nutlin-3a and Carcinoma--Squamous-Cell

Murine double minute 2 (MDM2) negatively regulates the activity of the p53 protein and plays a vital role in cell cycle arrest, apoptosis, and senescence mediated by p53. Nutlin-3, an antagonist of MDM2, is frequently used in anti-cancer studies. In many human tumors, nutlin-3 stabilizes p53 status and enhances p53 expression in cells with wild-type p53. However, the effect of nutlin-3 combined with radiotherapy on esophageal squamous cancer (ESCC) has not been reported. In this study, we examined whether nutlin-3 increases the radiosensitivity of ESCC in vitro and in vivo.We chose two cell lines, ECA-109 (wild-type p53) and TE-13 (p53 mutated), for the following experiments. Cell proliferation and clonogenic survival experiments showed that nutlin-3 inhibits the cell growth and colony formation of ECA-109 cells in a dose-dependent manner. Flow cytometry analysis showed that the apoptosis rate of ECA-109 cells co-treated with nutlin-3 and irradiation(IR) was significantly increased compared with cells treated with irradiation or nutlin-3 alone. Western blotting detected the expression of apoptosis-associated proteins in ECA-109 cells in response to nutlin-3 and irradiation. These effects were not evident in TE-13 cells. Xenograft mouse models indicated that nutlin-3 suppresses tumor growth and promotes radiosensitivity in the ESCC cell line ECA-109 in vivo. We have demonstrated that co-treatment of nutlin-3 with irradiation can significantly inhibit the growth and improve the radiosensitivity of ESCC cells with wild-type p53. The study suggests that nutlin-3 may be a potent therapeutic agent in conjunction with radiotherapy in ESCC.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Carcinoma, Squamous Cell; Cell Proliferation; Esophageal Neoplasms; Gamma Rays; Humans; Imidazoles; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Mutation; Piperazines; Proto-Oncogene Proteins c-mdm2; Radiation Tolerance; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

We evaluate whether p53-reactivating (p53RA) small molecules induce p53-dependent apoptosis in head and neck squamous cell carcinoma (HNSCC), a question that has not been previously addressed in head and neck cancer. PRIMA-1, CP-31398, RITA, and nutlin-3 were tested in four human HNSCC cell lines differing in TP53 status. Cell growth, viability, cell cycle progression, and apoptosis after treatment with p53RA small molecules individually or in combination with chemotherapeutic agents were assessed. Prominent p53 reactivation was observed in mutant TP53-bearing tumor cell lines treated with PRIMA-1 or CP-31398, and in wild-type TP53-bearing cell lines treated with nutlin-3. Cell-cycle arrest and apoptosis induced by p53RA small molecules were associated with upregulation of p21 and BAX, and cleavage of caspase-3. Nutlin-3 showed maximal growth suppression in tumor cells showing MDM2-dependent p53 degradation. High-dose treatment with p53RA small molecules also induced apoptosis in cell lines independent of p53 or MDM2 expression. In combination therapy, p53RA small molecules enhanced the anti-tumor activity of cisplatin, 5-fluorouracil, paclitaxel, and erlotinib against HNSCC cells. The p53RA small molecules effectively restored p53 tumor-suppressive function in HNSCCs with mutant or wild-type TP53. The p53RA agents may be clinically useful against HNSCC, in combination with chemotherapeutic drugs.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Combined Modality Therapy; Drug Screening Assays, Antitumor; Female; Furans; Genetic Therapy; Head and Neck Neoplasms; Humans; Imidazoles; Male; Piperazines; Pyrimidines; Quinuclidines; Tumor Suppressor Protein p53

Primary radiotherapy (RT) is a mainstay of treatment for laryngeal squamous cell carcinoma (LSCC). Although the cure rates for early (T1) vocal cord tumours are high, RT proves ineffective in up to a third of T3 carcinomas. Moreover, RT is associated with debilitating early- and late-treatment-related toxicity, thus finding means to de-escalate therapy, while retaining/augmenting therapeutic effectiveness, is highly desirable. p53 is a key mediator of radiation responses; we therefore investigated whether Nutlin-3, a small-molecule inhibitor of MDM2 (mouse double minute 2; an essential negative regulator of p53), might radiosensitise LSCC cells.. We performed clonogenic assays to measure radiosensitivity in a panel of LSCC cell lines (for which we determined p53 mutational status) in the presence and absence of Nutlin-3.. LSCC cells harbouring wild-type p53 were significantly radiosensitised by Nutlin-3 (P<0.0001; log-rank scale), and displayed increased cell cycle arrest and significantly increased senescence (P<0.001) in the absence of increased apoptosis; thus, our data suggest that senescence may mediate this increased radiosensitivity.. This is the first study showing Nutlin-3 as an effective radiosensitiser in LSCC cells that retain wild-type p53. The clinical application of Nutlin-3 might improve local recurrence rates or allow treatment de-escalation in these patients.

Topics: Animals; Carcinoma, Squamous Cell; Cell Proliferation; Cell Survival; Cellular Senescence; Genes, p53; Imidazoles; Laryngeal Neoplasms; Piperazines; Proto-Oncogene Proteins c-mdm2; Radiation Tolerance; Radiation-Sensitizing Agents