C
2005 Springer Science + Business Media, Inc. Manufactured in The Netherlands.
Ornithine decarboxylase prevents
methotrexate-induced apoptosis by reducing
intracellular reactive oxygen species production
C.-C. Huang, P.-C. Hsu, Y.-C. Hung, Y.-F. Liao, C.-C. Liu, C.-T. Hour, M.-C. Kao, G.J. Tsay, H.-C. Hung∗ and G.-Y. Liu∗
Tzu Hui Institute of Technology, Pingtung, Taiwan, R.O.C (C.-C. Huang); Department of Medicine, Da-Chien General Hospital, Miao-Li, Taiwan, R.O.C (P.-C. Hsu); Institute of Immunology, Chung Shan Medical University, Taichung, Taiwan, R.O.C (Y.-C. Hung, C.-C. Liu, G.-Y. Liu); Institute of Biochemistry, Kaohsiung Medical University, Kaohsiung, Taiwan, R.O.C (C.-T. Hour); Department of Life Sciences, National Chung-Hsing University, Taichung, Taiwan, R.O.C (Y.-F. Liao, H.-C. Hung); School of Medical, China Medical University, Taichung, Taiwan, R.O.C (M.-C. Kao); Department of Internal Medicine, Chung Shan Medical University Hospital, Taichung, Taiwan, R.O.C. (G.J. Tsay)
Methotrexate (MTX), a folate antagonist, was developed for the treatment of malignancies, and is currently used in rheumatoid arthritis (RA) and other chronic inflam-matory disorders. It has been proven in short-term and long-term prospective studies that low doses of MTX (0.75 mg/Kg/week) are effective in controlling the inflam-matory manifestations of RA. Low-concentrations of MTX achieve apoptosis and clonal deletion of activated pe-ripheral T cells. One of the mechanisms of the anti-inflammatory and immunosuppressive effects may be the production of reactive oxygen species (ROS). How-ever, the drug resistance of MTX in malignancies re-mains poorly understood. Ornithine decarboxylase (ODC) plays an important role in diverse biological functions, in-cluding cell development, differentiation, transformation, growth and apoptosis. In our previous studies, ODC over-expression was shown to prevent TNFα-induced apop-tosis via reducing ROS. Here, we also investigated one mechanism of MTX-induced apoptosis and of drug re-sistance as to the anti-apoptotic effects of ODC during MTX treatment. We found MTX could induce caspase-dependent apoptosis and promote ROS generation to-gether with disrupting the mitochondrial membrane po-tential (m) of HL-60 and Jurkat T cells. Putrescine and ROS scavengers could reduce MTX-induced apoptosis, which leads to the loss ofm, through reducing intra-cellular ROS. Overexpression of ODC in parental cells had the same effects as putrescine and the ROS scavengers. Moreover, ODC overexpression prevented the decline of Bcl-2 that maintainsm, the cytochrome c release and activations of caspase 9 and 3 following MTX
treat-∗Correspondence to: Prof. Guang-Yaw Liu, Institute of Immunol-ogy, Chung-Shan Medical University, No. 110, Sec. 1, Chien-Kuo N. Road, Taichung, Taiwan, ROC. Tel: (886)-4-2473-0022 (ext. 11709); Fax: (886)-4-23248172; e-mail: [email protected] and Prof. Hui-Chih Hung, Department of Life Sciences, National Chung-Hsing, University, No. 250, Kuo-Kuang Road, Taichung, Taiwan, ROC. Tel: 2284-0416 (ext. 505); Fax: (886)-4-22851856; e-mail: [email protected]
ment. The results demonstrate that MTX-induced apopto-sis is ROS-dependent and occurs along a mitochondria-mediated pathway. Overexpressed ODC cells are resis-tant to MTX-induced apoptosis by reducing intracellular ROS production.
Keywords: apoptosis; caspase; methotrexate; ODC; ROS. Abbreviations: MTX, methotrexate; ODC, ornithine
decar-boxylase; ROS, reactive oxygen species; m,
mitochon-drial membrane potential; DFMO, difluoromethylornithine; DCFH-DA, 2,7-dichlorofluorescin diacetate; z-VAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone.
Introduction
Methotrexate (MTX), the 4-amino, 10-methyl analogue of folic acid, is the most widely used antifolate against many malignancies in cancer chemotherapy, including leukemia, breast cancer, colorectal cancer, head and neck cancer, lymophma, osteogenic sarcoma, urothelial can-cer, and choriocarcinoma. It is also used in the treatment of nonmalignant disorders, such as psoriasis, rheumatoid arthritis and graft-versus-host diseases.1 Clinically, high doses of MTX cause tumor shrinkage.2–4 High doses of MTX induce apoptosis in several cell lines, including Chi-nese hamster ovary cells,5,6human leukemic T lympho-cytes such as CCRF-CEM and Jurkat T cells,6,7hepatoma cell lines8and keratinocytes.9Low doses of MTX also in-duce apoptosis of mitogen-activated CD4+and CD8+T cells, but not resting T cells. Furthermore, low doses of MTX lead to clonal deletion of activated T cells in mixed lymphocyte reactions.10,11
There are several mechanisms by which MTX-induced apoptosis have been investigated. MTX can induce the production of reactive oxygen species (ROS) and apoptosis
in Jurkat T cells via ROS.12Overexpression of Bcl-2 re-sults in increased resistance to cell destruction by MTX,13 and down-regulation of bcl-2 by synthetic bcl-2 antisense oligonucleotides resulting in a marked enhancement in the sensitivity of tumor cells to chemotherapeutic drugs.14 Bcl-2 is a protein anchored in the membrane of the mi-tochondria. It maintains mitochondrial membrane po-tential (m) by protecting apoptosis from any attacks.
It seems that mitochondria-mediated apoptotic pathway play a role in MTX-induced apoptosis. In addition, MTX activates specific caspases and induces apoptosis, which are blocked by zVAD-fluoromethyl ketone, a general cas-pase inhibitor.15Therefore, the caspase pathway may also play a role in this process.
Ornithine decarboxylase (ODC, EC 4.1.1.17), the first and rate-limiting enzyme of the polyamine biosynthetic pathway, decarboxylatesL-ornithine to form putrescine.16 ODC and polyamines (putrescine, spermidine and sper-mine) play an important role in several biological func-tions, including embryonic development, the cell cy-cle and proliferation,17 and in the origin and progres-sion of neoplastic diseases.18,19The polyamine pathway is an important target of therapeutic intervention in many cancers.17Polyamine deprivation enhances the efficacy of chemotherapy.20Measurement of ODC activity is useful in predicting the chemosensitivity to many anti-cancer drugs including MTX.21ODC and polyamines have para-doxical roles in preventing or enhancing apoptosis during different cell insults or cell lines.17 Inhibition of ODC activity by DFMO can induce apoptosis of HC11 mouse mammary epithelial cells, which is associated with a rapid increase in ROS concentration.22 Moreover, overexpres-sion of ODC promotes survival of human gastric cancer cells under stressful conditions, such as H2O2, radiation and some chemotherapeutic drugs including cisplatin, doxorubicin, paclitaxel, 5-flourouracil.23The purpose of the present study was to investigate whether ODC could prevent MTX-induced apoptosis. In addition, we aimed to determine exactly how and by what mechanism ODC prevented MTX-induced apoptosis.
Materials and methods
Chemicals and cell cultureMTX, ribonuclease A (RNase A), N-acetylcysteine (NAC), catalase, vitamin C, putrescine, benzylo-xycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk), acridine orange, propidium iodide (PI), 2, 7 -dichlorofluorescin diacetate (DCFH-DA), rhodamine 123 and HA14-1 were purchased from Sigma (St Louis, MO). The human promyelocytic leukemia HL-60 cells and Ju-rkat T cells were grown in 90% RPMI 1640 and 10% FBS (fetal bovine serum) obtained from Gibco BRL (Grand
Island, NY) at a temperature of 37◦C under a humidified, 5% CO2atmosphere.
Cell viability and acridine-orange staining
Cell numbers were counted using trypan blue exclusion assay. Morphological change and cell viability were de-tected by light microscopic observation. To identify apop-totic character upon MTX stimulation, 5× 104 cells in 10µl cell suspension were mixed with an equal volume of acridine orange solution (10 µg/ml) in phosphate-buffered saline (PBS) on each slide. Green fluorescence was detected by microscope as being between 500–525 nm (Olympus America, St Huntington, NY).24
Cloning of human ODC and cell transfection Parental cells were grown in RPMI 1640 medium sup-plemented with 10% heat-inactivated FBS for 3 h, and then the harvested cells were gently rinsed in PBS. Pu-rification of mRNA was carried out according to the sup-plier’s instructions (Mdbio, Taiwan) and the cDNA was synthesized by reverse transcriptase (Promega, Madison, WI). Polymerase chain reaction (PCR) amplification of the encoding region of the human ODC cDNA was per-formed with our designed primers derived from the hu-man ODC sequence. The PCR product was sequenced and sub-cloned to eukaryotic expression vector, pCMV-Tag (Novagen, Madison, WI). The plasmid of ODC expres-sion was constructed by inserting the BamHI-EcoRI 1,415 bp coding region fragment. Parental cells were transfected with WT-ODC (overexpressing ODC), m-ODC (frame-shift mutant) and DN-ODC (dominant-negative ODC) plasmids according to calcium phosphate-mediated trans-fections, respectively.25 Stably transfected cells were se-lected with the antibiotic G418 (400µg/ml). After ap-proximately 3 weeks, G418-resistant clones were isolated and analyzed individually for expression of ornithine de-carboxylase. The individual clones were examined for ex-pression of ODC by RT-PCR, immunoblotting and en-zyme activity assays. Overexpressed Bcl-2 cells were built as previously described.26
Site-directed mutagenesis of odc gene
To construct dominant-negative ODC (K69A/C360A-ODC; DN-ODC), site-directed mutagenesis was per-formed as described previously.27WT-ODC plasmid and two pairs of synthetic oligonucleotide primers contain-ing the desired mutations (point mutations to alanine at lysine 69 [K69A] and cysteine 360 [C360A] residues) were utilized and in vitro site-directed mutagenesis was
performed using pfu DNA polymerase (Promega) follow-ing the instructions from the manufacturer.
DNA fragmentation analysis
For this, 5× 106 cells were harvested in PBS and lysed overnight in a digestion buffer (0.5% sarkosyl, 0.5 mg/ml proteinase K, 50 mM Tris-HCl, pH 8.0 and 10 mM EDTA) at 55◦C. Subsequently cells were treated with 0.5 µg/ml RNase A for 2 h. The genomic DNA was extracted by phenol-chloroform-isoamyl alcohol extrac-tion and analyzed by gel electrophoresis at 50 volts for 90 min using 2% agarose. Approximately 20µg of ge-nomic DNA was loaded in each well, visualized under ultraviolet (UV) light and photographed.
ODC enzyme activity assay
ODC enzyme activity was assayed at 37◦C through measuring its product and putrescine as described previously28 with the following modification. Samples were suspended in ODC buffer (50µM EDTA, 25 µM pyridoxal phosphate and 2.5 mM DTT in 25 mM Tris HCl, pH 7.1) incubated with 2 nmole ofL-ornithine for 1 h, and the material was then spotted onto p81 phos-phocellulose (Whatman, Maidstone, England). Diamines were eluted from the dried papers by shaking at 37◦C for 60 min with 0.5 ml quantities of elution buffer (0.5 M magnesium chloride in 0.2 M boric acid-borax buffer, pH 8.4). Following this, samples were supplemented by 400µl of luminescence reagent (11.7 µg/ml luminal, 30µg/ml peroxidase type II (EC 1.11.1.7) and 67 mM glycine buffer, pH 8.6 (at 1:1:2.5, v/v/v) to each cuvette. By retaining the cuvettes in the dark for 30 min, the back-ground was able to measure using a TR 717 microplate luminometer (Perkin-Elmer, Foster, CA). Then 5µl of diamine oxidase (4.61µg/µl) (Sigma) were injected into each cuvette, and luminescence was recorded for 40 sec at 37◦C with the results being calculated according to the standard curve using putrescine.
Apoptotic sub-G1 analysis
For this, 1 × 106 cells were cultured in 35-mm petri dishes and incubated for 24 h. Cells were treated with MTX for time course and dose-dependence, then har-vested and washed with PBS, resuspended in 0.2 ml of PBS, fixed in 0.8 ml of ice-cold 100% ethanol at−20◦C overnight. The cell pellets were collected by centrifuga-tion, resuspended in 1 ml of hypotonic buffer (0.5% Triton X-100 in PBS and 0.5µg/ml RNase), and incubated at 37◦C for 30 min. Then, 1 ml of PI solution (50µg/ml)
was added and the mixture was allowed to stand on ice for 30 min. The nuclei were analyzed in a FACSCAN laser flow cytometer (Becton Dickenson, San Jose, CA).
Detection of intracellular reactive oxygen species (ROS)
Intracellular oxidative stress was assayed by measur-ing intracellular oxidation of 2, 7-dichlorofluorescin (DCFH).29,30 The resulting substrate is DCFH-DA, which easily diffuses into the cell and is then deacetylated by cellular esterases to the more hydrophilic, nonfluores-cent DCFH. ROS generation in the cell oxidizes DCFH to the fluorescent 2, 7-dichlorofluorescein (DCF). DCF flu-orescence was measured in a flow cytometer using the Cell Quest software (Becton Dickenson). In each study, 10000 events (cells) were counted. The results were presented as a percentage of the fluorescence intensity compared with the control sample.
Detection of mitochondrial membrane potential (m)
Mitochondrial potential was monitored by fluorescence of rhodamine 123.31,32Incubation was continued for 10 min with 10µM rhodamine 123. Finally, the cells were detached and fluorescence was measured in a flow cytome-ter. In each study, 10000 events (cells) were counted. Data were acquired and analyzed with Cell Quest software.
Immunoblotting
Mitochondrial and cytosolic proteins were isolated from cells after treatment with 1µM MTX for 24 h, as previ-ously described.33In order to extract mitochondrial pro-teins, cell (5 ×106) pellets were washed once with ice-cold PBS and resuspended with 50µl of mitochondrial buffer (25 mM Tris, pH 6.8, 1 mM EDTA, 1 mM DTT, 0.1 mM PMSF and 250 mM sucrose). To purify the to-tal proteins, cells were harvested and lysed in cold lysis buffer (10% v/v glycerol, 1% v/v Triton X-100, 1 mM sodium orthovanadate, 1 mM EGTA, 10 mM NaF, 1 mM sodium pyrophosphate, 20 mM Tris, pH 7.9, 100µM β-glycerophosphate, 137 mM NaCl, 5 mM EDTA, 1 mM PMSF, 10µg/ml aprotinin and 10 µg/ml leupoptin), ho-mogenized, centrifuged, and the supernatant was boiled in a loading buffer with an aliquot corresponding to 50 µg of protein being separated by SDS-PAGE. Af-ter blotting, the membranes were incubated with anti-cytochrome c, anti-Bcl-2, anti-caspase 3, anti-caspase 9, anti-Apaf-1, anti-PARP and anti-α-tubulin antibodies (Santa Cruz, Santa Cruz, CA) for 6 h and the second
antibody labeled with horseradish-peroxidase was incu-bated for 1 h. The antigen-antibody complexes were vi-sualized by the enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ).
Results
MTX induced caspase-dependent apoptosis, ROS generation and the loss of mitochondrial membrane potential (m)
We first investigated the cell death induced by MTX. HL-60 and Jurkat T cells were treated with 0, 0.1, 0.5,
Figure 1. MTX induced caspase-dependent apoptosis in HL-60 and Jurkat T cells. HL-60 and Jurkat T cells were treated with MTX 0,
0.1, 0.5, 1 or 10µM for the indicated times after having been treated with or without zVAD-fmk (500 nM) for 1 h. (A) HL-60 and Jurkat T cells were observed by fluorescence-microscope and counted for their percentage of apoptosis. (B) HL-60 cells were assayed by DNA fragmentation, microscope and flow cytometry with propidium iodide (PI) staining. The arrow indicates an apoptotic body. M, DNA ladder marker.
1 and 10 µM of MTX with or without zVAD-fmk, a total caspase inhibitor, for 24 h. Cells were observed by fluorescence-microscope and the percentage of apoptosis was recorded. We found MTX could induce apoptosis in a dose-dependent manner (Figure 1A). The morpho-logic changes were chromatin condensation, membrane blebbing and shrinkage, and apoptotic body formation. Then, cells were harvested for DNA gel electrophoresis and flow cytometry with propidium iodide (PI) stain-ing. Furthermore, there was a time course as indicated and dose-dependent increases in DNA fragmentation af-ter MTX treatment. We found zVAD-fmk (500 nM, pre-treated for 1 h before MTX) could block this phenomenon
(Figure 1B). For the experiment of flow cytometry with PI staining, the sub-G1 peak ratios were 3.1%, 41.8% and 11.2% in cells treated with vehicle, MTX (1µM) and MTX combined with fmk, respectively. zVAD-fmk could decrease the sub-G1 peak ratio after MTX treat-ment up to 82%. All of the aforetreat-mentioned morphologic and molecular changes are features typical of apoptosis.34 As shown in a previous study,15 MTX-induced apop-tosis could be blocked by z-VAD-fmk. All these data indicated that MTX could induce caspase-dependent apoptosis.
In order to measure intracellular ROS, HL-60 cells were treated with 0, 0.1, 0.5 and 1µM MTX for 1 h and then incubated with 5 µM DCFH-DA after MTX treatment at 37◦C. Intracellular ROS was quantified by flow cy-tometry, using a permeable cell fluorescent probe DCFH-DA. DCFH-DA is hydrolyzed by intracellular esterases to nonfluorescent 2, 7-dichlorofluorescin (DCFH), which is rapidly oxidized by ROS to generate the fluorescent 2, 7-dichlorofluorescein (DCF). DCF is very useful in quantifying overall oxidative stress in cells.35,36To assess m, rhodamine 123,31 a green fluorescent mitochon-drial dye, was measured by flow cytometry after MTX treatment for 24 h. The results of flow cytometric analy-sis showed there were dose-dependent increases of intra-cellular ROS generation and dose-dependent decreases of m in HL-60 cells after MTX treatment (Figure 2). These results indicated that MTX-induced apoptosis was coupled by the generation of ROS and the loss of m.
Figure 2. MTX caused increasing intracellular ROS and the loss of mitochondrial membrane potential (m). HL-60 cells were treated with MTX 0, 0.1, 0.5 or 1µM. Intracellular ROS andm were detected by flow cytometry by measuring the fluorescence of DCF (1 h) and rhodamine 123 (24 h), respectively. Cells were treated with H2O as a vehicle control. The production of ROS andmare expressed as the percentage of fluorescent cells in contrast with the control cells. Data are representative of at least three experiments.
Putrescine and ROS scavengers could reduce MTX-induced apoptosis and the loss ofm by reducing intracellular ROS
To clarify the role of ROS in the mechanism of MTX-induced apoptosis and the effects of putrescine, we pre-treated 1 mM putrescine and 10 mM NAC for 3 h. Other ROS scavengers, such as catalase (100 U) and vi-tamin C (1 mM) were pretreated for 1 h prior to 1µM MTX treatment. After MTX treatment for 24 h, all of the cells were observed by fluorescence-microscope. We found MTX could induce apoptotic morphologic changes, including chromatin condensation, membrane blebbing and shrinkage, and apoptotic body formation. However, these apoptotic morphologic changes occurred less if cells were pretreated with ROS scavengers or putrescine (data not shown). In the flow cytometric assay with PI fluo-rescence, we found that putrescine, vitamin C, NAC and catalase decreased the sub-G1 peak ratio following MTX treatment. However, inhibition of the endogenous ODC activity with DFMO increased the sensitivity to MTX-induced apoptosis (Figure 3). Among these ROS scav-engers, catalase was the most effective. It reduced the sub-G1 ratio by up to 55% and putrescine decreased the ratio by about 50% as compared with the vehicle.
Intracellular ROS andmwere determined by mea-suring DCF after treatment for 1 h and rhodamine 123 after treatment for 24 h with flow cytometry, respectively. Putrescine and NAC were shown to be capable of sig-nificantly decreasing intracellular ROS and maintaining
Figure 3. Putrescine and ROS scavengers reduced MTX-induced apoptosis. HL-60 cells were pretreated with NAC (10 mM), putrescine
(1 mM) or DFMO (1 mM) for 3 h, pretreated with vitamin C (1 mM) or catalase (100 U) for 1 h before MTX (1µM) treatment. After MTX treatment for 24 h, the sub-G1 peak ratio was measured by flow cytometry with PI fluorescence. Data are representative of at least three experiments. Cells were treated with H2O as a vehicle control. Vit C, vitamin C.
m after MTX treatment (Figure 4). Putrescine and ROS scavengers could prevent more than half of cells from undergoing apoptosis. Taken together, these results indi-cate MTX-induced apoptosis occurs mainly through the ROS-dependent pathway. Putrescine exerts a protective effect in MTX-induced apoptosis via decreasing intracel-lular ROS and maintaining them.
ODC overexpression could reduce intracellular ROS and prevent MTX-induced loss ofmand apoptosis
We further examined the role of ODC in MTX-induced apoptosis. We constructed ODC cDNA into a system of mammalian expression plasmid, pCMV-Tag and gener-ated cell lines overexpressing ODC in parental HL-60 and Jurkat T cells, named WT-ODC and JK-WT-ODC cells, respectively. Parental HL-60 and Jurkat T cells were transfected by its frame-shift mutant vector as a control, which were termed m-ODC and JK-m-ODC cells, re-spectively. At the same time, we also used the method of site-directed mutagenesis to construct dominant-negative ODC and build DN-ODC cells.37ODC was overexpressed in WT-ODC cells at the transcriptional and translational level, respectively (data not shown). Four hours after being stimulated by 10% FBS in parental HL-60, m-ODC, WT-ODC and DN-WT-ODC cells treated with or without 1 mM DFMO, WT-ODC cell expressed about two folds greater ODC enzyme activity than parental HL-60 and m-ODC
cells. This overexpression of ODC activity could be signif-icantly inhibited by DFMO (Figure 5). In addition, intra-cellular ROS had decreased more in WT-ODC cells than in m-ODC significantly after 10% FBS stimulation for 4 h (Figure 6A). One hour after MTX (1µM) treatment in the m-ODC and WT-ODC cells, significantly more intra-cellular ROS had accumulated in the m-ODC cells than in the WT-ODC cells (Figure 6B) This result revealed that ODC overexpression reduced intracellular ROS whether MTX induction had taken place or not. Twenty-four hours after MTX treatment, WT-ODC was more resistant to MTX-induced apoptosis than parental HL-60, m-ODC and DN-ODC cells in experiments of phase light mi-croscopy (data not shown) and flow cytometry with PI staining. The percentage of apoptotic sub-G1 in WT-ODC cells had decreased by approximately 20 and 34% as compared with total parental HL-60 cells subjected to 1 µM MTX treatment for 24 h and parental Jurkat T cells treated by 10µM MTX for 48 h, respectively (Fig-ure 6C and D). The protective effect of ODC in WT-ODC cells was reversed by DMFO (Figure 6E). In the experi-ment involvingm, WT-ODC cells had significantly less loss ofmthan parental HL-60, m-ODC and DN-DOC cells. In addition, DFMO, inhibiting the activity of ODC, blocked the maintaining ofmin WT-ODC cells (Figure 7A). MTX also induced the disruption ofm and apoptosis in JK-m-ODC cells. It achieved a similar level of disruption for the HL-60 cell system, but less for JK-WT-ODC cells (Figure 7B). Furthermore, WT-ODC cells prevented cytochrome c release from mitochondria to
Figure 4. Putrescine reduced intracellular ROS and the loss ofm after MTX treatment, as ROS scavengers. HL-60 cells were pretreated with NAC (10 mM) or putrescine (1 mM) for 3 h before MTX (1 mM) treatment. Intracellular ROS andmwere detected by flow cytometry with measuring fluorescence of DCF (A) and rhodamine 123 (B), respectively. Data are representative of at least three experiments.∗P<0.05 as compared to HL-60 cell only treated MTX. Put, putrescine.
Figure 5. ODC overexpression increased ODC activity, which
was reversed by DFMO. Parental HL-60, wild-type ODC (WT-ODC), frame-shift mutant ODC (m-ODC) and dominant-negative ODC (DN-ODC) cells were stimulated by 10% FBS with or with-out 1 mM DFMO. ODC enzyme activity was measured after 4 h of stimulation. Data are representative of at least three experiments. ∗P<0.05,∗∗P<0.01 and∗∗∗P<0.001.
cytosol after MTX treatment (Figure 7C). Based on these data, MTX-induced apoptosis seemed to occur through the mitochondria-mediated pathway. ODC overexpres-sion reduced ROS accumulation, and disruptedmand cytochrome c release after being induced by MTX. Simul-taneously, ODC overexpression reduced at least a part of
the apoptosis following MTX treatment of the HL-60 and Jurkat T cell systems.
ODC overexpression prevented the decline of Bcl-2 that maintainedmand reduced MTX-induced apoptosis
Following stimulation by 10% FBS, m-ODC, WT-ODC and DN-ODC cells were treated with or without MTX. ODC overexpression could efficiently prevent the decline of Bcl-2 following MTX untreatment (1.4 folds) and treat-ment (3 folds), respectively (Figure 8A). In addition, Bcl-2 inhibitor (HA14-1) enhanced the disruption ofm and apoptosis of WT-ODC cells after MTX treatment (data not shown). It was revealed that the mitochondria-mediated pathway perhaps plays an important role in MTX-induced apoptosis. Bcl-2 can maintainm, avoid cytochrome c release from the mitochondria and prevent apoptosis.38In previous studies, Bcl-2 overexpression was found to prevent MTX-induced apoptosis.39,40 To con-firm the importance of the mitochondria-mediated path-way in MTX-induced apoptosis, we transfected the bcl-2 gene (Bcl-2 cells) or its vector alone (vector cells) into parental HL-60 cells.26 Bcl-2 cells and vector cells were treated with or without MTX, and then harvested for flow cytometry with rhodamine 123 and PI staining. We
Figure 6. ODC overexpression reduced the intracellular ROS and MTX-induced ROS generation and apoptosis. After being stimulated
by 10% FBS for 0 or 4 h, m-ODC and WT-ODC cells were harvested for flow cytometry with measuring fluorescence of DCF (A). m-ODC and WT-ODC cells were treated with or without MTX (1µM) for 1 h, and then intracellular ROS was measured by flow cytometry (B). Parental, m-ODC, WT-ODC and DN-ODC cells were treated with or without MTX after 1 mM DFMO treatment or not for 3 h. Sub-G1 peak ratio was measured by flow cytometry with PI staining in HL-60 (C) and Jurkat T cells (D), respectively. Cells were observed and counted for the percentage of apoptosis by phase-construct microscope (E). Data are representative of at least three experiments.∗P<0.05, as compared to parental HL-60 or m-ODC cells.
Figure 7. ODC overexpression reduced the loss ofmand cy-tochrome c release. After being stimulated by 10% FBS, parental HL-60, m-ODC, WT-ODC and DN-ODC cells were treated with or without MTX (1µM) for 24 h after treated DFMO or not.mwas measured by flow cytometry with rhodamine 123 (A). Them of JK-m-ODC and JK-WT-ODC cells were detected by flow cy-tometry with rhodamine 123 (B). Cytoplasmic and mitochondrial proteins were separated and detected by immunoblotting with cy-tochrome c antibody (C).
found Bcl-2 cells had lost less m and sub-G1 peak ratio than their vector cells (Figure 8B and C). Indeed, our data revealed that ODC overexpression prevented the decline of Bcl-2 and Bcl-2 overexpression in parental
Figure 8. ODC overexpression prevented the decline of Bcl-2
which maintainedmand reduced MTX-induced apoptosis. Af-ter being stimulated by 10% FBS, m-ODC, WT-ODC and DN-ODC cells were treated with or without MTX (1µM) for 24 h and to-tal proteins were analyzed by immunoblotting with Bcl-2 and a-tubulin antibody (A). Parental HL-60 and overexpression of Bcl-2 cells were treated with and without MTX. Cells were harvested for measuringm(B) and sub-G1 peak ratio (C) by flow cytometry with rhodamine 123 and PI fluorescence, respectively. Data are representative of at least three experiments.
HL-60 cells decreasing the disruption ofmand apop-tosis induced by MTX. Therefore, maintaining m through the use of ROS scavengers, putrescine, ODC over-expression and Bcl-2 in parental cells are ways of prevent-ing MTX-induced apoptosis.
ODC overexpression prevented the activation of caspase 9, 3 and the formation of apoptosome after MTX treatment
According to the above results, cytochrome c was re-leased after MTX treatment in parental HL-60, m-ODC and DN-m-ODC cells, but to a lesser extent in WT-ODC cells. In addition, MTX-induced apoptosis in HL-60 cells seemed to occur mainly through the
Figure 9. ODC overexpression prevented the cleavage of
cas-pase 9, 3 and PARP and the generation of Apaf-1. After being stimulated by 10% FBS for 3 h, m-ODC, WT-ODC and DN-ODC cells were treated with or without MTX (1µM) for 24 h. Total pro-tein were extracted for immunoblotting with antibodies of caspase 9, Apaf-1, caspase 3 and PARP. Data are representative of at least three experiments.
mitochondria-mediated pathway. To clarify the down-stream of the ODC-involved mitochondria-mediated pathway after MTX-induced apoptosis, m-ODC, WT-ODC and DN-WT-ODC cells were treated with or without MTX. Total proteins from diverse cells were detected by immunoblotting with anti-Apaf-1, caspase 9, 3 and PARP (the substrate of caspase 3). In the WT-ODC cells, we found that Apaf-1 had decreased, the activation of cas-pase 9 and 3 were inhibited, and the cleavage of PARP was significantly more protected as compared with the m-ODC and DN-m-ODC cells after MTX treatment (Figure 9). The results indicated that ODC overexpression prevented the formation of apoptosome (the complex of Apaf-1, cy-tochrome c and caspase 9) and inhibited the activation of caspase 3 cascade during apoptotic progression.
Discussion
When cells are treated by MTX, extracellular MTX is transported into cells through at least two different energy-dependent mechanisms. One is a relatively low-affinity reduced folate transmembrane carrier in the mi-cromolar range and the other is a membrane-associated folate-binding protein with nanomolar affinity. Intracel-lular MTX is converted to polyglutamate forms by the binding of two to five of the polyglutamate groups, re-sulting in an increase in its intracellular half-life. MTX and its polyglutamate derivatives inhibit dihydrofolate reductase (DHFR), the major MTX target, and sev-eral folate-dependent enzymes such as thymidylate syn-thase (TS) and 5-amino-imidazole-4-carboxamide ribonu-cleotide (AICAR) transformaylase.41
The main pharmacological action of MTX is its inhibition of these enzymes during thymidine and purine
synthesis.42–45 Purine and pyrimidine nucleotides play critical roles in DNA and RNA synthesis as well as in membrane lipid biosynthesis and protein glycosylation. They are necessary for the development and survival of mature T lymphocytes. Pyrimidines control progression from early to intermediate S phase in the cell cycle. Inhibi-tion of pyrimidine synthesis by MTX is a way of inducing apoptosis of activated T lymphocytes.10,46 There are several reports demonstrating that apoptosis caused by an-ticancer drugs, including MTX, may be mediated via the CD95 system.6MTX-induced apoptosis may proceed via expression of CD95-L and ligation of the death receptor CD95 in leukemia T cell lines and hepatoma, gastric can-cer, colon cancan-cer, and breast cancer cell lines.6,8,47 How-ever, in activated human peripheral blood T-lymphocytes (PBL), MTX triggers apoptosis via a CD95-independent pathway.10 Here, we demonstrate another novel mecha-nism of MTX-induced apoptosis. That is, MTX induces caspase-dependent apoptosis in HL-60 and Jurkat T cells in a time- and dose-dependent manner. After MTX treat-ment, intracellular ROS accumulates in the initial one hour andmloss is detected 24 h later. ROS scavenges, such as NAC, vitamin C and catalase, could reduce the accumulation of intracellular ROS, prevent loss ofm, and protect at least part of the cells from apoptosis. These data provide evidence that MTX causesmdisruption and apoptosis in HL-60 and Jurkat T cells via ROS genera-tion. Overexpression of Bcl-2 also prevents MTX-induced disruption ofmand apoptosis. Furthermore, apopto-some formation, activation of caspase 3 and cleavage of PARP (substrate of caspase 3) are found after MTX treat-ment. There is evidence to suggest that the mitochondria-mediated pathway participates in MTX-induced apoptosis.
How can MTX induce the generation of ROS? One possible mechanism is that MTX inhibits the activ-ity of enzymes involved in enzyme defense mechanisms against ROS, including glucose-6-phosphate dehydro-genase, glucose-6-phosphogluconate dehydrodehydro-genase, glu-tathione reductase and gamma-glutamylcysteine syn-thetase. High doses of MTX decrease the cellular levels of glutathione and reduce the effectiveness of the antioxi-dant enzyme defense system.48It has been demonstrated that glutathione depletion induces ROS generation and disrupts m, resulting in mitochondrial cytochrome c release, caspase 3 activation, DNA fragmentation and finally apoptosis.49In another study, MTX increased the amount of hydrogen peroxide released by stimulated poly-morphonuclear neutrophils (PMNs) in a dose-dependent manner.50 Low doses of MTX also induced a time- and dose-dependent increase in cytosolic peroxide in U937 monocytes and Jurkat T cells. ROS generation by MTX is important for cytostasis in monocytes and cytotoxicity T cells.12Our results confirm previous studies and also show
that one of the mechanisms of MTX-induced apoptosis is through a ROS-dependent, mitochondria-mediated path-way.
It is known that overexpression of ODC can prevent cells from apoptosis for many insults, including H2O2, radiation and some chemotherapeutic drugs including cisplatin, doxorubicin, paclitaxel, 5-flourouracil.23 We provide the evidence that putrescine or overexpression of ODC can prevent MTX-induced apoptosis. Further-more, treatment with putrescine or overexpression of ODC in HL-60 and Jurkat T cells can decrease intra-cellular ROS without MTX treatment and reduce the ac-cumulation of ROS following the addition of MTX. Pu-trescine and overexpression of ODC also maintainm and prevent apoptosis following the performance of MTX treatment as a ROS scavenger, such as NAC, vitamin C, and catalase. These protective effects of ODC are not seen in DN-ODC cells and are blocked by DFMO. Through the mitochondria-mediated apoptotic pathway, WT-ODC prevented the formation of apoptosome and cy-tochrome c release from mitochondria to cytosol. More-over, it inhibited the activation of caspase 3 and the cleav-age of PARP (the substrate of caspase 3). A previous study suggested that the inherent ROS level might be determi-native in tumor cells as to their apoptotic susceptibility to As2O3.51Overexpression of ODC seems to reinforce the ability of the anti-oxidant defense system to decrease the inherent ROS level and enhance the resistance of tumor cells to anti-cancer drugs.
Furthermore, how can putrescine and ODC reduce ROS in vivo? Overexpression of ODC or treatment with pu-trescine in cells increases the concentration of polyamines. Spermine, one of the polyamines, can function directly as a free radical scavenger.52Inhibition of ODC by DFMO could increase intracellular ROS, and might lead to an imbalance in polyamine pools. Polyamine catabolism by polyamine oxidase (PAO) could continue and result in the production of ROS.53MTX also inhibits the synthe-sis of spermidine and spermine in stimulated lympho-cytes for patients with rheumatoid arthritis through in-hibition of the SAM-dependent pathway.54 Thus, over-expression of ODC possibly reduces ROS generation during polyamine catabolism, and overcomes the in-hibitory effects of MTX on the polyamine biosynthetic pathway.
In conclusion, low doses of MTX induce apopto-sis of HL-60 and Jurkat T cells in a time- and dose-dependent manner. MTX-induced apoptosis occurs via ROS-dependent and mitochondria-mediated pathways in at least part of the cells. Putrescine and overexpression of ODC can maintainm, avoiding cytochrome c release and the formation of apoptosome. This inhibits the ac-tivation of caspase 3 and prevents apoptosis, functioning as ROS scavengers. Our results explain why ODC activ-ity affects the chemosensitivactiv-ity of anti-cancer drugs and
supports the suggestion that ODC is an important tar-get for chemotherapeutic intervention in the combined treatment of cancers.
Acknowledgments
This study was supported by the grants from the National Science Council NSC 2320-B-040-048 and NSC 93-2745-B-040-005-URD, and by the Chung Shan Medical University grant CSMU 93-OM-A-104.
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