Evidence for the role of differentiation in the antiproliferative effects exerted by a flavonoid-rich extract of Citrus bergamia juice in an in vitro model of acute myeloid leukemia

Acute myeloid leukemia (AML) is a hematologic malignancy characterized by the arrest of normal differentiation of immature myeloid cells (blasts), which proliferate unceasingly, accumulating in the bone marrow and interfering with the normal process of hematopoiesis. In recent years, the use of drugs capable of inducing blast differentiation has brought significant improvements in the survival of leukemia patients. However, the so-called "differentiation therapy" is still limited to a few subtypes of leukemia and it is often associated with serious adverse effects as well as the onset of drug chemoresistance. Therefore, the search for innovative and safer therapeutic solutions, such as natural products, is absolutely necessary. In light of these considerations, the purpose of my PhD project was to evaluate the ability of a flavonoid-rich extract from bergamot juice (BJe) to exert antileukemic effects and to induce the differentiation of THP-1 leukemic monocytes, as well as the molecular mechanisms underlying these activities. The results of our study showed that BJe is capable of reducing THP-1 cell proliferation with a concentration- and time-dependent trend, accompanied by S-phase cell cycle blockage and induction of both extrinsic and intrinsic apoptotic pathways, as witnessed by cleavage of both caspase-8 and -9, which in turn activated caspase-3 and PARP, together with the modulation of BAX, Bcl-2 and p53 levels. The exposure of THP-1 cells to BJe induced the differentiation of leukemic cells, as shown by changes in cell adhesion, nitro blue tetrazolium (NBT) assay, and increased expression of differentiation-associated surface antigens such as CD11b, CD14, and CD68. In addition, we observed that the extract is able to modulate protein levels of autophagy-associated markers, including LC3 and Beclin-1, as well as to induce phosphorylation of mitogen-activated protein kinases (MAPKs), ERK, JNK and p38, suggesting a potential mechanism of action underlying the biological effect of BJe. In conclusion, the results of our study suggest that BJe reduces the proliferation and induces the differentiation of THP-1 cells by inducing both the apoptotic and autophagic machinery, suggesting MAPKs as possible cross-talk between the two processes and highlighting its potential in the area of differentiation therapy in AML.