Publication of the International Union Against Cancer
Int. J. Cancer: 107, 707–714 (2003) © 2003 Wiley-Liss, Inc.
AUTOCRINE MOTILITY FACTOR SIGNALING INDUCES TUMOR APOPTOTIC RESISTANCE BY REGULATIONS Apaf-1 AND CASPASE-9 APOPTOSOME EXPRESSION Arayo HAGA1*, Tatsuyoshi FUNASAKA1,3, Yasufumi NIINAKA2, Avraham RAZ3 and Hisamitsu NAGASE1 1 Department of Hygienics, Gifu Pharmaceutical University, Gifu, Japan 2 Maxillofacial Surgery, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan 3 Oncology, Pathology and Radiation Oncology, Barbara Ann Karmanos Cancer Institute, Wayne State University, School of Medicine, Detroit, MI, USA Autocrine motility factor (AMF) is a cytokine that regulates locomotion and metastasis of tumor cells. It is well known that expression levels of AMF secretion and its receptor (AMF R) are closely related to tumor malignancy and rheumatoid arthritis. We have established that AMF signaling induced anti-apoptotic activity and that human fibrosarcoma HT-1080 line that secreted high levels of AMF were resistant to drug-induced apoptosis. These cells did not express the apoptotic protease activating factor-1 (Apaf-1) and Caspase-9 genes that encode for the proteins that form the “apoptosome” complex. The disappearance of the Apaf-1 and Caspase-9 gene was recovered by a cellular signaling inhibitor of protein kinase C, phosphatidylinositol 3-phosphate kinase and mitogen-activated protein kinase of the in vitro cultured human fibrosarcoma HT-1080 line. Treatment with these inhibitors favored apoptotic cell death induced by anti-cancer drugs of the murine ascites Ehrlich line. Apoptotic resistance of tumor cells allows them to escape death from cancer chemotherapy, so an understanding of malignant anti-apoptotic activities is important. Antibodies against AMF induced Ehrlich ascites apoptosis in vitro, and effectively aided in vivo apoptosis induced by anti-cancer drugs. The results might indicate a novel route by which tumor cells protect themselves with products, such as AMF, and proliferate despite various stresses and chemical insults; AMF regulates expression of Apaf-1 and caspase-9 genes via a complex signaling pathway and indirectly regulates formation of the apoptosome. © 2003 Wiley-Liss, Inc. Key words: AMF; AMF-R; Apaf-1; Caspase-9; apoptosome
Cancer is a disease in which down-regulation of apoptosis often occurs, and this often appears to compromise chemotherapy using anti-cancer drugs. It is well known that abnormal regulation of many mitochondria-related factors, such as Bcl-2, cytochrome c, Bax, Bik, Mn SOD, APAF-1 and Caspase-9, is observed in cancer cells. For example, the lack or loss of apoptotic proteinase activating factor-1 (Apaf-1) was observed in metastatic melanomas, which can contend with chemotherapy and are unable to execute the typical apoptotic programme in response to p53 activation.1 Sensitivity to apoptosis induced by UV light dependent on its Apaf-1 deficiency level,2 and over-expression of Apaf-1 induced etoposide- or paclitaxel-sensitive apoptosis,3,4 have been reported in human leukemia cell lines. However, over-expression or experimental transduction of Apaf-1 and caspase-9 genes promoted the apoptotic sensitivities to radiation or the chemotherapeutic index of glioma cells.5–7 Thus, the expression levels of Apaf-1 and caspase-9 in tumor cells seem to be important factors in determining the malignancy of the cells, and control of their expression may produce a therapeutic effect in clinical cancer treatments such as radiation and anti-cancer drugs.8,9 Apaf-1 and caspase-9 are the core proteins of the “apoptosome” complex, which has an essential role in inducing mitochondrial programmed cell death.10 It is necessary for the activation of pro-caspase-9 that cytochrome c and dATP interact with Apaf-1 as cofactors, and then the activated caspase-9 turns on the effector caspase-3 that then kills the cell by proteolysis.11 Therefore, the
apoptosome is a key molecular event of programmed cell death in several diseases that express an unusual apoptotic regulation, such as cancer. The function of the apoptosome is controlled by multiple molecules, transforming growth factor-beta up regulation via cytochrome c release,12 heat shock protein 70 and 90 downregulation by binding to Apaf-1 and the formation of a cytosolic complex.13–15 However, it is poorly understood at the molecular level why Apaf-1 and caspase-9 are decreased in malignant cells and how they regulate the function of the apoptosome. We present novel evidence that Autocrine Motility Factor (AMF) induces the down-regulation of Apaf-1 and caspase-9 gene expression, leading to the apoptotic resistant phenotype in malignant cells. AMF is a cytokine that is secreted by tumor cells and promotes cell migration, and its expression is closely related to tumor progression and metastasis.16 AMF has also been identified as a phosphohexose isomerase (PHI/GPI), neuroleukin (NLK) and maturation factor (MF).17 PHI is a housekeeping enzyme of the Embden-Meyerhof pathway, which intracellularly catalyzes the isomerization between glucose 6-phosphate and fructose 6-phosphate, and AMF/PHI/NLK/MF is thus a poly functional molecule with intra- and extra-cellular functions. Recently AMF was implicated as a self-pathogenic antigen18 responsible for the onset of rheumatoid arthritis. Of note, both arthritis and the metastasis process involve migration of cells to the surrounding organs. The AMF-receptor, gp78 (AMF-R), is a 78 kDa glycoprotein on the cell surface, and its level has been found to be increased in a number of different malignancies, correlating with the metastatic potential.19 –21 Thus, the signaling of AMF and AMF-R is a major characteristic of tumor cell malignancy, and the following results imply a novel route to the suppression of Apaf-1 and caspase-9. Abbreviations: AMF, autocrine motility factor; AMF-R, autocrine motility factor receptor; Apaf-1, apoptotic proteinase activating factor-1; G-418, geneticin; GFX, GF109203X; GN, genistein; gp78, 78 kilo-Dalton glycoprotein; HRPO, horseradish peroxidase; MAPK, mitogen-activated protein kinase; NLK, neuroleukin; PD, PD98059; PHI, phosphohexose isomerase; PI3K, phosphatidylinositol 3-phosphate kinase; PKC, protein kinase C; PT, pertussis toxin; WO, Wortmannin. Grant sponsor: Japanese Grant-in-Aid for Scientific Research; Grant numbers: 13033032, 15790044; Grant sponsor: NIH; Grant number: R01CA51714. *Correspondence to: Department of Hygienics, Gifu Pharmaceutical University, 5-6-1 Mitahora-Higashi, Gifu 502-8585, Japan. Fax: ⫹81-58-237-5979. E-mail:
[email protected] Received 21May 2002; Revised 21 October 2002, 21 February 2003, 25 April 2003; Accepted 23 June 2003 DOI 10.1002/ijc.11449
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MATERIAL AND METHODS
Chemicals and antibodies G-418 and Wortmannin (WO) (Sigma Chemical Co., St. Louis, MO), GF 109203X (GFX) (Biomol Research Laboratory, Inc., Plymouth Meeting, PA), PD-98059 (PD) and genistein (GEN) (Wako, Osaka, Japan), H89 (Seikagaku Kogyo, Tokyo, Japan), pertussis toxin (PT) (List Biological Laboratories Inc., Campbell, CA) were used. Restriction enzymes etc. were obtained from
Takara (Osaka, Japan). All reagents and media for cell cultures were purchased from Gibco BRL (Grand Island, NY). Ac-DEVDMCA (Ac-Asp-Glu-Val-Asp-MCA) was purchased from Peptide Instruments, Inc. (Osaka, Japan). A mono-specific polyclonal antibody, anti-rh AMF, directed against human AMF was generated by immunization with a purified human recombinant AMF. SPF-New Zealand White rabbits were given a s.c. injection of 1.0 mg AMF in complete Freund’s
TABLE I – PRIMERS Target
Human AMF Mouse AMF Human Caspase-9 Mouse Caspase-9 Human APAF-1 Mouse APAF-1 Human -actin Mouse -actin
Sequence
dAATGCAGAGACGGCGAAGGAG dACGAGAAGAGAAAGGGGAGTCA dACCCCTCATGGTGACTGAAG dGGTCTGGACAGGGATGAGAA dCCATATGATCGAGGACATCCAG dGAAATTAAAGCAACCAGGCATCT dTGTTAAACCCCTAGACCACCTG dCATCTGGCTCAGAGTCACTGTC dCTTTTCCCTGGATTGGATTAAAG dATCTGGTGAAAATCTGCAGTGAT dTTCACGAGTTCGTGGCATATAG dTCACATCCCAAAGCCTTAAAGT dTGACGGGGTCACCCACACTGTGCCCATCTA dCTAGAAGCATTTGCGGTGGACGATGGAGGG dAGAGAGGTATCCTGACCCTGAAGTA dCATAGAGGTCTTTACGGATGTACAAC
Accession number
Product (amplification
K03515
1065 bp (676–1741)
M14220
747 bp (541–1288)
U60521
1107 bp (156–1263)
AB019600
754 bp (306–1060)
NM013229
750 bp (2056–2806)
AF013263
1281 bp (1651–2869)
M10278
660 bp (509–1169)
X03765
701 bp (104–805)
FIGURE 1 – Established transfectant lines of HT-1080. 1: HT-1080 parent line; 2: pBKCMV transfected (HT-1080/mock); 3: pBKCMV-AMF transfected (HT-1080/AMF). (a) Morphology. (b) AMF expression. a,b: Western analysis of conditioned media (a) and cell lysates (b) using the rabbit anti-AMF IgG as the primary antibody. c,d: RT-PCR against AMF mRNA (c) and beta-actin mRNA (d) expression. (c) Motile activity. Data were calculated using a phagokinetic track assay as described in the Material and Methods.
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THE AMF SUPPRESSES APOPTOSOME EXPRESSION TABLE II – APOPTOSIS-RELATED GENES LEVEL OF HT-1080/AMF CELLS COMPARED WITH A MOCK LINE Disappeared genes
Missing genes
Caspase 1 Caspase 4 Caspase 9 Apoptotic protease activating factor 1 Protease inhibitor 2 (anti-elastase) 37-kDa leucine rich repeat protein Dihydropyrimidine dehydrogenase Prostaglandin E synthase
Heat shock 70-kDa protein Death associated protein 6 Caspase 3 Tumor protein 53 binding protein DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 8 (RNA helicase) Tumor necrosis factor receptor superfamily member 10b Caspase 7 TNF receptor associated factor 2 increased genes Glutathione S transferase M4 Superoxide dismutase 2 mitochondrial Defender against cell death 1 Microsomal glutathione S transferase 1 Glutathione peroxidase 1 Glutathione S transferase like; glutathione transferase omega BCL2-related protein A1 BCL2 antagonist of cell death Cytochrome c-1 Superoxide dismutase 1 Same levels Caspase 6 Caspase 8 Hsp70 binding protein HSPC070 protein Apoptosis inhibitor 1 Apoptosis inhibitor 4 Fas (TNFRSF6) associated via death domain Tumor protein p53 BCL2/adenovirus E1B 19 kDa interacting protein 1 BCL2/adenovirus E1B 19 kDa-interacting protein 2 BCL2/adenovirus E1B 19 kDa interacting protein 3 BCL2 like 2 CASP2 and RIPK1 domain containing adaptor with death domain ATP synthase, H⫹ transporting, mitochondrial F0 complex, Subunit b, isoform 1 ATP binding cassette, sub-family C (CFTR/MRP), member 3
Ratio1
0.54 0.57 0.58 0.66 0.68 0.69 0.70 0.70 3.43* 3.09* 2.72* 2.77* 2.63* 2.25* 2.04* 1.88 1.86 1.80 1.28 1.11 1.09 0.91 1.02 0.99 1.17 0.79 0.86 0.83 1.01 1.28 1.29 1.31 1.23
Ratio numbers were calculated by “External control normalization” of ImaGene software (Takara Co., Ltd, Osaka, Japan). The ratio ⬍2.00 or ⬎0.50 means statistically significant increase or decrease (*p⬍0.01). 1
adjuvant, boosted at subsequent 3 week intervals and bled 3 days after the third boost. The specificity and titer were routinely monitored by ELISA assay, and the titer of this antiserum was at least 5,000.22 The rabbit anti-rh AMF IgG was purified using a Protein G Sepharose威 column (Pharmacia, Uppsala, Sweden). A rabbit polyclonal anti-Apaf-1 IgG was purchased from Stressgen (Victoria, Canada), and an anti-human/mouse Caspase-3 active, which recognized the amino acids 163–175 of Caspase-3, meaning that it did not detect or poorly detected the precursor form of Caspase-3, was from R&D Systems (Minneapolis, MN). A mouse monoclonal anti--actin IgG was obtained from Sigma Chemical Co. (St. Louis, MO). Horseradish peroxidase (HRP) or fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG, anti-mouse IgG and anti-rat Igs antibodies were purchased from Oncogene Research Products (Cambridge, MA). Cell cultures and gene transfections The human fibrosarcoma HT-1080 cell line was provided by the Health Science Research Resources Bank (H.S.R.R.B., Osaka, Japan). Two strains of the Ehrlich ascites tumor cells were obtained from the Cancer Cell Repository, Research Institute for Tuberculosis and Cancer, Tohoku University (Sendai, Japan) and Japanese Collection of Research Bioresources (J.C.R.B., Tokyo, Japan), respectively. All cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% heat-inactivated fetal bovine serum that was supplemented with nonessential amino acids. Cultures were maintained at 37°C in an air-5% CO2 incubator at constant humidity. Cells were harvested and passaged for experiments with 0.25% trypsin and 0.025% EDTA, and viability was monitored by trypan blue exclusion. To ensure maximal reproducibility, cultures were grown for no longer than 6 passages
after recovery from frozen stocks, and monitored to prevent mycoplasma contamination. Ten microgram purified AMF expression vectors,22–24 pBKCMV-AMF and pBK-CMV were conjugated with TransIT威-100 (PanVera Corp., Madison, WI) and dropped on HT-1080 monolayers, which were 70% confluent in 10 cm diameter dishes. After 4 hr, transfected cells were maintained with 10% FCS-DMEM containing 800 g/ml G-418. At least 12 visible colonies were picked up and the best clone was selected by its motile activity, as described below. Measurement of cell motility Random cell motility was measured by the phagokinetic track assay as described previously.22,25 Briefly, uniform carpets of gold particles were prepared on BSA-coated glass coverslips in 6-well culture plates and 3,000 cells in suspension culture were seeded per well. After allowing the cells to adhere for 1 hr at 37°C, the DMEM was replenished with fresh medium. After 16 hr, phagokinetic tracks were visualized using dark-field illumination in a Nikon diaphot inverted microscope at a magnification of ⫻200. The area cleared by at least 20 cells was measured using the NIH image analysis software. Gene expression analysis using a DNA chip Total RNA was isolated from HT-1080 transfectant cells using the TRIzol reagent (Gibco BRL, Grand Island, NY). The mRNA was purified using an mRNA purification kit (Amersham, Buckinghamshire, UK), and Takara Co., Ltd. (Osaka, Japan) was then requested to complete the analyses, including statistical transactions for which IntelliGeneTM Human Cancer CHIP Ver. 2.1 was used.
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HAGA ET AL.
FIGURE 2 – Anti-apoptotic ability of AMF mutant lines. HT-1080 and transfectant cells were exposed to 10 mM mitomycine C for 48 hr. Cell viability was then assayed using the MTT assay (a) and extracted cellular DNA migration in a 1.5% agarose gel (b). The mitochondrial apoptosis was assessed as the enzymic activity of caspase-9 (c), and the detection of the active form of Caspase-3 (d). (a) open circle, HT-1080 parent line; closed circle, pBKCMV transfected (HT-1080 / mock); closed square pBKCMV-AMF transfected (HT-1080/AMF). (b) Lane 1, 100 bp ladder; Lane 2, HT-1080 parent line; Lane 3, pBKCMV transfected (HT 1080/mock); Lane 4, pBKCMV-AMF transfected (Ht-1080/AMF). (c) Relative activities of caspase-9 with the Ac-DEVD-MCA substrate. (d) Lane 1, pBKCMV transfected (HT-1080/mock); Lane 2, pBKCMV-AMF transfected (HT-1080/AMF).
Semi quantitative RT-PCR Total RNA was prepared from HT-1080 transfectant cells using the RNA Isolation system (Promega, Madison, WI). The cDNA was synthesized from the total RNA by a reverse transcriptase
using random hexamers of a first-strand cDNA synthesis kit (Takara, Osaka, Japan). The target mRNA was then amplified by PCR with specific primers, as shown in Table I. The PCR conditions were as follows: 94°C for 5 min as an initial denaturation, 30
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THE AMF SUPPRESSES APOPTOSOME EXPRESSION
FIGURE 3 – Effect of intracellular signaling inhibitors on the expression of the apoptosome mRNAs. pBKCMV-AMF transfected HT1080 cells (HT-1080 / AMF) were cultured with 5 M GF109203X (GFX), 3 M H89, 100 nM Wortmannin (WO), 100 M genistein (GEN), 20 M PD 98059 (PD) or 150 ng/ml pertussis toxin (PT) for 12 hr. Total RNA was then extracted and the first strand cDNA was prepared and subjected to RT-PCR for apoptotic proteinase activating factor-1 (APAF) and caspase-9 (Casp-9) mRNA detection. Beta-actin mRNA amplification was used as a quantitative control. 1: untreated HT-1080/AMF; 2: untreated HT-1080 / mock line; 3: GFX; 4: H89; 5: WO; 6: GEN; 7: PD; 8: PT.
cycles of 94°C for 30 sec, 55°C for 60 sec and 72°C for 2 min, and then 72°C for 10 min as a final extension. The PCR products were then subjected to 1.0% agarose gel electrophoresis and the density of each band was analyzed using the NIH Image software. SDS-PAGE and western blotting Cells were lysed with 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10 mM EDTA, 1% of NP 40, Triton X-100, sodium deoxycholate, 1 mM PMSF and 100 U/ml aprotinin, and 20 g protein of the cell lysates were separated by 10% SDS-PAGE and blotted onto PVDF membranes (Bio-Rad, Hercules, CA). The membranes were blocked with 5% low-fat dried milk in PBS containing 0.02% Tween 20 overnight. The blocked membranes were incubated with anti-rhAMF (1/1,000), anti-Apaf-1 (1/500), anti-caspase-3 active (1/500) or anti--actin (1/ 500) as the primary antibody for 1 hr, and then with HRP-conjugated anti-rabbit or anti-mouse IgG (1/2,000) for 1 hr at room temperature. The labeled bands were revealed by 3,3⬘ diaminobenzine tetrahydrochloride (DAB) staining. Detection of apoptotic cell death Cells were lysed with 10 mM Tris-HCl, pH7.5, 10 mM EDTA, 0.5% Triton X-100, and then the RNA was digested with RNase A and the DNase was inactivated by the addition of proteinase K. The extracted DNA was concentrated, washed with alcohol and applied to agarose gel electrophoresis. Quantitative cell death was evaluated with the MTT assay. 3-(4,5-dimethylthiazol-2-yl) 2,5 diphenyl tetrazolium bromide (MTT)/PBS was added to cultured cells and incubated at 37°C. After 1 hr, the generated pigments were eluted by the addition of isopropanol and 0.04 N HCl, and detected by reading the OD at 570 nm. In vivo cell apoptosis was visualized by the TUNEL (TdTmediated dUTP-biotin nick end labeling) method using an In Situ Apoptosis Detection Kit (Takara Co. Ltd., Osaka, Japan). Briefly, Ehrlich cells grown in mice were fixed on a slide with paraformaldehyde, and endogenous peroxidase was blocked with H2O2. The DNA fragments were then marked with FITC-dUTP by TdT terminal transferase. Measurement of caspase activities Caspase substrate (100 mM), Ac-DEVD-MCA/DMSO, was added to 0.5 mg/ml protein of cell lysates and incubated at 37°C for 15 min. The reaction was then stopped by the addition of 175 mM CH3COOH and 1% sodium acetate. The fluorescence intensity was measured at an excitation of 340 nm and an emission of 460 nm.
FIGURE 4 – Anti-apoptotic ability of 2 different derivative cell lines of murine ascites tumor cells. (a) Total RNA of the Ehrlich lines was extracted and the first strand cDNA was prepared and subjected to RT-PCR for apoptotic proteinase activating factor-1 (Apaf-1), caspase-9 and AMF mRNA detection. Beta-actin mRNA amplification was used as a quantitative control. (b) Ehrlich lines were exposed to 20 M MMC for 48 hr and then the cell viability was assayed by extracted cellular DNA migration in a 1.5% agarose gel.
RESULTS
AMF-high secretion mutant line generated from HT-1080 It is well established that cell motility is an important step for tumor cell invasion into adjacent tissues and metastasis to distant organs. AMF is identical to the NLK cytokines and to the cytoplasmic phosphoenzyme phosphohexose isomerase (PHI).16,23 However, AMF/NLK/PHI/MF does not have the signal peptide that is critical for the classical endoplasmic reticulum (ER)-Golgi secretory pathway. Therefore, AMF/NLK/MF is presumably secreted via a novel alternative secretory pathway. To characterize this secretory mechanism of AMF/NLK/MF, we transfected the full-length cDNA of AMF into the human fibroblast cell line HT-1080 using the retroviral vector pBK-CMV. Two stable transfectant cell clones were isolated, HT-1080 vector as a mock cell line and HT-1080/AMF as an AMF-high expression line, which have the pBK-CMV vector only and pBK CMV-AMF, respectively. The sequence of pBK-CMV was detected in each of the cell lines (data not shown). As shown in Figure 1a, the edges of HT-1080/AMF cells were pointed and outgrowing compared to the parent cells, and they seemed to avoid coherence. Although the cellular AMF (PHI) levels were the same in all cells (Fig. 1b,c), the over expression of secreted AMF was manifested by its level in the conditioned medium of HT 1080/ AMF (Fig. 1b,a). This level of secreted AMF was reflected in their motile ability, since HT-1080/AMF exhibited 1.7 times higher locomotion than that of HT-1080 and mock cell lines (Fig. 1c). During the culture of these mutant cell lines, we found that HT-1080/AMF cells were resistant to physical stress and chemical stimulation. Therefore, we tested their gene expression using a DNA chip loaded with cancer-related genes. Significant changes in many apoptotic genes were detected between HT-1080/AMF and the mock cell line (Table II). Enzymes that are related to resistance against physical and physiological stresses, such as glutathione S-transferases and superoxide dismutases, were increased, while others that affect anti-apoptotic activities, such as heat shock proteins, were decreased. These changes seem to suggest that the physical and physiological resistance of HT-1080/AMF was caused by the apoptotic cascade. We noted the disappearance of caspase-9 and apoptotic protease activating factor-1 genes that encode the core proteins of the apoptosome, and both of which are significant factors in the mitochondrial apoptosis cascade.
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HAGA ET AL. TABLE III – EFFECT OF ANTIBODY AND SIGNAL INHIBITORS ON APOPTOTIC CELL DEATH1 Anti-AMF IgG
0
100 pM
100 nM
Surviving index
1.00
0.92 ⫾ 0.11
0.60 ⫾ 0.09 (p ⬍0.001)
Inhibitor (s)
—
Surviving index
1.00
GFX
0.63 ⫾ 0.12 (p ⬍0.01)
WO
PD
0.98 ⫾ 0.16
0.94 ⫾ 0.06
MIX
0.50 ⫾ 0.08 (p⬍0.001)
1 Antibody: 1 ⫻ 104 Ehrlich (⫹) cells were cultured with RPMI-1640 medium containing 1 % FCS for 12 hr. Then they were exposed to column purfied anti-AMF rabbit IgG for 72 hr. Signal inhibitors: 1 ⫻ 104 Ehrlich (⫹) cells were cultured with RPMI-1640 medium containing 1 % FCS for 12 hr. Then 5 M GFX, 100 nM WM, 20 M PD, or mixture of them (5 M GFX, 100 nM WM, 20 M PD) were added; 48 hr after, the signaling treated cells were exposed to 20 M MMC for 30 hr. Their survival was measured by MTT assay and significance (p value) was calculated by Student’s t-test.
To check the apoptotic death resistance of HT-1080/AMF, mitomycine C (MMC), which is known as an apoptosis-inducible anti-cancer drug, was used. Figure 2 indicates the anti-apoptotic ability of HT-1080/AMF against MMC. HT-1080/AMF exhibited a high drug resistance compared to the parent cell line (Fig. 2a), and degradation of chromatin DNA, which is a major characteristic of apoptotic cell death, was not observed in HT 1080/AMF after MMC treatment (Fig. 2b). Furthermore, caspase activity, which was detectable by the caspase-9 substrate, was markedly reduced compared to that of the mock cell lines (Fig. 2c). The proform (32 kDa) and the p17 subunit, which is the proteolytically active form of caspase-3, were both detected in the mock transfected cells but not in the HT-1080/AMF cells (Fig. 2d ). Apaf-1 was detected in the mock transfected cells but was barely detectable in the cell extracts from HT-1080/AMF cells (Fig. 2d). The signaling downstream of AMF-R for regulation of apoptosome expression To investigate the intracellular signaling following AMFAMF-R binding, we employed well-known inhibitors of intercellular signaling molecules, including PT (G-protein inhibitor), PD [mitogen-activated protein kinase (MAPK) inhibitor], WO [phosphatidylinositol 3 phosphate kinase (PI3K) inhibitor], GEN (tyrosine kinase inhibitor) and H89, GFX [protein kinases A and C (PKA and PKC) inhibitor]. To detect the mRNAs of apoptosome recovery, quantitative RT-PCR with beta-actin as an internal standard was performed and the result is shown in Figure 3. The Apaf-1 mRNA (corresponding to the 676 –1741 region of the cDNA) and caspase-9 mRNA (corresponding to the 156-1263 region of the cDNA) were hardly detected in the HT-1080/AMF line compared to the mock cell line (lanes 1 and 2, respectively), as was expected from the data of the DNA chip, although the gene regions loaded on the chip were different from these detected sequences. When HT-1080/AMF cells were treated with an inhibitor of PKC (GFX), PI3K (WO) or MAPK (PD), the disappearance of Apaf-1 and Caspase-9 mRNAs was fully recovered to the control levels observed in the mock cell line. The apoptotic effect of anti-AMF treatment on tumor cells We previously found 2 different murine ascites tumor cell lines, which were naturally derived from the Ehrlich cell line without any gene treatment:22,23 “Ehrlich ⫹,” which grows well in mice and induces abundant ascites, and “Ehrlich ⫺,” which grows poorly in mice and did not develop ascites. “Ehrlich ⫹” cells exhibited 100% lethality in mice, at almost any i.p. inoculum number. The secreted AMF level from “Ehrlich ⫹” cells was markedly higher than that of “Ehrlich ⫺” cells.22,23 The mRNA expression levels of the apoptosome components were also supported by the data of the HT-1080/AMF mutant cell clone as shown in Figure 4, i.e., the mRNAs of APAF-1 and Caspase-9 were hardly expressed in “Ehrlich ⫹” compared to the “Ehrlich ⫺” line (Fig. 4a). In addition, the “Ehrlich ⫹” line exhibited high resistance against apoptotic DNA degradation induced by MMC exposure, as expected (Fig. 4b).
FIGURE 5 – Effect of the anti-AMF IgG on the in vivo apoptosis of murine ascites tumor cells induced by an anti-cancer drug. “Ehrlich ⫹” cells (5 ⫻ 106) were i.p. injected into mice. Ten days later, mice were treated with 400 g of purified rabbit anti-AMF IgG, and 2 mg/kg of MMC was given after 6 hr of IgG treatment. The ascites tumor cells were removed after 24 hr of MMC treatment and apoptotic cell death was visualized by the TUNEL method under a confocal microscope. These cells were also subjected to RT-PCR analysis against apoptosome genes. 1: MMC treatment without IgG; 2: MMC treatment with IgG; 3: untreated background of the TUNEL method. 4: APAF-1 and caspase-9 expression in antibody-treated “Ehrlich ⫹” cells. a: untreated in vivo “Ehrlich ⫹”; b: MMC treated without IgG in vivo “Ehrlich ⫹” c: MMC treated with IgG in vivo “Ehrlich ⫹”.
To isolate the extracellular AMF, “Ehrlich ⫹” cells were exposed to the column-purified specific rabbit anti-AMF IgG in vitro. After 72 hr culture, 40% apoptotic cell death of “Ehrlich ⫹” was observed when 100 nM of IgG was added without an anti-cancer drug (Table III). DNA ladder formation was also recognized in IgG-treated “Ehrlich ⫹” (data not shown), and these results imply that the anti-AMF therapy can induce apoptosis itself. Furthermore, inhibition of the signaling between AMF-R and apoptosome gene expression promoted the apoptotic cell death caused by an anti-cancer drug. The results shown in Table II suggest that the inhibitor of PKC (GFX) was an effective helper chemical of the anti-cancer drug MMC, but the inhibitors of PI3K (WO) and MAPK (PD) treatment were not active in “Ehrlich ⫹” cell death.
THE AMF SUPPRESSES APOPTOSOME EXPRESSION TABLE IV – IN VIVO APOPTOTIC EFFECT OF ANTI-AMF IGG ON MURINE ASCITES Index
Background ⫹ MMC ⫹ MMC ⫹ IgG
1.00 2.82 ⫾ 0.29 9.35 ⫾ 0.81
Ehrlich (⫹) cells (/ mouse) (5 ⫻ 106) were i.p. injected into mice. 10 days after, mice were treated with 400 g of purified anti-AMF rabbit IgG and 2 mg/Kg of MMC was given after 6 hr of IgG treatment. The ascites tumor cells were removed after 24 hr of MMC treatment and apoptoic cell death was visualized by TUNEL method under-confocal laser scanning microscope. The FITC intensenes of more than 50 areas were calculated by NIH Image software.
However, the multiple use of GFX, WO and PD supported the MMC and half of the cells died. The “Ehrlich ⫹” ascites tumor-bearing mice were used to estimate the effect of anti-AMF treatment as an in vivo apoptosis model. The purified rabbit anti-AMF IgG injection exhibited the accumulation of malignant ascites induced by “Ehrlich ⫹” itself22–24 and was used again with the MMC treatment. In vivo cultured “Ehrlich ⫹” exhibited extensive apoptotic resistance against MMC, similar to the in vitro MMC exposure, i.e., it did not recognize a difference between the TUNEL background before and after the MMC procedure (Figs. 5,1 and 3,3). However, multiple DNA fragment formations were observed when ascites tumor-bearing mice were pretreated with the rabbit anti-AMF IgG as the fluorescence intensity marked with FITC dUTP (Fig. 5,2). The calculated data is shown in Table IV. Furthermore, the recovery of APAF-1 gene expression was also recognized in IgG-treated Ehrlich cells, but caspase-9 mRNA was not detected in in vivo cultured “Ehrlich ⫹” cells at all (Fig. 5,4c). DISCUSSION
AMF was originally identified from a human malignant melanoma cell line as a self producing factor that was able to enhance directed and random migration.27. AMF was genetically identified as a phosphohexose isomerase (PHI) intracellularly, and as NLK, which is known as a neurotrophic factor,28 and MF, which mediates the differentiation of human myeloid leukemic cells,29 extracellularly.17 Thus, the AMF/PHI/NLK/MF molecule exhibits multiple functions in various physiological situations, including cancer and autoimmune diseases. The results described in this study suggest a novel function of AMF and AMF-R signaling that causes apoptotic resistance, which agrees with the observations that the secretion level of AMF is closely related to the malignancy of clinical cancers.22–26 In cancer chemotreatment, drug resistance of cells is one of the most difficult problems and aberrant apoptotic behaviors are often recognized. The apoptotic resistance caused by the down-regulation of Apaf-1 and caspase-9 mRNA expression observed in this study in cells secreting high levels of AMF suggests a possible novel route of drug resistance in malignant cells, which agrees with the observation that metastatic melanomas often lose Apaf11. Two different cell types were used to estimate the anti-apoptotic mechanism related to AMF levels of malignant cells. One is an AMF-transfected human tumor model (HT 1080/AMF) and the other a natural derivative of the Ehrlich murine ascites tumor model (Ehrlich ⫹), which has shown high AMF expression and secretion compared to the original line.22 The anti-apoptotic ability of in vitro cultured “Ehrlich ⫹” seems to be poorer than HT-1080/ AMF, as shown in Figures 2 and 4. The original strain of Ehrlich provided by J.C.R.B. hardly ever secretes AMF23, i.e., the AMF secretion level appears almost the same between a mock line of
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HT-1080 and “Ehrlich ⫹.” This observation is supported by the result that the mRNA of Apaf-1 and Caspase-9 was clearly detected in the “Ehrlich ⫹” ascites line, although it was poor compared to the “Ehrlich ⫺” line as shown in Figure 4a. The intracellular signaling of AMF is initiated by binding to its receptor, AMF-R16.22–26 Motile signaling has been well investigated, for example, via a pertussis toxin (PT)-sensitive G-protein activation30 or small GTPase Rho.31 However, regulation of Apaf-1 and caspase-9 genes seems to be different from motile and AMF signaling, and might affect them indirectly because PT (G-protein inhibitor), which is considered to be the most upper stream of intercellular signalings, does not rescue of the expression of Apaf-1 and Caspase-9. The results shown in Figure 3 suggest that protein kinase C (PKC), phosphatidylinositol 3 phosphate kinase (PI3K) and mitogen-activated protein kinase (MAPK) are the signaling molecules of Apaf-1 and caspase-9 expression in high-AMF expression HT-1080 cells. The linkages of these signaling molecules are unclear, but in this model they appear to parallel Apaf-1 and Caspase-9, except for tyrosine kinase related signaling (Fig. 3, lane 6). These signaling molecules seem to be closely linked in a realistic tumor model (high-AMF secretion Ehrlich without transfection) because the signal inhibitors were not as effective at inducing apoptosis individually, except for PKC (inhibited by GFX), while the mixture of 3 was favored the anti-cancer drug as described in Table III. This result seems to contradict HT-1080/ AMF, which shows a higher apoptotic resistance compared to the “Ehrlich ⫹” line described above, and Figure 3 indicates the full rescue of Apaf-1 and Caspase-9 expression by these chemical inhibitors individually. Therefore sensitivity against these chemicals may be different between the HT-1080 line, which is cultured in adhesive, and the Ehrlich line, which is cultured in suspension. However, we considered that PKC, PI3K and MAPK should be related to the regulation of Apaf-1 or Caspase-9 expression in the high-AMF secretion tumor because effective apoptotic cell death was consequently induced in “Ehrlich ⫹” by using PKC inhibitor or a cocktail of chemicals. The down-regulation of Apaf-1 and caspase-9 mRNA levels reflects their protein levels, including their enzymic function and activities as shown in Figure 2c and d; with in vivo apoptosis is hardly recognized in MMC-treated tumor-bearing mice, as shown in Figure 5. This indicates that AMF-AMF-R signaling does not control the splicing of Apaf-1 and Caspase-9 or the enzymic activation of Procaspase-9. Recently, the promoter region of Apaf-1 has been elucidated, and E2F-1, one of the transcription factors, is a key molecule.32 Therefore, intracellular signaling regulated by AMF now have been connected to E2F-1. Since AMF is a malignant factor, it is probable that control of the AMF/AMF-R system should provide a new tool for cancer treatment targeting anti-metastatic and anti-apoptotic resistance. AMF activity is suppressible by monosaccharide, which is known as a PHI enzymic inhibitor,17,23–26 but it is unable to be used in vivo because of its cytotoxicity. However, immunological neutralization of external AMF can be achieved, as shown in Table III and Figure 5. Anti-AMF IgG can assist the in vivo apoptotic death influenced by MMC, and the antibody itself could induce 60%, death of a malignant Ehrlich cell line in vivo, as described in Table IV, suggesting that immunotherapy with anti-AMF together with chemotherapy could be an effective treatment. AMF might thus be considered as a new anti-metastatic and anti-apoptotic target molecule.
ACKNOWLEDGEMENTS
Our study was supported by a Japanese Grant-in-Aid for Scientific Research No 13033032 and No. 15790044 (to A. H.) and NIH Grant R01-CA51714 (to A. R.). A. R. is a recipient of the Paul Zuckerman Support Foundation for Cancer Research.
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