The SIRT2-p53 axis is involved in the antileukemic effect of bergamot juice extract and flavanones in an in vitro model of M5-subtype acute myeloid leukemia

Acute myeloid leukemia (AML) is one of the most alarming hematological malignancies, being frequently associated with the lowest survival rate compared to other types of leukemia. AML arises and progresses with the acquisition of new genetic and/or epigenetic modifications, responsible for its high genetic and clinical heterogeneity. Gene aberrations, such as the over-expression of SIRT2, an enzyme belonging to the histone deacetylases family, represent a key mechanism of AML pathogenesis. The SIRT2 upregulation was associated with proliferation and survival of AML cells and is particularly evident in AML patients belonging to the M5 subtype. Therefore, novel effective approaches and molecular targets are constantly researched to improve the life expentancy of AML patients. An ever-growing interest towards plant kingdom by the scientific community brought to light several bioactive molecules, including flavonoids, which stood out among others as potential anticancer agents. One of the most studied sources of this class of phytochemicals is Citrus fruits, whose species are widespread throughout the world. In particular, Citrus bergamia Risso (bergamot) has attracted the interest of the scientific community for its pharmacological properties, among which the anticancer properties demonstrated in a wide array of solid tumors, but not yet in the field of hematological malignancies, such as AML. Therefore, for the first time, we aimed at the investigation of the potential antileukemic activity of a flavonoid-rich extract of bergamot juice (BJe), along with the most common flavonones of the whole Citrus genus (i.e., naringenin – NAR, hesperetin -HSP, naringin – NRG, neohesperidin - NHP) in human leukemia THP-1 cell line, employed as in vitro model of AML-M5. Subsequently, acknowledged the SIRT2 crucial role in AML-M5 progression, we evaluated the potential inhibition of SIRT2 enzyme as an antileukemic mechanism, through in silico, cell-free and in vitro models. Our results demonstrated that BJe, NAR and HSP induced a significant reduction of THP-1 cell proliferation, without altering the growth of non-cancer peripheral blood mononuclear cells (PBMCs). The antiproliferative effects of BJe and the investigated flavanones were related to a cell cycle arrest in S phase and an induction of apoptosis. Both mechanisms were contextually confirmed at molecular level, where both BJe, NAR and HSP modulated the gene and protein expressions of S phase regulators, such as p21 and cyclin E1, as well as of apoptotic effectors, such as caspase 3. Interestingly, one of mechanisms responsible for the antiproliferative effects induced by BJe/NAR/HSP in leukemia THP-1 cells might also have been the inhibition of SIRT2 enzyme. This is because NAR and HSP were able to directly interact with the SIRT2 inhibitory site, as detected from molecular docking simulations. Moreover, both BJe, NAR and HSP, as well even more their equimolar combination (NAR-HSP) reduced the SIRT2 activity in the isolated enzyme (cell-free model). A SIRT2 inhibition by BJe, NAR, HSP and by their synergistic combination (NAR-HSP) was witnessed also in THP-1 cells, through an increase of acetylated p53, a specific substrate for SIRT2 deacetylation. Both BJe, NAR, and HSP were also able to reduce SIRT2 gene expression, yet only BJe significantly enhanced the gene and protein levels of the downstream factor p53. Our results suggested the role of BJe, NAR and HSP as potential antileukemic agents by targeting the SIRT2/p53 axis, which seems to be implied in both cell cycle arrest and apoptosis, induced in leukemia THP-1 cells. Therefore, BJe as well as the Citrus flavanones NAR and HSP were unveiled to be promising candidates in the AML-M5 managment by acting as SIRT2 inhibitors.