Construction, Expression, and Purification of p28 as a Cell-Penetrating Peptide ‎with Anticancer Effects on Burkitt’s Lymphoma Cell Line

Abstract

Background:The p28 is a small-sized cell-penetrating peptide derived from bacterial protein azurin and can function as a cancerspecific anti-proliferative agent. It can penetrate cancer tissues easily without involving the immune system, and increase the intracellular concentration of p53. Objectives: In this study, we have expressed and purified recombinant p28, then evaluated its anti-proliferative and pro-apoptotic effects on Raji cancer cell line. Methods: The p28 gene was amplified and cloned into pTZ57R cloning vector and was sequenced subsequently. Afterward, it was transformed into E. coli BL21 bacterial host by using pET-28a expression vector. Peptide purification was carried out using Ni-NTA chromatography system. Bradford, SDS-PAGE, and western blotting assays were applied to assess the concentration and expression level of the recombinant peptide. The proficiency of p28 in inhibition of tumor growth and induction of apoptosis in cancerous cells was investigated by evaluating the Raji and HEK-293 cells treated with different concentrations of p28. Results: The overexpression of the p28 peptide in the bacterial host was confirmed by SDS-PAGE and western blotting. Moreover, Bradford assay revealed desirable concentrations of the recombinant p28 before and after dialysis. The MTT and PE-Annexin V apoptosis assays indicated the specific function of p28 in impeding the proliferation of cancerous cells and triggering the apoptosis. Conclusions: The p28 induces apoptosis in cancerous cells but not in normal control cells. In summary, p28 is a non-immunogenic small peptide that can penetrate cancerous cells preferentially, impede the cell proliferation, and induce the apoptosis. Overall, these findings suggest p28 as a promising anticancer drug.

Gut Metabolites and Breast Cancer: The Continuum of Dysbiosis, Breast Cancer Risk, and Potential Breast Cancer Therapy

“…To date, the only bacteriocin to enter phase I of a clinical trial is azurin-p28 peptide, which is a drug used in chemotherapy treatment and it demonstrated substantial activity in preclinical pharmacological studies [ 39 , 40 ]. Preclinical research supported the evaluation of this compound in a clinical setting, and azurin-p28 was observed to show selective cytotoxicity to cancer cells and inhibited cancer cell growth via the induction of apoptosis and inhibition of cell proliferation [ 78 , 79 ]. The primary goal of a two-part registered phase I clinical study (NSC745104) was to observe and determine the optimal safe therapeutic dose and potential adverse effects of azurin-p28 in treating adult patients with solid tumours and pediatric patients with advanced or recurring CNS malignancies by inhibiting p53 ubiquitination [ 39 , 40 ].…”

p28 Bacterial Peptide, as an Anticancer Agent

“…In addition to the cancer cell lines listed above, the preferential entry and anti-proliferative effect of p28 are exhibited on other human cancer cell lines including ovarian cancer cells (SK-OV3 adenocarcinoma and ES-2 ovarian), fibrosarcoma (HT1080), leiomyosarcoma (HTB-88), osteosarcoma (TE85), and rhabdomyosarcoma (RD); pancreatic cancer (MIA-Paca2); Burkitt's lymphoma cell line (Raji and HEK-293); and neuroblastoma (IMR-32 (p53 wt ), and SK-N-BE2 (p53 mut ) (21,28,70,73).…”

Effects of Alkanna bracteosa extract on the expression level of HSP90α and HER2 genes in human gastric cancer cell line

“…The cancer cell deficiencies categorized into two types which including loss-of-function alterations affecting tumor genes and gain-of-function alterations affecting oncogenes (Teh & Fearon 2020 ). The cancer treatment modalities which are reported, including surgery, radiation therapy, and systemic treatment, as well as chemotherapy, targeted therapy, hormonal therapy, and immunotherapy (Abuei et al 2019 ; Miller et al 2019 ; Pirouzfar et al 2020 ). Although radio- and chemotherapy are the most common and non-invasive cancer treatment methods, it can cause serious damage to the normal cells (Mortezaee et al 2019 ).…”