Molecular Cancer Therapeutics Bridging the Lab and the Clinic in Cancer Medicine
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tang, L.
Right arrow Articles by Zhang, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tang, L.
Right arrow Articles by Zhang, Y.
Mol Cancer Ther. 2005;4:1250-1259
© 2005 American Association for Cancer Research

Mitochondria are the primary target in isothiocyanate-induced apoptosis in human bladder cancer cells

Li Tang and Yuesheng Zhang

Department of Chemoprevention, Roswell Park Cancer Institute, Buffalo, New York

Requests for reprints: Yuesheng Zhang, Department of Chemoprevention, Roswell Park Cancer Institute, Basic Science 711, Elm and Carlton Streets, Buffalo, NY 14263. Phone: 716-845-3097; Fax: 716-845-1144. E-mail: yuesheng.zhang{at}roswellpark.org


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Many isothiocysanates (ITC) are promising cancer-preventive agents, and induction of apoptosis is one of their underlying mechanisms of action. We recently found that caspase-9 was preferentially activated over other initiator caspases in human bladder cancer UM-UC-3 cells. We report here that caspase-9 activation is the major step leading to ITC-induced apoptosis in this cell line. More importantly, our results show that caspase-9 activation by the ITCs may result primarily from mitochondrial damage. Four common naturally occurring ITCs were studied, including allyl ITC, benzyl ITC (BITC), phenethyl ITC (PEITC), and sulforaphane. BITC and PEITC showed more potent mitochondria-damaging ability than the other two ITCs, correlating well with their stronger apoptosis-inducing potentials. Furthermore, BITC and PEITC damaged both the outer and inner mitochondrial membranes. Use of isolated mitochondria allowed us to establish that ITCs, and more importantly their major intracellular derivatives (glutathione conjugates) at concentrations that are readily achievable in cells, damage mitochondria, leading to the collapse of mitochondrial trans-membrane potential and release of cytochrome c. The mitochondria-damaging potencies of the ITCs correlate well with their lipophilicities. Bcl-2 family members are known to influence the stability of mitochondrial membrane. Our results show that the ITCs caused phosphorylation of Bcl-2, induced mitochondrial translocation of Bak, and disrupted the association of Bcl-xl with both Bak and Bax in mitochondrial membrane, indicating that ITC-induced mitochondrial damage results at least in part from modulation of select Bcl-2 family members.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Many isothiocyanates (ITC), some of which are abundantly available from various cruciferous vegetables, are promising cancer-preventive agents (1–4). Among their cancer-preventive mechanisms that have been discovered to date is the induction of apoptosis. Indeed, many lines of evidence show that induction of apoptosis by ITCs is not cell specific (5–10). Moreover, there also is evidence showing that ITCs are significantly more toxic to cancer cells than to normal cells, although it is not fully understood to what extent induction of apoptosis is responsible for the differential cytotoxic effects (11–13). These compounds may be especially useful for the prevention of bladder cancer, because orally ingested ITCs are rapidly and almost exclusively disposed in the urine, and bladder cancers originate overwhelmingly from the epithelia that directly face the urine stored in the bladder (4, 14–20). Four common dietary ITCs, including allyl ITC (AITC), benzyl ITC (BITC), phenethyl ITC (PEITC), and sulforaphane, have been extensively studied for their cancer preventive activities. Our recent study in cultured human bladder cancer UM-UC-3 cells showed that BITC and PEITC were more potent than AITC and sulforaphane in inducing apoptosis and activating caspases, including caspase-3, caspase-8, and caspase-9 (21). Interestingly, both BITC and PEITC were markedly more effective in activating caspase-9 than caspase-8, suggesting that caspase-9-related apoptotic pathway might play a more important role in the induction of apoptosis by the ITCs in these cells. It is noteworthy, however, that the role of a specific caspase in ITC-induced apoptosis may be cell specific. For example, Xu and Thornalley reported that caspase-8 activation played the major role and caspase-3 activation played a minor role in PEITC-induced apoptosis in HL60 cells (7), whereas caspase-3 activation was critical in PEITC-induced apoptosis in human cervical cancer HeLa cells (22).

Activation of caspase-9 requires the release of cytochrome c from the mitochondrial intermembrane space, which often is a sign of compromised mitochondrial membrane integrity. Indeed, our previous study in human leukemia HL60 cells (10), as well as those of Nakamura et al. in rat liver RL34 cells (23), revealed that AITC and/or BITC could rapidly induce the loss of mitochondrial trans-membrane potential ({Delta}{Psi}m). Nakamura et al. also reported that BITC induced cytochrome c release from isolated rat liver mitochondria (23). However, it is highly unlikely that the BITC concentration (200 µmol/L) used by these investigators can be achieved in cells, because ITCs, including BITC, are known to accumulate in cells mainly as dithiocarbamates, through conjugation with cellular thiols (glutathione in particular; refs. 24, 25). Consequently, ITC-derived dithiocarbamates should be examined for their effect on mitochondria. It was also unclear as to what extent ITC-induced mitochondrial damage in human bladder cancer cells relies on the modulation of mitochondria sensitizing or desensitizing Bcl-2 family proteins, some of which are known to be modulated by ITCs (see Discussion).

The studies reported herein document the critical importance of caspase-9 activation in ITC-induced apoptosis in UM-UC-3 cells. Evidence is provided to show that mitochondria are damaged by ITCs and more importantly by their intracellular metabolites at concentrations that can be readily achieved in cells and that ITC-induced mitochondrial damage leads to cytochrome c release, caspase-9 activation, and apoptosis. The role of Bcl-2 family proteins, including Bcl-2, Bcl-xl, Bad, Bak, and Bax, as well as the mitochondrial permeability transition pore, in mediating ITC-induced mitochondrial damage was also examined. Our results suggest that the action of ITCs on mitochondria in UM-UC-3 cells involves Bcl-2 family proteins.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
AITC, BITC, PEITC, and sulforaphane were purchased from LKT Laboratories (St. Paul, MN). Cyclosporin A and bongkrekic acid were purchased from A.G. Scientific (San Diego, CA) and EMD Biosciences (San Diego, CA), respectively. Caspase-9 inhibitor Z-LEHD-FMK was obtained from R&D Systems (Minneapolis, MN) and rhodamine 123 was purchased from Molecular Probes (Eugene, OR). All other chemicals were purchased from Sigma (St. Louis, MO).

Cell Culture
Human bladder cancer UM-UC-3 cells and their culture have been previously described (21). Normally, 1.5 x 106 cells were grown in a 10-cm dish with 10 mL McCoy's 5A medium containing L-glutamine and 10% fetal bovine serum for 48 hours and treated with an ITC at desired concentrations for 3 or 24 hours. Each ITC was dissolved in acetonitrile, the final concentration of which in the medium was 0.1% (v/v).

Western Blotting
Cells were treated with an ITC at 7.5, 15, and 30 µmol/L for 3 or 24 hours. The cells were then trypsinized, harvested by centrifugation (500 x g for 5 minutes at 4°C), and processed for preparation of whole cell lysates and cytosolic and mitochondrial fractions using published protocols with modifications (26). Briefly, for preparation of whole cell lysates, each cell pellet was washed twice with 10 mL ice-cold PBS, suspended in 200 µL of cell lysis buffer (Cell Signaling Technology, Beverly, MA) supplemented with 1 mmol/L phenylmethylsulfonyl fluoride, and sonicated by a Branson Model 450 sonicator. The lysates were centrifuged at 10,000 x g for 5 minutes at 4°C, and the supernatant was used for analysis. For the preparation of mitochondrial and cytosolic fractions, cells were harvested and washed as above. The cell pellets were suspended in buffer A (pH 7.5) containing 20 mmol/L HEPES-KOH, 10 mmol/L KCl, 1.5 mmol/L MgCl2, 1 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl fluoride, and a mixture of proteinase inhibitors (1 tablet of the complete mini inhibitor cocktail from Roche, (Indianapolis, IN) was added to 10 mL solution), which were incubated on ice for 15 minutes. After 15 strokes in a Kontes Dounce homogenizer, the homogenates were mixed with ~400 µL of 2.5 x buffer B (pH 7.5) containing 10 mmol/L HEPES-KOH, 210 mmol/L mannitol, 70 mmol/L sucrose, 0.1 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl fluoride, and the abovementioned proteinase inhibitor cocktail. After centrifugation at 1,000 x g for 10 minutes at 4°C, the supernatant fraction was transferred to another tube and centrifuged at 17,000 x g for 30 minutes at 4°C. The resulting supernatant fraction was collected as the cytosolic fraction, whereas the pellet containing the mitochondria was resuspended in buffer B. To prepare the medium fraction, cells were treated with an ITC as described above; the medium was then collected and centrifuged at 500 x g for 5 minutes at 4°C; the supernatant was supplemented with the proteinase inhibitor cocktail and was concentrated using a centrifugal filtration device (Amicon, Bedford, MA). Protein content of each sample was measured using the bicinchoninic acid assay kit from Pierce (Rockford, IL). Each sample (40–50 g proteins) was resolved by SDS-PAGE (8–15%) and blotted to polyvinylidene difluoride membranes, which were probed by specific antibodies and visualized using an enhanced chemiluminescence system (Amersham Biosciences, Piscataway, NJ). All antibodies were purchased from Cell Signaling Technology except anti-malate dehydrogenase (MDH) and anti-glyceraldehyde-3-phosphate dehydrogenase, which were obtained from Rockland (Gilbertsville, PA) and Chemicon (Temecula, CA), respectively.

Immunoprecipitation
Mitochondria were prepared as described above and used within 3 hours. The freshly isolated mitochondria were suspended in PBS at 1 mg protein equivalent/mL and incubated for 30 minutes at room temperature with 50 µmol/L BITC followed by centrifugation of the mixture at 17,000 x g for 15 minutes at 4°C. The resultant pellet was suspended in 200 µL of cell lysis buffer (Cell Signaling Technology) supplemented with 1 mmol/L phenylmethylsulfonyl fluoride and the protease inhibitor cocktail described above and sonicated. Lysates were incubated overnight at 4°C with an anti-Bcl-xl antibody (Cell signaling Technology), which was then mixed with 20 µL of protein A-agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA) and incubated for 3 hours at 4°C. The immunoprecipitated complexes were washed five times with lysis buffer and subjected to SDS-PAGE followed by immunoblotting using anti-Bcl-xl, anti-Bak, and anti-Bax antibodies (Cell Signaling Technology).

Measurement of Integrity of Cellular and Mitochondrial Membranes
At the end of an ITC treatment, the cells were harvested and washed with buffer as described above. Each cell pellet was resuspended in fresh medium at 5 x 105 cells/mL. Fluorescent propidium iodide (PI), a DNA-binding reagent, cannot penetrate a normal cell membrane but may stain cells with membrane damage. For the PI staining experiment, 1 mL cell suspension was mixed with 10 µg PI. After 5 minutes of incubation, each sample was immediately measured by flow cytometry for PI-stained cells. Rhodamine 123 was used to determine if {Delta}{Psi}m was lost. Rhodamine 123 is readily and selectively sequestered by normal mitochondria (cells showing strong fluorescence) but is removed from the latter when {Delta}{Psi}m is lost (cells showing greatly diminished fluorescence). Each above-described cell suspension (1 mL) was incubated with 10 µg rhodamine 123 for 30 minutes at 37°C; the cells were then washed twice with fresh medium, resuspended in an equal volume of fresh medium, and promptly measured by flow cytometry to determine fluorescence intensity of the rhodamine-treated cells. Ten thousand cells were examined in each sample, using a Becton Dickinson FACScan (BD Biosciences, San Jose, CA).

Measurement of {Delta}{Psi}m and Cytochrome c Release from Isolated Mitochondria
Measurement of {Delta}{Psi}m in isolated mitochondria was based on a previous report that the fluorescence of a rhodamine 123 solution is quenched upon addition of normal mitochondria that sequester the dye but recovers as the dye is leaked back out of the mitochondria when their {Delta}{Psi}m is lost (27). The mitochondria were prepared as described above, washed and resuspended in ice cold buffer B, and used within 3 hours. The protein concentration was measured using the bicinchoninic acid assay kit. To check {Delta}{Psi}m, isolated mitochondria (0.3 mg protein equivalent/mL) were suspended in buffer C (pH 7.5) containing 150 mmol/L sucrose, 5 mmol/L MgCl2, 5 mmol/L disodium succinate, 2.5 µmol/L rotenone, 5 mmol/L potassium phosphate, and 20 mmol/L HEPES-KOH (27) followed by incubation with 5 µmol/L rhodamine 123 for 5 minutes. A fluorescence reading of the solution was then promptly taken, and an ITC at a desired concentration was added to the solution. The fluorescence reading of the solution was taken again at 5, 10, 15, 20, 25, and 30 minutes after ITC addition. The experiment was carried out at room temperature, and the fluorescence of rhodamine 123 was measured at 538 nm (excitation, 485 nm). When cyclosporin A or bongkrekic acid was included in the experiment, mitochondria were pretreated with each compound for 10 minutes before ITC addition.

To measure cytochrome c release from mitochondria, freshly isolated mitochondria were suspended in PBS at 1 mg protein equivalent/mL and incubated for 30 minutes at room temperature with an ITC or a mixture of an ITC plus a 5-fold higher concentration of reduced glutathione (GSH). In the latter case, an ITC was mixed with GSH in PBS (pH 7.5) for 30 minutes at room temperature before addition of mitochondria. Based on spectroscopic changes previously described (28), all ITCs in the solution were found to be converted to the corresponding GSH conjugates (GS-ITC) before mitochondria treatment (results not shown). Thus, the mitochondria were exposed in effect to a GS-ITC at 0.5 mmol/L in the presence of 2 mmol/L GSH, mimicking the intracellular environment, where GSH concentration is in the millimolar range and ITCs are accumulated predominantly as GS-ITCs (24, 25). At the end of mitochondria treatment, each suspension was centrifuged at 17,000 x g for 15 minutes at 4°C, and the supernatant was examined for presence of cytochrome c by Western blot analysis.

Determination of Intracellular ITC Accumulation
The procedures involving cell exposure to an ITC, cell harvest, preparation of cell lysates, and measurement of total ITC content (free ITC plus its thiol conjugate) by the cyclocondensation assay were described previously (29). Intracellular fluid volume was determined using 14C-inulin and 3H2O (Moravek Biochemicals, Brea, CA) following a previously described procedure (29).

Statistics
Results are expressed as means ± SD (at least three determinations). Data were analyzed by one-way ANOVA followed by Dunnett's t test for separate comparisons with the control group. Differences were considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
ITC-Induced Apoptosis in UM-UC-3 Cells Was Mainly Mediated by Caspase-9
When UM-UC-3 cells were treated with either BITC or PEITC, caspase-9 was activated as early as 3 hours after the treatment started, although the activation was stronger at 24 hours (Fig. 1A). Caspase activation by BITC and PEITC was more significant at 15 than 7.5 µmol/L, but increasing the ITC concentration to 30 µmol/L did not lead to further caspase-9 activation, or in some cases even caused a decrease, which is in agreement with our previous results (21). Interestingly, as shown in Fig. 2C and 2D and described later, ITC treatment apparently causes the damage of cell surface membrane and the leakage of intracellular components into the extracellular space. Hence, it is likely that a majority of the cleaved caspases, especially cleaved caspase-3 (its molecular weight is relatively low: 17/19 kDa), leaked into the culture medium from cells treated with the highest ITC concentration. The activation of caspase-3 and the cleavage of poly(ADP-ribose) polymerase (PARP) follow a pattern similar to caspase-9 activation (Fig. 1A), in line with the fact that both caspase-3 and PARP are downstream of caspase-9. However, BITC- or PEITC-induced cleavage of PARP was substantially reduced in the presence of Z-LEHD-FMK (100 µmol/L), a caspase-9 specific inhibitor (Fig. 1B). Because our previous study showed that activation of caspases and cleavage of PARP closely correlate with the occurrence of apoptotic cells (terminal deoxynucleotidyl transferase–mediated nick end labeling–positive cells) in BITC- and PEITC-treated UM-UC-3 cells (21), the above results provide a clear indication that caspase-9 activation is a major step in mediating the induction of apoptosis in these cells by BITC and PEITC.



View larger version (49K):
[in this window]
[in a new window]
  		Figure 1. Effect of BITC and PEITC on caspase activation and PARP cleavage in UM-UC-3 cells. A, activation of caspases and cleavage of PARP. Cells were treated with BITC or PEITC at the specified concentrations for 3 or 24 h before they were harvested for analysis. ITCs were dissolved in acetonitrile (ACN). B, effects of Z-LEHD-FMK, a caspase-9-specific inhibitor, on ITC-induced PARP cleavage. Cells were pretreated with (+) or without (–) 100 µmol/L Z-LEHD-FMK for 30 min and were exposed to 15 µmol/L ITC or acetonitrile for 3 or 24 h in the presence of Z-LEHD-FMK. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to confirm the equal loading of samples.

 


View larger version (40K):
[in this window]
[in a new window]
  		Figure 2. Effect of ITCs on the integrity of cellular and mitochondrial membranes in UM-UC-3 cells. Cells were exposed to each ITC at 7.5, 15, 30 µmol/L, or to acetonitrile (ACN) for 3 or 24 h before analysis. A, loss of {Delta}{Psi}m, as determined by flow cytometry. Columns, means of three independent experiments; bars, SD. *, P < 0.05, versus control; **, P < 0.01, versus control. B, release of cytochrome c and MDH from mitochondria. The cytosolic fraction that was separated from ITC-treated cells was subjected to Western blot analysis for detection of cytochrome c and MDH. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the loading control. C, detection of cytochrome c in culture medium. Cells were treated with BITC or PEITC at 0 (acetonitrile), 15, or 30 µmol/L for 24 h. At the end of the treatment, medium was collected and concentrated for detection of cytochrome c by Western blot. D, damage of cell surface membrane. Cells treated with or without ITC were incubated with PI (10 µg/mL, 5 min) and flow cytometry was used to detect the cells with PI staining. Columns, means of three independent experiments; bars, SD. *, P < 0.05, versus control; **, P < 0.01, versus control.

 
Mitochondria Were Damaged by ITCs
Because caspase-9 activation is caused by the release of cytochrome c to the cytoplasm from mitochondria, we next examined the effect of ITCs on mitochondria. When UM-UC-3 cells were treated with BITC or PEITC at 7.5, 15, or 30 µmol/L, both compounds rapidly caused a loss of {Delta}{Psi}m in a dose-dependent manner (Fig. 2A). At 24 hours of treatment, 30 µmol/L BITC and PEITC caused 60% and 70% of the cells to lose their {Delta}{Psi}m, respectively. Even at 3 hours of treatment, BITC and PEITC at 30 µmol/L caused 54% and 35% of the cells to lose their {Delta}{Psi}m, respectively, indicating that mitochondrial damage occurred rapidly. In fact, mitochondrial damage in these cells could be observed after an ITC treatment for only 30 minutes (results not shown). In contrast, mitochondrial damage caused by AITC and sulforaphane at 7.5 to 30 µmol/L was rather limited (Fig. 2A), which correlates with the negligible activation of caspase-3 and casapse-9 and cleavage of PARP in these cells (results not shown). However, the weak effects of AITC and sulforaphane were dose related because incubation of cells with each ITC at 100 µmol/L for 24 hours resulted in 43% and 94% of the cells to lose their {Delta}{Psi}m, respectively. Clearly, the mitochondria-damaging potency varies with ITCs, and there is a correlation between loss of {Delta}{Psi}m and apoptosis caused by the ITCs.

We also examined whether ITC treatment would lead to cytochrome c release. It has been suggested that the collapse of {Delta}{Psi}m may not necessarily lead to cytochrome c release and apoptosis (30). However, both BITC and PEITC at 7.5 to 30 µmol/L caused the release of cytochrome c into the cytoplasm of UM-UC-3 cells in a manner that corresponds to the activation of caspases-9 and caspase-3 (Fig. 2B and Fig. 1A). More cytochrome c was detected in the cytoplasm when cells were treated with each ITC for 24 than for 3 hours. In addition, both ITCs at 15 µmol/L showed the strongest effect, which correlated well with the results of activation of the caspases. In line with the weak effect of both AITC and sulforaphane on {Delta}{Psi}m, little cytochrome c was detected in the cytoplasm of cells treated with either ITC at concentrations up to 30 µmol/L. Interestingly, MDH (35 kDa) was also detected in the cytoplasm of cells treated with either BITC or PEITC (Fig. 2B). Cytochrome c is known to exist in the mitochondrial intermembrane space, whereas MDH is normally found in the mitochondrial matrix. Although MDH is not known to be involved in the induction of apoptosis, detection of both MDH and cytochrome c in the cytoplasm indicates that BITC and PEITC damaged both the outer and inner mitochondrial membranes.

Cytochrome c level in the cytoplasm dropped when the concentration of BITC or PEITC was increased from 15 to 30 µmol/L (Fig. 2B). We questioned whether this drop was a reflection of damage to the cell surface membrane, which may result in leaking cytochrome c to the extracellular medium. Indeed, cytochrome c was found in the medium after cell treatment with BITC or PEITC (Fig. 2C), and the amount detected in the culture media increased as the ITC concentration was increased. To further confirm cell membrane damage, we used PI, a fluorescent DNA-binding reagent, which cannot penetrate an intact cell membrane. UM-UC-3 cells treated with BITC or PEITC showed a dose-dependent increase in the number of cells stained by PI (Fig. 2D). The number of cells stained with PI increased 6- and 4-fold, respectively, after treatment with either BITC or PEITC at 30 µmol/L for 24 hours. Moreover, ITC-induced cell membrane damage occurred rapidly, for the number of cells stained with PI was similar between 3 and 24 hours.

Modulation of Bcl-2 Family Members by ITCs
Because mitochondrial membrane integrity is known to be influenced by Bcl-2 family proteins, we wondered if ITCs modulated these proteins in UM-UC-3 cells. Two groups of Bcl-2 family members were examined: the antiapoptotic (mitochondrial membrane stabilizing) proteins, including Bcl-2 and Bcl-xl, which are normally located on the mitochondrial membrane and the proapoptotic (mitochondrial membrane destabilizing) proteins, including Bad, Bak, and Bax, which translocate to the mitochondria in response to apoptotic stimuli. When UM-UC-3 cells were treated with BITC or PEITC at 7.5, 15, or 30 µmol/L for 3 and 24 hours, the levels of Bcl-2, Bcl-xl, Bax, and Bak in whole cell lysates, as well as the levels of Bax and Bcl-2 in the mitochondria, did not change (Fig. 3A and B). Although total Bad levels seemed to increase in response to ITC treatment, its level in the mitochondria did not change, suggesting that Bad is unlikely to mediate ITC-induced mitochondrial damage. Phosphorylation of Bcl-2 is known to inactivate Bcl-2. However, increased phospho-Bcl-2 was detected only when cells were treated with each ITC at 7.5 µmol/L for 24 hours (Fig. 3A) but not at high ITC concentrations. Interestingly, whereas the total level of Bak in the cell did not change after ITC treatment, its level in the mitochondria was elevated by both ITCs, especially when the cells were treated by the compounds for 24 hours, indicating increased translocation of this protein to mitochondria, although it is unclear from where it is transferred. However, increase in mitochondrial Bak levels seem to reach a plateau at 15 µmol/L ITC and decline at a higher ITC concentration (Fig. 3B).



View larger version (66K):
[in this window]
[in a new window]
  		Figure 3. A and B, Western blot analysis of selected Bcl-2 family members in UM-UC-3 cells treated by BITC and PEITC. Cells were exposed to an ITC at 0 (acetonitrile, ACN), 7.5, 15, or 30 µmol/L for 3 or 24 h. Whole cell lysates and the mitochondrial fraction were analyzed. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the loading control. C, mitochondria were isolated from UM-UC-3 cells and treated with 50 µmol/L BITC for 30 min. The lysates were immunoprecipitated with anti-Bcl-xl antibody, and the immunoprecipitates were immunoblotted with anti-Bcl-xl, anti-Bak, and anti-Bax antibodies. Arrow, specific bands recognized by each antibody, and the top nonspecific IgG bands indicate an equal sample loading.

 
Because the mitochondria-destabilizing function of Bak and Bax are normally kept in check by association with Bcl-2 and Bcl-xl, we next asked if ITCs affected the association. Our immunoprecipitation experiment showed that treatment of isolated mitochondria with 50 µmol/L BITC (other ITCs not tested) for 30 minutes disrupted the association of Bcl-xl with both Bak and Bax (Fig. 3C), although similar BITC treatment did not seem to affect the interaction of Bcl-2 with Bak and Bax (results not shown). The exact reason for BITC-caused disassociation of the proteins is unknown but it reveals for the first time a novel mechanism by which Bcl-2 family proteins are modulated by ITC.

ITCs Damaged Isolated Mitochondria
We next asked if ITCs would induce similar changes in isolated mitochondria. When freshly isolated mitochondria from UM-UC-3 cells were added to a rhodamine 123 solution, the fluorescence of rhodamine 123 dropped significantly due to its uptake and sequestration by the mitochondria (Fig. 4A). When the rhodamine 123–loaded mitochondria were treated with BITC and PEITC at 15, 50, or 100 µmol/L, there was a dose- and time-dependent collapse of {Delta}{Psi}m as shown by fluorescence increase in the solution (due to rhodamine 123 leaking out of the mitochondria; Fig. 4B). In contrast, AITC and sulforaphane seemed much weaker in causing {Delta}{Psi}m to collapse, which again is correlated with their weak stimulation of apoptosis in UM-UC-3 cells (21). Moreover, the different potencies of each of the four ITCs in collapsing {Delta}{Psi}m of isolated mitochondria is also closely correlated with their effects on {Delta}{Psi}m in intact cells (Fig. 2A). The opening of mitochondrial permeability transition pore is believed to result in the collapse of {Delta}{Psi}m in response to various stimuli (31). Two types of mitochondrial permeability transition pore inhibitors, cyclosporin A (a ligand of cyclophilin D) and bongkrekic acid (a ligand of adenine nucleotide translocase) were used to explore whether the ITCs affected {Delta}{Psi}m by modulating the mitochondrial permeability transition pore. Both compounds were previously shown to inhibit mitochondrial permeability transition pore opening (32–34). When mitochondria were preincubated with the inhibitor for 10 minutes and then exposed to each ITC in the presence of the inhibitor, cyclosporin A at 10 µmol/L and bongkrekic acid at 50 µmol/L had no effect on BITC-induced loss of {Delta}{Psi}m (Fig. 4C). Addition of bongkrekic acid at 50 µmol/L seemed to enhance PEITC-induced loss of {Delta}{Psi}m, presumably due to drug toxicity. Although addition of cyclosporin A at 10 µmol/L seems to slightly protect the mitochondria against PEITC-induced damage, as reflected by a slight drop of fluorescence (Fig. 4C), this may not be a true protection, because the decrease was not statistically significant. In fact, when UM-UC-3 cells were pretreated with cyclosporin A at concentrations ranging from 2.5 to 50 µmol/L, neither the loss of {Delta}{Psi}m nor PARP cleavage, which were induced by BITC and PEITC, was attenuated (results not shown). Because the concentrations of cyclosporin A and bongkrekic acid used in the above experiments are apparently sufficient to inhibit cyclophilin D or adenine nucleotide translocase (35–39), our findings suggest that it is unlikely that these two mitochondrial permeability transition pore components are the targets of ITCs.



View larger version (36K):
[in this window]
[in a new window]
  		Figure 4. The effect of ITCs on isolated mitochondria. Mitochondria were freshly prepared from UM-UC-3 cells. A, quenching of rhodamine 123 fluorescence by mitochondria. The fluorescence of the incubation buffer containing 5 µmol/L rhodamine 123 was measured before (dark column) and after addition of mitochondria (0.3 mg protein equivalent/mL, open column). B, ITC-induced loss of {Delta}{Psi}m in isolated mitochondria. The rhodamine 123–loaded mitochondria were then incubated with an ITC at the specified concentrations for 30 min. The fluorescence of rhodamine 123 in the solution was measured every 5 min. The increase of fluorescence intensity in the solution indicates the loss of {Delta}{Psi}m. Each value is a mean of three measurements (SD ≤8% of the mean), and the presented values are adjusted by subtraction from control values. C, effect of mitochondrial permeability transition pore inhibitors on ITC-induced loss of {Delta}{Psi}m. Mitochondria were pretreated with or without 50 µmol/L bongkrekic acid (BA) or 10 µmol/L cyclosporin A (CsA) for 10 min followed by exposure to 100 µmol/L BITC or PEITC in the presence of the inhibitor for 30 min. The fluorescence intensity of rhodamine 123 in the solutions was measured every 5 min. Each value is a mean of three measurements (SD ≤4% of the mean). Again, the presented values are adjusted by subtraction from control values. D, release of cytochrome c from isolated mitochondria after ITC treatment. Mitochondria were exposed to an ITC at 50 µmol/L for 30 min. The incubation buffer was then separated from the mitochondria and subjected to Western blot analysis. E, effect of GS-ITCs in the presence of excess amount of GSH on the release of cytochrome c from isolated mitochondria. Mitochondria were incubated with 0.5 mmol/L of an GS-ITC in the presence of 2 mmol/L GSH for 30 min. Each GS-ITC conjugate was prepared in situ just before the addition of mitochondria. The incubation buffer was then separated from the mitochondria by centrifugation and analyzed by Western blot for detection of cytochrome c.

 
Exposure of mitochondria isolated from UM-UC-3 cells to BITC or PEITC (50 µmol/L, 30 minutes) also induced the release of cytochrome c (Fig. 4D). However, does this type of experiment really address what happens in cells? It is now well established that ITCs, including all four ITCs used in the current study, are accumulated 100- to 200-fold in cells within 1 to 3 hours (24, 25). Indeed, as shown in Fig. 5B, both BITC and PEITC were dramatically accumulated in UM-UC-3 cells. For example, total intracellular BITC and PEITC accumulation levels were 1.04 and 0.66 mmol/L, respectively, when cells were exposed to each ITC at 7.5 µmol/L for only 1 hour (Fig. 5B). Previous studies have shown that ITCs are accumulated in cells predominantly as GS-ITC through conjugation with GSH and the concentrations of free ITCs are relatively low in cells (24). It was therefore important to examine whether GS-ITCs at intracellularly achievable concentrations could cause cytochrome c release from mitochondria. Thus, each ITC at 0.5 mmol/L was preincubated with 2.5 mmol/L GSH at pH 7.5 for 30 minutes, then mitochondria were added and incubated for another 30 minutes. Based on spectroscopic changes of these solutions as mentioned before, it was found that all ITC molecules were converted to the corresponding GS-ITCs before mitochondria were added (results not shown); thus, the mitochondria were actually treated with 0.5 mmol/L GS-ITC in the presence of 2 mmol/L GSH, which mimics the intracellular environment. As shown in Fig. 4E, cytochrome c was released from the mitochondria treated with GS-BITC or GS-PEITC, whereas GS-AITC and GS-SF under similar conditions were ineffective. Furthermore, the rank order of the potencies of these compounds in damaging mitochondria in vitro matches the rank order of the potencies of the free ITCs in causing mitochondrial damage in treated UM-UC-3 cells. These results clearly indicate that exposure of cells to BITC and PEITC at 7.5 to 30 µmol/L directly damage mitochondria.



View larger version (21K):
[in this window]
[in a new window]
  		Figure 5. Total intracellular accumulation of ITCs in UM-UC-3 cells. A, standard curve relating the protein content in cell lysates to the intracellular fluid volume. The cell volume was determined as described before (27), using 3H2O with 14C-inulin. B, total intracellular ITC accumulation levels. Cells were exposed to an ITC at the specified concentrations for 1 h. Total intracellular amounts of ITCs were measured by the cyclocondensation assay (27) and calculated based on cell volume, using the standard curve shown above. Columns, means of three measurements; bars, ±SD. The cyclocondenation assay measures the total amount of both free ITC and its thiol conjugate (dithiocarbamate), including GS-ITC.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The four dietary ITCs showed very different apoptosis-inducing abilities in UM-UC-3 cells, providing a good model to study the targets of ITCs in the induction of apoptosis. Our current and previous results (21) show that BITC and PEITC are more potent inducers of apoptosis than AITC and sulforaphane. The ITCs induce apoptosis in UM-UC-3 cells primarily through the caspase-9-mediated pathway because in the presence of Z-LEHD-FMK, a specific caspase-9 inhibitor, PARP cleavage induced by the ITCs is largely abolished. The activation of caspase-9 apparently resulted from mitochondrial damage caused by these compounds. BITC and PEITC damaged both outer and inner mitochondrial membranes as shown by the release into cytoplasm of cytochrome c from the mitochondrial intermembrane space and MDH from the mitochondrial matrix (Fig. 2). Indeed, the abilities of the four ITCs in activating caspase-9 are closely correlated with their abilities in causing loss of {Delta}{Psi}m and mitochondrial cytochrome c release (compare the results in Fig. 1 with those in Fig. 2A and B). Similar effects of both BITC and PEITC on mitochondria were seen in the isolated mitochondria. Both ITCs at 15 to 100 µmol/L caused a dose- and time-dependent loss of {Delta}{Psi}m and cytochrome c release (Fig. 4). AITC and sulforaphane were much weaker in damaging the isolated mitochondria, correlating well with the results obtained from the whole cell experiments. The experimental system using isolated mitochondria also allowed us to show that the main intracellular metabolites of ITCs (GS-ITCs) caused mitochondrial damage, as cytochrome c was released after treatment of mitochondria for 30 minutes with 500 µmol/L GS-BITC or GS-PEITC in the presence of 2 mmol/L GSH (Fig. 4E). The treatment condition mimics the intracellular environment after cells are exposed to the ITCs.

Although Bcl-2 family proteins are widely known to affect the mitochondrial membrane integrity, neither BITC nor PEITC have any detectable effect on the expression of Bcl-2, Bcl-xl, Bak, and Bax in UM-UC-3 cells (Fig. 3). Both compounds elevated whole cell Bad levels but did not change its content in the mitochondria. However, BITC and PEITC markedly stimulated phosphorylation of Bcl-2, although the change occurred only when the cells were treated with each compound at 7.5 µmol/L for 24 hours (Fig. 3) but not at high ITC concentrations (15 and 30 µmol/L), which caused more damage to the mitochondria (Fig. 2A). Moreover, although total Bak levels in UM-UC-3 cells were not altered after ITC treatment, both BITC and PEITC apparently stimulated Bak translocation to mitochondria (Fig. 3). Choi and Singh recently reported that Bak as well as Bax play critical roles in apoptosis induction by sulforaphane in SV40-transformed mouse embryonic fibroblasts (40). However, increased translocation of Bak occurred when cells were treated with 7.5 to 15 µmol/L BITC or PEITC, and further increase in ITC concentration (30 µmol/L) resulted in a decrease in mitochondrial Bak levels although more cells suffered loss of {Delta}{Psi}m (compare results in Fig. 3B with those in Fig. 2A). These results show that the effect of ITCs on Bcl-2 and Bak is highly dependent on the concentrations of the compounds. It is noteworthy that published data show that the effect of ITCs on Bcl-2 family proteins varies with cell lines. Xiao et al. reported that AITC, which markedly induced apoptosis in human prostate cancer LNCaP cells and PC-3 cells, did not affect the expression of proapoptotic Bax and Bid, but decreased the expression of both Bcl-2 and Bcl-xl in these cells (12). However, both AITC and PEITC significantly activated Bid in HL60 cells (41), and BITC significantly induced the expression of Bax and phosphorylation of Bcl-2 in human T-cell leukemia Jurkat cells (42). Rose et al. also recently reported that PEITC induced conformational change and mitochondrial translocation of Bax in human hepatoma HepG2 cells and suggested that loss of {Delta}{Psi}m resulted from Bax-dependent membrane pore formation (43). Importantly, our results also show that ITC can cause the disassociation of Bcl-xl with Bak and Bax in the mitochondria, which likely frees the latter from inhibition by Bcl-xl, thus revealing a novel mechanism by which an ITC causes mitochondrial damage.

The mitochondrial permeability transition pore opening is known to lead to loss of {Delta}{Psi}m, and subsequently, osmotic swelling of the mitochondrial matrix, causing the rupture of the outer membrane and the release of cytochrome c (44, 45). The mitochondrial permeability transition pore is a preexisting channel at the contact site between the inner and outer mitochondrial membrane, primarily consisting of three components: voltage-dependent anion channel in the outer mitochondrial membrane, adenine nucleotide translocase in the inner mitochondrial membrane, and cyclophilin D in the matrix. Cyclosporin A and bongkrekic acid are known to bind to cyclophilin D and adenine nucleotide translocase, respectively, and inhibit the mitochondrial permeability transition pore opening. However, both cyclosporin A and bongkrekic acid in our hands did not inhibit the BITC- and PEITC-induced loss of {Delta}{Psi}m, suggesting that the ITCs likely do not modulate mitochondrial permeability transition pore, which is in line with what was reported by Rose et al. (43).

Several synthetic ITCs, including p-bromophenyl ITC, 4,4'-diisothiocyanatebiphenyl, and ß-naphthylmethyl ITC, were shown to act on isolated liver mitochondria as uncouplers with stimulation of ATPase activity (46). However, disruption of {Delta}{Psi}m by a mitochondrial uncoupler, such as carbonyl cynide m-chlorophenylhydrazone, was not followed by cytochrome c release and cell death, and additional triggers were required for induction of apoptosis (47, 48). It is not known if the ITCs in the current report also act as uncouplers. However, even if they do, this function alone may not be sufficient to rapidly induce mitochondrial damage. Exposure of cells to ITCs including BITC and PEITC also results in a rapid and dose-dependent increase of cellular reactive oxygen species levels in several cell lines (23, 49–51), including UM-UC-3 cells.1 Mitochondria are the major site of reactive oxygen species production, which may result from disruption of the respiratory chain (52–54). Although mitochondrial respiration was suppressed by several ITCs including BITC (23, 46, 55), it remains to be determined to what extent reactive oxygen species production mediates ITC-induced damage of mitochondrial membrane.

Interestingly, the potency of the four ITCs in damaging mitochondria (causing the loss of {Delta}{Psi}m, see Fig. 2A) seems to correlate with their lipophilicity (logP values), which was previously determined to be 3.2 (PEITC), 3.0 (BITC), 2.2 (AITC), and 0.3 (sulforaphane), respectively (25). The two aromatic ITCs (BITC and PEITC) are rather hydrophobic and potent mitochondria-damaging agents, whereas AITC, and sulforaphane in particular, are more water-soluble and show lower mitochondria-damaging potential. Certainly, higher lipid solubility facilitates the delivery of the compound to the membrane. In fact, Miko and Chance reported that the uncoupling activities of some ITCs positively correlated with their logP values (46). The importance of lipophilicity is also reflected by the damaging of the cell surface membrane. Both BITC and PEITC at 7.5 to 30 µmol/L caused dose-dependent damage to cell membrane (Fig. 2D), whereas AITC and sulforaphane showed much weaker effects at the same concentration range (results not shown).

In summary, the differential effects of the four dietary ITCs on apoptosis of UM-UC-3 cells are associated with their damaging effects on the mitochondria. Treatment of UM-UC-3 cells with BITC and PEITC at low micromolar concentrations caused the damage of both outer and inner mitochondrial membranes, leading to the release of cytochrome c into the cytoplasm, caspase-9 activation, and induction of apoptosis. Mitochondrial damage was caused by the main intracellular metabolites of ITCs (their glutathione conjugates). Increased phosphorylation of Bcl-2, mitochondrial translocation of Bak, and disruption of the association of Bcl-xl with both Bak and Bax in the mitochondria occur following ITC treatment, all of which likely contribute to mitochondrial damage and induction of apoptosis.


   Acknowledgments
 
We thank Joseph D. Paonessa for critically reading this article.


   Footnotes
 
Grant support: NIH grant CA80962.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Unpublished results. Back

Received 2/ 9/05; revised 6/ 1/05; accepted 6/14/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Zhang Y, Talalay P. Anticarcinogenic activities of organic isothiocyanates: chemistry and mechanisms. Cancer Res 1994;54:1976–81s.
  2. Hecht SS. Chemoprevention by isothiocyanates. J Cell Biochem 1995;22:195–209.[CrossRef]
  3. Hecht SS. Inhibition of carcinogenesis by isothiocyanates. Drug Metab Rev 2000;32:395–411.[CrossRef][Medline]
  4. Conaway CC, Yang Y, Chung FL. Isothiocyanates as cancer chemopreventive agents: their biological activities and metabolism in rodents and humans. Curr Drug Metab 2002;3:233–55.[CrossRef][Medline]
  5. Chiao JW, Chung FL, Kancherla R, Ahmed T, Mittelman A, Conaway CC. Sulforaphane and its metabolite mediate growth arrest and apoptosis in human prostate cancer cells. Int J Oncol 2002;20:631–6.[Medline]
  6. Fimognari C, Nusse M, Cesari R, Iori R, Cantelli-Forti G, Hrelia P. Growth inhibition, cell-cycle arrest and apoptosis in human T-cell leukemia by the isothiocyanate sulforaphane. Carcinogenesis 2002;23:581–6.[Abstract/Free Full Text]
  7. Xu K, Thornalley PJ. Studies on the mechanism of the inhibition of human leukaemia cell growth by dietary isothiocyanates and their cysteine adducts in vitro. Biochem Pharmacol 2000;60:221–31.[CrossRef][Medline]
  8. Huang C, Ma WY, Li JX, Hecht SS, Dong ZG. Essential role of p53 in phenethyl isothiocyanate-induced apoptosis. Cancer Res 1998;58:4102–6.[Abstract/Free Full Text]
  9. Gamet-Payrastre L, Li P, Lumeau S, et al. Sulforaphane, a naturally occurring isothiocyanate, induces cell cycle arrest and apoptosis in HT29 human colon cancer cells. Cancer Res 2000;60:1426–33.[Abstract/Free Full Text]
  10. Zhang Y, Tang L, Gonzalez V. Selected isothiocyanates rapidly induce growth inhibition of cancer cells. Mol Cancer Ther 2003;2:1045–52.[Abstract/Free Full Text]
  11. Gamet-Payrastre L, Lumeau S, Gasc N, Cassar G, Rollin P, Tulliez J. Selective cytostatic and cytotoxic effects of glucosinolates hydrolysis products on human colon cancer cells in vitro. Anticancer Drugs 1998;9:141–8.[Medline]
  12. Xiao D, Srivastava SK, Lew KL, et al. Allyl isothiocyanate, a constituent of cruciferous vegetables, inhibits proliferation of human prostate cancer cells by causing G2/M arrest and inducing apoptosis. Carcinogenesis 2003;24:891–7.[Abstract/Free Full Text]
  13. Musk SR, Johnson IT. Allyl isothiocyanate is selectively toxic to transformed cells of the human colorectal tumour line HT29. Carcinogenesis 1993;14:2079–83.[Abstract/Free Full Text]
  14. Chung FL, Morse MA, Eklind KI, Lewis J. Quantification of human uptake of the anticarcinogen phenethyl isothiocyanate after a watercress meal. Cancer Epidemiol Biomarkers Prev 1992;1:383–8.[Abstract]
  15. Mennicke WH, Gorler K, Krumbiegel G, Lorenz D, Rittmann N. Studies on the metabolism and excretion of benzyl isothiocyanate in man. Xenobiotica 1988;18:441–7.[Medline]
  16. Tang L, Zhang Y. Isothiocyanates in the chemoprevention of bladder cancer. Curr Drug Metab 2004;5:193–201.[CrossRef][Medline]
  17. Jiao D, Ho CT, Fioles P, Chung FL. Identification and quantification of the N-acetylcysteine conjugate of allyl isothiocyanate in human urine after ingestion of mustard. Cancer Epidemiol Biomarkers Prev 1994;3:487–92.[Abstract]
  18. Shapiro TA, Fahey JW, Wade KL, Stephenson KK, Talalay P. Chemoprotective glucosinolates and isothiocyanates of broccoli sprouts: metabolism and excretion in humans. Cancer Epidemiol Biomarkers Prev 2001;10:501–8.[Abstract/Free Full Text]
  19. Ye L, Dinkova-Kostova AT, Wade KL, Zhang Y, Shapiro TA, Talalay P. Quantitative determination of dithiocarbamates in human plasma, serum, erythrocytes and urine: pharmacokinetics of broccoli sprout isothiocyanates in humans. Clin Chim Acta 2002;316:43–53.[CrossRef][Medline]
  20. Ioannou YM, Burka LT, Matthews HB. Allyl isothiocyanate: comparative disposition in rats and mice. Toxicol Appl Pharmacol 1984;75:173–81.[CrossRef][Medline]
  21. Tang L, Zhang Y. Dietary isothiocyanates inhibit the growth of human bladder carcinoma cells. J Nutr 2004;134:2004–10.[Abstract/Free Full Text]
  22. Yu R, Mandlekar S, Harvey KJ, Ucker DS, Kong A-NT. Chemopreventive isothiocyanates induce apoptosis and caspase-3-like protease activity. Cancer Res 1998;58:402–8.[Abstract/Free Full Text]
  23. Nakamura Y, Kawakami M, Yoshihiro A, et al. Involvement of the mitochondrial death pathway in chemopreventive benzyl isothiocyanate-induced apoptosis. J Biol Chem 2002;277:8492–9.[Abstract/Free Full Text]
  24. Zhang Y. Role of glutathione in the accumulation of anticarcinogenic isothiocyanates and their glutathione conjugates by murine hepatoma cells. Carcinogenesis 2000;21:1175–82.[Abstract/Free Full Text]
  25. Zhang Y. Molecular mechanism of rapid cellular accumulation of anticarcinogenic isothiocyanates. Carcinogenesis 2001;22:425–31.[Abstract/Free Full Text]
  26. Liu X, Kim CN, Yang J, et al. Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell 1996;86:147–57.[CrossRef][Medline]
  27. Emaus RK, Grunwald R, Lemasters JJ. Rhodamine 123 as a probe of transmembrane potential in isolated rat-liver mitochondria: spectral and metabolic properties. Biochim Biophys Acta 1986;850:436–48.[Medline]
  28. Kolm RH, Danielson UH, Zhang Y, Talalay P, Mannervik B. Isothiocyanates as substrates for human glutathione transferases: structure-activity studies. Biochem J 1995;311:453–9.
  29. Zhang Y, Talalay P. Mechanism of differential protencies of isothiocyanates as inducers of anticarcinogenic phase 2 enzymes. Cancer Res 1998;58:4632–9.[Abstract/Free Full Text]
  30. Finucane DM, Waterhouse NJ, Amarante-Mendes GP, Cotter TG, Green DR. Collapse of the inner mitochondrial transmembrane potential is not required for apoptosis of HL60 cells. Exp Cell Res 1999;251:166–74.[CrossRef][Medline]
  31. Ly JD, Grubb DR, Lawen A. The mitochondrial membrane potential ({Delta}{Psi}m) in apoptosis; an update. Apoptosis 2003;8:115–28.[CrossRef][Medline]
  32. Henderson PJ, Lardy HA. An inhibitor of the adenine nucleotide translocase of mitochondria. J Biol Chem 1970;245:1319–26.[Abstract/Free Full Text]
  33. Broekemeier KM, Dempsey ME, Pfeiffer DR. Cyclosporin A is a potent inhibitor of the inner membrane permeability transition in liver mitochondria. J Biol Chem 1989;264:7826–30.[Abstract/Free Full Text]
  34. Halestrap AP, Davidson AM. Inhibition of Ca2+-induced large-amplitude swelling of liver and heart mitochondria by cyclosporin is probably caused by the inhibitor binding to mitochondrial-matrix peptidyl-prolyl cis-trans isomerase and preventing it interacting with the adenine nucleotide translocase. Biochem J 1990;268:153–60.[Medline]
  35. Narita M, Shimizu S, Ito T, et al. Bax interacts with the permeability transition pore to induce permeability transition and cytochrome c release in isolated mitochondria. Proc Natl Acad Sci U S A 1998;95:14681–6.[Abstract/Free Full Text]
  36. Kanno T, Sato E, Muranaka S, et al. Oxidative stress underlies the mechanism for Ca2+-induced permeability transition of mitochondria. Free Radic Res 2004;38:27–35.[CrossRef][Medline]
  37. Chinopoulos C, Starkov AA, Fiskum G. Cyclosporin A-insensitive permeability transition in brain mitochondria: inhibition by 2-aminoethoxydiphenyl borate. J Biol Chem 2003;278:27382–9.[Abstract/Free Full Text]
  38. Zamzami N, Susin SA, Marchetti P, et al. Mitochondrial control of nuclear apoptosis. J Exp Med 1996;183:1533–44.[Abstract/Free Full Text]
  39. Li M, Xia T, Jiang CS, Li LJ, Fu JL, Zhou ZC. Cadmium directly induced the opening of membrane permeability pore of mitochondria which possibly involved in cadmium-triggered apoptosis. Toxicology 2003;194:19–33.[CrossRef][Medline]
  40. Choi S, Singh SV. Bax and Bak are required for apoptosis induction by sulforaphane, a cruciferous vegetable-derived cancer chemoprevention agent. Cancer Res 2005;65:2035–43.[Abstract/Free Full Text]
  41. Xu K, Thornalley PJ. Signal transduction activated by the cancer chemopreventive isothiocyanates: cleavage of BID protein, tyrosine phosphorylation and activation of JNK. Br J Cancer 2001;84:670–3.[CrossRef][Medline]
  42. Miyoshi N, Uchida K, Osawa T, Nakamura Y. A link between benzyl isothiocyanate-induced cell cycle arrest and apoptosis: involvement of mitogen-activated protein kinases in the Bcl-2 phosphorylation. Cancer Res 2004;64:2134–42.[Abstract/Free Full Text]
  43. Rose P, Armstrong JS, Chua YL, Ong CN, Whiteman M. ß-phenylethyl isothiocyanate mediated apoptosis; contribution of Bax and the mitochondrial death pathway. Int J Biochem Cell Biol 2005;37:100–19.[CrossRef][Medline]
  44. Zamzami N, Kroemer G. The mitochondrion in apoptosis: how Pandora's box opens. Nat Rev Mol Cell Biol 2001;2:67–71.[CrossRef][Medline]
  45. Martinou JC, Green DR. Breaking the mitochondrial barrier. Nat Rev Mol Cell Biol 2001;2:63–7.[CrossRef][Medline]
  46. Miko M, Chance B. Isothiocyanates. A new class of uncouplers. Biochim Biophys Acta 1975;396:165–74.[Medline]
  47. Lim MLR, Minamikawa T, Nagley P. The protonophore CCCP induces mitochondrial permeability transition without cytochrome c release in human osteosarcoma cells. FEBS Lett 2001;503:69–74.[CrossRef][Medline]
  48. Nieminen AL, Saylor AK, Tesfai SA, Herman B, Lemasters JJ. Contribution of the mitochondrial permeability transition to lethal injury after exposure of hepatocytes to t-butylhydroperoxide. Biochem J 1995;307:99–106.
  49. Nakamura Y, Ohigashi H, Masuda S, et al. Redox regulation of glutathione S-transferase induction by benzyl isothiocyanate: correlation of enzyme induction with the formation of reactive oxygen intermediates. Cancer Res 2000;60:219–25.[Abstract/Free Full Text]
  50. Rose P, Whiteman M, Huang SH, Halliwell B, Ong CN. ß-Phenylethyl isothiocyanate-mediated apoptosis in hepatoma HepG2 cells. Cell Mol Life Sci 2003;60:1489–503.[CrossRef][Medline]
  51. Payen L, Courtois A, Loewert M, Guillouzo A, Fardel O. Reactive oxygen species-related induction of multidrug resistance-associated protein 2 expression in primary hepatocytes exposed to sulforaphane. Biochem Biophys Res Commun 2001;282:257–63.[CrossRef][Medline]
  52. Papa S, Skulachev VP. Reactive oxygen species, mitochondria, apoptosis and aging. Mol Cell Biochem 1997;174:305–19.[CrossRef][Medline]
  53. Turrens JF, Alexandre A, Lehninger AL. Ubisemiquione is the electron donor for superoxide formation by complex III of heart mitochondria. Arch Biochem Biophys 1985;237:408–14.[CrossRef][Medline]
  54. Fleury C, Mignotte B, Vayssiere JL. Mitochondrial reactive oxygen species in cell death signaling. Biochimie 2002;84:131–41.[Medline]
  55. Palmeira CM, Ferreira FM, Rolo AP, et al. Histological changes and impairment of liver mitochondrial bioenergetics after long-term treatment with {alpha}-naphthyl-isothiocyanate (ANIT). Toxicology 2003;190:185–96.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
L. Mi, Z. Xiao, B. L. Hood, S. Dakshanamurthy, X. Wang, S. Govind, T. P. Conrads, T. D. Veenstra, and F.-L. Chung
Covalent Binding to Tubulin by Isothiocyanates: A MECHANISM OF CELL GROWTH ARREST AND APOPTOSIS
J. Biol. Chem., August 8, 2008; 283(32): 22136 - 22146.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Epidemiol. Biomarkers Prev.Home page
L. Tang, G. R. Zirpoli, K. Guru, K. B. Moysich, Y. Zhang, C. B. Ambrosone, and S. E. McCann
Consumption of Raw Cruciferous Vegetables is Inversely Associated with Bladder Cancer Risk
Cancer Epidemiol. Biomarkers Prev., April 1, 2008; 17(4): 938 - 944.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
R. Munday, P. Mhawech-Fauceglia, C. M. Munday, J. D. Paonessa, L. Tang, J. S. Munday, C. Lister, P. Wilson, J. W. Fahey, W. Davis, et al.
Inhibition of Urinary Bladder Carcinogenesis by Broccoli Sprouts
Cancer Res., March 1, 2008; 68(5): 1593 - 1600.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
S. J. Thomson, K. K. Brown, J. M. Pullar, and M. B. Hampton
Phenethyl Isothiocyanate Triggers Apoptosis in Jurkat Cells Made Resistant by the Overexpression of Bcl-2.
Cancer Res., July 1, 2006; 66(13): 6772 - 6777.
[Abstract] [Full Text] [PDF]

Home page
 Molecular Cancer TherapeuticsHome page
L. Tang, Y. Zhang, H. E. Jobson, J. Li, K. K. Stephenson, K. L. Wade, and J. W. Fahey
Potent activation of mitochondria-mediated apoptosis and arrest in S and M phases of cancer cells by a broccoli sprout extract.
Mol. Cancer Ther., April 1, 2006; 5(4): 935 - 944.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
H. Kim, E. H. Kim, Y. W. Eom, W.-H. Kim, T. K. Kwon, S. J. Lee, and K. S. Choi
Sulforaphane Sensitizes Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL)-Resistant Hepatoma Cells to TRAIL-Induced Apoptosis through Reactive Oxygen Species-Mediated Up-regulation of DR5
Cancer Res., February 1, 2006; 66(3): 1740 - 1750.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow