doi: 10.1073/pnas.1222035110. Epub 2013 Jan 14.
Antitumor activity of a pyrrole-imidazole polyamide
Affiliations
- PMID:23319609
- PMCID: PMC3562772
- DOI: 10.1073/pnas.1222035110
Item in Clipboard
Antitumor activity of a pyrrole-imidazole polyamide
Fei Yang et al. Proc Natl Acad Sci U S A..
Display options
Format
Display options
Format
Abstract
Many cancer therapeutics target DNA and exert cytotoxicity through the induction of DNA damage and inhibition of transcription. We report that a DNA minor groove binding hairpin pyrrole-imidazole (Py-Im) polyamide interferes with RNA polymerase II (RNAP2) activity in cell culture. Polyamide treatment activates p53 signaling in LNCaP prostate cancer cells without detectable DNA damage. Genome-wide mapping of RNAP2 binding shows reduction of occupancy, preferentially at transcription start sites, but occupancy at enhancer sites is unchanged. Polyamide treatment results in a time- and dose-dependent depletion of the RNAP2 large subunit RPB1 that is preventable with proteasome inhibition. This polyamide demonstrates antitumor activity in a prostate tumor xenograft model with limited host toxicity.
Conflict of interest statement
The authors declare no conflict of interest.
Figures
Structure of polyamides1 and2.
Global effects of1 on RNAP2. Genome browser tracks of RPB1 occupancy from untreated, DHT-treated, and DHT +1-treated samples over (A) an AR-driven gene,KLK3 (PSA), and (B) a housekeeping gene,GAPDH. RNAP2 occupancy is mapped as reads per million. (C) Genomic RNAP2 occupancy at transcription start sites show comparable levels of enrichment for nontreated and DHT treated samples. Samples treated with DHT +1 exhibited much lower occupancy. (D) Genomic RNAP2 occupancy at enhancer regions is largely unchanged between the three treatment conditions. (E) Immunoblot of RPB1 protein in LNCaP cells treated with 1 μM doxorubicin (dox) for 16 h, or1 at 2 μM, 10 μM, and 20 μM for 48 and 72 h. (F) Quantitative RT-PCR measurement of RPB1 transcript levels after LNCaP cells are treated with 10 μM1 for the indicated times. Relative expression is normalized against nontreated cells. Data represent mean ± SD of biological quadruplicates.
Cytotoxicity of1 and2 and effects on RPB1. (A) Cytotoxicity of1 in LNCaP cells after incubation with1 for 72 h. Data represent mean ± SD. IC50 is calculated from three independent experiments and the error is a 95% confidence intervals. (B) Cell viability at 24 h of LNCaP cells treated with varying concentrations2 with and without proteasome inhibitor MG132 (3 μM, 24 h); proteasome inhibition reduces cytotoxicity of2. (C) Immunoblot of RPB1 protein in LNCaP cells treated with 10 μM2 for 12 h followed by 10 μM MG132 for 4 h. (D) Cytotoxicity of2 in LNCaP cells incubated with 10% FBS or with 0.5% FBS for 24 h. Serum starvation decreases percent of cells in the S phase from 8.5% to 4.4% (Fig. S2 ). Data represent mean ± SD.
Induction of p53 activity without evidence of DNA damage. (A) Induction of p53 target genes (GADD45A,MDM2,IGFBP3,P21,BAX) and DNA damage-inducible transcript 3 (DDIT3), by1 (10 μM) at 24, 48, and 72 h. Data represent the mean of four biological replicates and error bars represent SD. (B) Alkaline comet assay of LNCaP cells treated with vehicle, dox (5 μM, 4 h),1 (10 μM, 48 h). Error bars represents maximum and minimum; boxes represents the upper and lower quartiles and median. Representative comets for each treatment are shown. Effects of1 are indistinguishable from the nontreated control, but dox treatment significantly increases comet-tail percent of DNA.P = 0.00043. (C) DNA damage markers after treatment of LNCaP cells with1. There is no significant phosphorylation of DNA-PKcs, ATM, Chk2, p53, or γH2A.X. Accumulation p53 and PARP cleavage are observed. Data are representative of biological triplicates except for DNA-PKcs, which was in replicate.
Polyamide1 demonstrates antitumor activity in prostate cancer xenografts. (A) Male immunocompromised mice were engrafted with LNCaP cells and observed until tumors reached ∼100 mm3. Tumor-bearing mice were then treated with 20 nmol1 (n = 12) or vehicle (n = 13) by subcutaneous injections into the flank distal to the tumor once every 3 d for a total of three injections. Mice were killed and tumors resected and weighed 2 d after the final injection. Tumors from mice treated with1 were smaller (mean: 112 mg; median: 94 mg; range: 47–201 mg) than those of vehicle treated mice (mean: 310 mg; median: 292 mg; range: 173–440 mg). Error bars represents maximum and minimum; boxes represents the upper and lower quartiles and median.P = 1.6E-5. (B) Serum PSA measured by ELISA pre- and posttreatment. Serum PSA is lower in the posttreatment serum of mice treated with1 compared with vehicle.P = 0.024. (C) Selected tumors and histological stains of tumor cross-sections from mice treated with vehicle or1. (D) Treatment of LNCaP tumor bearing mice with1 increases serum uric acid compared with vehicle controls and polyamide-treated, nontumor-bearing mice.P = 3.2E-9.
References
- Derheimer FA, Chang CW, Ljungman M. Transcription inhibition: A potential strategy for cancer therapeutics. Eur J Cancer. 2005;41(16):2569–2576. - PubMed
- Jung Y, Lippard SJ. RNA polymerase II blockage by cisplatin-damaged DNA. Stability and polyubiquitylation of stalled polymerase. J Biol Chem. 2006;281(3):1361–1370. - PubMed
- Ljungman M, Zhang FF. Blockage of RNA polymerase as a possible trigger for u.v. light-induced apoptosis. Oncogene. 1996;13(4):823–831. - PubMed
- Ljungman M, Zhang FF, Chen F, Rainbow AJ, McKay BC. Inhibition of RNA polymerase II as a trigger for the p53 response. Oncogene. 1999;18(3):583–592. - PubMed
Publication types
MeSH terms
Substances
Associated data
- Actions
Grants and funding
LinkOut - more resources
Full Text Sources
Other Literature Sources
Medical
Molecular Biology Databases
Research Materials
Miscellaneous