Cell Penetrating ARF Peptide (26-44)

The forkhead box m1 (Foxm1) transcription factor is essential for initiation of carcinogen-induced liver tumors; however, whether FoxM1 constitutes a therapeutic target for liver cancer treatment remains unknown. In this study, we used diethylnitrosamine/phenobarbital treatment to induce hepatocellular carcinomas (HCCs) in either WT mice or Arf(-/-)Rosa26-FoxM1b Tg mice, in which forkhead box M1b (FoxM1b) is overexpressed and alternative reading frame (ARF) inhibition of FoxM1 transcriptional activity is eliminated. To pharmacologically reduce FoxM1 activity in HCCs, we subjected these HCC-bearing mice to daily injections of a cell-penetrating ARF(26-44) peptide inhibitor of FoxM1 function. After 4 weeks of this treatment, HCC regions displayed reduced tumor cell proliferation and angiogenesis and a significant increase in apoptosis within the HCC region but not in the adjacent normal liver tissue. ARF peptide treatment also induced apoptosis of several distinct human hepatoma cell lines, which correlated with reduced protein levels of the mitotic regulatory genes encoding polo-like kinase 1, aurora B kinase, and survivin, all of which are transcriptional targets of FoxM1 that are highly expressed in cancer cells and function to prevent apoptosis. These studies indicate that ARF peptide treatment is an effective therapeutic approach to limit proliferation and induce apoptosis of liver cancer cells in vivo.

Background: 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. Objective: Our objective is to study the anticancer and antimicrobial properties of ARF-derived peptides and their cell-penetrating and NrLS conjugates. Methods: 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. Results: 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. Conclusion: 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.