Affiliations of authors: Laboratory of Chemical Biology (HRP, YH, KS); Laboratory of Cell Growth and Regulation, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan (AT, YT, JY, TT); Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Toshima-ku, Tokyo, Japan (SS, TY, TT)
Correspondence to: Kazuo Shin-ya, PhD, Laboratory of Chemical Biology, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan (e-mail: kshin{at}iam.u-tokyo.ac.jp) or Takashi Tsuruo, PhD, Laboratory of Cell Growth and Regulation, Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan (e-mail: ttsuruo{at}iam.u-tokyo.ac.jp)
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ABSTRACT |
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INTRODUCTION |
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The UPR is initiated through signaling of the ER-localized transmembrane proteins ATF6, IRE1 (and, in some cells, IRE1
), and PERK (46). These signaling pathways produce several different active transcription factors that lead in turn to the coordinated expression of multiple UPR target genes (46). For example, ATF6 undergoes proteolytic cleavage to become an active transcription factor for UPR target genes (911), whereas IRE1
mediates the unconventional splicing of XBP1 mRNA, resulting in a potent UPR transcriptional activator (1215). PERK induces the expression of the transcription factor ATF4 by transiently inhibiting general protein synthesis (1618).
In the course of a screen for modulators of molecular chaperones, we recently isolated a novel compound from Streptomyces versipellis, designated versipelostatin (VST), that has a novel 17-membered macrocyclic structure with an -acyltetronic acid moiety (19). VST can inhibit transcription from a GRP78 promoter reporter construct (19). Here, we investigated the effect of VST on UPR activation in cells exposed to glucose deprivation or other UPR-inducing stressors by examining the expression of UPR target genes and their activators. We also examined the in vivo antitumor activity of VST.
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MATERIALS AND METHODS |
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VST was prepared as a stock solution of 10 mM in methanol or phosphate-buffered saline and stored at 20 °C (19). D-Glucose and 2-deoxyglucose (2DG) were purchased from Sigma (St. Louis, MO), dissolved in sterilized distilled water at stock concentrations of 200 mg/mL and 2 M, respectively, and stored at 20 °C. Tunicamycin and cycloheximide (CHX) were purchased from Nacalai Tesque (Kyoto, Japan), A23187 from Wako Pure Chemical Industries (Osaka, Japan), and MG132 from the Peptide Institute (Osaka, Japan). These compounds were dissolved in dimethyl sulfoxide (DMSO) at stock concentrations of 4 mg/mL, 10 mg/mL (CHX), 10 mM (A23187), and 10 mM (MG132) and were stored at 20 °C. All of the compounds were added to cell culture medium such that the solvent (methanol or DMSO) made up less than 0.5% of the volume of the culture medium.
Cell Lines
Human HT-29 colon cancer, HT1080 fibrosarcoma, and MKN74 stomach cancer cells were obtained from Dr. R. Shoemaker of the National Cancer Institute (National Institutes of Health, Bethesda, MD), the American Type Culture Collection (Manassas, VA), and IBL (Gunma, Japan), respectively. These cell lines were maintained in RPMI 1640 medium (containing 2 mg of glucose/mL; Nissui, Tokyo, Japan) supplemented with 10% heat-inactivated fetal bovine serum and 100 µg/mL of kanamycin and were cultured at 37 °C in a humidified atmosphere containing 5% CO2 (defined as "normal growth conditions"). Glucose-free RPMI 1640 medium was obtained from Invitrogen (San Diego, CA) and supplemented with 10% heat-inactivated fetal bovine serum as described previously (20,21). All in vitro experiments were performed using exponentially growing cells and were repeated at least twice.
Cell Treatments
To induce the UPR, we treated cells for various times with glucose deprivation by replacing the medium with glucose-free medium or by adding to glucose-containing culture medium one of the chemical stressors 2DG (20 mM), tunicamycin (TM) (5 µg/mL), or A23187 (1 µM). VST was added to the cells at various final concentrations immediately after they were placed in glucose-free medium or just before the chemical stressors were added to glucose-containing culture medium. In experiments involving CHX, it was also added to the cells in the same way as VST. In experiments that involved heat treatment, culture plates were floated on the water in a 42 °C water bath for 1 hour immediately after VST and 2DG were added to glucose-containing culture medium. In some experiments, we treated cells with the proteasome inhibitor MG132 at 10 µM during exposure to the UPR inducers.
Semiquantitative RTPCR Analysis
Total RNA was isolated from HT-29 and HT1080 cells by using an RNeasy Mini Kit with DNase digestion (Qiagen, Tokyo, Japan) and was converted to cDNA with SuperScript II reverse transcriptase (Invitrogen). The cDNAs for GRP78, GRP94, HSP70, XBP1, and G3PDH were then amplified by polymerase chain reaction (PCR) with PfuTurbo DNA polymerase (Stratagene, La Jolla, CA). The nucleotide sequences of the primer pairs (5' to 3') were GTATTGAAACTGTAGGAGGTGTC and TATTACAGCACTAGCAGATCAG for GRP78, TGAAAAGGCTGTGGTGTCTC and TCGTCTTGCTCTGTGTCTTC for GRP94, GCGCGACCTGAACAAGAGCATC and TCCGCTGATGATGGGGTTACAC for HSP70, CCTTGTAGTTGAGAACCAGG and GGGGCTTGGTATATATGTGG for XBP1, and TGAAGGTCGGAGTCAACGGATTTGGT and CATGTGGGCCATGAGGTCCACCAC for G3PDH. The reaction conditions (2125 cycles of 94 °C for 1 min, 60 °C for 1 min, and 72 °C for 2 min) had been predetermined (data not shown) to allow semiquantitative comparisons among cDNAs developed from identical reverse transcriptase reactions. The housekeeping gene G3PDH was amplified as an internal control. PCR products were separated by electrophoresis on 1.2% agarose gels (for analysis of GRP78, GRP94, HSP70, and G3PDH) or a 4%-20% polyacrylamide gel (for analysis of XBP1) and visualized with ethidium bromide staining.
Plasmids and Transfection
The pcFlag vector was produced by ligating an oligonucleotide DNA sequence encoding a FLAG epitope to the HindIII site of pcDNA3 (Invitrogen). The plasmids pATF6(F), which codes for full-length ATF6; pATF6(A), which corresponds to the active form of ATF6, i.e., amino acids 1373; pXBP1(U), which encodes the XBP1 protein corresponding to the unspliced mRNA; and pXBP1(S), which encodes the XBP1 protein corresponding to the spliced mRNA were created by inserting each cDNA [generated by PCR from untreated or 2DG-treated HT-29 cDNA, using the following primer pairs (5' to 3'): CATCCCAAGCTTTTGGGGGAGCCGGCTGGGGTTG and TGCTCTAGAGTCCCACGCTCAGTTTTCCAG for pATF6(F); CATCCCAAGCTTTTGGGGGAGCCGGCTGGGGTTG and TGCTCTAGACTAACTAGGGACTTTAAGCCTCTG for pATF6(A); and CATCCCAAGCTTTTGGTGGTGGTGGCAGCCGCGCCGAAC and TGCTCTAGAAGTCAAGGAAAAGGGCAACAG for pXBP1(U) and pXBP1(S)] in frame into the pcFlag vector at the HindIII/XbaI site. The pGRP78pro160-Luc plasmid was created by cloning the human GRP78 promoter region [nucleotides 160 to +7 relative to the start of transcription; generated by PCR, using the primer pair (5' to 3') CGGGGTACCGGAGCAGTGACGTTTATTGC and CATCCCAAGCTTTCGACCTCACCGTCGCCTACTC, from genomic DNA isolated from 293T cells with the QIAamp DNA Mini Kit (Qiagen)] into the KpnI/HindIII site of the pGL3-Basic vector (Promega, Madison, WI), which contains the firefly luciferase gene. pHSP70pro-Luc was created by inserting the human HSP70 promoter fragment (1.4 kb; upstream of +114 with respect to the start of transcription site), obtained by BglII/HindIII digestion of the Mammalian Heat Shock Expression Vector p2500-CAT (StressGen), into the BglII/HindIII site of the pGL3-Basic vector. pcDNA3.1/myc-His/lacZ was purchased from Invitrogen. The proper construction of all plasmids was confirmed by DNA sequencing. Transient transfections were performed using the FuGENE 6 Transfection Reagent (Roche Molecular Biochemicals, Indianapolis, IN) according to the manufacturers protocol.
Immunoblot Analysis
Cells were lysed in 1x sodium dodecyl sulfate (SDS) sample buffer (62.5 mM TrisHCl [pH 6.8], 2% SDS, 5% 2-mercaptoethanol, and 10% glycerol), and protein concentrations of the lysates were measured with a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA) (20,21). Equal amounts of proteins were resolved on a 4%-20% SDSpolyacrylamide gel and transferred by electroblotting to a nitrocellulose membrane (20,21). Immunoblots were probed with the following primary antibodies: mouse monoclonal anti-KDEL (for detection of GRP78 and GRP94; StressGen, Victoria, British Columbia, Canada); antiFLAG M2 (for detection of FLAG-tagged ATF6 and XBP1 proteins; Sigma); anti-Myc tag (for detection of Myc-tagged -galactosidase; Sigma); anti
-actin (for internal control; Sigma); anti-ATF6 (Active Motif, Carlsbad, CA); and rabbit polyclonal anti-CREB-2 (ATF4) (Santa Cruz Biotechnology, Santa Cruz, CA). Membranes were pretreated for 1 hour with TBST (50 mM TrisHCl [pH 7.5], 150 mM NaCl, and 0.1% Tween 20) containing 5% nonfat dry milk at room temperature and incubated for 14 hours with the above primary antibodies (1 : 500 for anti-ATF6 antibody and 1 : 1000 for the other antibodies) under the same conditions. The membranes were washed with TBST containing 5% nonfat dry milk at room temperature and incubated for 1 hour with horseradish peroxidaseconjugated sheep antimouse or donkey antirabbit immunoglobulin antibody (1 : 1000) (Amersham Pharmacia Biotech, Tokyo, Japan) under the same conditions. After washing with TBST, the specific signals were detected with an enhanced chemiluminescence detection system (Amersham Pharmacia Biotech).
Reporter Gene Assay
HT1080 cells (3 x 105 in each well of 12-well plates) were cultured overnight under normal growth conditions. The medium was then changed to antibiotic-free RPMI 1640 medium supplemented with 5% fetal bovine serum, transfection mixtures that contained 375 ng of the firefly luciferase genecontaining reporter plasmids pGRP78pro160-Luc, pHSP70pro-Luc, or pGL3 along with 125 ng of plasmid pRL-TK (Promega) (in which Renilla luciferase expression is under the control of the herpes simplex virus thymidine kinase promoter) as an internal control were added, and the cells were incubated for 8 hours at 37 °C in a CO2 incubator. In some experiments, the reporter plasmids were combined with 300 ng of plasmid mixtures containing pcFlag (mock transfection) with various amounts of pATF6(A), pATF6(F), or pXBP1(U). In the case of pXBP1(S) co-transfections, we used 1.7 ng of phRL-CMV (Promega) (in which Renilla luciferase expression is under the control of the cytomegalovirus promoter) as an internal control with 500 ng of pGRP78pro160-Luc. The medium was then replaced with fresh medium lacking plasmid DNA, and the cells were incubated under the same conditions for another 4 hours. The cells were then replated in 96-well plates (at 5 x 103 cells/well), cultured overnight, and treated for 18 hours with various concentrations of VST in the presence or absence of the UPR inducers. Heat treatment (42 °C) was carried out during the first 60 minutes of VST treatment. Ratios of firefly luciferase activity to Renilla luciferase activity (mean values with 95% confidence intervals from triplicate determinations) were determined using the dual luciferase kit (Promega).
Measurement of Protein Synthesis
Protein synthesis was assayed by measuring incorporation of [3H]alanine into trichloroacetic acid (TCA)insoluble material. Cells were seeded at 4 x103 or 8 x 103 in each well of a 96-well plate, cultured overnight under normal growth conditions, and then treated with various concentrations of VST for various times. During the last 1 or 2 hours of VST treatment, 2 µCi/mL of L-[2,33H]alanine (52 Ci/mmol; Amersham Pharmacia Biotech) was added to the cells. The cells were then recovered on glass filters (Molecular Devices, Tokyo, Japan) with a cell harvester device (Molecular Devices), and 1 mL/well of 5% TCA was passed through the filters, which were then washed with ethanol. The radioactivity on each filter was measured by scintillation counting. For cell counting, duplicate plates were treated with VST in the absence of [3H]alanine, and then the culture medium was replaced with fresh medium lacking VST. The cells were further cultured for an additional 2 hours under normal growth conditions, and cell number was determined with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma) assay (20). The amount of radioactivity incorporated was normalized to cell number, and relative radioactivity (mean values with 95% confidence intervals from triplicate determinations) was calculated.
Cell Viability Assays
Cells were seeded at 105 or 2 x 105 in 12- or six-well plates, cultured overnight, and treated with various concentrations of VST for various times. For the colony formation assay, cells were then diluted in fresh medium lacking VST, reseeded at 8 x 102 cells per well in six-well plates, and cultured under normal growth conditions for 78 days to form colonies (20,21). Cell survival (mean values with 95% confidence intervals from triplicate determinations) was calculated by setting the survival of control cells (i.e., not treated with VST) as 100%. IC50 values (concentration required for 50% inhibition of colony formation) were determined from doseresponse curves of colony formation inhibition. For the flow cytometry assays, the cells were fixed with 70% ethanol, treated with RNase, and stained with propidium iodide (22) or were stained directly (without ethanol fixation) with 7-amino-actinomycin D (PharMingen, San Diego, CA). Apoptotic and dead cells (mean values with 95% confidence intervals from triplicate determinations) were counted using a Beckman-Coulter flow cytometer.
The growth inhibition assay with the human cancer cell line panel and the COMPARE analysis were performed as described previously (2325). In brief, the cell line panel consists of the following 39 human cancer cell lines: lung cancer lines NCI3-H23, NCI-H226, NCI-H522, NCI-H460, A549, DMS273, and DMS114; colorectal cancer lines HCC-2998, KM-12, HT-29, HCT-15, and HCT-116; gastric cancer lines MKN-1, MKN-7, MKN-28, MKN-45, MKN-74, and St-4; ovarian cancer lines OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8, and SK-OV-3; breast cancer lines BSY-1, HBC-4, HBC-5, MDA-MB-231, and MCF-7; renal cancer lines RXF-631L and ACHN; melanoma line LOXIMVI; glioma lines U251, SF-295, SF-539, SF-268, SNB-75, and SNB-78; and prostate cancer lines DU-145 and PC-3. All cell lines were cultured in RPMI 1640 supplemented with 5% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 mg/mL) at 37 °C in humidified air containing 5% CO2. The GI50 (concentration required for 50% growth inhibition) of VST for each cell line was determined after 48 hours of drug treatment with a sulforhodamine B assay (2325). We then used the COMPARE algorithm to compare the GI50s of VST and each of more than 400 standard compounds (including various anticancer drugs and inhibitors of biological pathways) and assessed the correlations between the differential growth inhibition patterns of VST and each standard compound in the cell line panel by determining the Pearson correlation coefficient, as described previously (23,25).
MKN74 Xenograft Tumors
MKN74 cells were grown as subcutaneous tumors in nude mice, and 3 x 3 x 3mm tumor fragments were then inoculated subcutaneously into nude mice, as described (23). Therapeutic experiments (involving six mice per group) were started when tumors had grown to approximately 100 mm3 (day 0). VST was administered intravenously at 13.5 or 18 mg/kg of body weight per day on days 0, 1, and 2. The control group received phosphate-buffered saline. For combination treatment, cisplatin was administered intravenously at 7 mg/kg on day 2. The mice were weighed twice each week up to day 24 to monitor the toxic effects. Tumor volume was determined by measuring the length (L) and width (W) of each tumor twice a week, up to day 24, and was calculated as (L x W2)/2 (23).
Statistical Analysis
The statistical significance levels of differences in xenograft volume between groups of control and treated mice were evaluated using a one-way analysis of variance with Dunnetts test as described previously (21). Differences with P values less than .05 were deemed statistically significant using a two-tailed test between the groups of control and drug-treated mice.
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RESULTS |
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To examine the effect of VST on endogenous GRP78 gene expression, we carried out a semiquantitative reverse transcriptionpolymerase chain reaction (RTPCR) analysis of human colon carcinoma HT-29 and fibrosarcoma HT1080 cells that had been subjected to glucose deprivation for 18 hours in the presence or absence of VST. Whereas VST suppressed induction of GRP78 and GRP94 mRNA in glucose-starved HT-29 and HT1080 cells in a concentration-dependent manner (Fig. 1, a and b), it had no effect on GRP78 and GRP94 expression levels in cells grown in the presence of glucose (Fig. 1, b). Immunoblot analysis of lysates from the glucose-starved cells showed that VST also suppressed accumulation of the GRP78 and GRP94 proteins (Fig. 1, d).
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We also examined the effect of VST on human GRP78 promoter activity in HT1080 cells transiently transfected with a mammalian reporter gene plasmid (pGRP78pro160-Luc) that includes the 160 to +7 promoter region of GRP78 cloned immediately upstream of the firefly luciferase gene. This promoter region contains the cis-acting endoplasmic reticulum stress response element (ERSE), which is required for transcriptional activation in response to ER stress (26). Because global protein synthesis was affected by the UPR-inducing stimuli whether or not VST was present, we could not compare absolute firefly luciferase activities; instead, we co-transfected cells with a control plasmid that contained a Renilla luciferase gene and compared the ratios of the two luciferase activities. In HT1080 cells transfected with the pGRP78pro160-Luc plasmid, treatments with 2DG and TM increased relative firefly luciferase activity by approximately five- and sevenfold, respectively (Fig. 1, e). Consistent with the GRP78 mRNA analysis, VST suppressed 2DG-induced GRP78 promoter activity in a dose-dependent manner (Fig. 1, e, left) but had little effect on TM-induced GRP78 promoter activity, even at 30 µM, a concentration 10 times higher than that at which it suppressed 2DG-induced GRP78 promoter activity (Fig. 1, e, right). Under normal growth conditions, VST (at 130 µM) had no effect on GRP78 promoter activity. For use as a control, we transfected cells with the pGL3-Control vector, which contains simian virus 40 promoter and enhancer sequences driving firefly luciferase activity, and found that the promoter activity was not affected by VST alone, by each of chemical stressors 2DG and TM, or by combinations of the two compounds (Fig. 1, e).
To further define the specificity of the inhibitory effects of VST, we examined expression of HSP70 (HSPA1A, NM_005345), which represents a class of stress-inducible molecular chaperones distinct from those of the GRP family. HSP70 mRNA levels in HT1080 cells increased in response to glucose deprivation but decreased in response to 2DG and TM (Fig. 1, b and c). VST (at 3 or 10 µM) had no effect on the changes in HSP70 expression in the glucose-deprived and the TM-stressed cells. In the 2DG-stressed cells, VST at 10 µM strongly induced HSP70 gene expression. Thus, VST was incapable of inhibiting HSP70 gene expression under all conditions examined. We also determined the effect of VST on promoter activity of another HSP70 gene (HSPA6, NM_002155) by using HT1080 cells transfected with the mammalian reporter plasmid pHSP70pro-Luc. Although HSP70 promoter activity was inducible by treatment with heat (42 °C, 60 minutes), with 2DG, and with both together, VST (at 3 µM) had little effect on promoter activity under any of these conditions (Fig. 1, f). Taken together, these results indicate that VST selectively inhibits ERSE-dependent transcription during glucose deprivation.
Depletion of Spliced XBP1 by VST
The active form of ATF6 and the spliced form of XBP1 mediate ERSE-dependent transcriptional activation. To investigate whether VST inhibits the activation processes of ATF6 and XBP1, we transiently transfected HT1080 cells with plasmids containing cDNAs for full-length ATF6 [from pATF6(F)] or the unspliced XBP1 [from pXBP1(U)] and detected activation of each protein using immunoblot analysis (Fig. 2, a). To better detect activation, we included the proteasome inhibitor MG132 to prevent proteasomal degradation of the activated forms of each protein during exposure of cells to 2DG or TM. Using these experimental conditions, we were able to detect both the XBP1(S) and the ATF6(A) forms of these proteins (Fig. 2, a). VST completely suppressed the emergence of the spliced form of the XBP1 protein seen under 2DG stress conditions but not that seen under TM stress conditions (Fig. 2, a, upper panel). Moreover, VST did not inhibit production of the active form of the ATF6 protein under either stress condition (Fig. 2, a, lower panel).
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Immunoblot analysis of proteins from HT1080 cells transfected with each expression plasmid [pXBP1(S), pXBP1(U), pATF6(A), or pcDNA3.1/myc-His/lacZ (as a control)] showed that levels of the spliced XBP1 protein decreased more rapidly than those of the other proteins when VST was added to cells stressed by 2DG treatment (Fig. 2, d). Thus, VST can also operate at the post-translational level, thereby leading to complete loss of spliced XBP1 protein. Levels of unspliced XBP1 protein, as well as of the active form of the ATF6 protein, decreased more gradually in the presence of VST under 2DG stress conditions (Fig. 2, d), possibly as a result of active repression of protein synthesis in response to ER stress (28). Indeed, we estimated protein synthetic activity by measuring incorporation of tritiated alanine into protein. We found that overall protein synthesis was repressed by VST alone and by 2DG alone but was repressed to a much greater extent by the combination of VST and 2DG (Fig. 3, a). This effect was seen over all time periods of treatment examined (430 hours). However, VST alone had much less effect on global protein synthesis than the translation elongation blocker cycloheximide, which strongly inhibited protein synthesis in both the presence and absence of 2DG (Fig. 3, b). VST also enhanced protein synthesis repression during glucose deprivation but not during TM-induced stress (Fig. 3, c). Thus, VST enhanced translational repression and prevented UPR activation in hypoglycemic cells specifically.
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To address whether the depletion of spliced XBP1 protein induced by VST is involved in the inhibition of ERSE-dependent transcription, we co-transfected HT1080 cells with the reporter plasmid pGRP78pro160-Luc and pXBP1(S). Co-transfection of a relatively small amount (3 ng) of pXBP1(S), which encodes the spliced form of XBP1, was enough to enhance the basal reporter activity (i.e., in the absence of 2DG), and the activity was further increased by 2DG treatment (Fig. 4, a, left). Under these experimental conditions, 10 µM VST was required to achieve complete inhibition of 2DG-induced reporter gene activity in the pXBP1(S)-transfected cells, whereas 3 µM was sufficient in mock-transfected cells. Thus, ectopic expression of the spliced form of XBP1 conferred resistance to the inhibitory activity of VST against the ERSE-dependent transcription. By contrast, co-transfection with even large amounts (300 ng) of a plasmid encoding the unspliced form [pXBP1(U)] had little effect on basal reporter gene activity or VST sensitivity (Fig. 4, b).
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We also tested whether ectopic expression of either the full-length or active (shorter) form of ATF6 had any effect on the inhibitory activity of VST by using pGRP78pro160-Luc reporter assays (Fig. 4, c-f). Co-transfection of HT1080 cells with 300 ng of pATF6(F), which encodes the full-length form of ATF6, enhanced basal reporter gene activity (Fig. 4, c). Reporter gene activity was further increased by 2DG treatment, and this 2DG-induced activity was less sensitive to VST than that in mock-transfected cells (Fig. 4, c). The induction of VST resistance was dependent on the amount of transfected ATF6(F) plasmid and required at least 10 ng (Fig. 4, d), an amount that led to much higher expression of full-length ATF6 protein than the endogenous level seen in mock-transfected cells (Fig. 4, e). Co-transfection of HT1080 cells with pATF6(A), which encodes the active form of ATF6, also led to VST resistance in 2DG-treated cells (Fig. 4, f), suggesting that proteolytic activation of the full-length form of ATF6 mediates the induction of resistance in 2DG-treated cells overexpressing full-length ATF6. These results suggest that a relatively low level of expression of endogenous ATF6 is necessary for VST to effectively inhibit ERSE-dependent transcriptional activation in glucose-deprived cells.
Glucose-Regulated UPR-Inhibiting Activity
We next examined whether the UPR-inhibiting activity of VST was seen specifically in glucose-deprived cells. Immunoblot analysis of lysates from both HT1080 and HT-29 cells demonstrated that VST suppressed induction of GRP78 and GRP94 protein accumulation even in the presence of TM, as long as the cells were deprived of glucose (Fig. 5, a, left, and data not shown). GRP expression was inhibited at essentially the same VST concentrations in the presence or absence of TM, suggesting that the inhibitory activity of VST was strictly dependent on glucose deprivation. Indeed, the inhibition of TM-dependent GRP induction by VST was eliminated as the amount of glucose increased (Fig. 5, a, right).
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Selective Killing of Glucose-Deprived Cells
We next examined the effects of VST on cell viability. Under normal growth conditions, 24 hours of VST treatment of HT-29 cells had only a weak effect on cell viability, with approximately 30 µM VST required to inhibit colony formation by 50% (IC50). By contrast, VST was highly toxic in cells exposed to glucose-free or 2DG-containing medium, resulting in approximately 30-fold lower IC50 (1 µM) (Fig. 5, b, left and middle). Under hypoglycemic conditions, the cytotoxic activity of VST was associated with the inhibition of GRP expression (Fig. 1). By contrast, there was no consistent combined effect of VST with the chemical stressor TM (Fig. 5, b, right). HT1080 cells were similarly sensitized to VST under glucose starvation conditions, as indicated by flow cytometric assays of apoptotic cells showing sub-G1 DNA content (Fig. 5, c) and of dead cells stained with 7-amino-actinomycin D (Fig. 5, d and e). VST-induced cell death was not substantial within 24 hours (Fig. 5, c), however, indicating that inhibition of GRP expression preceded cell death. Furthermore, induction of cell death by VST was seen at glucose concentrations of 0.1 mg/mL or lower (Fig. 5, d), concentrations at which GRPs were induced in a VST-suppressible manner (Fig. 5, a, right). We also found that VST-induced cell death under glucose starvation was not influenced by the further addition of TM (Fig. 5, e). Collectively, therefore, these data indicate that VST selectively kills glucose-deprived cells by disrupting the UPR.
We evaluated antiproliferative activity of VST with a panel of 39 human cancer cell lines (2325). Across the entire cell line panel, the average VST concentration at which growth was inhibited by 50% (GI50) was 2.3 µM (for a 48-hour exposure). The COMPARE analysis revealed that the differential growth inhibition pattern of VST was unique, with little similarity to the patterns of more than 400 standard agents, which also suggests that VST has a unique mode of action. In the cell line panel analysis, VST had antiproliferative effects on most of the cell lines in the presence of glucose; the stomach cancer cell line MKN74 was one of the most sensitive lines, with a GI50 of 0.42 µM. MKN74 cells treated with VST for 48 hours showed little apoptosis under normal growth conditions; by contrast, when glucose was withheld for 48 hours, extensive apoptosis occurred (Fig. 6, a). Similarly, in the colony formation assay, a 24-hour treatment of VST was highly cytotoxic to glucose-deprived MKN74 cells (Fig. 6, b). Therefore, the strong cytotoxic effect of VST on MKN74 cells was evident only during glucose deprivation, although VST had some antiproliferative effects in the presence of glucose. At doses that resulted in strong cytotoxicity during glucose deprivation, VST also inhibited GRP78 induction in MKN74 cells (Fig. 6, c).
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DISCUSSION |
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In mammalian cells, the regulation of the UPR is a complicated process that involves three independent signaling pathways: ATF6, IRE1, and PERK. Indeed, cells defective for ATF6 cleavage show no induction of GRP78 mRNA but can still induce XBP1 mRNA expression (10,15). Cells deficient in IRE1 or both IRE1
and IRE1
display normal GRP78 induction and survive under conditions of ER stress (15,31,32). PERK/ cells show reduced survival, with defects in expression of various UPR target genes, but they do not show attenuated protein synthesis during ER stress (17,28). This complexity has been an obstacle to developing UPR-targeted drugs.
Nevertheless, it is likely that the production and/or quality of UPR transcriptional activators can be governed by a single signaling pathway, as seen in cells that lack functional GADD34, a downstream target gene product of the PERK signaling pathway (33). Indeed, GADD34-mutated cells show loss of GRP78 gene expression and impaired induction of the UPR transcriptional activators XBP1 and ATF4 (33). Furthermore, GADD34-mutated cells display persistent repression of protein synthesis and reduced cell survival in response to the chemical stressor thapsigargin (33), like cells treated with VST during glucose deprivation. The findings in GADD34 mutant cells indicate that disruption of the UPR, which leads to reduced cell survival during ER stress, can be achieved by mutation of a single gene and likely by a single compound. Therefore, the consistent observations between GADD34-mutated and VST-treated cells support a link between the cytotoxic and the UPR-inhibiting activities of VST during glucose deprivation. At present, however, we cannot rule out the possibility that mechanism(s) other than prevention of the UPR account for the biologic and therapeutic activity of VST. It will be important to clarify the precise mechanisms by which VST affects the UPR.
In addition to showing that the UPR is a potential target for cancer treatment, our findings have another important implication, namely that they provide insights into how to reduce resistance to chemotherapy. It is widely recognized that hypoglycemia, as well as hypoxia, induces resistance to chemotherapy, the principal problem in treating most common solid tumors (2,34,35). Indeed, under hypoglycemic conditions, a variety of human cancer cells are resistant to many clinically important antitumor drugs, as described previously (21,36). The induction of resistance is associated with the induction of the UPR target genes GRP78 and GRP94 (37,38). GRP78 and GRP94 show an anti-apoptotic function (7,39) and are induced in malignant cells (7,40,41). Therefore, selective inhibitors of the ER stress response, such as VST, may be useful to eliminate otherwise drug-resistant, hypoglycemic tumor cells. In addition, the severe hypoglycemic conditions under which VST becomes toxic are not observed in normal tissue. Thus, VST may be an attractive tool for exploring the potential of hypoglycemia-targeted therapy against solid tumors.
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NOTES |
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Supported in part by an Industrial Technology Research Grant program from New Energy and Industrial Technology Development Organization (NEDO) of Japan and by a Grant-in-Aid for Scientific Research on Priority Areas Cancer from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
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Manuscript received November 27, 2003; revised June 28, 2004; accepted June 29, 2004.
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