Anticancer and Antimicrobial Evaluations on Alternative Reading Frame (ARF) Peptides and their Derivatives

Alternative reading frame (ARF) protein up-regulates the intracellular level of a tumour suppressor protein, p53, by blocking MDM2 mediated p53 ubiquitination. The two homologous forms of ARF proteins are p19ARF in mice and p14ARF in humans. In our study, p19ARF-derived peptide ARF (26-44) and its cell-penetrating peptide conjugate Tat-ARF (26-44), p14ARF-derived peptide ARF (1-22), and its NrLS conjugate ARF (1-22)-NrLS were designed, and their anticancer properties were investigated.

Our objective is to study the anticancer and antimicrobial properties of ARF-derived peptides and their cell-penetrating and NrLS conjugates.

Peptides synthesized using solid-phase peptide synthesis (SPPS) were purified using RPHPLC and characterized using Bruker MALDI-TOF mass spectrometry. Cytotoxicity was evaluated on HeLa and BE(2)-C cells by cell viability IC₅₀ determination. Minimum inhibitory concentrations (MIC) were determined by the broth microdilution method. Morphological studies were carried out using SEM and TEM techniques, live/dead staining, ROS and Hoest staining.

Peptides Tat-ARF (1-22) and ARF (1-22)-NrLS exhibited potent cytotoxic effects, comparable to the known standard cisplatin. Cellular morphological studies showed signs of apoptosis which were confirmed by reactive oxygen species (ROS) generation and Hoechst nuclear staining. ARF peptides showed potent antimicrobial activities at low micromolar concentrations without haemolysis.

Tat modification improved the activity of ARF (26-44) by 9 folds against HeLa and 5 folds against BE(2)-C cells. NrLS modification of ARF (1-22) imparted 12 fold potency against HeLa and 2-fold potency against BE(2)-C cells. This study helps to further understand the effect of these peptides on MDM2 proteins and their role in the apoptosis signalling pathway.