Herein, we assessed the effect of Ferulic Acid (FA), a natural antioxidant with anti-cancer effect, on the human glioblastoma cells through molecular and Delayed Luminescence (DL) studies. DL, a phenomenon of ultra-week emission of optical photons, was used to monitor mitochondrial assessment. The effect of FA loaded in nanostructured lipid carriers (NLCs) was also assessed. To validate NLCs as a drug delivery system for glioblastoma treatment, particular attention was focused on their effect. We found that free FA induced a significant decrease in c-Myc and Bcl-2 expression levels accompanied by the apoptotic pathway activation. Blank NLCs, even if they did not induce cytotoxicity and caspase-3 cleavage, decreased Bcl-2, ERK1/2, c-Myc expression levels activating PARP-1 cleavage. The changes in DL intensity and kinetics highlighted a possible effect of nanoparticle matrix on mitochondria, through the involvement of the NADH pool and ROS production that, in turn, activates ERK1/2 pathways. All the effects on protein expression levels and on the activation of apoptotic pathway appeared more evident when the cells were exposed to FA loaded in NLCs. We demonstrated that the observed effects are due to a synergic pro-apoptotic influence exerted by FA, whose bioavailability increases in the glioblastoma cells, and NLCs formulation.Glioblastoma multiforme (GBM), also known as grade IV astrocytoma, represents the most prevalent and aggressive brain cancer. It is characterized by glial cells and has finger-like tentacles that infiltrate the brain, which make them very difficult to remove with surgical procedures. GBM exhibits a high level of resistance to conventional chemotherapy and radiotherapy, also due to the existence of blood-brain barrier (BBB), glioma stem cells and complex network of multiple modified signalling pathways 1 . The most frequent aberrant expression is represented by the dysregulation of extracellular signal-regulated protein kinase (ERK), which is associated with poor survival of the patients. The ERK isoforms (p42/44 or ERK1/ERK2) by interacting with specific phosphorylation substrates, play a pivotal role in the control of several cellular processes involved in proliferation, as well as activation of transcription factors, apoptosis and the control of cellular process 2,3 . In addition, the transcription factor c-Myc has been recognized as an important regulator of stem cell biology implicated with GBM malignancy and stemness 4 , as it contributes to proliferation, growth and survival of GBM stem cells 5 . GBM has been also related to the impairment of mitochondrial metabolic capacity, which leads to the alteration in energy production 6,7 and is characterized by an overexpression of Bcl-2 8 . This protein can regulate transition pores permeability of the outer mitochondrial membrane and block pro-apoptotic proteins 9 . Furthermore, it has been identified a novel www.nature.com/scientificreports www.nature.com/scientificreports/ interaction between Bcl-2 and (ADP-ribose) polymerase (PARP) 10 , t...

Menadione (MD) is an effective cytotoxic drug able to produce intracellularly large amounts of superoxide anion. Quercetin (QC), a widely distributed bioflavonoid, can exert both antioxidant and pro-oxidant effects and is known to specifically inhibit cell proliferation and induce apoptosis in different cancer cell types. We have investigated the relation between delayed luminescence (DL) induced by UV-laser excitation and the effects of MD, hydrogen peroxide, and QC on apoptosis and cell cycle in human leukemia Jurkat T-cells. Treatments with 500 μM H₂O₂ and 250 μM MD for 20 min produced 66.0 ± 4.9 and 46.4 ± 8.6% apoptotic cell fractions, respectively. Long-term (24 h) pre-exposure to 5 μM, but not 0.5 μM QC enhanced apoptosis induced by MD, whereas short-term (1 h) pre-incubation with 10 μM QC offered 50% protection against H₂O₂-induced apoptosis, but potentiated apoptosis induced by MD. Since physiological levels of QC in the blood are normally less than 10 μM, these data can provide relevant information regarding the benefits of flavonoid-combined treatments of leukemia. All the three drugs exerted significant effects on DL. Our data are consistent with (1) the involvement of Complex I of the mitochondrial respiratory chain as an important source of delayed light emission on the 10 μs-10 ms scale, (2) the ability of superoxide anions to quench DL on the 100 μs-10 ms scale, probably via inhibition of reverse electron transfer at the Fe/S centers in Complex I, and (3) the relative insensitivity of DL to intracellular OH• and H₂O₂ levels.