Inhibiting bladder tumor growth with a cell penetrating R11 peptide derived from the p53 C-terminus

Abstract

Urothelial carcinoma of the bladder (UCB) is the most common malignancy of the urinary tract, nearly half of which contains a mutation in TP53 gene. Hence, therapeutic approach by restoring functional p53 protein in cancer cells will be beneficial. Recent studies have demonstrated the inhibition of cancer cell growth by p53 reactivation using a peptide derived from the p53 C-terminus (p53C). However, the outcome of reactivating p53 in controlling bladder cancer development is limited by its efficiency and specificity of peptide delivery, especially in metastatic animal models. Herein, we report that the cell penetrating peptide (polyarginine, R11)-conjugated p53C can exhibit a preferential uptake and growth inhibit of UCB cells expressing either mutant or wild-type TP53 by the activation of p53-dependent pathway. R11-p53C peptide treatment of preclinical orthotopic and metastatic bladder cancer models significantly decreased the tumor burden and increased the lifespan without a significant cytotoxicity. Based on these results, we believe that R11-p53C peptide has therapeutic potential for primary and metastatic bladder cancer, and R11-mediated transduction may be a useful strategy for the therapeutic delivery of large tumor suppressor molecules to tumor cells in vitro and in vivo.

Keywords: bladder cancer; cell penetrating peptide; metastatic tumor; p53 C-terminus; targeted therapy.

Figure 1. Uptake of R11-p53C and its effects on growth inhibition

A. The specific uptake of R11-conjugated peptides was determined in T24 cells using fluorometry. B. The localization of R11-p53C peptide and its control peptides (R11-Con. and p53C) was detected in T24 cells by fluorescence microscope. C–D. Inhibiting the proliferation of tumor cells expressing mutant p53 (i.e., T24) or wild-type p53 (i.e., 253J). Cell viability was determined by MTT assay. Results represent three different experiments, each of which was performed in six repeats.

Figure 2. The effect of R11-p53C on p53-regulated gene expression and growth inhibition

A. Dose-dependent induction of G1 cell cycle arrest by R11-p53C in T24 cells. Results represent three different experiments, each of which was performed in triplicates. *P < 0.05. B. Total cellular RNA was collected from T24 cells exposed to 5 μM R11-p53C or R11-Con. then subjected to qRT-PCR for determining PUMA, p21, GADD45A and PIG3 at the indicated time. C. T24 cells were pre-treated with pifithrin-α 20 μM (or DMSO) before 5 μM R11-p53C or R11-Con treatment for 48 hrs then subjected to western blot analysis. D. T24 cells were pre-treated with pifithrin-α (20 μM) or DMSO before 5 μM R11-p53C or R11-Con treatment for 48 hrs then subjected to MTT assay. Data represent mean and standard deviation from three different experiments; each experiment was carried out in triplicates.

Figure 3. Systemic R11-p53C peptide administration inhibited orthotopical tumor growth

A–B. T24 orthotopical tumor growth after R11-p53C treatment. T24-luc cells were instilled into the bladder to develop a superficial bladder cancer model (n = 6/group) at Day 0 and randomized to receive 200 μl of peptides and control twice a week and repeated 3 weeks. Tumor growth was determined. C. The survival curves were determined in every group. *P < 0.05. D. Representative pictures of p21 and Ki-67 IHC staining in T24 orthotopical xenograft tissues with or without R11-p53C treatment. The scale bar represents 100 μm. Quantification analyses of IHC score were shown (right panel).

Figure 4. Systemic R11-p53C peptide administration inhibited T24 lung metastatic tumor growth

A. R11-p53C peptide can be specifically detected in the lung metastatic lesions. Nude mice were intravenously injected with T24 cells for 14 days (n = 6/group). Tissues were collected 30 mins after R11-p53C and control peptides injected. I DMSO, II p53C, III R11-Con, IV R11-p53C. B–C. The BLI intensity was serially monitored (B) and quantified (C) in every group. D. The long-term survival of mice harboring lung metastatic burden after R11-p53C/control peptides/DMSO treatment.

Figure 5. In vivo toxicity of R11-p53C peptide

A. Levels of RBC, WBC and CRP from mice injected with R11-p53C or control peptides pretreated 24 hrs by intraperitoneal injection. B. Serum levels of AST, ALT and creatinine from mice injected with R11-p53C (20 mg/kg) or control peptides pretreated 72 hrs by intraperitoneal injection. C. Mice weights were measured at 3 days after injected with R11-p53C or control peptides. D. Spleen weights were measured from mice injected with R11-p53C or control peptides treated for 3 weeks. Means ±SD shown (n ≥ 3). Background levels were 43 ± 14.3 U/l for AST, and 29 ± 4.2 U/l for ALT. ALT, alanine aminotransferase; AST, aspartate aminotransferase; WBC, white blood cell; RBC, red blood cell; CRP, C-reactive protein.