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Purpose:  Despite all the efforts for discovery of efficient anti-cancer therapeutics, cancer is still a major health concern worldwide. p28 is a bacterial small peptide which has been widely investigated due to its preferential cell internalization and anti-cancer activities. Intracellularly, p28 offers its anti-cancer traits by impeding the degradation of tumor-suppressor protein "p53". In this study, we investigated the potency of p28 in inducing apoptosis or decreasing cell viability in p53-null "HeLa" cell line.  Methods:  The coding sequence for p28 peptide was obtained from Pseudomonas aeruginosa by PCR amplification of the p28 gene. The coding gene was cloned in pET-28a vector and transformed into E. coli bacterial host. Subsequently, the expressed peptide was purified using Ni-NTA chromatography system and introduced into the target cells. The anti-proliferative and apoptotic activity of p28 on HeLa and HEK-293 cells were investigated using MTT and PEAnnexin V Flowcytometry assays.  Results:  Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting confirmed the expression of p28 peptide in the bacterial host. Bradford assay revealed a concentration of 0.05 mg/mL for the purified p28. MTT assay of cells treated with p28 at concentrations of 0, 0.5, 1, 2 and 2.5 µM indicated 24h viability values of 97%, 89%, 88%, 87% and 84% for HeLa cells, respectively. Data obtained from flowcytometry analyses revealed 24h apoptosis rate of 7.17%, 8.05%, 8.63% and 8.84% for HeLa cells treated with 0, 0.5, 1, and 2 µM p28, respectively.  Conclusion:  MTT and flowcytometry apoptosis assays suggest no statistically significant effect of p28 on the viability and apoptosis status of p53-null HeLa cells when results compared to data obtained from HEK-293 cells (P&gt;0.05). These results imply that anti-cancer efficacy of p28 is directly dependent on the presence of p53, suggesting p28 as an inappropriate therapeutic agent for treatment of cancers with negative p53 status.

The Effect of Bacterial Peptide p28 on Viability and Apoptosis Statusof P53-null HeLa Cells