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Experimental Therapeutics |
The 4th Department, Osaka Bioscience Institute, Osaka 565-0874 [H. M., S. H., A. K.]; Department of Urology, Faculty of Medicine, Kyoto University, Kyoto 606-8507 [H. M., Y. K., O. O.]; Department of Biomaterials Science, Faculty of Dentistry, Tokyo Medical and Dental University, Tokyo 113-8549 [H. N., H. I.]; and CREST, JST (Japan Science and Technology Corporation), Kawaguchi City, Saitama Prefecture 332-0012 [A. K.], Japan
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Development of cancer therapies depends on animal models that reproduce the clinical metastases that frequently affect the prognosis and quality of life of patients. Cancer cells have been injected into immunodeficient mice to reproduce cancer metastasis in animal models. Two major routes for this injection have been commonly used: i.v. or s.c. Ectopic models, however, have several disadvantages. For example, the i.v. injection model leaves out invasion, the initial, critical step in cancer metastasis, and in the s.c. injection model, the low incidence of metastasis limits assessment of therapeutic efficacy. Over the past decade, orthotopic inoculation models have been developed to overcome these disadvantages. Nowadays, several orthotopic models have succeeded in reproducing a high incidence of the metastasis similar to that observed in clinical cancers (3) . Likewise in prostate cancer, the orthotopic model provides a high incidence of metastasis to the lymph nodes, lungs, and bones, the major targets of metastasis in clinical cancers (4, 5, 6) . Orthotopic models, therefore, seem very useful for the development of effective therapies and are expected to become the new standard method (3) , although the real significance of the drug efficacies of therapies developed from these in vivo models in cancer research will remain to be validated through the clinical studies.
A recent epoch-making advance in cancer treatment is the ATRA3 therapy, which induces complete remission in most cases of APL (7 , 8) , but, thereafter, there are many cases of recurrence that result in death. For the treatment of this recurrent APL after ATRA treatment, As2O3 in the pharmacological range below 2 µM is reported to be dramatically effective (9) . Since the discovery of As2O3-induced cell death, its molecular mechanisms have been extensively studied, mainly in hematological cancers. The mechanisms of ATRA and As2O3 differ. ATRA induces differentiation in APL cells, whereas As2O3 mainly induces apoptosis (10) .
Until now, As2O3-induced apoptosis has been approached in three major apoptotic mechanisms by using pharmacological inhibitors: MAPKs, caspase, and ROS. MAPKs include JNK, p38, and ERK (11, 12, 13) . Of these, JNK and p38 belong to the SAPKs and have been investigated in As2O3-induced apoptosis (11 , 12) . An APL cell line, NB4, also undergoes As2O3-induced apoptosis through activation of the ASK1-SEK1-JNK kinase cascade in PML bodies.4 The second mechanism in As2O3-induced apoptosis has been postulated to be mediated through the cascade reaction of caspases, a family of aspartate-specific cysteine proteases (14 , 15) . Functionally, these caspases are divided into two subgroups: initiator (caspase-8, -9, and -10) and effector (caspase-3, -6, and -7) caspases (16) . Of the effector caspases, caspase-3 is the main molecule, and the other effectors, caspase-6 and -7, are called caspase-3-like caspases generically (16) . The last mechanism involves ROS such as hydrogen peroxide, superoxide, hydroxyl radicals, and nitric oxide (17) . In healthy organisms, ROS are inevitably generated through a respiratory chain of mitochondria but are scavenged by antioxidant defense systems. When this system is compromised, oxidative stress is considered to produce senescence and various diseases including cancer, inflammation, and neurodegenerative disorder. Recently, the generation of ROS has been reported to regulate As2O3-induced apoptosis (15 , 18 , 19) . Among these three mechanisms suggested in As2O3-induced apoptosis, the most critical mechanism in As2O3-induced apoptosis and the interactions between the mechanisms are still not clear, especially in solid cancers.
We used in vitro assays of androgen-independent prostate cancer cell lines and found that As2O3 induces apoptosis at high concentrations, and it inhibits growth at low ones. Analysis of intracellular signaling showed that the inhibition of ROS generation protected androgen-independent prostate cancer cells from As2O3-induced cell death, whereas the inhibitions of SAPKs and caspase did not. In in vivo experiments that used the murine orthotopic metastasis model of human androgen-independent prostate cancer, treatment with As2O3 inhibited tumor growth in both orthotopic and lymph nodal metastatic lesions. This treatment gave no signs of toxicity to major organs. These findings establish that As2O3 is a safe, promising treatment for androgen-independent prostate cancer and show that the ROS-scavenging system is a therapeutic target for the potentiation of As2O3-induced apoptosis.
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MATERIALS AND METHODS |
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Cell Culture and Cell Viability.
Cell lines PC-3, DU-145, and TSU-PR1 were cultured in RPMI 1640 plus 10% FCS. A growth inhibition assay was carried out to determine the growth inhibition effect of As2O3 using a cell-counting kit (Nacalai tesque), a cell proliferation assay modified from the MTT assay, in which WST-8, a tetrazolium salt, is used as the substrate (20)
. Briefly, 2.5 x 103 cells were plated per well in 100 µl of medium in 96-well microtiter plates. Cells were grown for 24 h, after which various concentrations of the test agents were added. After an additional 48 h of incubation, the medium was aspirated, and WST-8 solution was added. After incubation at 37°C, the absorbance of each well was determined in a microplate reader by the absorbance spectrophotometry at the wavelength of 450 nm. The assay gave an absorbance that correlated linearly with the number of cells and was not affected by As2O3 itself (data not shown). Cell growth was expressed as a percentage of the absorbance in the vehicle-treated control wells. Dose-response curves were drawn, and IC50, the concentration that inhibits 50% of the growth of control cells, were both calculated by the computer software, PRISM (GraphPad, San Diego, CA). These inhibitors were added 2 h before the addition of As2O3 in experiments with the enzyme inhibitors or NAC.
Colony Formation Assay.
Exponentially growing PC-3 cells (3 x 103 cells/well) in RPMI 1640–0.3% agar containing various concentrations of As2O3 (0, 0.5, 1, 2, 5, and 10 µM) were layered in 6-well culture dishes on top of a basal 0.6% agar layer containing the same concentrations of As2O3. Triplicate tests were made for each concentration. Three weeks later, cell colonies were stained with 1 ml of 1 mg/ml p-iodonitrotetrazolium violet (Sigma Chemical Co.). Colonies larger than 60 µM were counted under a phase-contrast microscope, and the results expressed as percentages of the control.
Assays for Apoptosis Detection.
Apoptosis was determined three ways. For nuclear morphology and mitochondrial transmembrane potential, cells that had been cultured on glass-bottom dishes were incubated with 1 µg/ml HOECHST 33342 and 10 µg/ml rhodamine 123 (Sigma Chemical Co.) at 37°C for 30 min and then was observed under a fluorescent microscope. For DNA flow cytometry, cells were trypsinized, washed with ice-cold PBS, fixed in 70% ethanol, and stored at 4°C for 60 min. After another wash with PBS, cells were incubated with 100 µg/ml DNA-free RNase at 37°C for 60 min and then stained at 4°C for 10 min with 50 µg/ml of propidium iodide. DNA-propidium iodide fluorescence was measured with FACScan (Becton Dickinson, San Jose, CA) with respective excitation and emission wavelengths of 488 and 620 nm. TUNEL assays were performed with an In Situ Cell Death Detection kit, Fluorescein (Roche Molecular Biochemicals, Mannheim, Germany) according to the vendor’s protocol with minor modifications.
Western Blotting.
One x 106 cells were incubated for the period indicated, harvested, and lysed in 150 mM NaCl, 1.0% NP40, 0.1% SDS, 0.5% deoxychorate 12 mM ß-glycerophosphate disodium salt hydrate, 1 mM sodium dihydrogenphosphate dihydrate, 5 mM sodium fluoride, 15 µg/ml approtinin, 2 mM dithiotreitol, 1 mM phenylmethylsulfonyl fluoride, and 1 mM sodium vanadate. Protein sample (10 µg each) were blotted through a standard method. Each blot was treated with primary antibodies purchased from New England Biolabs, Inc. (Beverly, MA) as follows: anti-phospho-specific p38 (Thr180/Tyr182), anti-p38, anti-phospho-specific SAPK/JNK (Thr183/Tyr185), anti- SAPK/JNK, anti-phospho-specific ERK1/2 (Ser217/221), anti-ERK1/2, anti-phospho-specific ATF2 (Thr71), anti-ATF2, anti-phospho-specific c-Jun (Ser73), anti-c-Jun, anti-phospho-specific MKK3/6 (Ser189/207), anti-MKK3, and anti-phospho-specific SEK1/MKK4 (Thr223). Anti- SEK1/MKK4 and anti-ASK1 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Primary antibody was detected by horseradish peroxidase-conjugated antibody (1:2000). Signals were detected by the enhanced chemiluminescence detection system (ECL; Amersham Pharmacia Biotech).
Immunocytochemistry.
After incubation with 50 µM As2O3 for 4 h in a slide chamber, cells were fixed for 10 min in 4% formaldehyde, treated for three min with 0.2% Triton X-100 in PBS for permeability, blocked for 30 min with 1% normal goat serum in PBS, then incubated at 4°C overnight with the first antibodies (1:1000 dilution): a mouse monoclonal anti-PML (Santa Cruz Biotechnology), a rabbit polyclonal anti-ASK1(Santa Cruz Biotechnology), and a rabbit polyclonal anti-phospho-specific SEK1/MKK4 (Thr223; New England Biolabs, Inc). Subsequently, the cells were incubated at room temperature for 3 h with Texas Red- or FITC-conjugated secondary antibody (1:500 dilution; Vector Laboratories) then were mounted in antifade solution with DAPI (Vector Laboratories). Labeled cells were analyzed by laser confocal microscopy.
Plasmids.
Rat dominant-negative SAPK/JNK was made by substitution of the phosphorylation sites Thr-183 and Tyr-185, respectively, with Val and Phe. For pGFP-DNSAPK/JNK, an entire DNSAPK/JNK-coding BglII was excised from pcDL-SR-DNSAPK/JNK and subcloned into the BglII site located 3' to the GFP coding region of pEGFPC1(Clontech, Palo Alto, CA). The resulting plasmid was named pGFP-DNSAPK/JNK. The plasmid that encodes the dominant-negative SEK, pGFP-DNSEK1 has been described previously (21)
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Transfection and Cell Death Assays.
DNA transfection was done with Lipofectamine Plus (Life Technology), 3.5 x 105 cells in a six-well dish being transfected with 1.0 µg of pEGFP vector containing each cDNA. Twenty-four h later, the medium with or without As2O3 was changed. Forty-eight h after transfection, cell death was determined by flow cytometry analysis with 7-AAD (Molecular Probe, Eugene, OR). 7-AAD (10 µg/ml) was used instead of propidium iodide because of better discrimination from the fluorescence of EGFP (22)
. Fluorescences of DNA-7-AAD and EGFP was measured with FACScan, using excitation and emission wavelengths of 488 and 620 nm, and 488 and 530 nm, respectively. The cell death rate was determined by dividing the number of 7-AAD- and EGFP-positive cells by the number of EGFP-positive cells.
Caspase-3-like Cleavage Activity Assay.
PC-3 cells were incubated with As2O3 for 8 h at 37°C after a 2-h pretreatment with 10 mM NAC, 20 µM z-DEVD-fmk and 20 µM SB203580. The caspase-3-like cleavage activity assay was done with ApoAlert caspase assay kits (Clontech) according to the manufacturer’s protocol. For the positive control, cells were treated for 8 h with anti-APO-1/Fas mouse monoclonal antibody (Bender MedSystems) after a 2-h pretreatment with 2.5 µg/ml cycloheximide with or without 20 µm z-DEVD-fmk.
Assays for ROS Detection.
Intracellular ROS accumulation was monitored with CM-H2DCFDA (Molecular Probes), which passively diffuses into cells and then is deacetylated by intracellular esterases. Hydrolyzed, oxidized CM-H2DCFDA emits green fluorescence at 529 nm. Briefly, after treatment with various concentrations of As2O3 and NAC, cells were incubated at 37°C for 30 min with serum-free medium containing 10 µM CM-H2DCFDA and then were observed under a fluorescent microscope. At the same time, fluorescence was monitored by FACScan with excitation at 488 nm and emission at 530 nm.
In Vivo Murine Studies.
The in vivo therapeutic effect of As2O3 was evaluated in the orthotopic inoculation mouse model as reported previously (6)
. Twenty-four 10-week-old male SCID mice (BALB/c; Charles River Japan Inc., Tokyo, Japan) were maintained in a specific, pathogen-free environment and handled in accordance with the Guidelines for Animal Experiments of Kyoto University. For the orthotopic inoculation, a transverse incision was made in the lower abdomen under i.p. anesthesia with pentobarbital sodium salt. After the abdominal wall muscles were split, the bladder and seminal vesicles were exposed then retracted anteriorly to reveal the dorsal prostate. Five x 105 PC-3 cells suspended in 20 µl of medium were carefully inoculated under the prostate capsule. Mice were divided into three subgroups according to the daily dose: Group 1 (n = 8), saline alone; Group 2 (n = 8), 2 mg/kg As2O3; Group 3 (n = 8), 5 mg/kg As2O3. The stock solution of As2O3 was diluted with physiological saline. i.p. administration was carried out from 3 days after surgery and continued every day for 32 days. Five weeks after inoculation, the mice were killed by deep anesthesia. Body weight measurement and blood sampling by cardiac aspiration were done immediately after anesthesia, and the peripheral blood cells were counted. Lymph node metastasis was examined by microscopy, and the number of grossly swollen lymph nodes larger than 0.5 mm in diameter were counted. After being weighed, the orthotopic tumor, swollen lymph nodes, lungs, livers, and kidneys were embedded in Tissue-Tek (Sakura Finetechnical Co., Ltd., Tokyo, Japan) and stored at -70°C until analysis. Serial sections were cut from each embedded specimen. One section of each specimen was stained with H&E. Additionally, in specimens from orthotopic tumors, another serial section underwent an in situ TUNEL assay with the Apoptosis in situ Detection kit (Wako Pure Chemical Industries, Ltd., Osaka, Japan) according to the manufacturer’s protocol.
Statistics.
An ANOVA and Fisher’s PLSD test for post hoc comparisons were used for the statistical analyses done with StatView software (Avacus Concepts). Ps of less than 0.05 were considered significant.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Sensitivity to As2O3 varied with the cell line, the respective IC50s of the TSU-PR1, PC-3, and DU-145 cell lines being 2.4, 2.5, and 4.8 µM. In the PC-3 cell colony formation assay, anchorage-independent growth was inhibited by As2O3 dose dependently (Fig. 1B)
. Remarkable inhibition (60% that of the control) was induced at 2 µM As2O3, and the IC50 was 3.3 µM (Fig. 1B)
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. Simultaneous rhodamine 123 staining detected a decrease in mitochondrial transmembrane potential (Fig. 2A)
, whereas nontreated PC-3 cells had healthy nuclei and bright rhodamine 123 staining, indicative of intact mitochondrial transmembrane potential (Fig. 2A)
. Apoptosis was made visible by TUNEL-positive nuclear staining which detected nuclear DNA fragmentation in cells treated with As2O3 (Fig. 2B)
. No signal was present in the nontreated cells. The relation between apoptosis and As2O3 dose was clearly shown by flow cytometry analysis. Dose-dependent induction of apoptosis was detected by DNA flow cytometry (Fig. 3A)
and by the flow-cytometric TUNEL assay (Fig. 3B)
in PC-3 cells treated for 36 h with As2O3. Time-dependent induction of apoptosis was also shown by DNA flow cytometry in PC-3 cells treated with 20 µM As2O3 (Fig. 3C)
. Treatment of PC-3 cells with low concentrations (
4 µM) of As2O3-induced cell cycle arrest at the G2-M phase, whereas fewer cells underwent apoptosis at these concentrations (Fig. 3D)
. The growth inhibition seen in Fig. 1A
, therefore, was mostly attributable to G2-M phase arrest. The respective IC50s of PC-3 cells was 2.5 µM at 48 h and 2.4 µM at 96 h. Time-dependent induction of apoptosis was not shown by DNA flow cytometry in PC-3 cells treated with 2 µM As2O3 until 96-h incubation (data not shown). These results indicate that As2O3 at high concentrations induces apoptosis in PC-3 cells but only causes growth inhibition at low concentrations.
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. Activation of p38 and JNK1/2 were also observed in the DU-145 cell line (data not shown). Other molecules in the signal transduction pathways of p38 and JNK were investigated. Of the transcription factors regulated by these two kinases, ATF2 and c-Jun were activated (Fig. 4C)
. Of the MKKs regulating p38 and JNK, MEKK3/6 was markedly activated (Fig. 4D)
, but there was no activation of SEK1 (Fig. 4D)
. ASK1, a member of the MKK kinase family regulating both MEKK3/6 and SEK1 (23)
, was not detected in the Western blots of the PC-3 cells (data not shown). p38, JNK1/2, and their downstream molecules, therefore, were activated, whereas, of the upstream regulators, the activation only of MEKK3/6 could be identified. The involvement of the PML body in As2O3-induced apoptosis of PC-3 cells, therefore, was investigated. Immunocytochemistry clearly showed that As2O3 induced an increase in the PML protein expression of PML bodies (Fig. 4E)
, but there was no colocalization of PML bodies with ASK1 or phosphorylated-SEK1 (data not shown).
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. Slight enhancement of p38 activation was detected (Fig. 5B)
, whereas there was remarkable enhancement of the activation of JNK1/2 and its downstream c-Jun (Fig. 5B)
. Next, DNA flow cytometry assay was used to evaluate whether p38 kinase is involved in As2O3-induced apoptosis. Few PC-3 cells, treated with SB203580 alone, underwent apoptosis (Fig. 5C)
, whereas addition of SB203580 to PC-3 cells treated with As2O3 produced an increase in the apoptotic fraction, which was not statistically significant (Fig. 5C)
. Addition of SB203580 to the DU-145 cells treated with As2O3 did not decrease the apoptotic fraction (data not shown). These findings indicate that the inhibition of p38 kinase enhances activation in the JNK-c-Jun signal cascade, which suggests that p38 may function as a survival signal mediator for protection against As2O3-induced cell death.
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Involvement of Caspase in As2O3-induced Apoptosis.
The role of caspase in As2O3-induced cell apoptosis was investigated. In the caspase-3-like cleavage activity assay, combined treatment with cycloheximide and anti-APO-1/Fas antibody induced marked activation, which was eliminated by the caspase-3-like protease specific inhibitor, z-DEVD-fmk (Fig. 6A)
. In contrast, treatment with As2O3 caused only a slight increase in caspase-3-like cleavage activity (Fig. 6A)
. The addition of z-DEVD-fmk returned the activity to the basal level, but few PC-3 cells were rescued by this addition from As2O3-induced apoptosis (Fig. 6B)
. These findings suggest that, in PC-3 cells, caspase-3-like proteases do not have major roles in the signaling pathway of As2O3-induced cell death. Next, signal cross-talk between caspase and SAPKs was investigated. Combined treatment of PC-3 cells with As2O3 and SB203580 had a limited effect on caspase-3-like cleavage activity (Fig. 6A)
. Similarly, in the Western blots, the addition of z-DEVD-fmk did not produce a remarkable change in the phosphorylated levels of p38 and JNK1/2 (data not shown). No signal cross-talk between caspase and these two SAPKs, therefore, was found.
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and flow cytometric analysis (Fig. 7A)
. This generation of ROS was completely suppressed by the addition of the antioxidant NAC (Fig. 7A)
. In addition, NAC, remarkably, protected PC-3 cells from As2O3-induced growth inhibition. Addition of 10 mM NAC increased the IC50 of As2O3 from 2.1 to 6.1 µM. Similarly, NAC dramatically inhibited As2O3-induced apoptosis in the flow cytometric TUNEL assay (Fig. 7B)
and DNA flow cytometry (Fig. 7C)
. Likewise, the generation of ROS and inhibition of As2O3-induced apoptosis by NAC were also observed in DU-145 cells (data not shown). Furthermore, this addition attenuated the As2O3-induced activation of caspase-3-like cleavage activity (Fig. 6A)
, of p38 (Fig. 7C)
and JNK (Fig. 7C)
, evidence that the suppression of ROS inhibits As2O3-induced apoptosis, and that ROS regulates the intracellular signal transduction of caspases and SAPKs.
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, orthotopic tumor growth and lymph node metastasis were inhibited markedly by As2O3 treatment. Dose-dependent inhibition of orthotopic tumor growth was statistically significant (Fig. 8B)
. Lymph node metastasis showed a tendency for dose-dependent inhibition as well, but the statistical difference was marginal (P = 0.06; Fig. 8C
). Neither apparent severe toxicity nor body weight loss occurred during the treatment period. No significant difference between groups was found for peripheral blood cell counts of RBCs and WBCs and platelets(data not shown). The histological examination showed there was no severe damage to the liver, lungs, or kidneys (data not shown). H&E staining could not identify any therapeutic effects between groups (Fig. 8D)
, but in situ TUNEL analysis of the tumor tissue showed a marked difference between the control and the As2O3-treated groups. More positive cells for in situ TUNEL analysis were identified in orthotopic tumors of As2O3-treated mice than in those of the control mice (Fig. 8D)
. These findings indicate that As2O3 induces both in vitro and in vivo tumor growth inhibition in the orthotopic murine metastasis model with no severe signs of toxicity.
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DISCUSSION |
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and 3
). In the in vivo study, carried out with our orthotopic murine metastasis model, As2O3 induced a marked inhibition of tumor growth in orthotopic tumors and a marginal inhibition in retroperitoneal lymph node metastases (Fig. 8)
. Results of this in vivo study showed there was no significant toxicity to major organs. Our findings indicate that As2O3 treatment may prove a novel, promising approach to the favorable treatment for androgen-independent prostate cancer. Another goal was to clarify the intracellular signaling in As2O3-induced apoptosis. Analysis showed that the suppression of ROS inhibited As2O3-induced apoptosis, whereas SAPKs and caspase did not. This suggests that drugs that produce intracellular ROS may be potent enhancers of As2O3-induced apoptosis. Since the discovery of the dramatic effects that As2O3 has on APL, several studies have investigated the use of As2O3 in the treatment for solid cancers that include neuroblastoma, gastric cancer, and head and neck cancer (11 , 14 , 24, 25, 26) . Except for one (26) , all of the studies were in vitro ones and showed antitumor effects that could not be compared with those of APL. As2O3 was reported to induce apoptosis or growth inhibition in each cell line, but concentrations used in those studies were higher than used in studies done on hematological cancers. At clinically obtainable As2O3 concentrations (<2 µM), all of the studies failed to induce total cell death. Likewise, in our study on prostate cancer cell lines, As2O3 induced apoptosis with reduced mitochondrial transmembrane potential at high concentrations but only growth inhibition at low ones. These facts suggest that to potentiate cytotoxicity of As2O3 at low concentrations, it is essential to develop the enhancer of As2O3 toxicity.
We, therefore, investigated the intracellular signaling mechanism of As2O3-induced apoptosis. Three major pathways have been proposed as the critical mechanism for As2O3-induced apoptosis: SAPKs, caspase, and ROS, but which mechanism actually is critical for As2O3-induced apoptosis, especially in solid cancers, has yet to be determined. In our study of PC-3 cells from prostate cancer, ROS proved to be the most important of the three signaling pathways. Similar results have been reported in another study done with CHO cells from ovarian cancer (15) . It also showed the association of ROS with caspase-dependent apoptosis (15) . A previous analysis of NB4 cells showed that As2O3-induced apoptosis occurs through the ASK1-SEK1-JNK kinase pathway regulated by the generation of ROS (manuscript in preparation4 and Ref. 19 ). These reports suggest that the three pathways are related rather than independent. Analysis of the relationship of the three pathways in androgen-independent prostate cancer cell lines of the interrelation of the three pathways showed that ROS is the regulator of the caspases and SAPKs, but no relationship between the caspases and SAPKs was found.
Whether SAPK activation is involved in apoptosis generally depends on the type of cells and stimuli (27) . In our study, p38 as well as JNK was activated in As2O3-induced apoptosis, but the inhibition of p38 did not protect androgen-independent prostate cancer cell lines from As2O3-induced apoptosis, and rather it activated the JNK-c-Jun pathway. This indicates that in androgen-independent prostate cancer cell lines, p38 is not involved in As2O3-induced apoptosis, and that some signaling cross-talk exists between p38 and JNK. We here assumed that the activation of JNK is critical to As2O3-induced apoptosis of PC-3 cells, but its roles could not be clearly determined from the overexpression of its dominant-negative forms. Unlike in NB4 cells,4 neither ASK1, SEK1 activation, nor their colocalization with PML was detected in PC-3 cells (data not shown). These differences between NB4 and PC-3 cells may complicate the interpretation of the role of JNK. Other studies have reported that JNK has a critical role in As2O3-induced apoptosis of both rat primary cortical neuron and the JB6 Cl41 mouse epidermal cell line (11 , 12) . Currently, selective inhibitors of JNK are commercially unavailable. Further investigation with JNK-specific inhibitors is required to clarify what that involvement is.
As stated, our in vitro analysis showed that low concentrations of As2O3 could induce growth inhibition alone but not apoptosis. To reproduce this in vitro growth inhibition in in vivo experiments, we administered As2O3 to an SCID mouse whose prostate had been inoculated with PC-3 cells. In a unique study of the in vivo therapeutic effect of As2O3 on solid cancer, As2O3 was very effective both for the tumor itself and the tumor-feeding vessels (26) . Although these findings may be very important experimentally, there must be two important points to be overcome to get clinical relevance. The first point is that a high dose (10 mg/kg) used for the single injection of As2O3 (26) was lethal in two of the three SCID mice used in our study (data not shown). Multiple administration at this dosage, therefore, must be lethal to all of the SCID mice. Less toxic agents and less invasive administration protocols are required in a clinical setting, especially for older patients with prostate cancer. In our murine study, the daily As2O3 dose was reduced by one-half, to 5 mg/kg, and continuous administration was shown to be safe over a 5-week period with no significant toxicity to the peripheral blood cells, liver, kidneys, or lungs. The other point was the therapeutic effects of As2O3 on metastases. Our orthotopic model overcomes this point by establishing sizable metastases in the retroperitoneal lymph nodes, and enables simultaneous evaluation of the compound’s therapeutic effects on primary and metastatic lesions. Although additional studies are required to determine correlations given the As2O3 dose, and serum and tissue concentrations of As2O3, we believe that our study is more clinically relevant and shows the feasibility of the clinical use of As2O3 for the treatment of androgen-independent prostate cancer.
We succeeded in reproducing in vitro growth inhibition in an in vivo orthotopic metastasis model, but monotherapy with As2O3 did not induce complete tumor disappearance. We think that this in vivo tumor growth inhibition indicates the limit of monotherapy with As2O3 because it could not induce total cell death because of its low intracellular concentrations. To obtain in vivo tumor disappearance without increasing the dose of As2O3, it would be necessary to develop potentiators of As2O3-induced apoptosis. Our study has shown that the generation of ROS has a central role in As2O3-induced apoptosis. Drugs that produce intracellular ROS or interfere ROS scavengers, therefore, are potential candidates for the enhancement of As2O3-induced apoptosis. In fact, L-buthionine sulfoximine, an intracellular glutathione-depleting agent, produces a large amount of ROS, and has been shown to greatly potentiate As2O3-induced apoptosis in other solid cancer cell lines (28) . In the future, combined therapy that uses As2O3 together with these drugs may prove useful for treating androgen-independent prostate cancer.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by Grants 10154203 (to Y. K.), 12215156 (to A. K.) from the Ministry of Education, Science, Culture and Sports, Japan; 11-10 (to Y. K.) from The Ministry of Health and Welfare of Japan; a grant from Sankyo Foundation of Life Science (to Y. K.), and a grant from the Japan Society for the Promotion of Science for Young Scientists (to H. M.). 
2 To whom requests for reprints should be addressed, at Department of Functional Biology, Kyoto University Graduate School of Biostudies, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan. Phone: 81-75-753-7675; Fax: 81-75-753-7676; E-mail: kakizuka{at}lif.kyoto-u.ac.jp
3 The abbreviations used are: ATRA, all-trans-retinoic acid; APL, acute promyelocytic leukemia; MAPK, Mitogen-activated protein kinase; ROS, reactive oxygen species; JNK, c-jun NH2-terminal protein kinase; ERK, extracellular-signal-regulated kinase; SAPK, stress-activated protein kinases; ASK1, apoptosis-signal regulating kinase; SEK1, SAPK-ERK kinase; PML, promyelocytic leukemia; z-DEVD-fmk, carbobenzoxyl-Asp-Glu-Val-Asp-fluoromethane; NAC, N-acetyl-L-cysteine; WST-8, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt; TUNEL, terminal deoxynucleotidyl transferase-mediated nucleotide nick-end labeling; DAPI, 4',6-diamidino-2-phenylindole; MKK, MAPK kinase; EGFP, enhanced GFP; 7-AAD, 7-aminoactinomycin D; Ac-DEVD-AFC, acetyl-Asp-Glu-Val-Asp-amino-7-amino-4-trifluoromethyl coumarin; CM-H2DCFDA, 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate; SCID, severe combined immunodeficient; GFP, green fluorescent protein.
4 S. Yasuda, S. Hori, H. M., and A. Kakizuka, manuscript in preparation.
Received 12/20/00. Accepted 5/18/01.
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REFERENCES |
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 A. Kimura, Y. Ishida, T. Hayashi, T. Wada, H. Yokoyama, T. Sugaya, N. Mukaida, and T. Kondo Interferon-{gamma} Plays Protective Roles in Sodium Arsenite-Induced Renal Injury by Up-Regulating Intrarenal Multidrug Resistance-Associated Protein 1 Expression Am. J. Pathol., October 1, 2006; 169(4): 1118 - 1128. [Abstract] [Full Text] [PDF]
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 B. Mukherjee, O. E. Salavaggione, L. L. Pelleymounter, I. Moon, B. W. Eckloff, D. J. Schaid, E. D. Wieben, and R. M. Weinshilboum GLUTATHIONE S-TRANSFERASE OMEGA 1 AND OMEGA 2 PHARMACOGENOMICS Drug Metab. Dispos., July 1, 2006; 34(7): 1237 - 1246. [Abstract] [Full Text] [PDF]
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 B. F. Taylor, S. C. McNeely, H. L. Miller, G. M. Lehmann, M. J. McCabe Jr., and J. C. States p53 Suppression of Arsenite-Induced Mitotic Catastrophe Is Mediated by p21CIP1/WAF1 J. Pharmacol. Exp. Ther., July 1, 2006; 318(1): 142 - 151. [Abstract] [Full Text] [PDF]
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 O. Tikhomirov and G. Carpenter Bax activation and translocation to mitochondria mediate EGF-induced programmed cell death J. Cell Sci., December 15, 2005; 118(24): 5681 - 5690. [Abstract] [Full Text] [PDF]
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 D. Douer and M. S. Tallman Arsenic Trioxide: New Clinical Experience With an Old Medication in Hematologic Malignancies J. Clin. Oncol., April 1, 2005; 23(10): 2396 - 2410. [Abstract] [Full Text] [PDF]
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 Y.-H. Kang, M.-J. Yi, M.-J. Kim, M.-T. Park, S. Bae, C.-M. Kang, C.-K. Cho, I.-C. Park, M.-J. Park, C. H. Rhee, et al. Caspase-Independent Cell Death by Arsenic Trioxide in Human Cervical Cancer Cells: Reactive Oxygen Species-Mediated Poly(ADP-ribose) Polymerase-1 Activation Signals Apoptosis-Inducing Factor Release from Mitochondria Cancer Res., December 15, 2004; 64(24): 8960 - 8967. [Abstract] [Full Text] [PDF]
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 Y. Moon, G. Park, Y. Kim, M. Kim, J. Lim, S. H. Pai, E. J. Lee, C. S. Kang, and K. Han Arsenic Trioxide (As2O3) Sensitivity of Carcinoma Cell Lines and Cancer Cells from Patients with Carcinomatosis Peritonei Ann. Clin. Lab. Sci., July 1, 2004; 34(3): 271 - 276. [Abstract] [Full Text] [PDF]
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 H. Maeda, S. Nagata, C. D. Wolfgang, G. L. Bratthauer, T. K. Bera, and I. Pastan The T Cell Receptor {gamma} Chain Alternate Reading Frame Protein (TARP), a Prostate-specific Protein Localized in Mitochondria J. Biol. Chem., June 4, 2004; 279(23): 24561 - 24568. [Abstract] [Full Text] [PDF]
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 T. Kurita-Ochiai, S. Amano, K. Fukushima, and K. Ochiai Cellular Events Involved in Butyric Acid-Induced T Cell Apoptosis J. Immunol., October 1, 2003; 171(7): 3576 - 3584. [Abstract] [Full Text] [PDF]
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Y.-L. Chen, S.-H. Chen, J.-Y. Wang, and B.-C. Yang Fas Ligand on Tumor Cells Mediates Inactivation of Neutrophils J. Immunol., August 1, 2003; 171(3): 1183 - 1191. [Abstract] [Full Text] [PDF]
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 S. Mathas, A. Lietz, M. Janz, M. Hinz, F. Jundt, C. Scheidereit, K. Bommert, and B. Dorken Inhibition of NF-{kappa}B essentially contributes to arsenic-induced apoptosis Blood, August 1, 2003; 102(3): 1028 - 1034. [Abstract] [Full Text] [PDF]
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T. Kanzawa, Y. Kondo, H. Ito, S. Kondo, and I. Germano Induction of Autophagic Cell Death in Malignant Glioma Cells by Arsenic Trioxide Cancer Res., May 1, 2003; 63(9): 2103 - 2108. [Abstract] [Full Text] [PDF]
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A. Verma, M. Mohindru, D. K. Deb, A. Sassano, S. Kambhampati, F. Ravandi, S. Minucci, D. V. Kalvakolanu, and L. C. Platanias Activation of Rac1 and the p38 Mitogen-activated Protein Kinase Pathway in Response to Arsenic Trioxide J. Biol. Chem., November 15, 2002; 277(47): 44988 - 44995. [Abstract] [Full Text] [PDF]
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 Y.-H. Ling, J.-D. Jiang, J. F. Holland, and R. Perez-Soler Arsenic Trioxide Produces Polymerization of Microtubules and Mitotic Arrest before Apoptosis in Human Tumor Cell Lines Mol. Pharmacol., September 1, 2002; 62(3): 529 - 538. [Abstract] [Full Text] [PDF]
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T. Hayashi, T. Hideshima, M. Akiyama, P. Richardson, R. L. Schlossman, D. Chauhan, N. C. Munshi, S. Waxman, and K. C. Anderson Arsenic Trioxide Inhibits Growth of Human Multiple Myeloma Cells in the Bone Marrow Microenvironment Mol. Cancer Ther., August 1, 2002; 1(10): 851 - 860. [Abstract] [Full Text] [PDF]
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, dotted line) and DU-145 (
, dashed and dotted line). Error bars, SD. B, inhibition of colony formation by As2O3. PC-3 cells were seeded in the top layer of a two-layer agar system in triplicate. Colony formation was determined after 21 days of continuous exposure to As2O3 by p-iodonitrotetrazolium violet staining. Error bars, SD.
