2014 Volume 37 Issue 4 Pages 633-641
GANT61 is a small-molecule inhibitor of glioma-associated oncogene 1 (GLI1)- and GLI2-mediated transcription at the nuclear level that exerts its effect by preventing DNA binding. It has been demonstrated to induce cell death against Ewing’s sarcoma family tumor (ESFT) cell lines in a dose-dependent manner. The most sensitive cell line was SK-N-LO, which expresses the EWS-FLI1 fusion gene. SK-N-LO cells treated with GANT61 showed cellular and nuclear morphological changes, including cell shrinkage, chromatin condensation and nuclear fragmentation, in a concentration-dependent manner, as visualized by Hoechst 33342 staining. Furthermore, annexin V-propidium iodide (PI) double-staining revealed a significant increase in the number of late apoptotic cells. GANT61 induced a significant decrease in the proportion of cells in the S phase. Significant decrease of the protein levels of GLI2, survivin, cyclin A and claspin, and significant increase of p21 expression was also observed in the cells treated with GANT61. Moreover, poly (ADP-ribose) polymerase (PARP) cleavage was observed, but no cleavage of caspase-3 or -7, or any change in the expressions of Bcl-2 or p53 were observed. These findings suggest that GANT61 induces cell death of SK-N-LO cells in a caspase-independent manner, by inhibiting DNA replication in the S phase.
The Ewing’s sarcoma family of tumor (ESFT) is classified as a group of undifferentiated small round cell tumors arising from the bones and soft tissues that includes Ewing’s sarcoma (ES), primitive neuroectodermal tumor (PNET), and Askin’s tumor. These malignant tumors are predominantly found in children and young adults, whose prognosis remains very poor in spite of a multidisciplinary treatment approach. Therefore, new therapeutic drugs are needed to treat these tumors. Tumors of the ESFT exhibit several specific chimeric genes caused by chromosomal translocations. The EWS-FLI1 fusion gene is detected in approximately 90% of ESFT, with the most common chromosomal translocation being t (11; 22) (q24; q12).1)
The hedgehog (Hh) signaling pathway plays an important role in organ development during embryogenesis. The Hh signal regulates the developmental process of the cerebellum, bone, gut, neural crest, and various organs.2) This signal is initiated by the Hh ligands, sonic hedgehog (Shh), Indian hedgehog (Ihh), and desert hedgehog (Dhh). In the absence of Hh ligands, patched (PTCH), a 12-transmembrane protein, inhibits Smoothened (SMO), a 7-transmembrane protein. Binding of Hh ligands to PTCH results in SMO being relieved from inhibition by PTCH. Stimulation of the pathway by SMO results in activation of the glioma-associated oncogene (GLI). GLI is a zinc finger transcription factor in the nucleus, three homologs of which have been identified in mammals. GLI1 and GLI2 are activators of the Hh signal. Activated GLI2 induces the expression of target genes such as GLI1, MYCN, and cyclin D. In contrast, GLI3 acts as a repressor of the Hh signal.3) Aberrant activation of the Hh signal has been implicated in several cancers, including basal cell carcinoma4) and medulloblastoma.5) GLI1 protein is a direct transcriptional target of EWS-FLI1 in ESFT.6,7) GANT58, an inhibitor of transcriptional activation by GLI1, has been reported to reduce cell proliferation in ES.8) However, there are few reports of the effect of GLI inhibitors on ESFT, and little is known about the underlying mechanism of action. Herein, we report our investigation of the mechanism of action of GLI inhibitors on ESFT, and propose that GLIs could be a new therapeutic target for the treatment of ESFT.
Cyclopamine and GANT61 were purchased from Wako Pure Chemical Industries, Ltd., Osaka, Japan. The chemical structures are shown in Fig. 1.
We used five ESFT cell lines. The characteristics of each cell line are shown in Table 1.9–11) The SCMC-ES1, NCR-EW2, SK-N-LO, and SK-N-MC cells were grown in RPMI 1640 medium (Life Technologies, Invitrogen, Carlsbad, CA, U.S.A.) supplemented with 10% fetal bovine serum (Thermo Scientific HyClone, Logan, UT, U.S.A.) and penicillin (100 U/mL)/streptomycin (0.1 mg/mL) at 37°C in a humidified incubator containing 95% air with 5% CO2. The SK-ES-111) cells were grown in McCoy’s 5a medium (Life Technologies, Invitrogen, U.S.A.) supplemented with 1.5 mM L-glutamine, 15% fetal bovine serum and penicillin (100 U/mL)/streptomycin (0.1 mg/mL) at 37°C in a humidified incubator containing 95% air with 5% CO2. We also used four neuroblastoma (NB) cell lines; IMR-32, LA-N-1, SK-N-SH (RIKEN cell bank, Ibaraki, Japan) and NB-39 (kindly provided by Dr. Toshimitsu Suzuki, Fukushima Medical University) were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum and penicillin (100 U/mL)/streptomycin (0.1 mg/mL) at 37°C in a humidified incubator containing 95% air with 5% CO2.
| Cell lines | Cell types | Chimeric genes | Origins |
|---|---|---|---|
| SCMC-ES1 | Ewing’s sarcoma | EWS (exon 7)–ERG (exon 6) | Bone |
| NCR-EW2 | Ewing’s sarcoma | EWS (exon 7)–FLI1 (exon 5) | Pelvis |
| SK-ES-1 | Ewing’s sarcoma | EWS (exon 7)–FLI1 (exon 5) | Bone |
| SK-N-LO | PNETa) | EWS (exon 7)–FLI1 (exon 6) | Brain |
| SK-N-MC | PNET Askin’s tumor | EWS (exon 7)–FLI1 (exon 6) | Brain |
a) PNET; primitive neuroectodermal tumor.
Cells were plated at a density of 1×104 cells /well on to 96-well flat-bottomed cell culture plates and cultured for 24 h. Then, each reagent (final concentration 1, 3, 10, 30, 100 µM) or dimethyl sulfoxide as vehicle (DMSO Sigma-Aldrich, St. Louis, MO, U.S.A.) was applied for 48 h. After the addition of 0.5% MTT solution at 10% of the volume of the medium in the well, incubation was continued for an additional 3 h. Stop solution (0.04 N HCl in isopropanol) was added at a volume of 100 µL to the culture medium in each well, and the absorbance values at 570 nm (peak) and 655 nm (trough) were measured after thorough pipetting to disperse the generated blue formazan. The experiments were repeated 3 times.
Hoechst 33342 StainingApoptotic nuclear morphology was observed by staining with Hoechst 33342. SK-N-LO cells were plated at a density of 2×105 cells/well onto 6-well flat-bottomed cell culture plates and cultured for 24 h. Then, GANT61 (final concentration 10, 30, 100 µM) was added and the incubation was continued for 48 h. Hoechst 33342 solution (final concentration, 0.001% of medium) was added to the wells for 15 min. The cells were then observed under a fluorescence microscope (IX-71 Olympus, Tokyo, Japan). The experiments were repeated 3 times.
Double Staining with Annexin V–Propidium IodideEarly apoptosis was detected using the Alexia Fluor® 488 Annexin V/Dead Cell apoptosis kit (Life Technologies, Invitrogen, U.S.A.). SK-N-LO cells were plated at a density of 1×106 cells/well onto 6-well flat-bottomed cell culture plates and cultured for 24 h. Then, GANT61 (final concentration 1, 3, 10, 30 µM) was added and the incubation was continued for 48 h. The cells were harvested and washed with ice-cold phosphate buffered saline (PBS). After discarding the supernatant, the cells were washed with annexin-binding buffer and stained by treatment of the cell suspension with Alexa Fluor® 488 Annexin V and propidium iodide (PI; final concentration 100 µg/mL) for 15 min. The cell samples were analyzed using a FC500 flow cytometer (Beckman Coulter, Brea, CA, U.S.A.), using the FL1 and FL4 range.
Proteome Profiler ArraySK-N-LO cells were plated at a density of 2×106 cells/5 mL/dish (3 dishes/sample) onto 60-mm cell culture dishes containing RPMI 1640 medium and cultured for 24 h. Then, GANT61 (30 µM) was added and the incubation was continued for 48 h. All immunodetection steps were performed utilizing a Proteome Profiler Array (R&D Systems, Minneapolis, MN, U.S.A.) in accordance with the manufacturer’s instructions. Briefly, the cells were harvested and lysed in lysis buffer. The array was incubated overnight with the diluted lysates (500 µg/250 µL) at 4°C on a rocking platform shaker. Reconstituted detection antibody cocktail conjugated with biotin and streptavidin–horseradish peroxidase (HRP) was added to each array. The blots were detected using an enhanced chemiluminescence (ECL) system (GE Healthcare, Tokyo, Japan) and imaged using a lumino image analyzer (LAS-1000; FUJIFILM, Tokyo, Japan). The density of the blots was analyzed using the National Institutes of Health (NIH) Image-J software.
Cell Cycle AnalysisSK-N-LO cells were plated at a density of 1×106 cells/well onto 6-well flat-bottomed cell culture plates and cultured for 24 h. Then, GANT61 (final concentration 30 µM) was added and the incubation was continued for 48 h. The cells were harvested and washed with ice-cold PBS. Cells were then fixed with ice-cold 70% ethanol for 2 h. After washing with PBS, the cells were treated with 0.25 mg/mL RNase solution for 30 min at 37°C. PI solution was added (final concentration 50 µg/mL) and the cells were incubated for 30 min at room temperature in the dark. The cell samples were analyzed with a FC500 flow cytometer using the FL3 range.
Western BlottingSK-N-LO cells were plated at a density of 2×106 cells/5 mL/dish onto 60-mm cell culture dishes containing RPMI 1640 medium and cultured for 24 h. Then, GANT61 (30 µM) was added for 0, 2, 4, 8, 24, or 48 h (0 h refers to untreated dishes). The cells were harvested, washed with Tris-buffered saline (TBS), and lysed in an extraction buffer containing 20 mM Tris–HCl (pH 8.0), 137 mM NaCl, 1% Nonidet P-40, 10% glycerol, protease inhibitor cocktail I (1 : 200; Sigma-Aldrich, U.S.A.), phosphatase inhibitor cocktail II (1 : 100; Sigma-Aldrich, U.S.A.), 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 mM dithiothreitol (DTT). The cells were disrupted by sonication twice for 30 s each, and the supernatants were obtained by centrifugation at 15000 rpm for 10 min at 0°C. Protein concentrations were determined with a Protein Assay Rapid Kit (Wako, Japan), using bovine serum albumin (BSA) as reference. The supernatants were mixed with sample buffer [0.24 M Tris–HCl (pH 6.8), 312 mM sodium dodecyl sulfate (SDS), 30% glycerol, 15% 2-mercaptoethanol and 1% bromophenol blue], and boiled for 3 min at 100°C. The supernatants (loading samples) were obtained by centrifugation at 15000 rpm for 1 min at 0°C. Loading samples containing 7.5 µg of total proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes (GE Healthcare, Japan). After being blocked with 5% skim milk, the membranes were probed with the primary antibodies overnight at 4°C. After another wash, the membranes were incubated with HRP–conjugated secondary antibodies for 1 h at room temperature. The blots were detected with an ECL system (GE Healthcare, Japan) and imaged using LAS-1000. The densities of the bands were analyzed using the NIH Image-J software. The primary antibodies were used as follows: rabbit anti-p21(C-19), goat anti-PTCH (G-9), rabbit anti-SMO (H-300), goat anti-SHH (N-19) and rabbit anti-cyclin A (H-432) (Santa Cruz Biotechnology, TX, U.S.A.), rabbit anti-survivin (#2808), rabbit anti-claspin (#2800), mouse anti-GLI1 (#2643), rabbit anti-caspase-3 (#9665), rabbit anti-cleaved caspase-3 (#9661), mouse anti-caspase-7 (#9494) and rabbit anti-Bcl-2 (#2876) (Cell Signaling Technology, Danvers, MA, U.S.A.), rabbit anti-GLI2 (ab26056; abcam, Cambridge, MA, U.S.A.), mouse anti-p53 (#610183), mouse anti-poly(ADP-ribose) polymerase (PARP) (#611038) and mouse anti-cyclin E (#551159) (BD Biosciences, San Jose, CA, U.S.A.), mouse anti-β-tubulin (T4026) (Sigma-Aldrich, U.S.A.).
Statistical AnalysisData are expressed as mean±S.E. (n=3). Significance testing was performed using one-way ANOVA, followed by Bonferroni’s test.
We examined the anticancer activities of antagonists of the Hh signal against ESFT cells by MTT assay (Figs. 2A, B). Treatment with GANT61, an inhibitor of GLI, for 48 h induced cell death in the ESFT cell lines in a concentration-dependent manner (Fig. 2B). In particular, GANT61 induced significant cytotoxicity in three of the five cell lines (SK-N-LO, SK-N-MC, and NCR-EW2) with IC50 values of 14.5 µM, 18.3 µM, and 18.1 µM, respectively (Table 2). The SK-N-LO and SK-N-MC cell lines are morphologically similar to the NB-like cell type.12) In contrast to GANT61, treatment for 48 h with cyclopamine, an inhibitor of SMO, had no effect on any of the ESFT cell lines (Fig. 2A). Next, we examined the anticancer activity of an antagonist of the Hh signal against four NB cell lines (LA-N-1, IMR32, NB39, and SK-N-SH) (Figs. 2C, D). GANT61 exerted cytotoxicity against the NB cell lines at a high concentration (Table 2). Moreover, treatment with cyclopamine for 48 h had no effect on any of the four cell lines other than SK-N-SH (Table 2).
Cell viability was measured by the MTT assay. Five ESFT (SCMC-ES1, NCR-EW2, SK-ES-1, SK-N-LO, and SK-N-MC) and four NB cell lines (LA-N-1, IMR32, NB39, and SK-N-SH) were treated with the indicated concentrations of the Hh antagonists [cyclopamine (A)(C), GANT61 (B)(D)] and dimethylsulfoxide (DMSO; vehicle control) for 48 h. Each plot shows the survival rate relative to the vehicle control. (−6 log M=1 µM, −5 log M=10 µM, −4 log M=100 µM).
| Compounds | Cytotoxicity IC50 (µM) | ||||||||
|---|---|---|---|---|---|---|---|---|---|
| ESFT cell lines | NB cell lines | ||||||||
| SCMC-ES1 | NCR-EW2 | SK-ES-1 | SK-N-LO | SK-N-MC | LA-N-1 | IMR-32 | NB-39 | SK-N-SH | |
| Cyclopamine | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 | 66.7 |
| GANT61 | 66.7 | 18.1 | >100 | 14.5 | 18.3 | 60.0 | 35.1 | >100 | 48.1 |
To detect whether GANT61 induced apoptosis in the most GANT61-sensitive cell line, SK-N-LO, we observed the cellular and nuclear morphological changes by Hoechst 33342 staining. As shown in Fig. 3, apoptosis, characterized by cell shrinkage, chromatin condensation and nuclear fragmentation, was observed in the SK-N-LO cells treated with GANT61 in a concentration-dependent manner, as well as a positive control [cisplatin (cis-diaminedichloro-platinum; CDDP)]. GANT61 was evaluated for its activity of inducing apoptosis in the SK-N-LO cells by Annexin V-PI double staining (Fig. 4A). Exposure of the membrane phospholipid, phosphatidylserine, to the external cellular environment is one of the earliest markers of apoptotic cell death. The early apoptotic population tended to increase following treatment with GANT61 at 1–10 µM. On the other hand, treatment with GANT61 at 30 µM significantly decreased the early apoptotic cell population (Fig. 4B left) and increased the late apoptotic and necrotic cell population (Fig. 4B right). These results suggest that GANT61 (30 µM) induces apoptotic cell death.
SK-N-LO cells were treated with GANT61 (10, 30, 100 µM) or cisplatin (CDDP) (30 µM) as a positive control for 48 h. Phase-contrast images (upper) and fluorescence images (lower) were obtained. Scale bar is 20 µm.
SK-N-LO cells were treated with GANT61 (1, 3, 10, 30 µM) or DMSO (as a vehicle control) for 48 h. B2: late apoptotic and necrotic cells, B3: live cells, B4: early apoptotic cells. (A) The vertical axis indicates PI (FL4 Log) and the horizontal axis indicates annexin V-Alexa Fluor 488 (FL1 Log). (B) Percentages of cell populations in each area of (A). ** p<0.01 versus vehicle control.
Expression of apoptosis-related proteins was examined by the Proteome Profiler Array in order to analyze the mechanisms related to cell death induced by GANT61 (Figs. 5A, B). SK-N-LO cells were either treated with DMSO (control) or with GANT61 (30 µM) for 48 h (Fig. 5A). As shown in Fig. 5B, striking decrease in the expressions of survivin and claspin was observed (43.0, 61.3% decrease, respectively) following treatment with GANT61 as compared with the expression levels in the control. In addition, marked increase of p21 protein expression was observed following treatment with GANT61 as compared with the expression level in the control (67.8% increase). However, cleaved caspase-3 and Bcl-2 were quite increased or decreased (Fig. 5B).
Multiple apoptosis-related proteins were detected using a Proteome Profiler Array in the SK-N-LO cells. GANT61 (30 µM) or DMSO (as a vehicle control) was applied for 48 h. (A) Array images. Blue arrows denote the change of p21 expression. White arrows denote the change of survivin expression. Yellow arrows denote the change of claspin expression. (B) Alteration rate as a percentage relative to the vehicle control.
We also examined the cell cycle status of SK-N-LO cells treated with GANT61 by flow-cytometric analysis. As shown in Fig. 6, treatment with GANT61 at 30 µM induced a significant decrease in the proportion of cells in the S phase (14.1±1.29% vs. 8.79±3.06%: Fig. 6C). This finding also suggests that GANT61 suppressed the S phase transition, however, the proportion of cells in the G0/G1 and G2/M phases did not increase (Figs. 6B, D) and a tendency towards increase in the number of cells in the subG1 phase was observed (Fig. 6A).
The cell cycle status was assessed by the DNA content using flow cytometry after propidium iodide (PI) staining. SK-N-LO cells were treated with GANT61 at 30 µM for 48 h. Each column shows the percentage of the gated cell populations relative to the total cell population. * p<0.05 versus vehicle control. (A) sub G1, (B) G0/G1, (C) S, D: G2/M.
To confirm the findings of the Proteome Profiler assay, we examined the expression levels of apoptosis-related proteins. SK-N-LO cells were exposed to GANT61 at the concentration of 30 µM for 0–48 h, followed by Western blot analysis (Figs. 7A–C). We examined the expressions of the Hh signal-related proteins (SMO, GLI1, GLI2, PTCH, and Shh: Fig. 7A). Expression of GLI2 was decreased after 48 h of GANT61 treatment, whereas GLI1 was quite weak between 0 and 48 h. Few significant changes in the expressions of SMO, Shh, and PTCH were observed following GANT61 treatment.
The SK-N-LO cells were treated with GANT61 (30 µM) for 0–48 h. The protein expression levels of proteins were assessed by Western blotting; β-tubulin was used as the loading control. (A) Hh signal-related proteins (SMO, GLI1, GLI2, PTCH and Shh). (B) Apoptosis-related proteins (caspase 3, cleaved caspase-3, caspase-7, cleaved caspase-7, Bcl-2, Pro-PARP, cleaved-PARP, survivin, p53, and p21). (C) Cell cycle-related proteins (claspin, cyclin A, and cyclin E).
We examined the expression levels of the apoptosis-related proteins (caspase-3, cleaved caspase-3, caspase-7, cleaved caspase-7, Bcl-2, PARP, survivin, p53, and p21) following GANT61 treatment of the SK-N-LO cells (Fig. 7B). The expression level of p21 was markedly increased after 24–48 h of GANT61 treatment. In contrast, the expression level of claspin decreased after 24–48 h of GANT61 treatment. Moreover, the expression level of survivin also decreased. No significant changes in the expression levels of caspase-3, -7, cleaved caspase-3, cleaved caspase-7, p53 or Bcl-2, although significant PARP cleavage was observed. Moreover, to confirm the data of the cell cycle analysis, we examined the expressions of cyclin A and cyclin E, cell cycle regulators in the S phase, following GANT61 treatment. The expression level of cyclin A decreased after 24–48 h of GANT61 treatment, while no change in the expression level of cyclin E was noted.
In this study, we initially investigated whether the Hh signaling pathway was involved in cell growth and cell death in ESFT. GANT61 induced significant cell death in the ESFT cell lines. GANT61 is a small-molecule inhibitor of GLI1- and GLI2-mediated transcription at the nuclear level that exerts its effect by preventing DNA binding.13) There are only a few reports of the anticancer activities of antagonists of the Hh signal against ESFT. In one previous study, GANT58 inhibited the transcription of GLI at the nuclear level, similar to GANT61,13) and dramatically reduced cell growth in the ES cell line A673 at the concentration of 5 µM.8) Our data also showed that the IC50 value of GANT61 was 14.5 µM for SK-N-LO, the most sensitive cell line (Table 2). SK-N-LO is morphologically characterized by a NB-like cell type. In contrast, NB cell lines not expressing the EWS-FLI1 fusion gene showed very weak sensitivity to GANT61. All the GANT61-sensitive cell lines (SK-N-LO, SK-N-MC, and NCR-EW2) expressed EWS-FLI1 fusion gene. In contrast, SCMC-ES1 cell lines which expressed the EWS-ERG fusion gene showed poor sensitivity. GLI1 is also known as one of the direct target genes of EWS-FLI1.6–8) Reduction of the protein level of GLI2, but not of GLI1 was observed in SK-N-LO cells treated with GANT61 (Fig. 7A). NB cell lines are reported to express high levels of GLI2, but not of GLI1, and GLI2 plays an important role as a transcription factor in the tumorigenicity of NB cells.14) Furthermore, from the point of view of the cell characteristics (Table 1), the GANT61-sensitive cell lines (SK-N-LO and SK-N-MC) were classified into NB-like cells and both were derived from the brain. In contrast, cell lines that showed low or no sensitivity to GANT61 (SCMC-ES1 and SK-ES-1) were classified into ES, and originated from the bone. GANT61 has been reported to induce apoptosis in Burkitt lymphoma,15) NB,16) myeloid leukemia17) and pancreatic cancer.18) Our findings suggest that GANT61 induced cell death especially in ESFT with NB-like cell types expressing EWS-FLI1, and GLI2 inhibition appears to be involved. Cyclopamine, an SMO inhibitor, had no effect on ESFT. Previous studies have shown that blockade of SMO activation by cyclopamine has little effect on EWS-FLI1-transformed cells6,7) or ESFT cell lines.8) Our findings also suggested that molecules upstream of the Hh signal may not contribute to ESFT cell proliferation.
We examined whether GANT61 induced apoptosis of ESFT cells. Apoptosis, which is characterized by cell shrinkage, chromatin condensation, and nuclear fragmentation, was observed in the SK-N-LO cells by Hoechst 33342 staining (Fig. 3). In addition, apoptotic inversion of the cell membrane and increase of permeability were also observed after GANT61 treatment. (Figs. 4A, B). Decrease of the protein expression level of survivin and cleavage of PARP were observed following treatment with GANT61 (Fig. 7B). Survivin is a member of the Inhibitor of Apoptosis Proteins (IAPs), and is expressed in most human cancer cells. Survivin inhibits apoptosis by inhibition of Smac/DIABLO and stabilization of the X-linked Inhibitor of Apoptosis Protein (XIAP), and promoting degradation of active caspase.19) PARP plays a major role in DNA repair and cell death. PARP is cleaved by a caspase during apoptosis.20) However, in our study, the Proteome Profiler assay did not reveal any changes in the expressions of Smac/DIABLO and XIAP after treatment with GANT61 (Fig. 5). Furthermore, the protein expression levels of caspase-3 and -7, and of cleaved caspase-3 and -7 also showed no change (Fig. 7B). These findings suggest that the suppression of survivin and PARP cleavage occurred in a caspase-independent manner. In Burkitt lymphoma15) and NB,16) GANT61 was shown to induce apoptosis in a caspase-dependent manner, while GANT61 induced caspase-independent apoptosis of the SK-N-LO cells in this study.
The protein level of p21, which is known as a cyclin-dependent kinase (CDK) inhibitor was remarkably increased following GANT61 treatment in this study. p21 plays a role in growth arrest after DNA damage, and its overexpression leads to G1, G2 or S-phase arrest.21) p21 was reported to be upregulated following knockdown of GLI2 in osteosarcoma22) and ovarian cancer.23) The protein level of p21 increased by inhibition of the Hh signal by cyclopamine in NB cells.14) These results suggest that the p21 protein level increase was mediated by GLI2 suppression.
In this study, GANT61 induced a significant decrease in the proportion of cells in the S phase. This finding suggested that GANT61 suppressed the transition to the S phase, and also effectively suppressed cell growth. However, no change in the proportion of cells in the G0/G1 phase was observed as compared to the control (Fig. 6B). Cyclin E expression is observed in the late G1-early S phase, and cyclin E/CDK2 plays a critical role in the G1 phase and in the G1-S phase transition.24) Cyclin A is expressed in the late G1 phase, and is involved in G1-S transition and DNA replication in the S phase.25) The protein expression level of cyclin A, but not of cyclin E was significantly decreased by treatment with GANT61. In a previous study, GANT61 induced transient cellular accumulation in the G1-S and early S phases, with elevated p21, cyclin E and cyclin A expressions.26) G1 arrest by cyclopamine was associated with a decrease of the cyclin A protein level along with an increase of p21 expression in ovarian cancer.23) The protein level of claspin decreased significantly after treatment with GANT61 for 24–48 h (Fig. 7B). Claspin is an S-phase checkpoint mediator, and mediates the checkpoint response to replication stress by facilitating ATM (ataxia-telangiectasia mutation)- and Rad3-related (ATR)-dependent phosphorylation of checkpoint kinase-1 (Chk-1).27,28) On the other hand, overexpression of claspin stimulated proliferation of cancer cells.29) Our findings suggested that GANT61 prevented DNA replication by inhibition of claspin in the S phase. Claspin has been shown to be cleaved by caspase-7 during apoptosis.30,31) In this study, no change in the protein expression level of caspase-7 was noted (Fig. 7B). An immunofluorescence study revealed that osteosarcoma cells expressing cyclin A contained claspin within their nuclei.32) Another study revealed transient expression of claspin with the initial elevation of cyclin A expression.33) We found that the decrease in the claspin protein expression level was correlated with a decrease of the cyclin A protein expression level. Moreover, cleaved PARP (24 kDa) increased after treatment with GANT61 in this study. Overexpression of the N-terminal peptide of PARP (24 kDa) is known to inhibit DNA repair.20) In this study, the mechanism of survivin suppression and PARP cleavage were not clear. Therefore, further investigations are required to elucidate the mechanism of action of GANT61.
In conclusion, we found that GANT61 induced apoptosis in ESFT cells expressing the EWS-FLI1 fusion gene in a caspase-independent manner, possibly via arresting DNA replication. Our findings indicate the GLI inhibitors as promising agents in the treatment of ESFT.
We thank to Dr. Yasuhide Hayashi (Gunma Children’s Medical Center) (SK-N-MC, SK-N-LO, and SCMC-ES1), Dr. Jun-ichi Hata (Keio University School of Medicine, Department of Pathology) (NCR-EW2) for providing the cell lines. This work was supported by a joint research Grant from Nihon University 2012–2013.
