The Effect of Bacterial Peptide p28 on Viability and Apoptosis Statusof P53-null HeLa Cells
| Autor: | Gholam Reza Rafiei Dehbidi, Ali Farhadi, Haniyeh Abuei, Farahnaz Zare, Abbas Behzad-Behbahani, Fatemeh Faghihi, Mohammad Pirouzfar |
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| Rok vydání: | 2019 |
| Předmět: |
p53
Cell Pharmaceutical Science Apoptosis HeLa 03 medical and health sciences 0302 clinical medicine medicine MTT assay Viability assay General Pharmacology Toxicology and Pharmaceutics 030304 developmental biology Gel electrophoresis 0303 health sciences Null HeLa cells biology Chemistry lcsh:RM1-950 p28 biology.organism_classification Molecular biology Blot lcsh:Therapeutics. Pharmacology medicine.anatomical_structure Viability Cell culture 030220 oncology & carcinogenesis Research Article |
| Zdroj: | Advanced Pharmaceutical Bulletin Advanced Pharmaceutical Bulletin, Vol 9, Iss 4, Pp 668-673 (2019) |
| ISSN: | 2251-7308 2228-5881 |
| Popis: | Purpose: Despite all the efforts for discovery of efficient anti-cancer therapeutics, cancer is stilla major health concern worldwide. p28 is a bacterial small peptide which has been widelyinvestigated 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 cellviability in p53-null "HeLa" cell line.Methods: The coding sequence for p28 peptide was obtained from Pseudomonas aeruginosaby PCR amplification of the p28 gene. The coding gene was cloned in pET-28a vector andtransformed into E. coli bacterial host. Subsequently, the expressed peptide was purified usingNi-NTA chromatography system and introduced into the target cells. The anti-proliferative andapoptotic activity of p28 on HeLa and HEK-293 cells were investigated using MTT and PEAnnexinV Flowcytometry assays.Results: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Westernblotting confirmed the expression of p28 peptide in the bacterial host. Bradford assay revealeda concentration of 0.05 mg/mL for the purified p28. MTT assay of cells treated with p28 atconcentrations 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 revealed24h 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 effectof p28 on the viability and apoptosis status of p53-null HeLa cells when results compared todata obtained from HEK-293 cells (P > 0.05). These results imply that anti-cancer efficacy of p28is directly dependent on the presence of p53, suggesting p28 as an inappropriate therapeuticagent for treatment of cancers with negative p53 status. |
| Databáze: | OpenAIRE |
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