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Blood, Vol. 95 No. 5 (March 1), 2000:
pp. 1773-1780
NEOPLASIA
From the Department of Physiology, National University of Singapore,
and The Bioscience Centre, National University of Singapore,
Singapore.
Induction of mitochondrial permeability transition (MPT) and
cytosolic translocation of cytochrome C are considered essential components of the apoptotic pathway. Hence, there is the realization that mitochondrial-specific drugs could have potential for use as
chemotherapeutic agents to trigger apoptosis in tumor cells. Recently,
we showed that photoproducts of merocyanine 540 (pMC540) induced tumor
cell apoptosis. In this study, we focused on identifying mitochondrial-specific compounds from pMC540 and studied their apoptotic potential. One purified fraction, C5, induced a drop in
mitochondrial transmembrane potential and cytosolic
translocation of cytochrome C in HL60 human leukemia cells. Moreover,
the addition of C5 to purified rat liver mitochondria induced MPT as
indicated by mitochondrial matrix swelling, which was completely
inhibited by cyclosporin A, an inhibitor of the inner-membrane
pore. Supernatant of C5-treated mitochondria showed a dose-dependent
increase in cytochrome C, which was also inhibited in the presence of
cyclosporin A, strongly indicating a direct effect on the
inner-membrane pore. Despite the strong mitochondrial reactivity, C5
elicited minimal cytotoxicity (less than 25%) against HL60 leukemia
and M14 melanoma cells because of inefficient caspase activation.
However, prior exposure to C5 significantly enhanced the apoptotic
response to etoposide or the CD95 receptor. Thus, we demonstrate that
MPT induction and cytochrome C release by the novel compound C5, in the
absence of effective caspase activation, is insufficient for triggering
efficient apoptosis in tumor cells. However, when used in combination
with known apoptosis inducers, such compounds could enhance the
sensitivity of tumor cells to apoptosis.
(Blood. 2000;95:1773-1780)
There is convincing evidence to implicate mitochondria
in the execution of the apoptotic pathway. At least 3 mitochondrial-specific events have been well defined in cells
undergoing apoptosis, namely, loss of mitochondrial transmembrane
potential ( Tumor cell lines
Purification of photo-oxidation products from pMC540
Determination of mitochondrial   m as described elsewhere.11 HL60 cells
(1 × 106) were exposed to 50 to 150 µg/mL C5 for
8 hours; this was followed by the loading of cells with DiOC6
(40 nmol/L for 15 minutes. at 37°C). As a
positive control, cells were incubated at the same time with uncoupling
agent CICCP (100 µmol/L), a protonophore that disrupts the
m. Cells were then washed twice with
1 × phosphate-buffered saline (PBS) and immediately analyzed in
an Epics Profile (Coulter, Hialeah, FL) flow cytometer with the
excitation set at 488 nm. At least 10 000 events were collected per
sample, and data were analyzed by the WINMDI software (University of
Massachusetts, Amherst, MA). In a separate set of experiments, 50 µg
purified rat liver mitochondria were exposed to 100 µg/mL C5 for 1 hour at 25°C, followed by incubation for 15 minutes at 37°C
with 40 nmol/L DiOC6. After 2 gentle washes with
1 × PBS, mitochondria were analyzed by flow cytometry as above.
Detection of cytosolic C For the determination of Cyt C, HL60 cells (30 × 106) were treated with 50 to 150 µg/mL C5 for 18 hours and cytosolic fractions were obtained. Briefly, cells were washed twice with ice-cold PBS, pH 7.4, which was followed by centrifugation at 200g for 5 minutes. The cell pellet was then resuspended in 600 µL extraction buffer containing 200 mmol/L mannitol, 68 mmol/L sucrose, 50 mmol/L PIPES-KOH, pH 7.4, 50 mmol/L KCl, 5 mmol/L EGTA, 2 mmol/L MgCl2, and 1 mmol/L dithiothreitol and protease inhibitors (Complete Cocktail; Boehringer Mannheim, Mannheim, Germany). After 30 minutes' incubation on ice, cells were homogenized with a dounce homogenizer, and the homogenate was spun at 14 000g for 15 minutes. Supernatants were removed and stored at 80°C until analysis by gel electrophoresis for
Cyt C. In a separate set of experiments, M14 cells
(2 × 103) were grown on coverslips, exposed to 150 µg/mL C5, fixed with methanol:acetone (1:1 vol/vol), and incubated
for 2 hours at 37°C with 1:150 dilution (in 3% bovine serum
albumin) of monoclonal anti-Cyt C antibody (clone 7H8.2C12; Pharmingen,
San Diego, CA) as described previously.19 After 3 washes
with 1 × PBS + 1% FBS, cells were exposed to a 1:20
dilution of antimouse fluorescein isothiocyanate-conjugated IgG
(Pharmingen, San Diego, CA) for 1 hour, washed twice, and analyzed by
confocal microscopy (NUMI Core Facility, NUS, Singapore). Cytosolic C
was defined as diffuse cytoplasmic staining compared with punctate
mitochondrial Cyt C staining obtained with nontreated control cells.
Preparation of rat liver mitochondria Mitochondria were isolated from rat liver (albino rats, Wistar strain) as described elsewhere.20 Briefly, liver cells were homogenized in 10 mL buffer A (0.3 mol/L sucrose, 5 mmol/L TES, 0.2 mmol/L EGTA, pH 7.2 with KOH), and centrifuged at 2000g for 10 minutes at 4°C. The supernatant (S1) was removed and the pellet was resuspended in 10 mL buffer A and centrifuged at 2000g for10 minutes at 4°C. The supernatant obtained (S2) was then mixed with S1 and centrifuged at 8000g for 10 minutes at 4°C. The pellet was then resuspended in 1 mL buffer A and loaded on top of a Percoll gradient (60%, 30%, 18%) prepared in buffer A and centrifuged at 8000g for 10 minutes at 4°C. Mitochondria were then separated from nonmitochondrial membranes and nonfunctional organelles, collected at the 30%/60% interface, and washed with 10 vol buffer A at 8000g for 10 minutes at 4°C to wash off the Percoll. Mitochondria were then resuspended in 2 mL buffer A and kept at 4°C with gentle stirring. All experiments with isolated mitochondria were performed within 4 hours of the preparation.Mitochondrial swelling and release of Cyt C Large-amplitude mitochondrial swelling was determined spectroscopically by the loss of absorbance at 540 nm as described elsewhere.15 To induce mitochondrial swelling 200 µmol/L diethyl pyrocarbonate (DEPC; Sigma, St. Louis, MO) was used as a positive control.13 Where indicated, 10 µmol/L cyclosporin A (CsA; Sigma, St. Louis, MO) was added to the mitochondria before the addition of DEPC or C5.Western blotting for Cyt C Fifty micrograms cytosolic protein extracts or supernatants from mitochondria treated with C5 or 0.2% Triton-X treated mitochondrial pellets were subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes using a Trans-blot SD semidry system (Bio-Rad Laboratories, Hercules, CA). Membranes were then blocked overnight with 5% dry milk in TBST (50 mmol/L Tris/HCl, pH 7.4, 150 mmol/L NaCl, 0.1% Tween 20). Exposure of blocked membranes to primary anti-Cyt C antibody 7H8.2C12 was accomplished at room temperature for 1 hour using an antibody dilution of 1:5000 made in TBS + 0.05% Tween 20 and 1% bovine serum albumin. After 3 washes with TBST, the membranes were exposed to 1:5000 dilution of goat antimouse IgG-horseradish peroxidase (HRP) conjugate supplied as 0.8 mg/ml (Pierce, Rockford, IL) for 1 hour and washed 3 times with TBST. Chemiluminescence was detected using the SuperSignal Substrate Western Blotting Kit (Pierce, Rockford, IL).Determination of caspases 3, 8, 2, and 9 activity Activation of intracellular caspases 3 and 8 was assayed by the ApoAlert Fluorescent Assay Kits (Clontech, Palo Alto, CA), whereas activity of caspases 2 and 9 was measured using AFC-conjugated substrates supplied by Bio-Rad Laboratories (Hercules, CA). HL60 cells (1 × 106 cells/mL) were exposed to C5 (50-150 µg/mL), washed twice with 1 × PBS, resuspended in 50 µL chilled cell lysis buffer (provided by the supplier), and incubated on ice for 10 minutes. Fifty microliters 2 × reaction buffer containing 10 mmol/L dithiothreitol and 6 µL fluorogenic caspase-specific substrate (DEVD-AFC for caspase 3, IETD-AFC for caspase 8, VDVAD-AFC for caspase 2, and LEHD-AFC for caspase 9) was added to each sample, and the samples were incubated at 37°C for 30 minutes. As a comparison, HL60 cells were exposed to 100 µg/mL of the potent caspase-inducing photoproduct C2, which has been described in a recent report,19 and caspase assays were performed as below. In a separate set of experiments, HL60 and M14 cells (1 × 106/mL) were treated with 40 µmol/L etoposide or 0.5 µmol/L staurosporine for 8 hours, and lysates were analyzed for caspase 3 activity in the presence or absence of 1 µmol/L caspase 3-specific inhibitor DEVD-CHO. Protease activity was determined by measuring the relative fluorescence intensity at 505 nm after excitation at 400 nm using a spectrofluorometer (Luminescence Spectrometer LS50B; Perkin Elmer, Buckinghamshire, UK). Results are shown as fold increase in activity relative to untreated control cells. Cell lysates obtained from C5- or C2-treated HL60 cells were also analyzed for cleavage of poly-adenosine diphosphate ribose polymerase (PARP) by 10% SDS-PAGE and Western blotting using a monoclonal anti-PARP antibody (clone C2-10 from Pharmingen, San Diego, CA). Detection of the 116-kd or the cleaved 85-kd bands, or both, was carried out using HRP-conjugated anti-mouse IgG as described in "Western Blotting for Cyt C."Detection of processed caspase 3 Processing of procaspase 3 was assessed by either flow cytometry using a phycoerythrin-conjugated rabbit polyclonal antiactive caspase 3 antibody (Pharmingen) or by Western blot analysis of processed caspase 3 (17 kd) using 1:750 dilution of a polyclonal rabbit anticaspase 3 antibody (Pharmingen). The HRP-conjugated goat antirabbit IgG (DAKO, Carpenteria, CA) was used at 1:2000 dilution as the secondary antibody, and chemiluminescence was detected using the SuperSignal Substrate Western Blotting Kit (Pierce) as described above. For flow cytometry, HL60 cells (1 × 106) were treated with 150 µg/mL C5 or 100 µg/mL C2 in the presence or absence of caspase 3-specific inhibitor DEVD-CHO (1 µmol/L) for 12 hours, washed with PBS, fixed, and permeabilized as described elsewhere.21 The cell pellets were then resuspended in 20 µL antiactive caspase 3 antibody for 20 minutes at 25 °C, washed twice with PBS + 1% FBS, and at least 10 000 events were analyzed by flow cytometry with the detection wavelength set at 585 nm.Determination of antitumor activity HL60 and M14 cells were treated with increasing concentrations of C5 (50-150 µg/mL) for 18 hours. HL60 cells were plated at 1 × 105/well in a 96-well plate, and viability was determined by the MTT assay. Ten microliters of 5 mg/mL MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) was then added to each well of cells and incubated for 4 hours at 37°C. Elution of the precipitate was performed with 100 µL dimethyl sulfoxide + 10 µL Tris-glycine buffer. Cell viability was calculated from the absorption values obtained at 570 nm using an automated ELISA reader. M14 cells were plated at 2 × 104/well in RPMI/5%FBS and allowed to attach overnight. Medium was then removed and replaced with fresh medium containing C5. After an 18-hour incubation, wells were aspirated and 50 µL 0.75% crystal violet solution containing 50% ethanol, 0.25% NaCl, and 1.75% formaldehyde was added to each well for 10 minutes. Cells were then washed with water and air dried, and the dye was eluted with PBS/1% SDS. Cell viability was measured by dye absorbance at 590 nm on an automated ELISA reader. To evaluate the ability of C5 to sensitize tumor cells to apoptosis triggered by either etoposide or the cell surface receptor CD95 (Fas/Apo1), M14 cells or M14TF4 cells expressing a chimeric receptor consisting of the extracellular portion of the tumor necrosis factor (TNF)- receptor p55 and the entire
cytoplasmic region of the CD95 receptor22 (generous gift
from Dr Clément) were exposed to 40 µmol/L etoposide or 10 ng/mL rTNF- for 8 hours in the presence or absence of 150 µg/mL
C5, and caspase 3 activity was determined as described above. To
ascertain whether the measured activity was caspase 3-specific, cells
were exposed to 1 µmol/L DEVD-CHO before the addition of the
apoptotic triggers, and lysates were analyzed for caspase 3 activity.
Cell-free system of apoptosis To generate a cell-free system of apoptosis, cytosolic extracts from HL60 cells (50 × 106) were prepared as described above. 150 µg of the cytosolic extracts were then incubated for 6 hours at 30°C with supernatants obtained from C5 or C2-treated mitochondria (30 µL 1 mg/mL mitochondria) or 1-10 µmol/L purified Cyt C (Sigma, St. Louis, MO) in the presence of 1 mmol/L dATP (Sigma). Samples were then analyzed for caspase 3 activation by enzyme activity assay and by Western blotting of processed caspase 3 as described above.
Purification of C5 from pMC540 In a previous study, we showed that tumor cells undergo caspase-mediated cell death after exposure to a mixture of photoproducts generated by the photoactivation of MC540.18 Because pMC540 is a mixture of photoproducts, and for it to achieve potential as a clinical chemotherapeutic agent, the biologically active component(s) in the mixture have to be identified. The conditions used for photoactivation of MC540 were identical to those described in our in vitro studies demonstrating caspase-dependent antitumor activity of pMC540.18 After photoactivation in ethanol, the solvent was evaporated and the mixture of photo-oxidation product was analyzed by TLC on aluminum sheets coated with fluorescent silica gel. TLC solvent system 1 yielded 2 fractions that have been described elsewhere,19 and solvent system 2 yielded a product at Rf 0.25, termed C5, a highly photosensitive intermediate product with 2 absorbance peaks at 230 nm and 270 nm in methanol, which accounted for approximately 60% of the photoactivated mixture.C5 induces a drop in mitochondrial m secondary to the induction of mitochondrial
permeability transition.9 Therefore, we set out to identify
the fraction(s) purified from pMC540, which triggered changes in
m of tumor cells and induced cytosolic translocation
of Cyt C. Our results showed that purified fraction C5 induced a
significant drop in the mean m of HL60 cells as shown
in Figure 1A. Recent observations suggest
that mitochondrial release of pro-apoptotic factors, such as Cyt C, is
dependent on a drop in m secondary to opening of the
inner membrane MPT pore. Having shown that C5 induced a drop in
mitochondrial m, we next investigated the ability of
C5 to trigger the release of apoptogenic factors from mitochondria of tumor cells. One of the factors released from the mitochondria during
apoptosis is Cyt C, which can then activate downstream caspases and
amplify the apoptotic signal. Thus, we tested the potential of C5 to
induce Cyt C translocation to the cytosol of HL60 and M14 cells.
Cytosolic extracts prepared from C5-treated HL60 cells were subjected
to SDS-PAGE and Western blot analysis for Cyt C. Indeed, our results
showed that whereas no detectable Cyt C was found in the cytosolic
extract of untreated cells, C5 triggered the translocation of Cyt C to
the cytosols of HL60 cells, as shown in Figure 1B. To alleviate
concerns that the process of homogenization could have resulted in
artifactual "release" of Cyt C in HL60 cells, we assessed the
translocation of Cyt C in intact cells after exposure to C5. To do so,
M14 cells (2 × 103) were grown on coverslips,
exposed to 150 µg/mL C5, fixed with methanol:acetone (1:1 vol/vol),
incubated with monoclonal anti-Cyt C antibody, and analyzed by confocal
microscopy as described in "Materials and methods." Analysis of
M14 cells by confocal microscopy showed a punctate pattern of staining
for Cyt C in untreated control cells, suggesting mitochondrial
localization. However, cells exposed to C5 exhibited bright green,
diffuse cytoplasmic staining, indicating the translocation of Cyt C to
the cytosol (Figure 1C). These data clearly suggested a
mitochondrial-specific activity for C5 and stimulated us to focus on
elucidating the mechanism of action of C5 and its potential as an
antitumor agent.
C5 induces mitochondrial permeability transition and opening of CsA-sensitive MPT pore Two major hypotheses have been experimentally suggested for the mechanism of Cyt C release from the mitochondria during apoptosis. One is dependent on a drop inm secondary to opening of
the inner-membrane MPT pore,23 and the other contends that
Cyt C release is independent of a decrease in
m.2 MPT pore opening results in volume
dysregulation of the mitochondria from the hyperosmolality of the
matrix, causing the matrix space to expand. Inhibitors of MPT pore
opening, such as CsA and bongkrekic acid, appear to block
apoptosis in some systems,10,24,25 implying a general role.
However, some studies have provided evidence that Cyt C release can
occur independent of any detectable loss in mitochondrial m.2 Encouraged by our findings that C5
triggered the release of mitochondrial Cyt C in HL60 cells, we set out
to investigate whether this cytosolic translocation of Cyt C resulted
from a direct effect on mitochondrial membrane structures, specifically the MPT pore. To accomplish that, we purified rat liver mitochondria and asked whether C5 induced mitochondrial swelling secondary to MPT
pore opening and whether Cyt C release was dependent on opening of the
pore. Freshly isolated mitochondria (0.5 mg) were exposed to 150 µg
C5 under conditions previously shown to be conducive for swelling
induced by activators of MPT, such as Ca++ and
DEPC.13 As expected, the addition of DEPC (0.2 mmol/L) to
isolated mitochondria resulted in mitochondrial matrix swelling, measured by a decrease in absorbance at 540 nm using a double-beam spectrophotometer, which was completely inhibited by prior addition of
CsA (10 µmol/L), an MPT pore inhibitor (Figure
2A). As with DEPC, the addition of C5 to
fresh mitochondria resulted in a rapid loss of absorbance, which was
completely inhibited by the earlier addition of CsA (Figure 2B).
Furthermore, incubation of purified mitochondria with C5 (100 µg) for
1 hour resulted in alkalinization of the matrix milieu from pH 7.5 to
more than 8.5 (data not shown); matrix alkalinization has been shown to
facilitate the opening of the MPT pore, whereas acidification impedes
pore opening.26 Thus, a possible mechanism by which C5
triggers opening of the MPT pore could be by alkalinization of the
matrix environment. To provide further impetus to these findings, we
directly assessed the ability of C5 to induce Cyt C release from
isolated mitochondria and to determine whether it was dependent on
opening of the MPT pore and a drop in mitochondrial
m. Purified mitochondria (0.5 mg) was incubated with
150 µg/mL C5 in the presence or absence of MPT pore inhibitor CsA (10 µmol/L). Mitochondria were then gently pelleted, and supernatants
were used for Western blot analysis of Cyt C. Our results showed that
C5 triggered the release of Cyt C from purified mitochondria, which was
completely inhibited by CsA (Figure 3A).
Moreover, similar to our observations with tumor cells, incubation of
purified mitochondria for 30 minutes with C5 induced a drop in
mitochondrial m, which was completely inhibited by
CsA, as shown in Figure 3B. These results demonstrate that C5 directly
targets the MPT pore and induces mitochondrial swelling in a
CsA-inhibitable manner, supporting a mitochondrial-specific mode of
action for this purified fraction.
C5 lacks antitumor activity because of inefficient caspase activation Stimulated by the findings that C5 effectively targeted the mitochondrial pore and induced translocation of Cyt C, we next assessed the antitumor potential of C5. If mitochondrial events were directly involved in triggering the execution of drug-induced apoptosis, then C5 should make an excellent antitumor agent. Hence, we investigated the ability of C5 to trigger cell death in HL60 leukemia and M14 melanoma cells. Our results showed that exposure of both cell lines to C5 did not trigger significant cell death (less than 25%) as shown in Figure 4A, which did not increase significantly on longer incubation for 48 hours (data not shown). We were intrigued by the findings that despite its ability to induce mitochondrial permeability transition and cytosolic translocation of Cyt C, C5 was an inefficient inducer of cell death in both tumor cell lines. Because of the central role of caspase proteases in the execution of the apoptotic program, we argued that this low antitumor activity could be caused by the inability of C5 to trigger caspase activation efficiently. Therefore, we assayed HL60 cells after exposure to C5 (50-150 µg/mL) for caspase 3 activity, 1 of the executioner caspases extensively studied in drug-induced apoptosis. Indeed, lysates obtained from C5-treated HL60 cells showed minimal activation of caspase 3 compared to the potent caspase-inducing photoproduct C2 (Figure 4B), the antitumor activity of which has been well described in a recent communication.19 The lack of caspase 3 activity was further reinforced by the absence of the processed form (17 kd) of caspase 3 in HL60 cells treated with C5, unlike cells treated with C2 (Figure 4C). Prior exposure of cells to DEVD-CHO completely inhibited the processing of caspase 3 in C2-treated HL60 cells, as shown in Figure 4C. Furthermore, exposure of HL60 cells to C5 resulted in insufficient activation of other upstream caspases, such as caspase 8, 2, and 9, compared to C2 (Figure 5A). The inability of C5 to activate intracellular caspases was also evidenced by the absence of PARP cleavage in lysates of C5-treated HL60 cells, a hallmark of apoptotic cells (Figure 5B). To ensure that the absence of executioner caspase 3 activity in response to C5 was not the result of low expression of the caspase in HL60 and M14 cells, we used 2 commonly used apoptotic stimuli, etoposide and staurosporine, to trigger apoptosis in tumor cells. Both apoptotic stimuli induced efficient activation of caspase 3 in both cell lines, which were completely inhibited by prior incubation of cells with DEVD-CHO as shown in Figure 6. We also ruled out the possibility of C5 as a caspase inhibitor by exposing HL60 cells to etoposide for 8 hours and then by caspase 3 activity assay in the presence of either the specific inhibitor DEVD or C5. Although DEVD completely inhibited caspase 3 activity, C5 had no effect on etoposide-induced caspase 3 activity (data not shown). These data clearly indicate that despite triggering MPT pore opening and release of Cyt C, the low antitumor activity of C5 could be attributed to an inefficient activation of the caspase cascade. Thus, compounds that can target mitochondria and at the same time induce activation of caspases (upstream or downstream) could efficiently activate the apoptotic pathway in tumor cells. These findings highlight the importance of drug-induced caspase activation in the effectiveness of antitumor agents.
C5-treated mitochondrial supernatants lack caspase 3 processing activity In light of our findings, compounds such as C5 that are potent inducers of mitochondrial permeability transition and Cyt C release may not prove effective if they do not sufficiently activate the caspase cascade. To substantiate our observations, we next asked whether supernatants obtained from C5-treated purified mitochondria could trigger activation and processing of caspase 3 in cytosolic extracts from HL60 cells in a cell-free system of apoptosis. Therefore, 150 µg cytosolic extracts were incubated with the supernatant from C5- or C2-treated mitochondria for 6 hours at 37°C in the presence of 1 mmol/L dATP. Moreover, to demonstrate that the cell-free system was functioning, HL60 cytosols were also incubated with increasing concentrations of purified Cyt C (0.1-10 µmol/L) under similar conditions. Interestingly, whereas the C5-treated mitochondrial supernatant was unable to activate caspase 3, the C2-treated supernatant induced processing/activation of caspase 3 in HL60 cytosols (Figure 7). Incubation of cytosols with relatively high concentrations of purified Cyt C (10 µmol/L) also triggered caspase 3 processing/activation, as shown in Figure 7, confirming the integrity of the apoptotic machinery in the cytosolic preparation. There are 2 reasons we think these observations are significant. First, as shown in our recently reported findings,19 purified photoproduct C2 also induces the release of Cyt C in the cytosol of tumor cells, albeit by mechanisms independent of the MPT pore opening. Indeed, analysis of supernatants derived from isolated mitochondria treated with C5 or C2 showed no significant difference in the amount of Cyt C released shown in Figure 7. Therefore, the fact that the C2-treated mitochondrial supernatant was able to induce activation/processing of caspase 3 in HL60 cytosol, unlike C5, suggests the release of a factor "X" in addition to Cyt C from C2-treated mitochondria. Could it be the simultaneous release of Cyt C and apoptosis-inducing factor or another mitochondrial proapoptotic protein that determines the effectiveness of drug-induced caspase activation? Second, the concentration of exogenous Cyt C (10 µmol/L) required to activate caspase 3 in the cell-free system is perhaps many times higher than could be attained in vivo in the cytosol in response to a given anticancer agent. Hence, the ability of a drug to trigger cytosolic translocation of Cyt C may not independently induce effective caspase activation by itself, but it may help to amplify the response to other apoptotic stimuli.
Simultaneous exposure to C5 amplifies drug-induced apoptotic signaling We have thus far demonstrated that in the absence of direct caspase activation, Cyt C translocation may not be able independently to activate the caspase pathway and to trigger effective apoptosis in tumor cells. However, the release of apoptogenic factors such as Cyt C from the mitochondria could certainly help to amplify the apoptotic process once the caspase(s) have been activated. Having shown the inability of C5 to activate intracellular caspases efficiently despite triggering cytosolic translocation of Cyt C in tumor cells, we questioned whether Cyt C release in C5-treated tumor cells could amplify the apoptotic signal in response to known inducers of apoptosis. Therefore, we investigated the usefulness of C5 as an adjuvant to etoposide and CD95 (Fas-Apo-1)-mediated apoptosis in tumor cells. Indeed, our results showed that prior exposure of M14 cells to 150 µg/mL C5 (2 hours) significantly enhanced caspase 3 activation in response to 8 hours of treatment with 40 µmol/L etoposide, which was completely inhibited in the presence of caspase 3 inhibitor DEVD-CHO (Figure 8). Similarly, M14TF4 cells expressing a chimeric receptor consisting of the extracellular portion of the TNF- (p55) and the entire cytoplasmic region of the CD95 receptor showed significantly higher caspase 3 activation when incubated for 2 hours with C5 and then by 8-hour incubation with rTNF- (10 ng/mL) than either of the drugs alone, as shown in Figure 8. These data strongly argue in support of our
hypothesis that prior exposure to C5 serves to amplify
significantly the apoptotic signal, perhaps because of its ability to
the trigger cytosolic translocation of Cyt C. Hence, drugs similar to
C5, despite having poor antitumor activity, could prove beneficial as
part of a combination chemotherapy protocol to enhance tumor cell
sensitivity to apoptosis by providing essential amplification factor(s)
such as Cyt C.
The authors thank Dr Marie-Véronique Clément for useful discussions.
Submitted May 14, 1999; accepted October 29, 1999.
J.L.H. and M.A.S. contributed equally to this work.
Reprints: Shazib Pervaiz, Department of Physiology, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260; e-mail: phssp{at}leonis.nus.edu.sg.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
1. Decaudin D, Marzo I, Brenner C, Kroemer G. Mitochondria in chemotherapy-induced apoptosis: a prospective novel target of cancer therapy [review]. Int J Oncol. 1998;12:141-152[Medline] [Order article via Infotrieve]. 2. Bossy-Wetzel E, Newmeyer DD, Green DR. Mitochondrial cytochrome c release in apoptosis occurs upstream of DEVD-specific caspase activation and independently of mitochondrial transmembrane depolarization. EMBO J. 1998;17:37-49[Medline] [Order article via Infotrieve].
3.
Fulda S, Scaffidi C, Susin SA, et al.
Activation of mitochondria and release of mitochondrial apoptogenic factors by betulinic acid.
J Biol Chem.
1998;273:33,942-33,948 4. Kluck RM, Martin SJ, Hoffman BM, Zhou JS, Green DR, Newmeyer DD. Cytochrome c activation of CPP32-like proteolysis plays a critical role in a Xenopus cell-free apoptosis system. EMBO J. 1997;16:4639-4649[Medline] [Order article via Infotrieve].
5.
Susin SA, Lorenzo HK, Zamzami N, et al.
Mitochondrial release of caspase-2 and -9 during the apoptotic process.
J Exp Med.
1999;189:381-394 6. Troyan MB, Gilman VR, Gay CV. Mitochondrial membrane potential changes in otseoblasts treated with parathyroid hormone and estradiol. Poultry Sci. 1997;233:247-280. 7. Uehara T, Kikuchi Y, Nomura Y. Caspase activation accompanying cytochrome c release from mitochondria is possibly involved in nitric oxide-induced neuronal apoptosis in SH- SY5Y cells. J Neurochem. 1999;72:196-205[Medline] [Order article via Infotrieve].
8.
Zamzami N, Marchetti P, Castedo M, et al.
Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo.
J Exp Med.
1995;181:1661-1672
9.
Zamzami N, Marchetti P, Castedo M, et al.
Sequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early programmed cell death.
J Exp Med.
1995;182:367-377
10.
Zamzami N, Susin SA, Marchetti P, et al.
Mitochondrial control of nuclear apoptosis.
J Exp Med.
1996;183:1533-1544
11.
Marzo I, Brenner C, Zamzami N, et al.
The permeability transition pore complex: a target for apoptosis regulation by caspases and Bcl-2-related proteins.
J Exp Med.
1998;187:1261-1271
12.
Marzo I, Brenner C, Zamzami N, et al.
Bax and adenine nucleotide translocator cooperate in the mitochondrial control of apoptosis.
Science.
1998;281:2027-2031 13. Nicolli A, Petronilli V, Bernardi P. Modulation of the mitochondrial cylosporin A-sensitive permeability transition pore by matrix pH: evidence that the pore open-closed probability is regulated by histidine protonation. J Biol Chem. 1993;32:4461-4465.
14.
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;95:14,681-14,686
15.
Bernardi P, Vassanelli S, Veronese P, Colonna R, Szabo I, Zoratti M.
Modulation of the mitochondrial permeability transition pore.
J Biol Chem.
1992;267:2934-2939
16.
Bernardi P, Veronese P, Petronilli V.
Modulation of the mitochondria cyclosporin A-sensitive permeability transition pore.
J Biol Chem.
1993;268:1005-1010 17. Bernardi P, Petronilli V. The permeability transition pore as a mitochondrial calcium release channel: a critical appraisal. J Bioenerg Biomembr. 1996;28:131-138[Medline] [Order article via Infotrieve]. 18. Pervaiz S, Hirpara JL, Clement M-V. Caspase proteases mediate apoptosis induced by anti-cancer agent preactivated MC540 in human tumor cell lines. Cancer Lett. 1998;27:1-12.
19.
Pervaiz S, Seyed MA, Hirpara JL, Clément MV, Loh KW.
Purified photoproducts of merocyanine 540 trigger cytochrome C release and caspase 8-dependent apoptosis in human leukemia and melanoma cells.
Blood.
1999;93:4096-4108 20. Petit PX, O'Connor JE, Grunwald D, Brown SC. Analysis of the membrane potential of rat- and mouse-liver mitochondria by flow cytometry and possible applications. Eur J Biochem. 1990;194:389-397[Medline] [Order article via Infotrieve].
21.
Clément MV, Hirpara JL, Chawdhury SH, Pervaiz S.
Chemopreventive agent resveratrol, a natural product derived from grapes, triggers CD95 signaling-dependent apoptosis in human tumor cells.
Blood.
1998;92:996-1002 22. Clément M-V, Stamenkovic I. Superoxide anion is a natural inhibitor of Fas-mediated cell death. EMBO J. 1996;15:216-225[Medline] [Order article via Infotrieve].
23.
Bradham CA, Qian T, Streetz K, Trautwein C, Brenner DA, Lemasters JJ.
The mitochondrial permeability transition is required for tumor necrosis factor alpha-mediated apoptosis and cytochrome c release.
Mol Cell Biol.
1998;18:6353-6364
24.
Marchetti P, Castedo M, Susin SA, et al.
Mitochondrial permeability transition is a central coordinating event of apoptosis.
J Exp Med.
1996;184:1155-1160 25. Marchetti P, Hirsch T, Zamzami N, et al. Mitochondrial permeability transition triggers lymphocyte apoptosis. J Immunol. 1996;157:4830-4836[Abstract]. 26. Bernardi P, Vassanelli S, Veronese P, Colonna R, Szabo I, Zoratti M. Modulation of the mitochondrial permeability transition pore: effect of protons and divalent cations. J Biol Chem. 1992;267:2934-2939.
27.
Steemans M, Goossens V, Van de Craen M, et al.
A caspase-activated factor (CAF) induces mitochondrial membrane depolarization and cytochrome c release by a nonproteolytic mechanism.
J Exp Med.
1998;188:2193-2198
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  S. Caffieri, F. Di Lisa, F. Bolesani, M. Facco, G. Semenzato, F. Dall'Acqua, and M. Canton The mitochondrial effects of novel apoptogenic molecules generated by psoralen photolysis as a crucial mechanism in PUVA therapy Blood, June 1, 2007; 109(11): 4988 - 4994. [Abstract] [Full Text] [PDF]
|
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 Y. Tan, C. Wu, T. De Veyra, and P. A. Greer Ubiquitous Calpains Promote Both Apoptosis and Survival Signals in Response to Different Cell Death Stimuli J. Biol. Chem., June 30, 2006; 281(26): 17689 - 17698. [Abstract] [Full Text] [PDF]
|
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Y. Tan, N. Dourdin, C. Wu, T. De Veyra, J. S. Elce, and P. A. Greer Ubiquitous Calpains Promote Caspase-12 and JNK Activation during Endoplasmic Reticulum Stress-induced Apoptosis J. Biol. Chem., June 9, 2006; 281(23): 16016 - 16024. [Abstract] [Full Text] [PDF]
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Y. Sun, S. Orrenius, S. Pervaiz, and B. Fadeel Plasma membrane sequestration of apoptotic protease-activating factor-1 in human B-lymphoma cells: a novel mechanism of chemoresistance Blood, May 15, 2005; 105(10): 4070 - 4077. [Abstract] [Full Text] [PDF]
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 K. A. Ahmad, K. B. Iskandar, J. L. Hirpara, M.-V. Clement, and S. Pervaiz Hydrogen Peroxide-Mediated Cytosolic Acidification Is a Signal for Mitochondrial Translocation of Bax during Drug-Induced Apoptosis of Tumor Cells Cancer Res., November 1, 2004; 64(21): 7867 - 7878. [Abstract] [Full Text] [PDF]
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K. A. Ahmad, M.-V. Clement, I. M. Hanif, and S. Pervaiz Resveratrol Inhibits Drug-Induced Apoptosis in Human Leukemia Cells by Creating an Intracellular Milieu Nonpermissive for Death Execution Cancer Res., February 15, 2004; 64(4): 1452 - 1459. [Abstract] [Full Text] [PDF]
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M. Milella, Z. Estrov, S. M. Kornblau, B. Z. Carter, M. Konopleva, A. Tari, W. D. Schober, D. Harris, C. E. Leysath, G. Lopez-Berestein, et al. Synergistic induction of apoptosis by simultaneous disruption of the Bcl-2 and MEK/MAPK pathways in acute myelogenous leukemia Blood, May 1, 2002; 99(9): 3461 - 3464. [Abstract] [Full Text] [PDF]
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N. J. Torok, H. Higuchi, S. Bronk, and G. J. Gores Nitric Oxide Inhibits Apoptosis Downstream of Cytochrome c Release by Nitrosylating Caspase 9 Cancer Res., March 1, 2002; 62(6): 1648 - 1653. [Abstract] [Full Text] [PDF]
|
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 M. J. Gomez-Lechon, E. O'Connor, J. V. Castell, and R. Jover Sensitive Markers Used to Identify Compounds That Trigger Apoptosis in Cultured Hepatocytes Toxicol. Sci., February 1, 2002; 65(2): 299 - 308. [Abstract] [Full Text] [PDF]
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 M. Li, X. Wu, and X.-C. Xu Induction of Apoptosis in Colon Cancer Cells by Cyclooxygenase-2 Inhibitor NS398 through a Cytochrome c-dependent Pathway Clin. Cancer Res., April 1, 2001; 7(4): 1010 - 1016. [Abstract] [Full Text]
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 S. PERVAIZ Reactive oxygen-dependent production of novel photochemotherapeutic agents FASEB J, March 1, 2001; 15(3): 612 - 617. [Abstract] [Full Text] [PDF]
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O. Salvucci, M. Carsana, I. Bersani, G. Tragni, and A. Anichini Antiapoptotic Role of Endogenous Nitric Oxide in Human Melanoma Cells Cancer Res., January 1, 2001; 61(1): 318 - 326. [Abstract] [Full Text]
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J. L. Hirpara, M.-V. Clement, and S. Pervaiz Intracellular Acidification Triggered by Mitochondrial-derived Hydrogen Peroxide Is an Effector Mechanism for Drug-induced Apoptosis in Tumor Cells J. Biol. Chem., January 5, 2001; 276(1): 514 - 521. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
