Tabriz University of Medical Sciences Advanced Pharmaceutical Bulletin 2228-5881 9 4 2019 10 24 The Effect of Bacterial Peptide p28 on Viability and Apoptosis Status of P53-null HeLa Cells 668 673 10.15171/apb.2019.078 EN Haniyeh Abuei https://orcid.org/0000-0002-2044-0666 Abbas Behzad-Behbahani https://orcid.org/0000-0001-5917-920X Fatemeh Faghihi https://orcid.org/0000-0001-7374-3970 Ali Farhadi https://orcid.org/0000-0002-2271-670X Gholam Reza Rafiei Dehbidi Mohammad Pirouzfar https://orcid.org/0000-0002-8061-2503 Farahnaz Zare https://orcid.org/0000-0002-3746-4621 Journal Article 10.15171/apb.2019.078 2019 04 12 2019 06 18 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>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.