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The search of new pharmacological targets with original mechanism of action within the ubiquitin-proteasome pathway is still a goal to be reached in oncopharmacology. Modification by phosphorylation/dephosphorylation has been found to be involved in cancer and to regulate functional activity of proteasome. Until now, phosphorylated forms of alpha subunits of the 20S human proteasome have been mostly reported. Here, we have rationally designed a polyclonal antibody specifically directed against a phosphorylated peptide sequence bearing the beta7 subunit Ser249 residue of the human 20S proteasome. This anti-beta7 phosphoSer249 antibody appeared to be a probe of choice to detect the presence of a phosphorylated isoform of the beta7 subunit of the human 20S proteasome using mono or two-dimensional gel electrophoresis. PhosphoSer249 was sensitive to acid phosphatase treatment of native 20S proteasome. Dephosphorylation affected the peptidylglutamyl-peptide hydrolyzing activity whereas the chymotrypsin-like and trypsin-like activities remained unchanged. A comparative analysis between human normal and tumor cells showed a differential expression of the phosphoSer249 beta7 isoform with a significantly lower detection in the proteasome isolated from tumor cells, suggesting its possible use as a biomarker.

The pharmacologic properties of ajoene, the major sulfur-containing compound purified from garlic, and its possible role in the prevention and treatment of cancer has received increasing attention. Several studies demonstrated that induction of apoptosis and cell cycle blockade are typical biologic effects observed in tumor cells after proteasome inhibition. The proteasome is responsible for the degradation of a variety of intracellular proteins and plays a key role in the regulation of many cellular processes. The aim of the present work was therefore to explore the effects of ajoene on the proteasome activities. In vitro activities of 20S proteasome purified from human erythrocytes on fluorogenic peptide substrates specific for trypsin-like, chymotrypsin-like and peptidylglutamyl peptide hydrolyzing activities revealed that ajoene inhibited the trypsin-like activity in a dose- and time-dependent manner. Further, the ability of 20S proteasome to degrade the OVA(51-71) peptide, a model proteasomal substrate, was partially but significantly inhibited by ajoene. In addition, when human leukemia cell line HL60 was treated with ajoene, both trypsin- and chymotrypsin-like activities were affected, cells arrested in G2/M phase and total amount of cytosolic proteasome increased. All these data clearly indicate that ajoene may affect proteasome function and activity both in vitro and in the living cell. This is a novel aspect in the biologic profile of this garlic compound giving new insights into the understanding of the molecular mechanisms of its potential antitumor action.