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Photodynamic Therapy (PDT) is a minimally invasive radiation therapy currently used in treatment of various cancerous and pre-malignant diseases. Photodynamic therapy with 5-aminolaevulinic acid (5-ALA/PDT) induces DNA damage following photoactivation of Protoporphyrin IX (PpIX), the photosensitizer generated from the endogenous metabolism of 5-ALA. The occurrence of DNA damage could represent one of the last causes of cell death induced by PDT. We have dissected the molecular effectors involved in the DNA damage induced by 5-ALA/PDT in several cell lines: 1. two lung adenocarcinoma cell lines, namely H1299 (p53-/-) and A549 wild type (p53+/+); 2. two sub-clones of the same cell line HCT-116, that differ for p53 expression (p53+/+ and p53-/-) and 3. a prostate adenocarcinoma cell line, PC3 (p53-/-). We have focused our attention on p53, which controls the DNA damage response and on the efflux regulator ATP binding cassette transporter G2 (ABCG2). The levels and the activity of these two gene products are relevant for the survival in cells exposed to photodynamic treatment. Cell cycle, DNA damage (phosphorylated γ-H2AX and Comet assay) and ABCG2 levels were measured before or after irradiation. We found that all cell lines, except A549 and PC3, underwent extensive DNA damage upon PDT. The resistance of A549 and PC3 cells to photodynamic DNA damage was due to the high levels of ABCG2 expression upon irradiation. In fact, these cells displayed high levels of DNA damage signatures and underwent death when the ABCG2 was inhibited. Analysis of ABCG2 expression shows a complex regulation at transcriptional and post-transcriptional levels and its expression was dependent on oxidative stress. On the other hand, p53 expression or activity did not exert a significant effect on 5-ALA-induced cell death. Taken together, all these findings provide novel information on the role of the ABCG2 transporter in oxidative stress and a new tool to manipulate resistance to photodynamic therapy.

The methanol extracts of the aerial part of four ethnomedicinal plants of Mediterranean region, two non-seed vascular plants, Equisetum hyemale L. and Phyllitis scolopendrium (L.) Newman, and two Spermatophyta, Juniperus communis L. (J. communis) and Cotinus coggygria Scop. (C. coggygria), were screened against four human cells lines (A549, MCF7, TK6 and U937). Only the extracts of J. communis and C. coggygria showed marked cytotoxic effects, affecting both cell morphology and growth. A dose-dependent effect of these two extracts was also observed on the cell cycle distribution. Incubation of all the cell lines in a medium containing J. communis extract determined a remarkable accumulation of cells in the G2/M phase, whereas the C. coggygria extract induced a significant increase in the percentage of G1 cells. The novelty of our findings stands on the observation that the two extracts, consistently, elicited coherent effects on the cell cycle in four cell lines, independently from their phenotype, as two of them have epithelial origin and grow adherent and two are lymphoblastoid and grow in suspension. Even the expression profiles of several proteins regulating cell cycle progression and cell death were affected by both extracts. LC-MS investigation of methanol extract of C. coggygria led to the identification of twelve flavonoids (compounds 1-11, 19) and eight polyphenols derivatives (12-18, 20), while in J. communis extract, eight flavonoids (21-28), a alpha-ionone glycoside (29) and a lignin (30) were found. Although many of these compounds have interesting individual biological activities, their natural blends seem to exert specific effects on the proliferation of cell lines either growing adherent or in suspension, suggesting potential use in fighting cancer.

Photofrin/photodynamic therapy (PDT) at sub-lethal doses induced a transient stall in proteasome activity in surviving A549 (p53(+/+)) and H1299 (p53(-/-)) cells as indicated by the time-dependent decline/recovery of chymotrypsin-like activity. Indeed, within 3 h of incubation, Photofrin invaded the cytoplasm and localized preferentially within the mitochondria. Its light activation determined a decrease in mitochondrial membrane potential and a reversible arrest in proteasomal activity. A similar result is obtained by treating cells with Antimycin and Rotenone, indicating, as a common denominator of this effect, the ATP decrease. Both inhibitors, however, were more toxic to cells as the recovery of proteasomal activity was incomplete. We evaluated whether combining PDT (which is a treatment for killing tumor cells, per se, and inducing proteasome arrest in the surviving ones) with Bortezomib doses capable of sustaining the stall would protract the arrest with sufficient time to induce apoptosis in remaining cells. The evaluation of the mitochondrial membrane depolarization, residual proteasome and mitochondrial enzymatic activities, colony-forming capabilities, and changes in protein expression profiles in A549 and H1299 cells under a combined therapeutic regimen gave results consistent with our hypothesis.

Despite the successful deployment of efficacious vaccines and therapeutics, the development of novel vaccines for SARS-CoV-2 remains a major goal to increase vaccine doses availability and accessibility for lower income setting. We report here on the kinetics of Spike-specific humoral and T-cell response in young and old volunteers over 6 months follow-up after a single intramuscular administration of GRAd-COV2, a gorilla adenoviral vector-based vaccine candidate currently in phase-2 of clinical development. At all three tested vaccine dosages, Spike binding and neutralizing antibodies were induced and substantially maintained up to 3 months, to then contract at 6 months. Potent T-cell responses were readily induced and sustained throughout the study period, with only minor decline. No major differences in immune response to GRAd-COV2 vaccination were observed in the two age cohorts. In light of its favorable safety and immunogenicity, GRAd-COV2 is a valuable candidate for further clinical development and potential addition to the COVID-19 vaccine toolbox to help fighting SARS-CoV-2 pandemic.

Although photodynamic therapy (PDT), a therapeutic approach that involves a photosensitizer, light and O2, has been principally considered for the treatment of specific types of cancers, other applications exist, including the treatment of infections. Unfortunately, PDT does not always guarantee full success since it exerts lethal effects only in cells that have taken up a sufficient amount of photosensitizer and have been exposed to adequate light doses, conditions that are not always achieved. Based on our previous experience on the combination PDT/chemotherapy, we have explored the possibility of fighting bacteria that commonly crowd infected surfaces by combining PDT with an antibiotic, which normally does not harm the strain at low concentrations. To this purpose, we employed 5-aminolevulinic acid (5-ALA), a pro-drug that, once absorbed by proliferating bacteria, is converted into the natural photosensitizer Protoporphyrin IX (PpIX), followed by Gentamicin. Photoactivation generates reactive oxygen species (ROS) which damage or kill the cell, while Gentamicin, even at low doses, ends the work. Our experiments, in combination, have been highly successful against biofilms produced by several Gram positive bacteria (i.e., Staphylococcus aureus, Staphylococcus epidermidis, etc.). This original approach points to potentially new and wide applications in the therapy of infections of superficial wounds and sores.