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In this study we investigated corresponding precursor and active forms of a p53 small molecule inhibitor for effect on temozolomide (TMZ) anti-tumor activity against glioblastoma (GBM), using both in vitro and in vivo experimental approaches. Results from in vitro cell viability analysis showed that the cytotoxic activity of TMZ was substantially increased when GBMs with wild-type p53 were co-treated with the active form of p53 inhibitor, and this heightened cytotoxic response was accompanied by increased PARP cleavage as well as elevated cellular phospho-H2AX. Analysis of the same series of GBMs, as intracranial xenografts in athymic mice, and administering corresponding p53 inhibitor precursor, that is converted to the active compound in vivo, yielded results consistent with the in vitro analyses: i.e., TMZ + p53 inhibitor precursor co-treatment, of three distinct wild-type p53 GBM xenografts, resulted in significant enhancement of TMZ anti-tumor effect relative to treatment with TMZ alone, as indicated by serial bioluminescence monitoring as well as survival analysis (p < 0.001 for co-treatment survival benefit in each case). Mice receiving intracranial injection with p53 null GBM showed similar survival benefit from TMZ treatment regardless of the presence or absence of p53 inhibitor precursor. In total, our results indicate that the p53 active and precursor inhibitor pair enhance TMZ cytotoxicity in vitro and in vivo, respectively, and do so in a p53-dependent manner.
Attempts and approaches at manipulating p53 activity in treating human cancer have been numerous and diverse. For example, viral-mediated introduction and expression of wild-type TP53 in p53-defective tumor cells has been extensively investigated for more than a decade, including through clinical trial activity (1). Alternative approaches for increasing tumor cell wild-type p53 activity include the use of small molecules that promote p53 transcription, and the use of compounds that inhibit p53’s interaction with mdm2 (2).
Perhaps because of it being counter to conventional thinking about the role of tumor suppressor genes in cancer etiology, as well as being counterintuitive regarding the way in which tumor suppressor genes are viewed in relation to the treatment of cancer, there has been relatively little research directed towards the development of anti-tumor therapeutic strategies that include a p53 inhibitory component. Indeed, as a monotherapy, such a treatment approach could promote increased tumor cell proliferation and decreased tumor cell apoptosis. However, the potential consequences of attempted cell cycling by tumor cells with damaged DNA, resulting from genotoxic therapy with concurrent inhibition of p53, are interesting to consider. In fact, results from several studies, involving in vitro investigation of tumor cell lines, support enhanced cytotoxic chemotherapeutic response in association with p53 inhibition (3–6). Furthermore, with respect to GBM, the p53 small molecule inhibitor pifithrin-α, which was identified nearly a decade ago in association with a chemical library screen (7), has been shown to enhance in vitro cytotoxic effect of temozolomide (TMZ), a DNA alkylator, as well as the cytotoxic effect of chloroethylating nitrosoureas such as carmustine (8, 9).
In addition to reasons described above, in vivo investigation of p53 small molecule inhibitors, as part of a cancer treatment strategy, has been hindered due to limitations imposed by physical properties of the pifithrin-α reference compound (10). Recently, however, derivatives of the reference compound were described with respect to their potential in vivo use (11). In the current study we have tested one of these compounds, using an intracranial GBM xenograft therapy-response model, and present results indicating its enhancement of TMZ anti-tumor activity in vivo, and in a manner that is dependent on tumor cell p53 status.
GBM xenografts used in this study have been previously described (12,13), as has the modification of xenografts for bioluminescence imaging (13). Culturing of xenograft cells were as non-adherent neurospheres in neurobasal media (Invitrogen, San Diego, CA), while U87 cells (American Type Culture Collection) were propagated as monolayer cultures in DMEM supplemented with 10% fetal calf serum. Temozolomide (TMZ: obtained as Temodar from Schering-Plough, Kenilworth, NJ) and active form p53 inhibitor (cyclic pifithrin-α p-nitro, Calbiochem, San Diego, CA) were dissolved in dimethyl-sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO) as 20 and 5 mM stock solutions, respectively. For bioluminescence viability analysis, cells were treated with DMSO, TMZ (added to concentrations of 50 or 100 μM), p53 inhibitor (concentration of 10 μM), or a combination of TMZ and p53 inhibitor, with chemical agents added to media 1x/day for 3 consecutive days. Cell culture specimens were examined for bioluminescence signal using a Xenogen imaging system (Caliper Life Sciences, Alameda, CA), following the addition of 25 μl of 20 mg/ml sodium luciferin (Gold Biotechnology, St. Louis, MO) in phosphate buffered saline (PBS, Invitrogen).
U87 cells were treated 1x/day for 3 days with DMSO only, 10 μM p53 inhibitor, 50 or 100 μM TMZ, or 50 or 100 μM TMZ + 10 μM p53-inhibitor. At 1, 4, and 7 days following final treatment the cells were harvested, washed with PBS, and fixed with cold 70% ethanol. Cells were stained with propidium iodide and examined by flow-cytometry (BD LSR II, Becton-Dickinson, Franklin Lakes, NJ), with results analyzed using FlowJo software (Ashland, OR).
Primary antibodies used for immunoblot analysis (previously described: see reference 13) were for detection of PARP (Cell Signaling Tech, Danvers, MA), phospho-histone H2AX (Cell Signaling Tech), p53 (Sigma-Aldrich), p21 (Santa Cruz Biotech, Santa Cruz, CA), MGMT (R&D Systems, Minneapolis, MN), or beta-tubulin (Millipore, Danvers, MA). Secondary antibodies used were either anti-mouse or anti-rabbit (Cell Signaling Tech), or anti-goat (Santa Cruz Biotech).
Anyalysis of MGMT promoter methylation was as described by Esteller et al (14). PCR products were resolved in 4.5% agarose gels (NuSieve 3:1, Lonza, Inc., Allendale, NJ), and were subsequently stained using ethidium bromide.
Procedures used for intracranial tumor therapy-response experiments, including monitoring of tumor growth and response to therapy by bioluminescence imaging, have been previously described (13). Treatment groups for the experiments reported here were as follows: three days intraperitoneal DMSO and oral suspension vehicle (OraPlus, Paddock Laboratories, Minneapolis, MN) by gavage (control group); three days 10 mg/kg TMZ in oral suspension vehicle for GBM 12, GBM 14 and U87, and 50 mg/kg for GBM 26; three days 0.25 mg p53 inhibitor precursor (pifithrin-α p-nitro, Calbiochem) in DMSO by intraperitoneal injection; or 3 days combination TMZ and p53 inhibitor as indicated for their administration as single agents.
GBM xenograft explant cultures were incubated with TMZ +/− cyclic pifithrin-α p-nitro (Supplementary Figure 1: see reference 11) to assess in vitro effects of this p53 inhibitor on tumor cell TMZ cytotoxic response. Results for p53 wild-type (p53wt) GBM 26 showed substantial decreases in viable cell number resulting from TMZ + p53 inhibitor co-treatments, in relation to cells treated with TMZ alone (Figures 1A, 1B). Importantly, cyclic pifithrin-α p-nitro alone showed no anti-tumor effect. In contrast to the results for GBM 26, GBM 12 cells, which express no endogenous p53 (p53null: see reference 12), showed similar cytotoxic response to TMZ irrespective of the presence or absence of the p53 inhibitor (Figure 1C). As shown by others (11), cyclic pifithrin-α p-nitro inhibits DNA damage-associated induction of the negative cell cycle regulator p21, but only in p53wt cells (Figure 1D). Also, and consistent with the p53 small molecule inhibitor treatments, pre-treating cells with p53 siRNA enhanced TMZ cytotoxic response of GBM 26 cells, relative to pretreating cells with non-specific siRNA, whereas GBM 12 cells responded similarly to TMZ whether pre-incubated with p53 or non-specific siRNA (Supplementary Figure 2).
To further evaluate cyclic pifithrin-α p-nitro for in vitro activity, as well as to contrast results from the use of this compound with those previously reported for the pifithrin-α reference compound (Supplementary Figure 1C; see reference 8), U87 cells were incubated with TMZ +/− cyclic pifithrin-α p-nitro and examined by flow cytometric cell cycle analysis. Similar to results reported for the reference compound (8), higher sub-G1 cell fractions were observed in samples treated with TMZ + cyclic pifithrin-α p-nitro, relative to TMZ treatment alone. Sub-G1 ratios for U87 cells subjected to co-treatment vs. treatment with 100 μM TMZ only were 1.97, 2.25, and 1.54, at days 1, 4, and 7 following final treatment, respectively (Figures 2A, 2B). Consistent with the GBM xenograft explant culture cytotoxic response results described above (Figure 1), results from U87 flow cytometry analysis showed little difference in cell cycle distributions between U87 treated with cyclic pifithrin-α p-nitro vs. mock-treated control cells (Figure 2A).
Immunoblot analysis of U87 cells incubated with TMZ +/− cyclic pifithrin-α p-nitro showed increased PARP cleavage resulting from combined treatment, in comparison with cells treated with the same concentration of TMZ alone (Figure 2C), thereby suggesting increased caspase activity in association with the enhanced cytotoxic response to TMZ + p53 inhibitor. Heightened apoptotic response to combination treatment was also indicated by increased phospho-H2AX in cells incubated with TMZ + p53 inhibitor, relative to cells incubated with the same concentration of TMZ alone. Levels of p53 protein were similar between culture treatments (Figure 2C), consistent with the inhibitory activity of pifithrin-α compounds involving suppression of existing p53 activity, rather than suppression of p53 expression.
Previous study of pifithrin-α p-nitro derivatives has revealed that 2-aminothiazole salt cyclization (Figures 1A, 1B) is required for the formation of active p53 inhibitor, and that this cyclization occurs spontaneously in vivo, albeit over a period of several hours (11). Because active form p53 inhibitors have relatively short half-lives in vivo, it has been suggested that the kinetics of inactive, open form precursor conversion to corresponding cyclized, active form compound can be used to advantage for achieving prolonged cell exposure to active state inhibitors in vivo, and thereby achieve prolonged p53 inhibitory effect (11). In accord with this line of reasoning, we investigated the open form precursor of cyclic pifithrin-α p-nitro (Supplementary Figure 1A) for effect on TMZ in vivo anti-tumor activity, using a previously-described intracranial GBM xenograft therapy-response model (13). Mice injected with p53wt tumor cells (GBM 14, GBM 26, and U87), or with cells from p53null GBM 12, were monitored for luminescence increases indicative of log phase growth of injected cells, at which time TMZ (gavage) and p53 inhibitor precursor (intraperitoneal injection) were administered singularly or concurrently 1x/day for three consecutive days (treatment periods indicated by vertical gray bars, Figures 3A, 3B). For mice injected with p53wt GBM, survival was significantly extended by combined TMZ + p53 inhibitor treatment, relative to TMZ treatment alone, and this effect was observed for three p53wt tumors (p < 0.001 for each: Figure 3A, Table 1). Survival benefits were consistent with corresponding results from bioluminescence monitoring, which revealed prolonged decrease of p53wt intracranial tumor luminescence in mice receiving TMZ + p53 inhibitor (Figure 3B). It is worth noting that p53 inhibitor precursor enhancement of TMZ cytotoxicity was observed irrespective of p53wt GBM sensitivity to TMZ alone, as TMZ resistant GBM 26, with unmethylated MGMT promoter and highly expressed MGMT protein, as well as GBM 14 and U87, in which MGMT promoter methylation is accompanied by a lack of detectable MGMT protein (Figures 3C, 3D), all showed significantly increased response to combination treatment. Conversely, mice with p53null GBM 12 intracranial tumor did not experience significant survival benefit from combined TMZ + p53 inhibitor precursor treatment, in comparison to mice receiving treatment with TMZ only (Figure 3A, Table 1).
Research for examining potential clinical applications of p53 small molecule inhibitors has often involved experimental paradigms that address the protection of normal cells, rather than determining effects on tumor cell response to therapy that includes p53 inhibition. Specifically, there is considerable interest regarding whether p53 small molecule inhibitors can be used to spare normal cells from p53-mediated apoptosis that occurs from oxidative stress during stroke, renal injury, or cardiac arrest (15), as well as from the genotoxic effects of cancer treatments (7). Small molecule inhibition of p53 in tumor cells, for enhancing the anti-cancer effects of therapy, has not attracted much attention, even though there is ample, related evidence in support of this concept. This support, however, primarily stems from the use of viral gene product expression (4) and RNA interference (3, 6) to suppress tumor cell p53 function, and the perspective of the associated studies has been to investigate the role of p53 as a determinant of tumor cell response to cytotoxic therapy, rather than to pursue p53 as a target for augmenting the effects of therapy.
In the current study we have contrasted p53wt vs. p53null GBM xenograft response to cytotoxic therapy that includes the use of a small molecule inhibitor of p53. Significantly extended survival benefit from combination therapy was evident among series of mice with intracranial p53wt GBM, whereas mice with intracranial p53null GBM did not experience survival benefit from combination therapy. The issue regarding the response of p53 missense mutants to p53 inhibitor treatment is complex due to the diversity of p53 mutations in human cancer, the distinct transcriptional effects of individual p53 mutations, and the transcription-independent cellular activities of mutant p53 (16–18). Current studies are in progress to address the variability of p53 missense mutant GBM response to p53 inhibitor augmented cytotoxic therapy.
There are numerous candidates to consider as mediators of p53 inhibitor enhanced cytotoxic response, with our results currently supporting more extensive investigation of p53 inhibitor mediated suppression of p21 (Figure 1D), as well as investigation of apoptotic response effectors that are inferred by our preliminary results (Figure 2; Supplementary Figure 3). There are, of course, additional aspects of this cytotoxic response to be investigated, one of which is indicated in a recent report showing that p53 inhibition can suppress TMZ associated induction of MGMT (19). In the case of the tumor cell sources used here, this could be a contributing factor associated with p53 inhibitor enhancement of TMZ cytotoxicity against GBM 26, for which MGMT is unmethylated and expressed, but is probably less likely for GBM 14 and U87 tumors, for which MGMT is presumably silenced through MGMT promoter methylation (Figure panels 3C and 3D).
In addition to investigating the molecular biology of pathways and proteins associated with enhanced TMZ cytotoxic effect through concurrent p53 inhibition, the identification of surrogate markers that can be used to assess p53 inhibitor activity in tumors is critically important. The ability to analyze tumors for temporal variations in the expression and/or activity of biomarkers that respond to p53 inhibition, in combination with corresponding analysis of tumor extracts for temporal variations in p53 inhibitor content (e.g., through use of HPLC), will provide key information regarding inhibitor pharmacodynamics for optimizing combination therapy dosing and sequencing.
Results presented here provide some basis for considering the subgroup of GBM that would benefit from therapy that includes p53 inhibition. As concerns this point, it is significant that p53 inhibition enhances TMZ cytotoxicity in p53wt tumors irrespective of their sensitivity to TMZ monotherapy. Consequently, this therapeutic approach could conceivably benefit the two-thirds of GBM patients whose tumors are of wild-type p53 status (20). Looking beyond the GBM-specific interests of this report, it seems plausible that the results of our study are generalizable to other types of cancer, as well as to other types of genotoxic therapy.
An additional observation associated with this study is that TMZ + p53 inhibitor treatment is without adverse effect, as evidenced by autopsy analysis of animal subjects that showed no abnormal cellular proliferation in tissue from mice receiving TMZ + p53 inhibitor treatment (e.g., gastrointestinal tract: supplementary Figure 4A). This, in combination with the long term survival of mice that received TMZ + p53 inhibitor (see GBM 14 results in Figure 3A), as well as the similarity in group body weight patterns for TMZ only and TMZ + p53 inhibitor treated mice (Supplementary Figure 4B), suggest a lack of acute as well as of long-term effects of this therapy. Nonetheless, the simultaneous administration of a DNA damaging agent with an inhibitor of DNA repair could promote undesirable systemic side effects, including the development of independent cancers in other organs. To address this concern, future research should perhaps include strategies for the local delivery of inhibitor to tumor, in order to minimize side effects resulting from combination therapy. Irrespective of the methods of delivery that are used, the long-term consequences of this therapeutic approach will need to be investigated in detail.
Supported by NIH Grants NS049720, CA097257, CA108961, CA127716, CA100011