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. 2010 Aug 15;70(16):6566-76.
doi: 10.1158/0008-5472.CAN-10-0942. Epub 2010 Aug 3.

Targeting wild-type and mutant p53 with small molecule CP-31398 blocks the growth of rhabdomyosarcoma by inducing reactive oxygen species-dependent apoptosis

Jianmin Xu  1 , Laura Timares, Clay Heilpern, Zhiping Weng, Changzhao Li, Hui Xu, Joseph G Pressey, Craig A Elmets, Levy Kopelovich, Mohammad Athar
Affiliations

Targeting wild-type and mutant p53 with small molecule CP-31398 blocks the growth of rhabdomyosarcoma by inducing reactive oxygen species-dependent apoptosis

Jianmin Xu et al. Cancer Res. .

Abstract

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-G(0) and G(1) 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.

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Figures

Figure 1
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 hours then stained with propidium iodide (PI) for immediate flow cytometric analysis. A, Loss of viable cells. Two parameter psuedocolor density plots of total un-gated events for forward 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 upper left corner of each un-gated panel. The small circular gate encompasses viable cells. The percentage of cells is displayed in the lower right corner of each un-gated panel. A graph summarizing the % of viable cells within the single cell gate is shown at the bottom. B, Increased 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 percentage of Annexin V positive cells within PI negative (early apoptosis) and PI positive (late apoptosis) subsets are shown in the upper left and right quadrants, respectively. A graph displaying total apoptotic and dead cells (% Annexin + PI) is shown at the bottom. The percentage of PI stained cells is shown in histogram, and a summary graph is shown at the bottom. Note the inverse correlation of viable cells (panel for A), indicated by light scatter properties (small gate) and apoptotic cells (central and right panel graphs in B). C, G1 phase block of cell cycle. A204 cells were incubated with the indicated concentration of CP-31398for 24 hrs and DNA 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 ± SEM (n=3) for the percent of cells in each phase. *Student's t-Test (2-sided) P < 0.05. 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 2
Figure 2. CP-31398 induces apoptosis in A204 cells by translocating p53 to mitochondria, and alters mitochondrial membrane potential (MP) in A204 cells which can be blocked by Cyclosporin A (CsA) pretreatment
A, IF staining showing co-localization of p53 with MitoTracker, which stains mitochondria; B, IF staining showing that CsA blocks CP-31398–induced p53 localization to mitochondria; C, CsA blocked CP-31398–induced cytochrome c release in the cytoplasm; and D, CP-31398-mediated alterations in MP are attenuated by CsA treatment. For experiments described in A to C, A204 cells were treated with PBS (control) or CP-31398 (20μg/ml) for various time intervals. However, for experiments described in D, cells were treated with PBS (control) or CP-31398 (20 and 40 μg/ml) for 15 minutes, and then stained with JC-1 dye. Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was used as positive control. CsA (1μM) pretreatment was done for 15 min prior to CP treatment. Each value represents mean ± S.D. of three independent experiments. a P < 0.05 when compared to control; b P < 0.01 when compared to control; c P < 0.05 when compared to 20μg/ml CP-31398 treatment.
Figure 3
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 pre-treating 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 mM NAC; B, Pretreatment of CsA blocked CP-31398–induced generation of ROS. ROS generation was measured by using the ROS-detecting fluorescent dye, DCF-DA on a flow cytometry. The corresponding linear diagram of FACScan is also shown (n=3, mean ± S.D; **P < 0.01). Cells were treated with CP-31398 (20μg/ml) for 15min. Each value represents mean ± S.D. of three independent experiments (n=3, *P < 0.01).
Figure 4
Figure 4. CP-31398 treatment reduces the growth xenograft tumors and augments mesenchymal-epithelial transition 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 indicated by red arrow and apoptosis indicated by green arrow); C, TUNEL, IF and IHC 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 MMP2/9, snai, slug, twist and fibronectin in xenograft tumors. D1 represents CP-31398 daily treatments (24h intervals) whereas D2 represents twice daily (12h intervals) treatments. Mice were treated over 4 weeks beginning treatment at the time of tumor cell inoculation. Each value represents mean ± S.E. of 10 mice (*P < 0.05, **P < 0.01).
Figure 5
Figure 5. CP-31398 treatment induces SOX9 expression which co-localizes in cells showing induction of p21 but does not co-localize with TUNEL-positive cells in A204 xenograft tumors
A, CP-31398-induced expression of SOX9 (red arrows) which does not co-localize with TUNEL-positive (green arrows) cells; and B, CP-31398-induced expression of SOX9 that co-localizes with p21 expression(yellow arrow); C, Western blot showing the kinetics of SOX9 and p21 induction in A204 cells treated with CP-31398 (20μg/ml) for various time intervals. Insets show a 40X magnification.
Figure 6
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 24h interval whereas D2 represents twice daily treatments at 12h interval. B, Histology of tumors developed in vehicle or CP-31398-treated mice (insets show mitotic figures with red arrows and apoptosis with green arrows). IHC and TUNEL staining showing that CP-31398-treatment reduces proliferation as assessed by PCNA, 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, 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 weeks starting at the time of tumor inoculation. Each value represents mean ± S.E. of 5 mice.
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