Anti-cancer activity of Citrus bergamia essential oil and its constituents in in vitro experimental models of paediatric neoplasms

Cancer represents one of the leading causes of death worldwide. In paediatric subjects, leukaemia and brain tumours showed the highest incidence in 2020. The plant kingdom has always been a great source for bioactive compounds for treating several illnesses, including cancer, and Citrus fruits stand out among the others. For my PhD project, I focused on the anti-proliferative activity of the essential oil (BEO) of Citrus bergamia (bergamot) Risso & Poiteau and its constituents in in vitro experimental models of paediatric neoplasms. The first step of the project was performing the quali-quantitative characterization of BEO and its furocoumarin-free fraction (BEO-FF). The chemical profile of these plant matrices showed that they are composed of a volatile part (< 95%) rich in monoterpenes, and a non-volatile one, containing coumarins, results in line with those present in literature. The second step of my PhD project originated from a previous assumption by which bergamottin (BRG) and 5-geranyloxy-7-methoxycoumarin (5-G-7-MOC), two coumarins found in BEO, have been claimed to be relevant in the anti-proliferative effects exerted by BEO in the SH-SY5Y human neuroblastoma cells. Therefore, this step was designed to verify this and to assess the mechanisms underlying the anti-proliferative effect of both compounds. The results of this step demonstrate that BRG and 5-G-7-MOC are able to reduce the proliferation of SH-SY5Y cells, inducing apoptosis and increasing sub-G0/G1 phase cell population. Furthermore, the pro-oxidant activity of the two coumarins was demonstrated, which increased reactive oxygen species and consequently reduced mitochondrial membrane potential. Moreover, BRG and 5-G-7-MOC were able to modulate apoptosis-related factors both at protein and gene level. As a final point, we evaluated the synergistic effect of their combination, finding that the highest synergy was observed at a concentration ratio similar to observed in the whole BEO, supporting our initial hypothesis. These results deepen the knowledge on the effect of BRG and 5-G-7-MOC in SH-SY5Y cells, stressing the relevance of their cooperation in achieving this effect. Acknowledged the clinical relevance of multi-drug resistance (MDR) in cancer treatment, the last step of my PhD project was to evaluate the effectiveness of BEO and BEO-FF in acute leukemic lymphoblasts and in their doxorubicin-resistant counterpart (i.e., CCRF-CEM and CEM/ADR5000, respectively). Both fractions showed similar antiproliferative activity in both cell lines, as showed by cell viability assays. Flow cytometric analyses indicated that this effect was achieved by inducing apoptosis, although with different extent for BEO and BEO-FF, and confirmed by the increase of population in sub-G0/G1 phase, meaning hypodiploidy. To assess their role against MDR, likewise for coumarins present in BEO, both bergamot fractions were combined with doxorubicin at different ratios. If in CCRF-CEM cells neither BEO nor BEO-FF elicited any relevant effect, in CEM/ADR5000 cells, the whole BEO brought a strong synergistic effect, restoring the sensitivity of resistant cells to doxorubicin. Therefore, these results corroborate the hypothesis that a phytocomplex, like BEO, can be an appropriate weapon to fight MDR exploiting their collateral sensitivity, a phenomenon by which cells resistant to a specific antineoplastic agent may be particularly sensitive to another compound or a multitude of them.