Radiosensitization by a novel therapy with p16 functioning peptide in prostate cancer

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The resistance of prostate cancers to radiation therapy has been linked to low expression of p16, which is an upstream regulator of the retinoblastoma (RB) pathway. p16 plays an important role to enhance the radiosensitivity either through the p53 or Bcl-2 pathway. A novel therapy with p16 functioning peptide, as the molecular targeting agent, seems to be less toxic and more compatible because it is one of the endogenous key molecules in human cells. Herein, we aim to confirm whether the p16 peptide conjugated with Wr-T transporter peptide combined with irradiation could render the prostate cancer cells more susceptible to the effects of radiation. A panel of human prostate cancer cell lines (PC-3 and DU-145: p16 wild type, LNCaP: p16 deletion) were subjected to irradiation with or without treatment of p16 (8uM)/Wr-T (5uM) conjugate. We performed cytotoxic, clonogenic, and cell cycle analyses in vitro and then assessed the in vivo efficacy, on tumor volume, in the PC-3 xenograft mouse tumor model by treating with the p16/Wr-T conjugate (p16: 20nmol/Wr-T: 40nmol) and radiotherapy (5Gy).
 In all of the prostate cancer cell lines, treatment with p16 /Wr-T conjugate significantly inhibited cell growth (0Gy (PC-3): control; 0%, p16; 11.0%, p16/Wr-T conjugate; 25.9%) and resulted in G1 cell cycle accumulation. Furthermore, combining irradiation at 5 Gy demonstrated enhanced in vitro killing efficacy (5Gy (PC-3): control; 14.9%, p16; 24.4%, and p16/Wr-T conjugate; 56.9%). Similarly, clonogenic survival results yielded consistent findings. We also confirmed effective incorporation of the peptide into cells by using FITC-labeled p16 peptide and fluorescence microscopy. Combination treatment with p16 /Wr-T conjugate and radiation significantly inhibited PC-3 xenograft tumors by approximately 30% over the control group. Combination therapy demonstrated increases of cellular apoptotic rates as well as decreases in proliferation rates as measured by the TUNEL method and PCNA immunohistochemistry, respectively.
 p16 functioning peptide conjugated with Wr-T could be a promising molecular targeting therapy against prostate cancer as well as a novel candidate for rendering cancer cells to become more radiosensitized.