ABSTRACT
Mutation in the tumor suppressor gene p53 is the most frequent molecular defect in endometrial carcinoma (EC). Recently, CP-31398, a p53-stabilizing compound, has been indicated to possess the ability to alter the expression of non-p53 target genes in addition to p53 downstream genes in tumor cells. Herein, we explore the alternative mechanisms underlying the restoration of EC tumor suppressor function in mutant p53 by CP-31398. A p53-mutated EC cell was constructed in AN3CA cells with restored or partial loss of Slug using lentiviral vectors, followed by treatment with 25 µM CP-31398. A p53-independent mechanism of CP-31398 was confirmed by the interaction between mouse double minute 2 homolog (MDM2) and Slug AN3CA cells treated with IWR-1 (inhibitor of Wnt response 1). Furthermore, the AN3CA cells were treated with short hairpin RNA against Slug, Wnt-specific activators (LiCl) or inhibitors (XAV-939) followed by CP-31398 treatment. Moreover, AN3CA cell proliferation and apoptosis were examined. A tumorigenicity assay was conducted in nude mice. CP-31398 could promote the apoptosis of p53-mutated EC cells, while Slug reversed this effect. Slug ubiquitination was found to occur via binding of Slug to MDM2 in AN3CA cells. We found that CP-31398 increased the GSK-3ß, p-Slug, Puma, Wtp53, and Bax expressions whereas Wnt, Mtp-53, Slug, Bcl-2, and Ki-67 expressions were decreased. However, these findings were reversed following the activation of the Wnt pathway and overexpression of Slug. Finally, the in vivo experimental evidence confirmed that CP-31398 with depleted Slug suppressed tumor growth by downregulating the Slug. Collectively, CP-31398-regulated Slug downregulation represses the p53-mutated EC via the p53/Wnt/Puma pathway.
Subject(s)
Apoptosis/drug effects , Cell Proliferation/drug effects , Endometrial Neoplasms/drug therapy , Pyrimidines/pharmacology , Tumor Suppressor Protein p53/metabolism , Animals , Cell Line, Tumor , Endometrial Neoplasms/metabolism , Female , Humans , Mice, Nude , Proto-Oncogene Proteins c-mdm2/drug effects , Proto-Oncogene Proteins c-mdm2/metabolism , Tumor Suppressor Protein p53/geneticsABSTRACT
CP-31398, a styrylquinazoline, emerges from a screen for therapeutic agents that restore the wild-type DNA-binding conformation of mutant p53 to suppress tumors in vivo, but its effects on cervical cancer (CC) remain unknown. Hence, this study aimed to explore the effects CP-31398 has on the CC cells and to investigate whether it is associated with paired box 2 (PAX2) expression. CC cells were treated with different concentrations of CP-31398 (1, 2, 4, 6, 8, and 10 µg/ml) to determine the optimum concentration using fluorometric microculture cytotoxicity assay. After constructing the sh-PAX2 vector, CC cells were transfected with sh-PAX2 or treated with CP-31398. The effects of CP-31398 or PAX2 silencing on CC cell proliferation, apoptosis, invasion, and migration were evaluated. Epithelial mesenchymal transition (EMT)-related genes such as E-cadherin, vimentin, N-cadherin, snail, and twist in CC cells were detected. Tumor formation experiment in nude mice was performed to observe tumor growth. The optimum concentration of CP-31398 was 2 µg/ml. PAX2 was overexpressed in CC cells. CC cells treated with CP-31398 or treated with sh-PAX2 inhibited proliferation, invasion, and migration but promoted apoptosis with decreased PAX2 expression. The EMT process in CC cells was also reversed after treatment with CP-31398 or sh-PAX2. Moreover, the tumor formation experiment in nude mice revealed the inhibitory activity of CP-31398 in CC tumor in nude mice by suppressing PAX2. Our results provide evidence that CP-31398 could inhibit EMT and promote apoptosis of CC cells to curb CC tumor growth by downregulating PAX2.