipitously in hepatocytes after Br-A23187 and t-BuOOH treatment to induce necrotic cell death (Nieminen et al., 1995; Qian et al., 1999
). ATP depletion-dependent necrotic killing is independent of caspase activation, since pan-caspase inhibition does not prevent necrotic cell killing induced by the MPT, although identical caspase inhibition does prevent MPT-dependent apoptosis (Kim et al., 2003
). The lack of caspase activation in models of necrotic killing of hepatocytes is likely the conseqence of the requirement for dATP or ATP for caspase 3 activation via cytochrome c/APAF-1/caspase 8 (Budihardjo et al., 1999
). Accordingly in the present work, we did not pursue measurements of caspase activity during necrotic cell killing since caspase activation is not implicated in ATP depletion-dependent necrosis.
Without causing increased cell killing by itself, salicylate also enhanced the tumoricidal activity of etoposide in MCF-7 human breast tumor cells and TRAIL in PLC/PRF/5 human hepatoma cells (Fig. 7). Etoposide is a topoisomerase II inhibitor whose activity leads to p53 activation and mitochondria-mediated apoptotic cell death by so-called intrinsic pathways (Beck et al., 2001
; Chresta et al., 1996
). Etoposide-dependent tumor cell killing has recently been linked to onset of the MPT (Karpinich et al., 2002
). TRAIL is a naturally occurring tumoricidal ligand that induces apoptosis of nascent transformed cells by activating receptor-mediated extrinsic pathways to apoptosis (Srivastava, 2001
). TRAIL-induced killing of hepatoma cells is mediated by the MPT (Kim et al., 2002
). The enhancement by salicylate of tumor cell killing by both intrinsic and extrinsic apoptotic pathways and hepatocyte killing by dissimilar agents indicates a robustness of this potentiating effect of salicylate. In these diverse models of cell killing, the one common feature is the involvement of the MPT, which supports the conclusion that salicylate enhances cytotoxicity by decreasing the threshold for MPT onset.
Taken together, the data show that salicylate enhances onset of the MPT caused by three different classes of inducers in hepatocytescalcium ionophore, oxidant stress, and TNF-. Additionally, salicylate enhances apoptosis in tumor cells through both intrinsic and extrinsic pathways. Rather than acting as a direct inducer, salicylate appears to lower the threshold for MPT induction by these inducers. Thus, salicylate may subtlely potentiate apoptosis and acute cytotoxicity induced by subthreshold concentrations of various cytotoxic agents. The present studies utilized 1 mM salicylate, which is in the high range of salicylate serum concentrations after therapeutic aspirin administration. However, since aspirin is administered orally, the liver and other gastrointestinal organs may be exposed to salicylate concentrations substantially higher than serum levels.
Chronic administration of small doses of aspirin has beneficial effects, for example by decreasing platelet aggregation and the incidence of heart attacks and strokes. Aspirin and other NSAIDS also decrease the incidence of colorectal cancer (Shiff and Rigas, 1997). The mechanisms for reduction of colorectal cancer by NSAIDS are only partially understood. NSAIDS induce caspase-dependent apoptosis in different cell types, an effect that may be mediated by salicylate inhibition of NF-
B activation (Kopp and Ghosh, 1994
; Yamamoto et al., 1999
). However, salicylate inhibition of NF-
B activation is unlikely to account for enhanced TNF-
-mediated apoptosis by salicylate, since NF-
B in our experiments was already profoundly inhibited by expression of an I
B superrepressor. Thus, our results implicate a new pathway by which salicylate sensitizes apoptosis through augmentation of the MPT. This pro-apoptotic effect may enhance the elimination of neoplastic and preneoplastic cells in the early stages of carcinogenesis.
In conclusion, salicylate enhanced both necrotic and apoptotic cell death in hepatocytes and tumor cells. This enhancement seems mediated by a salicylate-induced decrease in the threshold for onset of the MPT. Promotion by salicylate of MPT-mediated apoptosis may contribute to protection by aspirin against colorectal and other cancers and possibly enhance anticancer drug cytotoxicity.
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ACKNOWLEDGMENTS |
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NOTES |
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2 These authors contributed equally to this work.
3 To whom correspondence should be addressed at Department of Cell Biology and Anatomy, University of North Carolina at Chapel Hill, Campus Box 7090, 236 Taylor Hall, Chapel Hill, NC 27599-7090. Fax: (919) 966-7197. E-mail: lemaster{at}med.unc.edu.
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