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NEOPLASIA
From the Arizona Cancer Center, Department of
Microbiology and Immunology, and Department of Pathology, University of
Arizona, Tucson.
Imexon is a cyanoaziridine derivative that has antitumor activity
in multiple myeloma. Previous studies have shown that imexon induces
oxidative stress and apoptosis in the RPMI 8226 myeloma cell line. This
study reports that imexon has cytotoxic activity in other malignant
cell lines including NCI-H929 myeloma cells and NB-4 acute
promyelocytic leukemia cells, whereas normal lymphocytes and U266
myeloma cells are substantially less sensitive. Flow cytometric
experiments have shown that imexon treatment is associated with the
formation of reactive oxygen species (ROS) and the loss of
mitochondrial membrane potential ( Imexon (4-imino-1, 3-diazabicyclo-[3.1.0]
hexan-one) is a 2-cyanoaziridine derivative that has been extensively
studied as an immunomodulator and an anticancer agent. Imexon was shown
to be active in a variety of animal tumor models, in tumor cell lines, and in humans.1,2 Among 10 fresh human tumor types,
multiple myeloma was the most sensitive to imexon with a median
inhibitory concentration of 50% (IC50) of 1µM at 10 to
14 days using colony forming assays.3 Importantly, in
pilot phase I trials imexon was well tolerated by cancer
patients.2 No myelosuppression, renal dysfunction, or
elevation of hepatic enzyme was observed after imexon treatment in
humans.2 In the absence of antiemetics, nausea and
vomiting were the major toxicities associated with intravenous
administration of imexon.2 The lack of myelosuppression after imexon treatment was confirmed also in animal studies with mice
and dogs.1
We have recently shown that the cytotoxic mechanism of imexon action in
RPMI 8226 myeloma cells involves thiol depletion, oxidative stress, and
apoptosis.4 This activity requires an aziridine ring. The
activation of imexon is believed to involve aziridine ring opening and
subsequent binding to sulfhydryl groups of cysteine
residues.5 This results in the depletion of cellular thiols, the induction of oxidative stress, and apoptosis.4 Whether thiol depletion is causal or a marker of activity is not known.
Interestingly, imexon also induces gross alterations in mitochondrial
ultrastructure, but not in other cellular organelles.4 Moreover, oxidative damage of DNA was observed primarily in the cytoplasm and not in the nucleus, suggesting that mitochondria could be
targets of the drug.
It is well established that mitochondria are important regulators of
apoptosis and undergo major changes during apoptotic cell
death.6-10 These changes include opening of the
mitochondrial megachannel known as the permeability transition pore,
leading to disruption of the mitochondrial membrane potential
( Based on these data, the major goal of the current studies was to
investigate whether imexon induces mitochondrial ultrastructural and
biochemical alterations that are characteristic of the apoptotic cell
death pathway in RPMI 8226 myeloma cells. We also investigated whether
inhibition of normal superoxide production in mitochondria inhibits
imexon-induced cytotoxicity. In addition, we investigated the effects
of imexon on peripheral blood lymphocytes, acute promyelocytic leukemia
NB-4 cells, and NCI-H929 and U266 myeloma cell lines. The results show
that imexon induces changes in mitochondrial morphology, a reduction of
the mitochondrial membrane potential, and cytochrome c release. These
changes are consistent with drug-induced mitochondrial oxidation and
apoptotic signaling.
Chemicals
Cell cultures and viability assays
Cellular dehydrogenase activity, which is considered to reflect mitochondrial function and cell viability, was measured by a microculture tetrazolium (MTT) assay that is based on the ability of normally functioning mitochondria to reduce the dye, MTT (3-(4,5,-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), to a blue formazan.17 Bright-field microscopy studies For morphologic studies, the RPMI 8226 cells were pretreated with 50 µM TTFA, an inhibitor of complex II,18 for 16 hours. The cells were then treated with 50 µM TTFA and 180 µM imexon simultaneously for 48 hours. Untreated RPMI 8226 myeloma cells and cells treated with 50 µM TTFA or 180 µM imexon only were included as controls. The cells were cytospun on slides using a Cytospin 2 centrifuge (Shandon, Pittsburgh, PA), then fixed with 100% methanol for 2 minutes at room temperature, air-dried, and then stained with DiffQuick stain (Gibco-BRL Products). The cells were morphologically evaluated for apoptosis by bright-field microscopy (100 × oil immersion). The criteria used to identify apoptotic cells included chromatin condensation, formation of apoptotic bodies, and cellular shrinkage as described by Payne and coworkers.19Transmission electron microscopy and morphometric studies Mitochondrial morphologic changes and effects of imexon on cellular organelles were evaluated by transmission electron microscopy of RPMI 8226 cells. After treatment with 180 µM imexon for various time periods, the cells (1 × 106) were fixed with 3% glutaraldehyde made up in 0.1 M cacodylate buffer (pH 7.2). The cells were then postfixed in 1% osmium tetroxide, dehydrated in a graded series of ethanols, and embedded in epoxy resin. Ultrathin sections were evaluated for mitochondrial morphologic changes using a Philips CM12 transmission electron microscope (Eindhoven, The Netherlands).Cytofluorometric determination of   m and ROS levels after imexon treatment. Also, RPMI
8226 cells pretreated with 10 mM NAC for 3 hours and then incubated with 180 µM imexon for 48 hours were included.
The lipophilic cationic dye, MitoTracker Red (CMXRos), which is
concentrated in intact mitochondria was used along with flow cytometry
analysis to detect changes in the
Oxidative damage in the imexon-treated cells was assessed by staining with the membrane permeable dye, dihydroethidium (HE), which is oxidized to the fluorescent intercalator, ethidium, by cellular oxidants, particularly superoxide radicals.22 The oxidative conversion of HE to ethidium is then measured by flow cytometry. Cells (0.5 × 106/mL) were stained at a final concentration of 2 µM HE for 30 minutes at 37°C. The cells were then centrifuged for 5 minutes at 750g, the supernatant was removed, and the cells were resuspended in 500 µL PBS and kept on ice. The cells were then analyzed on a flow cytometer (BD FACScan, excitation: 488 nm, emission: 620 nm). Preparation of S-100 fraction and Western blot analysis for cytochrome c Cytosolic fractions were isolated according to the method of Vander Heiden and coworkers.23 Briefly, RPMI 8226 cells (2 × 108) were resuspended in 0.24 mL ice-cold buffer A (20 mM Hepes, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA,1 mM DTT, 17 µg/mL phenylmethylsulfonyl fluoride [pH = 7.4]). Cells were incubated on ice and after 30 minutes, sucrose solution (1 M) was added to achieve a final sucrose concentration of 250 mM. Cells were then immediately homogenized in a ground glass homogenizer (Kontes Glass, Vinaland, NJ) and centrifuged for 10 minutes at 750g to remove unlysed cells and nuclei. The supernatant was then centrifuged at 10 000g for 25 minutes. The resulting pellet containing the mitochondrial fraction was resuspended in buffer A containing 250 mM sucrose. The 10 000g supernatant was then centrifuged at 100 000g for 60 minutes to yield the cytosolic fraction in the resulting supernatant. The protein concentrations were determined according to the method of Smith and colleagues.24 A Laemmli sample buffer25 was then added to samples and boiled for 5 minutes. Protein aliquots were loaded (30 µg/lane) on 15% sodium dodecyl sulfate-polyacrylamide gel for size fractionation by electrophoresis. The proteins were then blotted onto Immobilon-P PVDF transfer membrane (Millipore, Bedford, MA) at 100 mA overnight. Membranes were blocked with 5% milk proteins in Tris buffer saline/0.05% Tween (TBST) and immunostained with mouse anticytochrome c monoclonal antibody (1:500, Pharmingen, San Diego, CA). The membranes were washed and incubated with goat antimouse IgG antibody conjugated to horseradish peroxidase (1:40 000, Pierce, Rockford, IL). Antibody complexes were detected using the enhanced chemiluminescence detection system (Amersham, Pharmacia Biotech, Piscataway, NJ). To estimate the apparent molecular mass of proteins, kaleidoscope prestained standards from Biorad (Biorad Laboratories, Richmond, CA) were used. Individual protein band densities were analyzed by the Eagle Eye II Video Still System (Stratagene, La Jolla, CA).Semiquantitative polymerase chain reaction method DNA damage in mitochondrial DNA and the nuclear hprt (hypoxanthine phosphoribosyltransferase) gene was assessed using a semiquantitative polymerase chain reaction (PCR) according to the previously described method of Yakes and associates.26 DNA was isolated from imexon-treated RPMI 8226 cells with the QIAamp isolation kit (Qiagen, Valencia, CA) according to the protocol supplied by the manufacturer with the following modifications. The RPMI 8226 cells were washed with PBS, resuspended in 200 µL PBS, and lysed at 50°C in the presence of proteinase K and the buffer provided with the Qiagen kit. The concentrations of total DNA were determined using a Picogreen dsDNA quantification kit and a fluorescent plate reader (Fluorolite FPM-2, Jolley Consulting and Research, Grayslake, IL).Semiquantitative PCRs were performed in a Mastercycler gradient 5331 (Eppendorf Scientific, Hamburg, Germany) with a GeneAMP-XL PCR kit (PerkinElmer, Norwalk, CT). Reaction mixtures contained 20 ng DNA, 1 × XL buffer II, 100 ng/µL bovine serum albumin (BSA; Boehringer Mannheim, Mannheim, Germany), 1.2 mM Mg(AOC)2, 0.4 µM primers, and 200 µM dNTPs. The total volume of the reaction mixture was 50 µL. The mock tube without DNA was added as a control. The reaction was initiated by adding 1 U rTth polymerase XL, (recombinant DNA polymerase blend of Thermus thermophilus and Thermus litoralis DNA polymerases, Perkin-Elmer) when samples had reached a temperature of 75°C. The thermocycler profile was: initial denaturation for 2 minutes at 94°C followed by 32 cycles of 94°C denaturation for 17 seconds, and then 65.8°C primer extension for 12 minutes. A final extension was performed at 72°C for 10 minutes. The nuclear region analyzed for DNA damage included a 10.4- kb fragment from the hprt gene (GenBank accession no. J00205) encompassing exons 2-5 amplified by using primers, 5'-TGG GAT TAC ACG TGT GAA CCA ACC-3' (sense), and primer, 5'-GCT CTA CC TCT CCT CTA CCG TCC-3' (antisense). The mitochondrial DNA region analyzed for damage included an 8.9-kb fragment of the mitochondrial genome (GenBank no. J01415) amplified by using primers, 5'-TCT AAG CCT TAT TCG AGC CGA-3' (sense) and 5'-TTT AT GCG GAG ATG TTG GAT GG-3' (antisense). Aliquots of PCR products were resolved on a 1% agar gel. The gel was stained with ethidium bromide and the band densities were evaluated with the Eagle Eye II Still Video System (Stratagene).
Cytotoxicity in malignant cell lines and normal lymphocytes Imexon reduced viability in all malignant cell lines examined, but different cell lines exhibited distinct sensitivities to imexon. The IC50 of imexon measured by MTT at 48 hours was in the 30- to 40-µM range in RPMI 8226, NCI-H929 myeloma cells, and NB-4 acute promyelocytic leukemia cell line. However, the U266 myeloma cell line was not affected by imexon at these concentrations (Table 1). The IC50 of imexon in U266 cells was 419 ± 36.8 µM.
To evaluate the effects of imexon on lymphocytes, we studied cytotoxic effects of imexon in human unstimulated lymphocytes and in lymphocytes stimulated with PHA for 3 days. Unstimulated lymphocytes as well as lymphocytes stimulated with PHA are partially protected against imexon cytotoxic effects. The IC50 of imexon at 48 hours measured by MTT assay was 125.8 ± 12.5 µM and 75.3 ± 6.5 µM for unstimulated lymphocytes and lymphocytes stimulated with PHA, respectively. Morphologic changes in myeloma cells after imexon treatment Because RPMI 8226 myeloma cells are known to be sensitive to imexon, this cell line was used to test whether imexon treatment is associated with time-dependent morphologic changes in mitochondria. Transmission electron microscopy revealed that mitochondria of imexon-treated cells are enlarged compared to control cells.4 To investigate the time course of morphologic changes induced by imexon, the cells were treated with 180 µM imexon for 0, 4, 8, 16, 24, and 48 hours (Figure 1). In untreated cells normal mitochondria with cristae and calcific bodies are found. Treatment of RPMI 8226 cells with 180 µM imexon for 4 hours (Figure 1B) and 8 hours (Figure 1C) leads to the formation of megamitochondria; however, no damage is observed in other cellular organelles. Also, no calcific bodies are detected after imexon treatment. The enlargement of mitochondria was not consistent with swelling that accompanies classic necrosis, because no flocculent densities were observed in the mitochondrial matrix and no evidence of other organelle swelling was observed.27,28 The formation of lipid droplets (Figure 1B,D) in association with mitochondria was also observed after imexon treatment. Mitochondria of cells treated with 180 µM imexon for 16 hours were electron dense with abundant cristae (Figure 1D) indicating high cellular adenosine triphosphate (ATP) demand and associated oxidative phosphorylation. When RPMI 8226 myeloma cells were exposed to 180 µM imexon for 24 and 48 hours, typical features of apoptotic cell death were detected in the majority of cells (data not shown).
Release of cytochrome c from the mitochondria into the cytoplasm in imexon-treated RPMI 8226 cells Immunoblots of cytosolic cytochrome c from imexon-treated RPMI 8226 cells indicate that imexon induces a substantial release of cytochrome c from mitochondria into the cytosol in a time-dependent manner (Figure 2). Continuous treatment of RPMI 8226 myeloma cells with 180 µM imexon caused release of cytochrome c into the cytoplasm, first observed at 8 hours and continuing to increase up to 24 hours after imexon was added (Figure 2).
Changes in the m and an increase in cellular oxidants and whether
these changes are correlated with sensitivity to imexon.
The lipophilic cation, CMXRos, accumulates in the mitochondrial matrix
by the electrochemical gradient according to the physicochemical principle of the Nernst equation. In control cells, the concentration of cations will be 2 to 3 logs higher in the mitochondrial matrix than
in the cytosol.34 The staining of RPMI 8226 myeloma cells with CMXRos revealed that imexon induced disruption of the
Imexon treatment is also associated with an increase in the levels of ROS.4 Figure 3B shows a representative flow cytometry experiment of RPMI 8226 human myeloma cells stained with HE to detect ROS (primarily superoxide) after treatment with imexon for 48 hours. Myeloma cells exposed to 200 µM tert-butylhydroperoxide (tbhp) for 30 minutes were included as a positive control. An increased fraction of cells staining with HE was observed after treatment with 45 µM imexon and this fraction expanded as the concentration of imexon was increased (Figure 3B). Similarly, treatment with 180 µM imexon for various time periods resulted in a time-dependent increase in the fraction of cells experiencing oxidative stress (Figure 4B). At 4 hours there was no change in ROS, but at 8 hours a significantly increased fraction of cells staining with HE was observed (P < .05). Longer treatments with imexon resulted in commensurately increased levels of ROS. Mitochondrial membrane potential was also significantly reduced after
treatment with 180 µM imexon for 48 hours in the NCI-H929 and NB-4
cells (Figure 5A). These cell lines were
shown to be highly sensitive to imexon effects. In contrast, treatment
with 180 µM imexon for 48 hours did not induce dramatic loss of
Inhibition of imexon-induced cytotoxicity by the mitochondrial inhibitor TTFA Theonyltrifluoroacetone inhibits superoxide production in the mitochondrial complex II of the electron transport chain. This compound (50 µM) was not toxic in myeloma cells as measured by eosin Y staining (data not shown). Myeloma cells pretreated overnight with 50 µM TTFA and then simultaneously treated with 180 µM imexon and 50 µM TTFA for 48 hours showed reduction in imexon-induced cytotoxicity. Morphologic changes observed in control cells, imexon-treated cells, cells exposed to TTFA only, and cells treated with imexon and TTFA are shown in Figure 6A. Two hundred cells per slide in each treatment group were evaluated for characteristic features of apoptosis by bright-field microscopy (100 × oil immersion). The majority of cells (90.5% ± 2.9%) exposed to 180 µM imexon for 48 hours exhibit typical features of apoptosis, including chromatin condensation, cell shrinkage, and cytoplasmic blebbing. In contrast, only 57.8% ± 5.2% of the RPMI 8226 cells pretreated overnight with 50 µM TTFA and then treated with 180 µM imexon and 50 µM TTFA simultaneously for 48 hours display characteristic apoptotic features (P < .05). In the untreated RPMI 8226 cells and cells treated with 50 µM TTFA, we found 3.7% ± 2.3% and 18.4% ± 5.3% apoptotic cells, respectively.
Mitochondrial DNA damage A semiquantitative PCR assay was used to detect imexon-induced DNA damage in mitochondria or in the nucleus. DNA lesions such as strand breaks, base modification, or apurinic sites will block DNA polymerase activity. Thus, the amount of amplified product will be decreased in PCRs using such damaged templates. The data show that imexon exposure, at low concentrations for 48 hours, induced a loss of 8.9-kb amplified product of the mitochondrial genome. However, amplification of a nuclear 10.4-kb fragment of the hprt gene was not affected (Figure 7). These results indicate that imexon damages mitochondrial DNA but not nuclear DNA. Higher concentrations of imexon (180 µM exposure for 48 hours) induced changes in both mitochondrial and nuclear DNA. A bifunctional aziridine containing the DNA alkylator, AZQ, was used as a positive control in this study. As expected, AZQ treatment (2.7 µM) for 24 hours or 48 hours induced a loss of both mitochondrial and nuclear PCR products in the same proportion (Figure 7). Thus, there was not preferential damage of mitochondrial DNA with a bifunctional aziridine alkylator.
Imexon is a monoaziridine compound originally studied for
immune-enhancing effects on lymphocytes.35,36 Several
studies clearly demonstrated imexon activity against a variety of fresh human tumors and tumor cell lines in culture.3,37 The
antitumor effect of imexon was also shown in vivo with inhibition of
large cell lymphoma development in severe combined immunodeficient
mice.38 However, the precise mechanism of imexon action
was unknown. In previous studies, we have shown that imexon induces
oxidative stress and apoptosis in RPMI 8226 myeloma
cells.4 Data presented here demonstrate that in RPMI 8226 myeloma cells imexon causes mitochondrial alterations associated with
the apoptotic cell death pathway. These changes include mitochondrial
enlargement, the loss of Mitochondria have been shown to play a major role in programmed cell death. Moreover, these organelles have a central position in the control of cell survival because they are necessary for the generation of energy required for cell function. Mitochondria consume large amounts of molecular oxygen for generating the energy required for the synthesis of ATP from adenosine diphosphate (ADP). However, continued consumption of oxygen by mitochondria routinely leads to the generation of ROS such as superoxide anion, organic peroxides, hydrogen peroxide, or hydroxyl radical, depending on the number of electrons transferred to molecular oxygen.39 Such oxidants can cause cell damage if not detoxified by antioxidant systems. It has been suggested in a number of studies that formation of ROS is a common scheme in some pathways of apoptosis.40-42 Oxidants and compounds that are capable of depleting GSH or damaging the cellular antioxidant defense system can directly induce or potentiate apoptosis.29,43,44 On the other hand, antioxidants such as N-acetyl-L-cysteine, Trolox, or butylated hydroxyanisole can inhibit apoptosis.40,45 Due to high cellular GSH levels, the GSH redox system represents one of the most important cellular defense systems against oxidative stress, particularly in mitochondria. GSH is synthesized solely in the cytoplasm from glutamine, glycine, and cysteine and can be transported into the mitochondria and the nucleus. Importantly, mitochondria from most mammalian cells do not contain catalase, an enzyme that plays a crucial role in the detoxification of hydrogen peroxide in extramitochondrial compartments. In our previous report,4 it was shown in RPMI 8226 myeloma cells that (1) imexon can bind cysteine and glutathione in vitro, (2) imexon treatment is associated with decreased levels of cellular thiols in myeloma cells, and (3) imexon induces oxidative damage of cytosolic nucleotides and apoptosis. Thus, we speculated that after exposure to imexon, endogenous antioxidant defense systems in myeloma cells are compromised and the cellular ability to scavenge ROS is reduced. This can lead to increased endogenous production of ROS in mitochondria, leading to oxidative stress and the induction of apoptosis. The oxygen radicals produced in mitochondria can escape detoxifying pathways and induce various cellular injuries characterized by protein inactivation, DNA damage, and lipid peroxidation. Although some ROS diffuse from mitochondria to damage more distant cellular components, the half-life of most radicals is short. It is, therefore, conceivable that mitochondria may be affected by ROS to the greatest extent. One of the first consequences induced by imexon treatment in RPMI 8226 myeloma cells involves morphologic alteration of the mitochondria and formation of ROS. The significant enlargement of mitochondria was observed after imexon treatment and may represent an attempt by the cell to dilute the ROS by enlarging the area occupied by the ROS.46 This hypothesis is also supported by the fact that imexon affected mitochondrial DNA (mtDNA), but not nuclear DNA. Data presented here from MTT and flow cytometric studies also indicate
that sensitivity to imexon highly correlates with the loss of
mitochondrial membrane potential and formation of ROS. For example,
increased levels of ROS and loss of That the generation of ROS comprises a crucial event in imexon action is also supported by our previous finding that antioxidants such as N-acetyl-L-cysteine protect against imexon-induced cytotoxicity.4 In agreement with these previous experiments the data from flow cytometry experiments indicate that NAC treatment inhibits formation of imexon-related ROS and loss of mitochondrial membrane potential. Furthermore, in this paper we have shown that TTFA-induced inhibition of the electron transport from succinate dehydrogenase (complex II) partially reduces imexon-induced apoptosis in RPMI 8226 cells. The sensitivity of imexon in RPMI 8226 myeloma cells and the lack of myelotoxicity could be explained by imexon effects on mitochondria. The generation of ROS leads to the consequences associated with apoptosis such as release of cytochrome c from mitochondria to cytosol and morphologic features of apoptosis. We have shown that imexon in RPMI 8226 cells induces translocation of cytochrome c from mitochondria into the cytosol that can be detected as early as at 8 hours (Figure 2). However, the current findings do not explain why imexon selectively
inhibits growth of RPMI 8226, NCI-H929 myeloma cells, and NB-4 acute
promyelocytic leukemia cells, yet U266 myeloma cells are relatively
resistant to imexon effects. Interestingly, U266 cells have been shown
to be insensitive to the effects of dexamethasone and
interferon- In summary, this study highlights the mitochondrial effects of imexon in human myeloma cells. These effects are unique among existing anticancer agents. They support previous reports showing imexon effectiveness in various malignancies and low toxicity in limited human phase I/II trials conducted in Europe. Imexon is a promising chemotherapeutic agent and should be investigated further for potential in vivo activity against multiple myeloma.
Supported by grants CA 23074 and 17094 (to R.T.D.) from the National Institutes of Health (NIH), Bethesda, Maryland, and CA7176 (to M.M.B.) from the National Cancer Institute (NCI), NIH. K.D. and M.E.T. were partially supported by grant CA09213 from the NCI.
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.
Reprints: Robert T. Dorr, Arizona Cancer Center, 1515 N Campbell Ave, Tucson, AZ, 85724; e-mail: bdorr{at}azcc.arizona.edu.
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Top
Abstract
Introduction
80°C. A stock solution of NAC (200 mM) was prepared in PBS, titrated with NaOH to pH 7.2, and filter
sterilized. The solution of TTFA was prepared in dimethyl sulfoxide
(DMSO; 10 mM), diluted in PBS to 1 mM concentration, and filter
sterilized. All other chemicals were the highest purity available and
were obtained from Sigma unless noted otherwise.
m, and cytochrome c release
from the mitochondria into the cytosol. In addition, we investigated
the activity of imexon in several other myeloma cells, acute
promyelocytic leukemia cells, and peripheral blood lymphocytes.
Interestingly, the results in different cell lines show that imexon
sensitivity correlates with the extent of mitochondrial changes after
imexon treatment.
.