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¹ Department of Clinical Chemistry and Laboratory Medicine, Kyushu University Graduate School of Medical Sciences, Fukuoka; ² Center for Gene Research, Yamaguchi University, Yamaguchi; ³ Department of Immunology, Juntendo University School of Medicine; ⁴ Division of Clinical Immunology, Advanced Clinical Research Center, Institute of Medical Science, University of Tokyo; and ⁵ Department of Biochemistry and Research Institute of Immunological Treatment, Tokyo Medical University, Tokyo, Japan

Requests for reprints: Dongchon Kang, Department of Clinical Chemistry and Laboratory Medicine, Kyushu University Graduate School of Medical Sciences, 3-1-1 Maidashi, Higashi, Fukuoka 812-8582, Japan. Phone: 81-92-642-5749; Fax: 81-92-642-5772. E-mail: kang{at}mailserver.med.kyushu-u.ac.jp

	Abstract

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2-Amino-4,4-dihydro-4,7-dimethyl-3H-phenoxazine-3-one (Phx-1) has been developed as a novel phenoxazine derivative having an anticancer activity on a variety of cancer cell lines as well as transplanted tumors in mice with minimal toxicity to normal cells. We examined the effects of Phx-1 on Jurkat cells, a human T cell line. Phx-1 inhibited proliferation of the cells in a dose-dependent manner but hardly induced cell death, suggesting that Phx-1 acts primarily as an antiproliferative reagent but not as a cytocidal drug. Phx-1 enhanced tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)-induced apoptotic cell death about 100-fold. Tumor necrosis factor , which alone does not induce cell death of Jurkat cells, caused apoptosis in combination with Phx-1. These enhancements of cell death were not due to up-regulation of the death receptors. Phx-1 decreased serum-induced phosphorylation of Akt, a kinase involved in cell proliferation and survival, and inhibited complex III of mitochondrial respiratory chain. Considering that both TRAIL and Phx-1 have only marginal cytotoxicity to most normal cells, Phx-1 may provide an ideal combination for cancer therapy with TRAIL.

	Introduction

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Tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL), a member of the TNF family, interacts with the death receptors, DR4 and DR5, and initiates a caspase-mediated cascade resulting in apoptosis. TRAIL induces apoptosis in a wide range of tumor cell lines, but not in most normal cells. Thus, TRAIL may represent a suitable ligand as an anticancer therapeutic drug (1). Sensitivity to TRAIL is enhanced by combination treatments with either chemotherapeutic agents or irradiation (2, 3). Almost all of the chemotherapeutic agents used in such combinations are also toxic to normal cells, and therefore it is important to find reagents that are not toxic to normal cells but synergize with TRAIL.

Actinomycin D, which shows a strong antitumor activity by intercalating DNA (4, 5), contains a phenoxazine structure. However, phenoxazines which have thus far been chemically synthesized have little water solubility, or hardly exert antitumor effects (6). Tomoda et al. have synthesized a novel water-soluble phenoxazine compound, 4-dihydro-4,7-dimethyl-3H-phenoxazine-3-one (Phx-1, Fig. 1; ref. 7). Phx-1 is produced by the reaction of 2-amino-5-methylphenol with human or bovine hemoglobin. Phx-1 inhibits the proliferation of a variety of cultured cell lines such as human epidermoid carcinoma cells (8), Meth A tumor cells (9), human lung carcinoma cell lines (10), leukemia cell lines K562, HL-60, and HAL-01 (11), human endometrial adenocarcinoma cell lines EN and KLE cells (12). In addition to the in vitro effects, Phx-1 also reduces the growth of tumor cells in mice that are transplanted with the cells (9, 11). As Phx-1 has an antitumor activity in a variety of tumor cells but little cytotoxicity to normal cells (9, 11), this compound may have a potential as a novel anticancer drug. This compound, unlike actinomycin D, does not intercalate DNA, and hence suppresses the proliferation of tumor cells in a manner distinct from actinomycin D. Currently, however, little is known of how Phx-1 acts on the tumor cells.

View larger version (18K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Structure of Phx-1 and Phx-2.

	Materials and Methods

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Reagents
Phx-1 (13) and -2 (14) were synthesized and purified as described previously. TNF was purchased from Sigma (St. Louis, MO). Purified TRAIL was kindly given by Myung-Shik Lee (Samsung Medical Center; ref. 15). Anti-human TNF receptor type I-biotin, anti-human TNF receptor type II-biotin, anti-human IGF receptor I-phycoerythrin, and streptavidin-phycoerythrin were from Becton Dickinson (Mountain View, CA). For detection of TRAIL receptors, biotin-labeled anti-DR4 (DJR1) and anti-DR5 (DJR2) mouse monoclonal antibodies were used (16). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt were from Dojin (Kumamoto, Japan).

Cell Count
Jurkat cells, a human T cell leukemic cell line, were cultured in RPMI 1640 (Sigma) containing 10% fetal bovine serum (FBS) at 37°C under 95% air/5% CO₂. Jurkat cells (1 x 10⁵ cells in 2 mL medium) were seeded onto a well of a six-well plate and grown in the presence or absence of Phx. An aliquot (10 µL) was taken at each day and viable cells that were not stained with a trypan blue dye were counted.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt assays were done according to the manufacturer's instructions (Dojin). Briefly, 3 x 10⁴ cells in 100 µL were plated into a 96-well plate and incubated with the indicated reagents for 24 hours. For the former, 50 µL of 5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was added onto each well and the cells were incubated for another 4 hours. After removing the medium, the resulting insoluble dye was solubilized by adding 100 µL of DMSO. The absorbance was measured at 570 to 650 nm. For the latter, the medium was replaced by 100 µL of PBS and 10 µL of the 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt solution. The cells were incubated for another 1 hour. The absorbance was measured at 450 to 650 nm.

Phosphorylation of Protein Kinases
Jurkat cells were grown to 70% confluence in RPMI containing 10% FBS. After 48 hours incubation without serum, the cells were incubated for 30 minutes in RPMI without serum containing 50 µmol/L Phx-1 and then incubated with 10% FBS for the indicated time. The cells were washed with PBS twice and the total cell lysates were prepared (17). Eight micrograms of the lysates were used for detection of phosphorylation of protein kinases by immunoblotting: Akt with rabbit anti-Akt antibodies (Cell Signaling, Beverly, MA), phosphorylated Akt with rabbit anti-phospho-Ser⁴⁷³-Akt antibodies (Cell Signaling), extracellular signal-regulated protein kinase (ERK) with rabbit anti-ERK2 (Santa Cruz, Santa Cruz, CA), phosphorylated ERK with mouse anti-phospho-ERK (E-4; Santa Cruz), p38 mitogen-activated protein kinase (p38MAPK) with rabbit anti-p38MAPK (Cell Signaling), and phosphorylated p38MAPK with rabbit anti-phospho-p38MAPK (Thr¹⁸⁰/Thr¹⁸²; Cell Signaling; ref. 18).

Mitochondrial Membrane Potential
The mitochondrial membrane potential was analyzed with JC-1, a lipophilic cationic fluorescence dye, using a mitochondrial membrane potential detection kit, MitoProbe (Molecular Probes, Eugene, OR). JC-1 enters mitochondria and changes its emission light from green (FL-1) to red (FL-2) as the mitochondrial membrane become more polarized. Jurkat cells were incubated with the indicated reagents for 24 hours, The cells were then washed with PBS, resuspended in RPMI/2% FBS containing 2.0 µmol/L JC-1 at 1 x 10⁶ cells/mL for 30 minutes at 37°C. After washing with RPMI/2% FBS, the cells were resuspended in PBS and analyzed on a FACSCalibur flow cytometer (Becton Dickinson). Ten thousand events were recorded and analyzed with a CellQuest program (Becton Dickinson).

Apoptosis
After 24-hour incubation of Jurkat cells with the indicated reagents, binding of Annexin V-FITC and staining of propidium iodide were measured for assessment of early and late apoptosis, respectively, using an Annexin V-FITC apoptosis detection kit (BD Biosciences, Palo Alto, CA).

Cell Surface Receptors
After 12-hour incubation of Jurkat cells with the indicated reagents, the cells were washed once with PBS, resuspended in PBS/2% FBS containing the indicated phycoerythrin-labeled or biotin-labeled antibodies against cell surface receptors, and incubated for 45 minutes at 4°C. The cells were washed with PBS and incubated, if necessary, with the streptavidin-phycoerythrin. The fluorescence of phycoerythrin was analyzed on a FACSCalibur flow cytometer.

Mitochondrial Enzyme Activities
Activities of complex I (NADH-ubiquinone oxidoreducatse), complex II (succinate-ubiquinone oxidoreducatse), and complexes II and III (succinate-cytochrome c oxidoreductase) were measured as described by Trounce et al. (19). Mitochondria were prepared from Jurkat cells by differential centrifugation and following sucrose density gradient separation as described previously (20).

	Results

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Effects of Phx on Proliferation of Human Leukemia T Cells
We first examined if Phx-1 inhibits proliferation of Jurkat cells. Jurkat cells were grown in medium containing 0, 20, 50, and 100 µmol/L Phx-1. The cells that were not stained with a trypan blue dye were directly counted. Phx inhibited the cell proliferation in a dose-dependent manner (Fig. 2A). Phx-1 strongly inhibited the cell growth at 50 µmol/L and completely inhibited at 100 µmol/L. Most cells were trypan blue–negative, i.e., alive at 50 µmol/L for 3 days. Less than 2% of the dead cells were trypan blue–positive on day 1 even in the presence of 100 µmol/L Phx-1, suggesting that Phx-1 is primarily antiproliferative but not cytocidal on Jurkat cells. More than 50% of the cells were trypan blue–positive at day 3 in the presence of 100 µmol/L Phx-1.

View larger version (22K): [in this window] [in a new window] [Download PPT slide]   Figure 2. Effects on growth of Jurkat cells. A, Jurkat cells were cultured in the presence of various concentrations of Phx-1. The trypan blue–negative viable cells were counted daily. Bars, 1 SD from 3 independent experiments. B, expression of IGF receptor I (IGFR-I). After 24 h of incubation with 50 µmol/L Phx-1, the cells were incubated with (solid line) or without (broken line) biotin-labeled anti-IGF receptor I and then incubated with FITC-labeled streptavidin. The incubation with Phx increased nonspecific fluorescent signals due to fluorescence of Phx-1 per se (compare broken lines, top and bottom). The apparent increase in specific signals after Phx incubation (compare solid lines, top and bottom) exactly corresponded with the increase in the nonspecific signals, indicating that there is no increase in expression of IGFR-I. C, phosphorylation of Akt. Jurkat cells were preincubated for 30 min in the FBS-free medium containing 50 µmol/L Phx-1 and then incubated with 10% FBS for the indicated time. The total cell lysates were used for the detection of Akt and phosphorylated Akt (p-Akt) by immunoblotting. D, phosphorylation of ERK and p38MAPK was examined as in (C). p-ERK, phosphorylated ERK; p-p38MAPK, phosphorylated p38MAPK.

Phosphorylation of Akt by serum addition, a protein kinase involved in cell survival and proliferation signals, was strongly inhibited at 50 µmol/L Phx-1 (Fig. 2C) and also at 20 µmol/L to a similar extent (results not shown). On the other hand, phosphorylation of ERK, another protein kinase involved in cell survival and proliferation signals, was not affected (Fig. 2D, top). Phosphorylation of p38MAPK, which is mainly located downstream of inflammatory cytokine receptors, was barely changed by the serum addition or by Phx-1 (Fig. 2D, bottom). These results are consistent with a recent report that Phx-1 inhibited antigen-induced phosphorylation of Akt but not that of ERK or p38MAPK in B cells (23).

Synergistic Effects on TRAIL-Induced Cell Death
Next, we examined if Phx-1 affects cytotoxicity of TRAIL. Jurkat cells were incubated with various concentrations of TRAIL for 24 hours and then the living cell number was estimated by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay that is based on the activity of mitochondrial succinate dehydrogenase. The number of living cells decreased in a dose-dependent fashion (Fig. 3A, top). Most cells died at 1,000 ng/mL TRAIL. When the cells were coincubated with TRAIL and 50 µmol/L Phx-1, cell death was enhanced by two orders of magnitude; most cells died at 10 ng/mL TRAIL in the presence of 50 µmol/L Phx-1. Phx-1 alone decreased the number of living cells to about 70% of the control (Fig. 3A, top) but we observed essentially no trypan blue–positive cells as described above. To adjust this antiproliferative effect, we expressed the cell number as relative changes (Fig. 3A, bottom). The marked enhancement of cell death with Phx-1 was clearly shown in this method of presentation. A TRAIL receptor, DR5, was not increased by Phx-1 (Fig. 3B), suggesting that this enhancement of cell death was not mediated by the increase in DR5. Another TRAIL receptor, DR4 was not expressed on the Jurkat cells (results not shown).

View larger version (23K): [in this window] [in a new window] [Download PPT slide]   Figure 3. Synergistic effects of Phx-1 and TRAIL. Jurkat cells were incubated with the indicated reagents for 24 h. A, the cell number was estimated by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Three independent experiments were done. Top, one representative experiment of the three independent experiments. Points, mean; bars, 1 SD. Bottom, the amount of cells at day 0 was expressed as 100%. Points, mean; bars, ±SD. B, the amount of cell surface DR5 was analyzed as in Fig. 2B. C, early (Annexin V-positive) and late (propidium iodide–positive) apoptotic cells were analyzed. The value in each compartment indicates the percentage of distribution of cells. D, the mitochondrial membrane potential (JC-1 red) was estimated.

TRAIL or Phx-1 alone hardly decreased mitochondrial membrane potential but their combination decreased it (Fig. 3D), suggesting that the apoptosis is mediated at least in part by mitochondrial dysfunction.

Effects on TNF-Mediated Cell Death
It is well-known that TNF alone, a death receptor-mediated apoptotic factor, hardly induced cell death of Jurkat cells (Fig. 4A). TNF-induced cell death in combination with Phx-1 (Fig. 4A), whereas TNF receptor 1 was not up-regulated by Phx-1 on the cell surface (Fig. 4B). The TNF receptor 2 was not expressed on the Jurkat cells (results not shown). This cell death was apoptotic because Annexin V-positive cells were increased by the combinational incubation (Fig. 4C). The mitochondrial membrane potential was also decreased by the incubation with TNF and Phx-1 (Fig. 4D).

View larger version (29K): [in this window] [in a new window] [Download PPT slide]   Figure 4. Synergistic effects of Phx-1 and TNF. A, Jurkat cells were incubated with the indicated reagents for 24 h and then cell numbers were measured as in Fig. 3A. Columns, mean; bars, ± SD. TNF, TNF (20 ng/mL); CHX, cycloheximide (2 µg/mL); Phx, Phx-1 (50 µmol/L). B, the cell surface TNF receptor 1 (TNFR-1) was measured. C, the apoptotic cells were estimated as in Fig. 3C. D, the mitochondrial membrane potential was estimated as in Fig. 3D.

View larger version (29K): [in this window] [in a new window] [Download PPT slide]   Figure 5. Respiratory chain. A, activities of complex II and complexes II and III. Mitochondria were prepared from Jurkat cells. The mitochondria were incubated with various concentrations of Phx-1 at 37°C for 5 min and then activities of complex II and complexes II and III were measured. Columns, mean; bars, ± SD. B, effects on 143B 0 cells. The wild-type 143B (+) and mtDNA-less 143B (0) cells were incubated for 24 h with the indicated reagents. The cell number was estimated by a 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt assay in which lactate dehydrogenase activity is measured because 0 cells are respiration-incompetent. Bars, 1 SD. Phx (+), 50 µmol/L Phx-1.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Tomoda et al. have synthesized a novel phenoxazine derivative, Phx-1, which shows antitumor effects. Phx could be a promising anticancer drug owing to its minimal side effects. In this work, we have shown that Phx-1 has an antiproliferative activity with little cytocidal activity, suggesting that the primary action of Phx-1 on Jurkat cells is antiproliferation. Higher doses of Phx-1 and longer incubation caused cell death. 3-Amino-1,4-dihydro-4,8-dimethyl-2H-phenoxazine-2-one (Phx-2, Fig. 1), which is a chemical isomer of Phx-1, at 100 µmol/L showed essentially the same effects on cell proliferation, TRAIL- and TNF-induced apoptosis as did Phx-1 at 50 µmol/L (results not shown).

Under the conditions where Phx-1 showed only antiproliferative effects (Figs. 2A and 3C), Phx-1 markedly enhanced TRAIL-induced cell death of Jurkat cells (Fig. 3A). In addition, similar to cycloheximide, Phx-1 induced cell death with TNF (Fig. 4A). Because these cell deaths were apoptotic, Phx-1 may potentiate the apoptotic death signals. This hypothesis is compatible with the experiments of 143B cells. The respiration-competent 143B cells were completely resistant to TRAIL, whereas the respiration-incompetent mtDNA-less (0) 143B cells acquired weak sensitivity. Phx-1 could not induce cell death of respiration-competent 143B cells even in the presence of 1,000 ng/mL TRAIL but enhanced the cell death of the respiration-incompetent 143B cells (Fig. 5B), supporting the idea that Phx-1 hardly elicits apoptotic signals but strongly augments the death signals once elicited. This augmentation may occur within a cell because there was no up-regulation of cell surface receptors such as TNF and TRAIL receptors. Phx-1 inhibited the phosphorylation of Akt, a protein kinase that can work both for cell proliferation and cell survival. It is reported that Akt-inhibition enhances TRAIL-induced apoptosis in glioma cells (24), whereas Akt-overexpression can inhibit TRAIL-induced apoptosis (25). Thus, the inhibition of the Akt pathway may contribute to the antiproliferation as well as to the cell death enhancement by Phx-1 although it is not yet certain to what extent the inhibition of Akt contributes to the two actions of Phx-1.

2-Methoxyestradiol, a naturally occurring metabolite of estradiol, also enhances cell death by TRAIL (26). This enhancement is exerted at least in part by increasing the expression of DR5 and by suppressing the HIF-1 (26). Phx-1 did not increase the expression of DR5 (Fig. 3B). Overexpression of constitutive active HIF-1 in Jurkat cells (27) did not affect the cell death with TRAIL plus Phx-1 (results not shown). Thus, Phx-1 may enhance the TRAIL-induced cell death in a different manner from 2-methoxyestradiol.

The inhibitor of apoptosis protein family (IAP) inhibits caspase-3, which is a major final executing caspase of apoptosis, and thus negatively regulates apoptosis. In recent reports, several small molecule antagonists of IAPs potentiated TRAIL-induced cell death (28–30). TNF-elicited death signaling may be antagonized by IAPs when TNF is applied alone (31). Cycloheximide restores the TNF-induced cell death probably by suppressing this IAP-mediated inhibition of apoptosis because second mitochondria-derived activator of caspases (Smac), an inhibitor of IAPs, substituted cycloheximide for the TNF-induced cell death (29). Considering that Phx-1 also caused TNF-induced apoptosis instead of cycloheximide, Phx-1 may act on a pathway leading to suppression of IAPs, for example by enhancing the release of Smac from mitochondria. In this context, it should be noted that Phx-1 inhibits complex III. This inhibition could modulate mitochondrial functions. In fact, Phx-1 synergistically decreased the mitochondrial membrane potential in combination with TRAIL or TNF. The inhibition of the respiratory chain may contribute to the antiproliferation as well as apoptosis enhancement, at least in respiration-competent cells, although mitochondrial respiration is not necessarily required for the enhancement of apoptosis.

Higher concentrations of Phx-1 are required for in vitro inhibition of the respiratory chain than for that of the proliferation or phosphorylation of Akt. One possibility is that Phx-1 may accumulate in mitochondria in vivo. Cationic and lipophilic reagents are concentrated in mitochondria in vivo due to the mitochondrial inside-negative membrane potential. Phx-1 has an amino base on an aromatic ring (Fig. 1) and so might be cationic and lipophilic in a cell to some extent. Another possibility is that Phx-1 is metabolized in a cell and converted to a more effective substance. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine, which produces an experimental model of Parkinson's disease in animals, is a good example of the above situation. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine is oxidized in glial cells to 1-methyl-4-phenylpyridinium ion. This metabolite is concentrated in mitochondria due to its cationic and lipophilic nature and then inhibits the respiratory chain (32).

In conclusion, Phx-1 inhibited proliferation of Jurkat cells on its own and enhanced cell death in combination with TRAIL or TNF. Phx-1 and its related compounds may be useful for an understanding of the regulation of TRAIL- and TNF-induced apoptosis. Moreover, because TRAIL or Phx is not cytotoxic to most normal cells, their synergistic activation of apoptosis would provide an ideal combination for cancer therapy.

	Footnotes

 
Grant support: Supported in part by the Naito Foundation and Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Technology, Sports, and Culture of Japan.

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

Received 3/10/05; revised 4/13/05; accepted 4/29/05.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Yagita H, Takeda K, Hayakawa Y, Smyth MJ, Okumura K. TRAIL and its receptors as targets for cancer therapy. Cancer Sci 2004;95:777–83.[CrossRef][Medline]
2. Shankar S, Srivastava RK. Enhancement of therapeutic potential of TRAIL by cancer chemotherapy and irradiation: mechanisms and clinical implications. Drug Resist Updat 2004;7:139–56.[CrossRef][Medline]
3. Wajant H, Pfizenmaier K, Scheurich P. TNF-related apoptosis inducing ligand (TRAIL) and its receptors in tumor surveillance and cancer therapy. Apoptosis 2002;7:449–59.[CrossRef][Medline]
4. Goldberg IH, Friedman PA. Specificity in the mechanism of action of antibiotic inhibitors of protein and nucleic acid synthesis. Pure Appl Chem 1971;28:499–524.[Medline]
5. Wadkins RM, Jares-Erijman EA, Klement R, Rudiger A, Jovin TM. Actinomycin D binding to single-stranded DNA: sequence specificity and hemi-intercalation model from fluorescence and 1H NMR spectroscopy. J Mol Biol 1996;262:53–68.[CrossRef][Medline]
6. Motohashi N, Mitscher LA, Meyer R. Potential antitumor phenoxazines. Med Res Rev 1991;11:239–94.[CrossRef][Medline]
7. Tomoda A, Hamashima H, Arisawa M, Kikuchi T, Tezuka Y, Koshimura S. Phenoxazinone synthesis by human hemoglobin. Biochim Biophys Acta 1992;1117:306–14.[Medline]
8. Ishida R, Yamanaka S, Kawai H, et al. Antitumor activity of 2-amino-4,4 -dihydro-4 , 7-dimethyl-3H-phenoxazine-3-one, a novel phenoxazine derivative produced by the reaction of 2-amino-5-methylphenol with bovine hemolysate. Anticancer Drugs 1996;7:591–5.[Medline]
9. Mori H, Honda K, Ishida R, Nohira T, Tomoda A. Antitumor activity of 2-amino-4,4-dihydro-4, 7-dimethyl-3H-phenoxazine-3-one against Meth A tumor transplanted into BALB/c mice. Anticancer Drugs 2000;11:653–7.[CrossRef][Medline]
10. Abe A, Yamane M, Tomoda A. Prevention of growth of human lung carcinoma cells and induction of apoptosis by a novel phenoxazinone, 2-amino-4,4-dihydro-4,7-dimethyl-3H-phenoxazine-3-one. Anticancer Drugs 2001;12:377–82.[CrossRef][Medline]
11. Shimamoto T, Tomoda A, Ishida R, Ohyashiki K. Antitumor effects of a novel phenoxazine derivative on human leukemia cell lines in vitro and in vivo. Clin Cancer Res 2001;7:704–8.[Abstract/Free Full Text]
12. Nakada T, Isaka K, Nishi H, et al. A novel phenoxazine derivative suppresses proliferation of human endometrial adenocarcinoma cell lines, inducing G2M accumulation and apoptosis. Oncol Rep 2003;10:1171–6.[Medline]
13. Tomoda A, Arai S, Ishida R, Shimamoto T, Ohyashiki K. An improved method for the rapid preparation of 2-amino-4,4a-dihydro-4a,7-dimethyl-3H-phenoxazine-3-one, a novel antitumor agent. Bioorg Med Chem Lett 2001;11:1057–8.[CrossRef][Medline]
14. Tomoda A, Arisawa M, Koshimura S. Oxidative condensation of 2-amino-4-methylphenol to dihydrophenoxazinone compound by human hemoglobin. J Biochem (Tokyo) 1991;110:1004–7.[Abstract/Free Full Text]
15. Kim JY, Kim YH, Chang I, et al. Resistance of mitochondrial DNA-deficient cells to TRAIL: role of Bax in TRAIL-induced apoptosis. Oncogene 2002;21:3139–48.[CrossRef][Medline]
16. Uno K, Inukai T, Kayagaki N, et al. TNF-related apoptosis-inducing ligand (TRAIL) frequently induces apoptosis in Philadelphia chromosome-positive leukemia cells. Blood 2003;101:3658–67.[Abstract/Free Full Text]
17. Mizukami Y, Kobayashi S, Uberall F, Hellbert K, Kobayashi N, Yoshida K. Nuclear mitogen-activated protein kinase activation by protein kinase c during reoxygenation after ischemic hypoxia. J Biol Chem 2000;275:19921–7.[Abstract/Free Full Text]
18. Mizukami Y, Okamura T, Miura T, et al. Phosphorylation of proteins and apoptosis induced by c-Jun N-terminal kinase 1 activation in rat cardiomyocytes by H(2)O(2) stimulation. Biochim Biophys Acta 2001;1540:213–20.[Medline]
19. Trounce IA, Kim YL, Jun AS, Wallace DC. Assessment of mitochondrial oxidative phosphorylation in patient muscle biopsies, lymphoblasts, and transmitochondrial cell lines. Methods Enzymol 1996;264:484–509.[Medline]
20. Kang D, Nishida J, Iyama A, et al. Intracellular localization of 8-oxo-dGTPase in human cells, with special reference to the role of the enzyme in mitochondria. J Biol Chem 1995;270:14659–65.[Abstract/Free Full Text]
21. Gao S, Takano T, Sada K, et al. A novel phenoxazine derivative suppresses surface IgM expression in DT40 B cell line. Br J Pharmacol 2002;137:749–55.[CrossRef][Medline]
22. Baier TG, Jenne EW, Blum W, Schonberg D, Hartmann KK. Influence of antibodies against IGF-I, insulin or their receptors on proliferation of human acute lymphoblastic leukemia cell lines. Leuk Res 1992;16:807–14.[CrossRef][Medline]
23. Enoki E, Sada K, Qu X, et al. The phenoxazine derivative Phx-1 suppresses IgE-mediated degranulation in rat basophilic leukemia RBL-2H3 cells. J Pharmacol Sci 2004;94:329–33.
24. Puduvalli VK, Sampath D, Bruner JM, Nangia J, Xu R, Kyritsis AP. TRAIL-induced apoptosis in gliomas is enhanced by Akt-inhibition and is independent of JNK activation. Apoptosis 2005;10:233–43.[CrossRef][Medline]
25. Chen X, Thakkar H, Tyan F, et al. Constitutively active Akt is an important regulator of TRAIL sensitivity in prostate cancer. Oncogene 2001;20:6073–83.[CrossRef][Medline]
26. Mooberry SL. Mechanism of action of 2-methoxyestradiol: new developments. Drug Resist Updat 2003;6:355–61.[CrossRef][Medline]
27. Makino Y, Nakamura H, Ikeda E, et al. Hypoxia-inducible factor regulates survival of antigen receptor-driven T cells. J Immunol 2003;171:6534–40.[Abstract/Free Full Text]
28. Schimmer AD, Welsh K, Pinilla C, et al. Small-molecule antagonists of apoptosis suppressor XIAP exhibit broad antitumor activity. Cancer Cell 2004;5:25–35.[CrossRef][Medline]
29. Li L, Thomas RM, Suzuki H, De Brabander JK, Wang X, Harran PG. A small molecule Smac mimic potentiates TRAIL- and TNF-mediated cell death. Science 2004;305:1471–4.[Abstract/Free Full Text]
30. Rosato RR, Dai Y, Almenara JA, Maggio SC, Grant S. Potent antileukemic interactions between flavopiridol and TRAIL/Apo2L involve flavopiridol-mediated XIAP downregulation. Leukemia 2004;18:1780–8.[CrossRef][Medline]
31. Wrzesien-Kus A, Smolewski P, Sobczak-Pluta A, Wierzbowska A, Robak T. The inhibitor of apoptosis protein family and its antagonists in acute leukemias. Apoptosis 2004;9:705–15.[CrossRef][Medline]
32. Singer TP, Ramsay RR. Mechanism of the neurotoxicity of MPTP. An update. FEBS Lett 1990;274:1–8.[CrossRef][Medline]

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