Restoration of p53 Pathway by Nutlin-3 Induces Cell Cycle Arrest and Apoptosis in Human Rhabdomyosarcoma Cells

Abstract

Purpose: Seventy to eighty percent of rhabdomyosarcoma (RMS) tumors retain wild-type p53.The tumor suppressor p53 plays a central role in inducing cell cycle arrest or apoptosis in response to various stresses. p53 protein levels are regulated by MDM2 through ubiquitin-dependent degradation. In this study, we evaluated whether nutlin-3, a recently developed small-molecule antagonist of MDM2, has an effect on p53-dependent cell cycle arrest and apoptosis in cultured human RMS cell lines. Experimental Design: Five RMS cell lines with different p53 statuses and MDM2 expression levels were treated with nutlin-3. Gene expression patterns, cell viability, cell cycle, and apoptosis after nutlin-3 treatment, and antitumor activity of combination treatment with vincristine or actinomycin D were assessed. Results: Significant p53 activation was observed in wild-type p53 cell lines after nutlin-3 treatment. p53 activation led to cell cycle arrest in parallel with increased p21expression. Furthermore, these cell lines underwent p53-dependent apoptosis, concomitant with elevation of proapoptotic genes and activation of caspase-3. The effect of nutlin-3 was almost the same in terms of half maximal inhibitory concentration and apoptosis whether or not MDM2 was overexpressed. Nutlin-3 did not induce either cell cycle arrest or apoptosis in p53 mutant cell lines. A combination of vincristine or actinomycin D with nutlin-3 enhanced the antitumor activity in RMS cell lines with wild-type p53. Conclusions: Nutlin-3 effectively restored p53 function in both normal MDM2 expression and MDM2 overexpression RMS cell lines with wild-type p53. p53 restoration therapy is a potential therapeutic strategy for refractory RMS with wild-type p53.

Nutlin-3a Is a Potential Therapeutic for Ewing Sarcoma

“…However, because the synergistic effects of Nutlin-3a in combination with conventional cytotoxic agents were only observed in vitro using limited dose ranges of each of these agents, detailed preclinical pharmacokinetic studies of these combinations will be required. Nutlin-3a has been shown to synergize with these drugs in other sarcoma types, with previously reported CIs similar to those reported in this study at optimal dosages (15,16). Unfortunately, these previous studies were limited to investigations of synergy at a single dose of these agents.…”

“…Nutlin-3a disrupts the p53-MDM2 interaction by blocking the p53-binding pocket of MDM2, resulting in rapid stabilization of biologically active p53 protein (13). Promising results from several preclinical studies have clearly showed the therapeutic potential of Nutlin-3a in a variety of tumor types with wild-type p53, including liposarcoma (14), rhabdomyosarcoma (15), osteosarcoma (13), synovial sarcoma (16), neuroblastoma (17), retinoblastoma (18), and leukemia (19)(20)(21). In an effort to accelerate clinical implementation of p53 activators, Lane and colleagues screened a library of clinically approved drugs and successfully identified actinomycin D (ActD) as a compound that mimics the action of Nutlin-3a when administered at nongenotoxic "low doses" (22).…”

Nutlin-3a Efficacy in Sarcoma Predicted by Transcriptomic and Epigenetic Profiling

“…This is supported by several preclinical in vitro and mouse xenograft studies, which showed that Nutlin-3a can effectively activate the TP53 pathway, resulting in cell-cycle arrest or apoptosis in Ewing sarcoma (17,34), liposarcoma (7), osteosarcoma (8), rhabdomyosarcoma (35), and synovial sarcoma (36).…”

Analysis of TP53 gene expression and p53 level of human hypopharyngeal FaDu (HTB-43) head and neck cancer cell line after microRNA-181a inhibition

“…Real-time RT-PCR was performed using the Rotor-Gene SYBR Green PCR kit (Qiagen, Hamburg, Germany) in a Fast Real-time PCR System (Rotor Gene 6000). p53 mRNA was amplified using previously described primer pairs: 5'-AGA GTC TAT AGG CCC ACC CC -3'(forward) and 5'-GCT CGA CGC TAG GAT CTG AC-3' (reverse), and GAPDH 5'-CAT GGG GAA GGT GAA GGT CGG A-3' (forward) and 5'-TTG GCTCCC CCC TGC AAA TGA G -3' (reverse) (Miyachi et al, 2009). Relative target mRNA levels were determined using the delta-delta CT method (Schmittgen and Livak, 2008), with the formula given below (Livak and Schmittgen, 2001), and threshold value of 0.2, where the results were expressed in fold changes of treated cells compared with untreated cells.…”