Photodynamic therapy using the natural photosensitizer Berberine (BBR) and its derivatives NAX induces apoptosis in human astrocytoma-established cells.

Glioblastoma (GBM) is the most common and aggressive malignant brain tumor in adults. Despite many standard therapeutic approaches including surgical removal, radiation, and chemotherapy, GBM remains a poor prognosis neoplasm with a high rate of recurrence. Therefore, in clinical oncology GBM treatments became a challenging task. Photodynamic therapy (PDT) is a promising anticancer strategy involving light and photosensitizing (PS) designed to elicit cell death. Once the light irradiates the PS, a photochemical reaction is triggered resulting in the generation of reactive oxygen species (ROS) which are highly cytotoxic for the cells. In this work, the use of BBR as a potential photosensitizer agent in PDT was extensively investigated for the treatment of GBM. Taking advantage of BBR and blue light sources, a BBR-PDT scheme was developed in human-established astrocytoma cell lines. The data confirmed that BBR was an efficient PS agent and its association with PDT could be a novel strategy for high malignant gliomas. In detail, the BBR-PDT scheme preferentially induced the activation of the intrinsic apoptotic pathway, through alteration in mitochondrial membrane potential, massive ROS accumulation, and the subsequent activation of the caspases pathway. The development of new BBR formulations or derivatives, with the same biological activity capable of improving its pharmacokinetic limits and absorption, may have great potential for cancer treatments. Therefore, different compounds based on BBR structure effective at lower concentrations accomplished with a stronger biological activity were investigated. Various attempts have been made to design modified BBR compounds effective in various therapies. Particularly, a new class of compounds, called NAX, characterized by the presence of a (hetero)aromatic moieties linked to position 13 of the parent-BBR skeleton through a hydrocarbon linker of variable length and functionality has been developed. This hydrocarbon linker works in a condition that eventually creates a geometric propensity for further non-covalent aromatic interactions with cellular targets. In the present thesis, molecular mechanisms behind the NAX- enhanced cytotoxic effect with and without the adjuvant of PDT were deeply investigated. Among the different BBR derivatives studied, the focus was mainly on a promising NAX candidate, called NAX111. This active compound exhibited a remarkable lysosome co-localization compared with BBR. NAX111 exerted its cytotoxic action by targeting lysosomes and inducing their functional impairment resulting in early autophagy blockade with further activation of the apoptotic pathway. Therefore, NAX111, with the adjuvant of PDT, produced its cytotoxic action by early blocking of autophagy, as a direct consequence of the impairment of the degradative activity of lysosomes. These results took into consideration a newly possible active role of lysosomes on apoptosis- dependent cell death. Unlike BBR, NAX111 by targeting lysosomes instead of mitochondria provided insights into a more effective PDT protocol even at low PS concentrations. Further studies are needed to confirm the involvement of lysosomes in the induction of apoptotic cell death and to understand the consequences of oxidative damage caused in the new target organelle. Thus, precise targeting of organelles and thus activation of specific cell death mechanisms could contribute to the development of novel therapies against GBM.