Rhabdomyosarcoma (RMS) is a common soft-tissue sarcoma of childhood in need of more effective therapeutic options. The expression of p53 in RMS is heterogeneous such that some tumors are wild-type whereas others are p53 mutant. The small molecule CP-31398 modulates both the wild-type and the mutant p53 proteins. Here, we show that CP-31398 blocks the growth of RMS cells that have either wild-type or mutant p53 status. In wild-type A204 cells, CP-31398 increased the expression of p53 and its downstream transcriptional targets, p21 and mdm2; enhanced the expression of apoptosis-related proteins; and reduced proliferation biomarkers. Flow profiling of CP-31398–treated cells indicated an enhancement in sub-G0 and G1 populations. CP-31398 inhibited proliferation in a manner associated with co-induction of SOX9 and p21. Apoptosis induced by CP-31398 occurred with translocation of p53 to mitochondria, leading to altered mitochondrial membrane potential, cytochrome c release, and reactive oxygen species release. In vivo, CP-31398 decreased the growth of tumor xenografts composed of wild-type or mutant p53 tumor cells, increasing tumor-free host survival. Our findings indicate that the ability of CP-31398 to modulate wild-type and mutant p53 results in the inhibition of RMS growth and invasiveness. Cancer Res; 70(16); 6566–76. ©2010 AACR.

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma in pediatrics, with an incidence of 4.6 cases per million children (1). Embryonal (ERMS) and alveolar (ARMS) RMS, the two major histologic subtypes of RMS, carry distinct clinical features (2). ERMS tumors are more common among young children, typically occurring in the head, neck, and genitourinary tract. ERMS tumors are generally more sensitive to chemotherapy and radiation. In contrast, ARMS tumors are more common in adolescents, often occurring in the extremities. Patients with ARMS and undifferentiated sarcoma carry a less favorable prognosis than patients with ERMS tumors (3). The overall 5-year survival rate for children with RMS is ∼64% for cases diagnosed from 1985 to 1994 (1, 2). The majority of RMS tumors occur sporadically, but a subset of tumors develop in patients with cancer predisposition syndromes such as Li-Fraumeni (4).

Using multiagent chemotherapy, surgery, and radiation, the outcome for patients with favorable features has steadily improved (3). However, the prognosis for metastatic and relapsed RMS tumors remains very poor (5). RMS therapies beyond cytotoxic chemotherapy are desperately needed. During the last decade, efforts were made to use tumor suppressor p53 as a major target of drug development for blocking the pathogenesis and progression of various cancers (6). It is well known that p53 is mutated in more than 50% of all human cancers including RMS (7). In the remaining 50% where p53 is not mutated and remains wild-type, the signaling downstream of p53 is frequently interrupted (8, 9). Wild-type p53 is usually not accumulated in the cells due to its short half life (<30 minutes). Therefore, attempts to increase transcriptionally active p53 either by enhancing the stability of wild-type p53 or by reverting mutant p53 to its wild-type conformation with its ability to block cell cycle progression and induce apoptosis has been considered as an important approach in cancer treatment. In this regard, CP-31398, a styrylquinazoline, can restore a wild-type–associated epitope (monoclonal antibody 1620) on the DNA-binding domain of the mutant p53 protein (1014). Furthermore, CP-31398 not only restores p53 functions in mutant p53-expressing cells but can also significantly increase the protein level and promote the activity of wild-type p53 in multiple human cancer cell lines, leading to cell cycle arrest or apoptosis (14). The putative mechanism by which CP-31398 enhances the protein levels of wild-type p53 includes blockade of ubiquitination and degradation of p53 without interrupting the physical association between p53 and MDM2 in vivo (14).

In this study, we investigated the chemotherapeutic effects of CP-31398 in a poorly differentiated RMS cell line, A204, which carries wild-type p53 (15, 16), and the ERMS cell line RD, which carries mutant p53. Our results show that CP-31398 induces p53-dependent cell cycle arrest and apoptosis in both A204 and RD cells. CP-31398–induced transcriptional activation of p53 is evident by the induction of its downstream targets, p21, mdm2, and puma, in both of these cells. The induction of apoptosis involved the mitochondrial translocation of p53 followed by the release of cytochrome c and activation of caspase-3. Parenteral administration of CP-31398 reduced the growth of xenograft tumors that developed following the s.c. inoculation of A204 or RD cells. Our data indicate that CP-31398 can be highly effective in promoting diminution of the growth and invasiveness of RMS tumors carrying wild-type or mutant p53.

Antibodies and reagents

Primary antibodies (Supplementary Table S1; Santa Cruz Biotechnology); horseradish peroxidase–secondary antibodies (Pierce) and Alexa Fluor 488 or 596 secondary antibodies (eBioscience); MitoTracker Red CMXRos (Invitrogen); Apoptosis Detection Kit (Roche Applied Science); JC-1 dye (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl benzimidazolocarbocyanine iodide) staining kit (Molecular Probes, Inc.); and cyclosporine A (CsA), N-acetyl-cysteine (NAC), and 2′,7′-dichlorofluorescein diacetate (DCF-DA; Sigma) were purchased.

Cell culture

The ERMS A204 cells (wild-type p53) and the ERMS RD cells (mutant p53) were obtained from the American Type Culture Collection. A204 cells were cultured in McCoy's 5A medium whereas RD cells were cultured in DMEM (Hyclone), supplemented with 10% fetal bovine serum, 100 units/mL of penicillin, and 100 μg/mL of streptomycin at 37°C in a humidified atmosphere of 5% CO2.

Western blotting, immunofluorescence, and immunohistochemistry

Western blotting, immunofluorescence, and immunohistochemistry analyses were done as described previously (11).

Terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling

Terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining was performed according to the vendor's guidelines.

Flow cytometric analysis of cell cycle

Flow cytometry was done using Becton Dickinson FACSCan and cell cycle was analyzed using FlowJo (8.8.6) Watson pragmatic analysis software.

Mitochondrial membrane potential assay

Cells from various treatments were trypsinized, resuspended in 200 nmol/L JC-1 solution, and incubated in a 5% CO2 incubator at 37°C and then analyzed by flow cytometry. Carbonyl cyanide 3-chlorophenylhydrazone (5 μmol/L) was used to completely disrupt mitochondrial potential as a positive control (11).

Measurement of intracellular reactive oxygen species generation

The cells were incubated with 10 μmol/L DCF-DA at 37°C for 15 minutes. The intracellular reactive oxygen species (ROS) mediates oxidation of DCF-DA to the fluorescent DCF. The cells were then harvested and suspended in PBS and analyzed by flow cytometry.

Tumor xenograft study

Six- to eight-week-old female Nu/Nu mice from the National Cancer Institute were used in this study. Animals were divided into three groups of 10 mice each. Each mouse from all three groups received 2 × 106 cells in 200 μL of PBS s.c. in each flank. Twenty-four hours after the inoculation of cells, group 1 mice received an i.p. injection of vehicle, whereas group 2 and group 3 mice received i.p injections of CP-31398 (2 mg/mouse) at 12- or 24-hour intervals, respectively, for 5 consecutive days per week for 1 month. Tumors were measured by digital calipers and tumor volumes calculated using the formula volume = length × width × height/2, plotted as a function of days on test.

Statistics

Statistical analysis was performed using Microsoft Excel software. The significance between the two test groups was determined using Student's t test, and P < 0.05 was considered as significant.

Treatment of A204 cells with CP-31398 induces G1 cell cycle arrest and apoptosis in RMS A204 cells

To characterize the effects of CP-31398 in A204 cells in vitro, cells were grown in 60-mm plates and treated with 0, 10, 20, and 40 μg/mL of CP-31398 for 24 hours, and then samples were analyzed by flow cytometry. We observed a dose-dependent increase in apoptosis of A204 cells following CP-31398 treatment (Fig. 1A–C). This was indicated by an accumulation of events with light scatter properties consistent with apoptotic cells and debris and a reduction in events with viable cell light scatter properties (Fig. 1A). This correlated with an increased percentage of cells stained with Annexin V and propidium iodide (PI; Fig. 1B). In the viable cell gate, we observed a dose-dependent accumulation of cells in the G1 phase (up to 38% more than control) following exposure to lower concentrations of CP-31398. At the highest concentration, CP-31398–induced apoptosis occurred in cells in all phases of the cell cycle equally, as no significant differences in the cell cycle distribution of the remaining viable cells as compared with control were discernable (Fig. 1C, graph).

Figure 1.

CP-31398 (CP) treatment blocks cell cycle progression and induces apoptosis in RMS A204 cells carrying wild-type p53. Cells were incubated in the presence of CP at the indicated concentrations for 24 h and then stained with PI for immediate flow cytometric analysis. A, loss of viable cells. Two-parameter psuedocolor density plots of total ungated events for forward scatter and side scatter properties. Note the CP dose–dependent accumulation of dead cells and apoptotic debris to the left side of the panel. The large circular gate includes both live and dead single cells, excluding small debris. The percentage of gated total cellular events is shown in the top left corner of each ungated panel. The small circular gate encompasses viable cells. The percentage of cells is displayed in the bottom right corner of each ungated panel. A graph summarizing the percentage of viable cells within the single cell gate is shown at the bottom. B, increase in apoptotic cells. Two-parameter bitmap displays of gated cells (indicated by arrows in A) analyzed for Annexin V and PI staining (left) and histograms for PI alone (right). The percentages of Annexin V–positive cells within PI-negative (early apoptosis) and PI-positive (late apoptosis) subsets are shown in the top left and top right quadrants, respectively. A graph displaying total apoptotic and dead cells (% Annexin + PI) is shown at the bottom. The percentages of PI-stained cells are shown in the histograms, and a summary graph is shown at the bottom. Note the inverse correlation of viable cells (A), indicated by light scatter properties (small gate), and apoptotic cells (central and right graphs in B). C, G1-phase block of cell cycle. A204 cells were incubated with the indicated concentrations of CP-31398 for 24 h and DNA was stained with PI for cell cycle analysis using the Watson pragmatic model (results at the top of each panel). The summary graph below shows the mean percent of cells ± SEM in each phase (n = 3). *, P < 0.05, two-sided Student's t test. D, kinetics of CP-mediated induction of p53-related, apoptosis-related, and cell cycle–related protein expression. Western blot analyses of A204 cell lysate proteins following exposure to 20 μg/mL CP for different times (as indicated).

Figure 1.

CP-31398 (CP) treatment blocks cell cycle progression and induces apoptosis in RMS A204 cells carrying wild-type p53. Cells were incubated in the presence of CP at the indicated concentrations for 24 h and then stained with PI for immediate flow cytometric analysis. A, loss of viable cells. Two-parameter psuedocolor density plots of total ungated events for forward scatter and side scatter properties. Note the CP dose–dependent accumulation of dead cells and apoptotic debris to the left side of the panel. The large circular gate includes both live and dead single cells, excluding small debris. The percentage of gated total cellular events is shown in the top left corner of each ungated panel. The small circular gate encompasses viable cells. The percentage of cells is displayed in the bottom right corner of each ungated panel. A graph summarizing the percentage of viable cells within the single cell gate is shown at the bottom. B, increase in apoptotic cells. Two-parameter bitmap displays of gated cells (indicated by arrows in A) analyzed for Annexin V and PI staining (left) and histograms for PI alone (right). The percentages of Annexin V–positive cells within PI-negative (early apoptosis) and PI-positive (late apoptosis) subsets are shown in the top left and top right quadrants, respectively. A graph displaying total apoptotic and dead cells (% Annexin + PI) is shown at the bottom. The percentages of PI-stained cells are shown in the histograms, and a summary graph is shown at the bottom. Note the inverse correlation of viable cells (A), indicated by light scatter properties (small gate), and apoptotic cells (central and right graphs in B). C, G1-phase block of cell cycle. A204 cells were incubated with the indicated concentrations of CP-31398 for 24 h and DNA was stained with PI for cell cycle analysis using the Watson pragmatic model (results at the top of each panel). The summary graph below shows the mean percent of cells ± SEM in each phase (n = 3). *, P < 0.05, two-sided Student's t test. D, kinetics of CP-mediated induction of p53-related, apoptosis-related, and cell cycle–related protein expression. Western blot analyses of A204 cell lysate proteins following exposure to 20 μg/mL CP for different times (as indicated).

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CP-31398 induces the expression of p53 and its downstream target proteins

The induction of the cell cycle G1 block and apoptosis by CP-31398 was correlated with the results of Western blot analyses. As shown in Fig. 1D, CP-31398 slightly enhanced p53 levels in A204 cells detectable over a period of 24 hours, which may be due to the stabilization of p53 in addition to the enhanced transcription of this protein (6). We further tested its effects on the expression of p53-dependent downstream target genes p21 and mdm2. The expression of these proteins was upregulated at 3, 6, and 12 hours after treatment with CP-31398. It is known that p21 is required for G1 cell cycle arrest. Next, we assessed the effect of CP-31398 on the expression of proapoptotic and other cell cycle regulatory proteins in these cells. A time- and dose-dependent (data not shown) increase in proapoptotic proteins such as Apaf1, Bax, Bcl2, Bad, and caspase-3 was observed. Consistently, poly(ADP-ribose) polymerase (PARP) cleavage was also induced. CHK1/2 and Cdc2 levels were increased, whereas cyclin E expression was decreased. Accumulation of p53 and induction of p21, mdm2, and CHK1/2 proteins were detected and often peaked at the earliest time points of 3 to 6 hours, whereas increased levels of the apoptotic pathway proteins caspase-3, Bad and Puma and PARP cleavage peaked at 12 hours. The enhanced CHK1/2 expression may, in part, be due to the induction of the DNA damage–dependent ATM/ATR–regulated signaling pathway. However, this needs to be investigated in further detail, which is beyond the scope of this article. Overall, these results are consistent with CP-31398–mediated stabilization of wild-type p53 protein conformation and activation of p53 downstream functions that block cell cycle and apoptosis in RMS A204 cells.

CP-31398 induces mitochondrial translocation of mutant p53, resulting in cytochrome c release and apoptosis

Next, we investigated whether CP-31398 can enhance translocation of p53 to mitochondria. It is known that wild-type p53 migrates to mitochondria in response to genotoxic insult (17) and alters mitochondrial membrane potential (MP; ref. 11). This leads to the release of cytochrome c from mitochondria to cytoplasm followed by apoptosis induction. In this experiment, we used MitoTracker red dye to stain mitochondria of vehicle- or CP-31398–treated A204 cells. The localization of p53 in mitochondria (stained in green) was confirmed by the orange to yellow color in the overlay. Thus, CP-31398 enhanced p53 mitochondrial localization (Fig. 2A) whereas no such effect was discernible in vehicle-treated control cells at 15 minutes.

Figure 2.

CP-31398 induces apoptosis in A204 cells by translocating p53 to mitochondria and alters MP in A204 cells, which can be blocked by CsA pretreatment. A, immunofluorescence staining showing colocalization of p53 with MitoTracker, which stains mitochondria. B, immunofluorescence staining showing that CsA blocks CP-31398–induced p53 localization to mitochondria. C, CsA blocks CP-31398–induced cytochrome c release in the cytoplasm. D, CP-31398–mediated alterations in MP are attenuated by CsA treatment. For the experiments in A to C, A204 cells were treated with PBS (control) or CP-31398 (20 μg/mL) for various time intervals. However, for the experiments in D, cells were treated with PBS (control) or CP-31398 (20 and 40 μg/mL) for 15 min and then stained with JC-1 dye. Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was used as positive control. CsA (1 μmol/L) pretreatment was done for 15 min before CP treatment. Columns, mean of three independent experiments; bars, SD. a, P < 0.05, compared with control; b, P < 0.01, compared with control; c, P < 0.05, compared with 20 μg/mL CP-31398 treatment.

Figure 2.

CP-31398 induces apoptosis in A204 cells by translocating p53 to mitochondria and alters MP in A204 cells, which can be blocked by CsA pretreatment. A, immunofluorescence staining showing colocalization of p53 with MitoTracker, which stains mitochondria. B, immunofluorescence staining showing that CsA blocks CP-31398–induced p53 localization to mitochondria. C, CsA blocks CP-31398–induced cytochrome c release in the cytoplasm. D, CP-31398–mediated alterations in MP are attenuated by CsA treatment. For the experiments in A to C, A204 cells were treated with PBS (control) or CP-31398 (20 μg/mL) for various time intervals. However, for the experiments in D, cells were treated with PBS (control) or CP-31398 (20 and 40 μg/mL) for 15 min and then stained with JC-1 dye. Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was used as positive control. CsA (1 μmol/L) pretreatment was done for 15 min before CP treatment. Columns, mean of three independent experiments; bars, SD. a, P < 0.05, compared with control; b, P < 0.01, compared with control; c, P < 0.05, compared with 20 μg/mL CP-31398 treatment.

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CsA blocks CP-31398–mediated mitochondrial localization of p53 and consequent induction of apoptosis in A204 cells

CsA is known to be a potent and specific inhibitor of mitochondrial membrane permeability pore transition (MPT; refs. 18, 19). We used this agent to determine whether MPT blockade in A204 cells inhibits CP-31398–mediated p53 mitochondrial translocation and the associated alterations such as cytochrome c release and induction of apoptosis. As shown in Fig. 2B, CsA pretreatment blocked mitochondrial translocation of p53. Concomitantly, mitochondrial cytochrome c release into the cytoplasm was also diminished (Fig. 2C). In addition, CP-31398–mediated apoptosis was abrogated.

CP-31398 alters mitochondrial membrane potential

To confirm that CP-31398 alters MP, we used JC-1 dye, which accumulates in mitochondria and forms aggregates that emit red-orange fluorescence when exposed to light at 590 nm. Formation of these aggregates requires normal MP. With altered MP following the membrane depolarization, the dye remains as a monomer in the cytoplasm and shows green fluorescence (20, 21). The ratio of red/green fluorescence is a function of MP. CP-31398 decreased dose-dependently the MP in A204 cells (Fig. 2D). However, as expected, CsA pretreatment blocked changes in CP-31398–induced MP.

CP-31398 triggers ROS generation, which mediates apoptosis in A204 cells

ROS are considered to play an important role in apoptosis in various cell types (22, 23). To investigate whether CP-31398 stimulated ROS generation in A204 cells, we measured intracellular ROS levels using a ROS-detecting fluorescence dye, DCF-DA. Generation of ROS was evidenced by the increased intensity of DCF fluorescence. Following treatment of A204 cells with CP-31398, an increased generation of ROS could be observed at 12 and 24 hours. The percentage of DCF-positive cells was 1.5%, 36.4%, and 52.3% at 0, 12, and 24 hours, respectively (Fig. 3A). However, the ROS scavenger NAC (5 mmol/L) markedly decreased the level of ROS to ∼6% at 24 hours. Furthermore, the enhanced ROS production was significantly reduced by pretreating cells with CsA (Fig. 3B), suggesting that the major source of CP-31398–mediated ROS may be the mitochondria.

Figure 3.

CP-31398 induces ROS and ROS-dependent cell death in A204 cells. A, CP-31398 stimulated the generation of ROS, which was blocked by pretreating cells with NAC. Cells were cultured in medium containing CP-31398 (20 μg/mL) for 0, 12, and 24 h in the absence or presence of 5 mmol/L NAC. B, pretreatment with CsA blocked CP-31398–induced generation of ROS. ROS generation was measured with the ROS-detecting fluorescent dye DCF-DA by flow cytometry. The corresponding linear diagram of FACScan is also shown (n = 3; mean ± SD; **, P < 0.01). Cells were treated with CP-31398 (20 μg/mL) for 15 min. Each value represents the mean ± SD of three independent experiments (n = 3; **, P < 0.01).

Figure 3.

CP-31398 induces ROS and ROS-dependent cell death in A204 cells. A, CP-31398 stimulated the generation of ROS, which was blocked by pretreating cells with NAC. Cells were cultured in medium containing CP-31398 (20 μg/mL) for 0, 12, and 24 h in the absence or presence of 5 mmol/L NAC. B, pretreatment with CsA blocked CP-31398–induced generation of ROS. ROS generation was measured with the ROS-detecting fluorescent dye DCF-DA by flow cytometry. The corresponding linear diagram of FACScan is also shown (n = 3; mean ± SD; **, P < 0.01). Cells were treated with CP-31398 (20 μg/mL) for 15 min. Each value represents the mean ± SD of three independent experiments (n = 3; **, P < 0.01).

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CP-31398 reduces the growth of xenograft tumors developed by A204 RMS cells in nude mice

To investigate whether CP-31398 affects the growth of A204 RMS xenograft tumors in nude mice, we injected CP-31398 i.p. (2 mg/mouse daily or twice daily, every 12 hours) to nude mice over a period of 4 weeks after inoculating them with A204 cells. Tumor volume was measured every other day. Animals were sacrificed after 4 weeks of treatment. We found that tumor growth in the CP-31398 treatment groups was reduced significantly in a dose-dependent manner as compared with the vehicle-treated control group as shown in Fig. 4A. The tumor volume in the control group was 277 ± 47 mm3, whereas it was reduced to 127 ± 41 mm3 in the CP-31398 daily treatment group and to 68 ± 24 mm3 in the CP-31398 twice-daily treatment group (P < 0.05 and P < 0.01, respectively). At early time points (day 14 and day 18), the differences between the two doses of CP-31398 were found to be significant (P < 0.05), but at later time points (day 22 and day 30), these differences became less significant. As compared with the vehicle-treated controls, the histology of tumors in the CP-31398 treatment groups showed less mitotic figures (Fig. 4B) and large necrotic areas. In addition, we observed remarkably decreased proliferating cell nuclear antigen (PCNA) and cyclin E staining with a dramatic increase in the number of TUNEL-positive cells. We also found a concomitant reduction in Bcl2 expression in tumors excised from the CP-31398–treated groups (Fig. 4C).

Figure 4.

CP-31398 treatment reduces the growth xenograft tumors and augments MET in nude mice developed by inoculating A204 cells carrying wild-type p53. A, CP-31398 reduces the volume of xenograft tumors. B, histology of tumors developed in vehicle- or CP-31398–treated mice. Inset shows mitotic figures (red arrow) and apoptosis (green arrow). C, TUNEL, immunofluorescence, and immunohistochemical staining for apoptosis, Bcl2, PCNA (arrow indicates positive nuclear staining), cyclin E (arrow indicates positive nuclear staining), and E-cadherin expression (data showing that CP-31398 treatment reduces proliferation and increases E-cadherin expression in xenograft tumors). D, CP-31398 reduces the expression of MMP-2/MMP-9, snai, slug, twist, and fibronectin in xenograft tumors. D1 represents CP-31398 daily treatments (24-h intervals) whereas D2 represents twice-daily (12-h intervals) treatments. Mice were treated over 4 wk, beginning treatment at the time of tumor cell inoculation. Each value represents the mean ± SE of 10 mice (*, P < 0.05; **, P < 0.01).

Figure 4.

CP-31398 treatment reduces the growth xenograft tumors and augments MET in nude mice developed by inoculating A204 cells carrying wild-type p53. A, CP-31398 reduces the volume of xenograft tumors. B, histology of tumors developed in vehicle- or CP-31398–treated mice. Inset shows mitotic figures (red arrow) and apoptosis (green arrow). C, TUNEL, immunofluorescence, and immunohistochemical staining for apoptosis, Bcl2, PCNA (arrow indicates positive nuclear staining), cyclin E (arrow indicates positive nuclear staining), and E-cadherin expression (data showing that CP-31398 treatment reduces proliferation and increases E-cadherin expression in xenograft tumors). D, CP-31398 reduces the expression of MMP-2/MMP-9, snai, slug, twist, and fibronectin in xenograft tumors. D1 represents CP-31398 daily treatments (24-h intervals) whereas D2 represents twice-daily (12-h intervals) treatments. Mice were treated over 4 wk, beginning treatment at the time of tumor cell inoculation. Each value represents the mean ± SE of 10 mice (*, P < 0.05; **, P < 0.01).

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CP-31398 treatment augments mesenchymal-epithelial transition in A204 xenograft tumors

To confirm whether CP-31398 alters the invasive growth of these tumors, we investigated its effects on the expression of mesenchymal-epithelial transition (MET) pathway–related proteins (24) in A204 xenograft tumors using immunofluorescence assay. In CP-31398–treated tumors, the expression of matrix metalloproteinase (MMP)-2/MMP-9, snail, slug, and twist was found to be reduced (Fig. 4D). Parallel to these observations, we noticed a concomitant decrease in the expression of fibronectin with an increase in E-cadherin expression (Fig. 4C).

CP-31398 treatment induces SOX9 expression in xenograft tumors developed by A204 cells in nude mice

CP-31398 induced the expression of SOX9 in xenograft tumors, which does not consistently colocalize with TUNEL-positive cells (Fig. 5A). These results suggest that SOX9 does not target the cells destined to die. However, the observed increase of SOX9 and p21 expression in CP-31398–treated A204 xenograft tumors (Fig. 5B) indicates their role in the blockade of tumor growth because these tumors are significantly smaller in size compared with vehicle-treated controls. These results are also supported by Western blot analysis of CP-31398–treated A204 cells showing an identical pattern of SOX9 and p21 induction kinetics (Fig. 5C). This is consistent with previous reports where SOX9 was shown to bind with the promoters of p21 and induce its expression, thereby reducing tumor growth (25).

Figure 5.

CP-31398 treatment induces SOX9 expression, which colocalizes with cells showing induction of p21 but does not colocalize with TUNEL-positive cells in A204 xenograft tumors. A, CP-31398–induced expression of SOX9 (red arrows) that does not colocalize with TUNEL-positive (green arrows) cells. B, CP-31398–induced expression of SOX9 that colocalizes with p21 expression (yellow arrow). C, Western blot showing the kinetics of SOX9 and p21 induction in A204 cells treated with CP-31398 (20 μg/mL) for various time intervals. Magnification, ×40 (insets).

Figure 5.

CP-31398 treatment induces SOX9 expression, which colocalizes with cells showing induction of p21 but does not colocalize with TUNEL-positive cells in A204 xenograft tumors. A, CP-31398–induced expression of SOX9 (red arrows) that does not colocalize with TUNEL-positive (green arrows) cells. B, CP-31398–induced expression of SOX9 that colocalizes with p21 expression (yellow arrow). C, Western blot showing the kinetics of SOX9 and p21 induction in A204 cells treated with CP-31398 (20 μg/mL) for various time intervals. Magnification, ×40 (insets).

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CP-31398 reduces the growth of xenograft tumors developed by RD RMS cells in nude mice

To determine whether CP-31398 affects the growth of mutant p53–expressing RMS, we examined the growth of RD RMS xenograft tumors in nude mice. We administered CP-31398 i.p. (2 mg/mouse daily or twice daily, every 12 hours) over a period of 11 weeks after inoculating these animals with RD cells (injected s.c. into right and left flanks). Tumor volume was measured every 5 days. Animals were sacrificed after 11 weeks of treatment. We observed that tumor growth in the CP-31398 treatment groups was reduced significantly (P < 0.05) as compared with the vehicle-treated control group in a dose-dependent manner, as shown in Fig. 6A. The tumor volume in the control group was found to be 778 ± 180 mm3, whereas it was reduced to 389 ± 99 mm3 in the CP-31398 once-daily treatment group and to 240 ± 109 mm3 in the CP-31398 twice-daily treatment group. At day 70, the differences between these two doses of CP-31398 were significant (P = 0.0369), but at the termination of experiment, these differences were no longer statistically significant. As compared with the vehicle-treated controls, the histology of tumors in CP-31398 treatment groups showed a larger area of necrosis. The tumor cells manifested less mitotic figures and a remarkable decrease in staining of the proliferation biomarkers PCNA and cyclin E (Fig. 6B). The CP-31398–treated tumors also showed a substantially increased number of TUNEL-positive cells. To show that these effects of CP-31398 are specifically dependent on p53, we treated RD cells with various concentrations of CP-31398 and assessed the levels of p53 and its downstream transcription target genes, p21, mdm2, and Apaf1. We observed that CP-31398 treatment stabilizes the levels of p53 and enhances the expression of p21, mdm2, and Apaf1 (Fig. 6C). In addition, consistent with immunohistochemistry and TUNEL data in xenograft tumors, CP-31398 treatment reduces the expression of cyclin E and enhances PARP cleavage.

Figure 6.

CP-31398 treatment reduces the growth of xenograft tumors developed by RD cells carrying mutant p53 in nude mice. A, CP-31398 reduces the volume of xenograft tumors (P < 0.05). D1 represents CP-31398 daily treatments at 24-h intervals, whereas D2 represents twice-daily treatments at 12-h intervals. B, histology of tumors developed in vehicle- or CP-31398–treated mice. Insets show mitotic figures (red arrows) and apoptosis (green arrows). Immunohistochemical and TUNEL staining showing that CP-31398–treatment reduces proliferation as assessed by PCNA and cyclin E expression (arrow indicates positive nuclear staining) and induces apoptosis (as assessed by an increase in TUNEL-positive cells) in xenograft tumors. C, Western blot analysis showing that CP-31398 induces stabilization of p53 and enhances the expression of its downstream target genes. In addition, it induces apoptosis-related genes and reduces the expression of proliferation marker proteins. Mice were treated over 11 wk, starting at the time of tumor inoculation. Each value represents the mean ± SE of five mice.

Figure 6.

CP-31398 treatment reduces the growth of xenograft tumors developed by RD cells carrying mutant p53 in nude mice. A, CP-31398 reduces the volume of xenograft tumors (P < 0.05). D1 represents CP-31398 daily treatments at 24-h intervals, whereas D2 represents twice-daily treatments at 12-h intervals. B, histology of tumors developed in vehicle- or CP-31398–treated mice. Insets show mitotic figures (red arrows) and apoptosis (green arrows). Immunohistochemical and TUNEL staining showing that CP-31398–treatment reduces proliferation as assessed by PCNA and cyclin E expression (arrow indicates positive nuclear staining) and induces apoptosis (as assessed by an increase in TUNEL-positive cells) in xenograft tumors. C, Western blot analysis showing that CP-31398 induces stabilization of p53 and enhances the expression of its downstream target genes. In addition, it induces apoptosis-related genes and reduces the expression of proliferation marker proteins. Mice were treated over 11 wk, starting at the time of tumor inoculation. Each value represents the mean ± SE of five mice.

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p53 is known to be a potent tumor suppressor and functions as a tetrameric transcription factor by binding to specific DNA sequences and transactivating or repressing a large number of target genes involved in cell cycle regulation and apoptosis (26). Therefore, it is considered to be an important drug target for blocking cancer growth. In tumors carrying mutant p53, use of small molecules such as PRIMA-1, capable of refolding mutant p53 to its wild-type conformation, may be effective in tumor regression (27, 28). In tumors where p53 is not mutated, agents such as Nutlins have been shown to be effective in blocking tumor progression (29). By disrupting mdm2 binding to p53, Nutlins enhance the levels of p53 protein in tumors carrying wild-type p53 (30). Mdm2 is a ubiquitin ligase that degrades wild-type p53 by its polyubiquitination (31). In this study, we used CP-31398, a chemical agent that has both an ability to revoke wild-type functions of mutant p53 and a potential to induce wild-type p53 (32, 33). Earlier, we showed that CP-31398 is highly efficacious against the induction of skin cancer carrying mutant p53 (11). Because the expression of p53 in RMS is heterogeneous, we investigated the chemotherapeutic effects of CP-31398 on the growth of human xenograft tumors developed by A204 cells carrying wild-type p53 as well as tumors developed by RD cells carrying mutant p53 (homozygous 742 C>T; R248W missense mutation; ref. 34).

It is known that increase in wild-type p53 induces proteins that block cell cycle progression and subsequently induce apoptosis (35, 36). Consistent with previous observations, we observed increased p53 levels and the induction of downstream transcriptional target genes of p53, such as p21, mdm2, puma, etc., in CP-31398–treated A204 and RD cells. This is further confirmed by the observed cell cycle arrest and apoptosis in RMS cells that manifest augmented PARP cleavage, reduced Bcl2, and enhanced Bax expression. The effects may be due to the stabilization of p53 and enhanced transcriptional activity of p53. Similarly in CP-31398–treated nude mice, we observed smaller tumors that showed enhanced numbers of TUNEL-positive cells and reduced expression of Bcl2. These results suggest that these p53-dependent effects of CP-31398 on cells in culture and in xenograft tumors are almost identical (37, 38). Interestingly, we also observed enhanced tumor-free survival of these animals, suggesting the potential of CP-31398 in the treatment of RMS.

To probe the molecular mechanism by which CP-31398 invokes apoptosis in A204 cells, we investigated its effects on the migration of p53 to the mitochondria. It is known that wild-type p53 migrates to mitochondria where it disrupts permeability pore potential and activates mitochondria-regulated intrinsic apoptotic pathways characterized by the release of mitochondrial proteins into the cytoplasm (39). Our observation that CP-31398 treatment induces mitochondrial localization of p53 in A204 cells suggests mitochondria-regulated apoptosis as the underlying mechanism in the therapeutic response of CP-31398. This was verified using CsA, a potent blocker of mitochondrial membrane pore transition. CsA blocked CP-31398–mediated p53 mitochondrial localization, alterations in MPT, and apoptosis induction in these cells. Similar results were obtained for RD cells. The involvement of mitochondria in CP-31398–mediated killing of RMS cells was further confirmed by observations in this study that CP-31398 treatment enhances ROS production; that NAC, a cell-permeable antioxidant, affords protection against CP-31398–mediated cell death; and that NAC and CsA manifest similar protective effects in CP-31398–treated A204 cells. These data suggest the possibility that CP-31398 may induce ROS production through mitochondrial membrane disruption and that ROS play a crucial role in the induction of CP-31398–mediated apoptosis.

p53 is known to play a role in altering the expression of proteins that regulate the balance between epithelial and mesenchymal phenotypes and thus determine the invasiveness and metastatic potential of cancer cells (40). It is known that wild-type p53 suppresses cancer cell invasion by inducing MDM2-mediated slug degradation (41). Slug is a member of the Snail family of transcription repressors and is capable of repressing E-cadherin expression thereby triggering EMT (42). The observations in this study that CP-31398 reduces the expression of mesenchymal markers such as fibronectin, slug, snai, and twist with a concomitant decrease in the expression of matrix-degrading enzymes, MMP-2/MMP-9, and an increase in E-cadherin suggest that CP-31398–mediated wild-type p53 induction dampens the invasiveness of A204 xenograft tumors and acts by altering the MET. This is confirmed by the observed decrease in the expression of proliferation biomarkers PCNA and cyclin E in these tumors. The mechanism(s) by which CP-31398 may reduce proliferation of RMS cells remains largely undefined. Recently, it has been shown that SOX9 overexpression downregulates melanoma cell proliferation through direct and indirect stimulation of the p21 promoter (25). However, our observation that SOX9 and p21 are coexpressed and that SOX9 induction does not occur in cells undergoing apoptosis is consistent with a novel mechanism by which CP-31398 may inhibit proliferation in wild-type p53–positive RMS cells. A similar efficacy of CP-31398 in abrogating the growth of xenograft RMS tumors developed in nude mice by inoculating A204 or RD cells suggests that restoring wild-type functions of mutant p53 is equally efficacious in tumor regression as activating wild-type p53. Together, these data suggest that CP-31398 has potential to block the growth of human RMS irrespective of the mutational status of p53. In summary, our data indicate that small molecular weight compounds such as CP-31398 can be highly effective in diminishing the growth and invasiveness of RMS tumors and in enhancing tumor-free survival.

No potential conflicts of interest were disclosed.

Grant Support: NIH grants R01 ES015323, NO1-CN-43300, and P30 AR050948.

We thank the National Cancer Institute for kindly providing CP-31398 (lot 10960).

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

1
Ries
LAGSM
,
Gurney
JG
,
Linet
M
,
Tamra
T
,
Young
JL
,
Bunin
GR
, editors.
Cancer incidence and survival among children and adolescents: United States SEER Program 1975-1995, National Cancer Institute, SEER Program
.
Bethesda (MD)
:
NIH Pub
; 
1999
.
2
Arndt
CA
,
Crist
WM
. 
Common musculoskeletal tumors of childhood and adolescence
.
N Engl J Med
1999
;
341
:
342
52
.
3
Meza
JL
,
Anderson
J
,
Pappo
AS
,
Meyer
WH
. 
Analysis of prognostic factors in patients with nonmetastatic rhabdomyosarcoma treated on intergroup rhabdomyosarcoma studies III and IV: the Children's Oncology Group
.
J Clin Oncol
2006
;
24
:
3844
51
.
4
Li
FP
,
Fraumeni
JF
 Jr
. 
Rhabdomyosarcoma in children: epidemiologic study and identification of a familial cancer syndrome
.
J Natl Cancer Inst
1969
;
43
:
1365
73
.
5
Carli
M
,
Colombatti
R
,
Oberlin
O
, et al
. 
European intergroup studies (MMT4-89 and MMT4-91) on childhood metastatic rhabdomyosarcoma: final results and analysis of prognostic factors
.
J Clin Oncol
2004
;
22
:
4787
94
.
6
Bassett
EA
,
Wang
W
,
Rastinejad
F
,
El-Deiry
WS
. 
Structural and functional basis for therapeutic modulation of p53 signaling
.
Clin Cancer Res
2008
;
14
:
6376
86
.
7
Felix
CA
,
Kappel
CC
,
Mitsudomi
T
, et al
. 
Frequency and diversity of p53 mutations in childhood rhabdomyosarcoma
.
Cancer Res
1992
;
52
:
2243
7
.
8
Hollstein
M
,
Rice
K
,
Greenblatt
MS
, et al
. 
Database of p53 gene somatic mutations in human tumors and cell lines
.
Nucleic Acids Res
1994
;
22
:
3551
5
.
9
Woods
YL
,
Lane
DP
. 
Exploiting the p53 pathway for cancer diagnosis and therapy
.
Hematol J
2003
;
4
:
233
47
.
10
Foster
BA
,
Coffey
HA
,
Morin
MJ
,
Rastinejad
F
. 
Pharmacological rescue of mutant p53 conformation and function
.
Science
1999
;
286
:
2507
10
.
11
Tang
X
,
Zhu
Y
,
Han
L
, et al
. 
CP-31398 restores mutant p53 tumor suppressor function and inhibits UVB-induced skin carcinogenesis in mice
.
J Clin Invest
2007
;
117
:
3753
64
.
12
Marx
J
. 
Oncology. Recruiting the cell's own guardian for cancer therapy
.
Science
2007
;
315
:
1211
3
.
13
Rippin
TM
,
Bykov
VJ
,
Freund
SM
,
Selivanova
G
,
Wiman
KG
,
Fersht
AR
. 
Characterization of the p53-rescue drug CP-31398 in vitro and in living cells
.
Oncogene
2002
;
21
:
2119
29
.
14
Wang
W
,
Takimoto
R
,
Rastinejad
F
,
El-Deiry
WS
. 
Stabilization of p53 by CP-31398 inhibits ubiquitination without altering phosphorylation at serine 15 or 20 or MDM2 binding
.
Mol Cell Biol
2003
;
23
:
2171
81
.
15
Barlow
JW
,
Wiley
JC
,
Mous
M
, et al
. 
Differentiation of rhabdomyosarcoma cell lines using retinoic acid
.
Pediatr Blood Cancer
2006
;
47
:
773
84
.
16
Bache
M
,
Dunst
J
,
Wurl
P
, et al
. 
G2/M checkpoint is p53-dependent and independent after irradiation in five human sarcoma cell lines
.
Anticancer Res
1999
;
19
:
1827
32
.
17
Erster
S
,
Mihara
M
,
Kim
RH
,
Petrenko
O
,
Moll
UM
. 
In vivo mitochondrial p53 translocation triggers a rapid first wave of cell death in response to DNA damage that can precede p53 target gene activation
.
Mol Cell Biol
2004
;
24
:
6728
41
.
18
Marques-Santos
LF
,
Coqueiro
VM
,
Rumjanek
VM
. 
Cyclosporin A does not protect the disruption of the inner mitochondrial membrane potential induced by potassium ionophores in intact K562 cells
.
Cell Biol Int
2006
;
30
:
197
204
.
19
Tay
VK
,
Wang
AS
,
Leow
KY
,
Ong
MM
,
Wong
KP
,
Boelsterli
UA
. 
Mitochondrial permeability transition as a source of superoxide anion induced by the nitroaromatic drug nimesulide in vitro
.
Free Radic Biol Med
2005
;
39
:
949
59
.
20
Smiley
ST
,
Reers
M
,
Mottola-Hartshorn
C
, et al
. 
Intracellular heterogeneity in mitochondrial membrane potentials revealed by a J-aggregate-forming lipophilic cation JC-1
.
Proc Natl Acad Sci U S A
1991
;
88
:
3671
5
.
21
Di Lisa
F
,
Blank
PS
,
Colonna
R
, et al
. 
Mitochondrial membrane potential in single living adult rat cardiac myocytes exposed to anoxia or metabolic inhibition
.
J Physiol
1995
;
486
:
1
13
.
22
Huang
J
,
Wu
L
,
Tashiro
S
,
Onodera
S
,
Ikejima
T
. 
Reactive oxygen species mediate oridonin-induced HepG2 apoptosis through p53, MAPK, mitochondrial signaling pathways
.
J Pharmacol Sci
2008
;
107
:
370
9
.
23
Simon
HU
,
Haj-Yehia
A
,
Levi-Schaffer
F
. 
Role of reactive oxygen species (ROS) in apoptosis induction
.
Apoptosis
2000
;
5
:
415
8
.
24
Gentile
A
,
Trusolino
L
,
Comoglio
PM
. 
The Met tyrosine kinase receptor in development and cancer
.
Cancer Metastasis Rev
2008
;
27
:
85
94
.
25
Passeron
T
,
Valencia
JC
,
Namiki
T
, et al
. 
Upregulation of SOX9 inhibits the growth of human and mouse melanomas and restores their sensitivity to retinoic acid
.
J Clin Invest
2009
;
119
:
954
63
.
26
Levine
AJ
,
Momand
J
,
Finlay
CA
. 
The p53 tumour suppressor gene
.
Nature
1991
;
351
:
453
6
.
27
Liang
Y
,
Besch-Williford
C
,
Hyder
SM
. 
PRIMA-1 inhibits growth of breast cancer cells by re-activating mutant p53 protein
.
Int J Oncol
2009
;
35
:
1015
23
.
28
Zache
N
,
Lambert
JM
,
Wiman
KG
,
Bykov
VJ
. 
PRIMA-1(MET) inhibits growth of mouse tumors carrying mutant p53
.
Cell Oncol
2008
;
30
:
411
8
.
29
Miyachi
M
,
Kakazu
N
,
Yagyu
S
, et al
. 
Restoration of p53 pathway by nutlin-3 induces cell cycle arrest and apoptosis in human rhabdomyosarcoma cells
.
Clin Cancer Res
2009
;
15
:
4077
84
.
30
Zhang
L
,
Zhang
J
,
Hu
C
, et al
. 
Efficient activation of p53 pathway in A549 cells exposed to L2, a novel compound targeting p53-2 interaction
.
Anticancer Drugs
2009
;
20
:
416
24
.
31
Coutts
AS
,
Adams
CJ
,
La Thangue
NB
. 
p53 ubiquitination by Mdm2: a never ending tail?
DNA Repair (Amst)
2009
;
8
:
483
90
.
32
Tanner
S
,
Barberis
A
. 
CP-31398, a putative p53-stabilizing molecule tested in mammalian cells and in yeast for its effects on p53 transcriptional activity
.
J Negat Results Biomed
2004
;
3
:
5
.
33
Bullock
AN
,
Fersht
AR
. 
Rescuing the function of mutant p53
.
Nat Rev Cancer
2001
;
1
:
68
76
.
34
Taylor
AC
,
Shu
L
,
Danks
MK
, et al
. 
P53 mutation and MDM2 amplification frequency in pediatric rhabdomyosarcoma tumors and cell lines
.
Med Pediatr Oncol
2000
;
35
:
96
103
.
35
Conzen
SD
,
Gottlob
K
,
Kandel
ES
, et al
. 
Induction of cell cycle progression and acceleration of apoptosis are two separable functions of c-Myc: transrepression correlates with acceleration of apoptosis
.
Mol Cell Biol
2000
;
20
:
6008
18
.
36
Chen
F
,
Chang
D
,
Goh
M
,
Klibanov
SA
,
Ljungman
M
. 
Role of p53 in cell cycle regulation and apoptosis following exposure to proteasome inhibitors
.
Cell Growth Differ
2000
;
11
:
239
46
.
37
Takimoto
R
,
Wang
W
,
Dicker
DT
,
Rastinejad
F
,
Lyssikatos
J
,
el-Deiry
WS
. 
The mutant p53-conformation modifying drug, CP-31398, can induce apoptosis of human cancer cells and can stabilize wild-type p53 protein
.
Cancer Biol Ther
2002
;
1
:
47
55
.
38
Luu
Y
,
Bush
J
,
Cheung
KJ
 Jr.
,
Li
G
. 
The p53 stabilizing compound CP-31398 induces apoptosis by activating the intrinsic Bax/mitochondrial/caspase-9 pathway
.
Exp Cell Res
2002
;
276
:
214
22
.
39
Mihara
M
,
Erster
S
,
Zaika
A
, et al
. 
p53 has a direct apoptogenic role at the mitochondria
.
Mol Cell
2003
;
11
:
577
90
.
40
Kurrey
NK
,
Jalgaonkar
SP
,
Joglekar
AV
, et al
. 
Snail and slug mediate radioresistance and chemoresistance by antagonizing p53-mediated apoptosis and acquiring a stem-like phenotype in ovarian cancer cells
.
Stem Cells
2009
;
27
:
2059
68
.
41
Wang
SP
,
Wang
WL
,
Chang
YL
, et al
. 
p53 controls cancer cell invasion by inducing the MDM2-mediated degradation of Slug
.
Nat Cell Biol
2009
;
11
:
694
704
.
42
Bolos
V
,
Peinado
H
,
Perez-Moreno
MA
,
Fraga
MF
,
Esteller
M
,
Cano
A
. 
The transcription factor Slug represses E-cadherin expression and induces epithelial to mesenchymal transitions: a comparison with Snail and E47 repressors
.
J Cell Sci
2003
;
116
:
499
511
.

Supplementary data