Anticancer activity of cationic porphyrins in melanoma tumour-bearing mice and mechanistic in vitro studies

Background Porphyrin TMPyP4 (P4) and its C14H28-alkyl derivative (C14) are G-quadruplex binders and singlet oxygen (1O2) generators. In contrast, TMPyP2 (P2) produces 1O2 but it is not a G-quadruplex binder. As their photosensitizing activity is currently undefined, we report in this study their efficacy against a melanoma skin tumour and describe an in vitro mechanistic study which gives insights into their anticancer activity. Methods Uptake and antiproliferative activity of photoactivated P2, P4 and C14 have been investigated in murine melanoma B78-H1 cells by FACS, clonogenic and migration assays. Apoptosis was investigated by PARP-1 cleavage and annexin-propidium iodide assays. Biodistribution and in vivo anticancer activity were tested in melanoma tumour-bearing mice. Porphyrin binding and photocleavage of G-rich mRNA regions were investigated by electrophoresis and RT-PCR. Porphyrin effect on ERK pathway was explored by Western blots. Results Thanks to its higher lipophylicity C14 was taken up by murine melanoma B78-H1 cells up to 30-fold more efficiently than P4. When photoactivated (7.2 J/cm2) in B78-H1 melanoma cells, P4 and C14, but not control P2, caused a strong inhibition of metabolic activity, clonogenic growth and cell migration. Biodistribution studies on melanoma tumour-bearing mice showed that P4 and C14 localize in the tumour. Upon irradiation (660 nm, 193 J/cm2), P4 and C14 retarded tumour growth and increased the median survival time of the treated mice by ~50% (P <0.01 by ANOVA), whereas porphyrin P2 did not. The light-dependent mechanism mediated by P4 and C14 is likely due to the binding to and photocleavage of G-rich quadruplex-forming sequences within the 5\u2032-untranslated regions of the mitogenic ras genes. This causes a decrease of RAS protein and inhibition of downstream ERK pathway, which stimulates proliferation. Annexin V/propidium iodide and PARP-1 cleavage assays showed that the porphyrins arrested tumour growth by apoptosis and necrosis. C14 also showed an intrinsic light-independent anticancer activity, as recently reported for G4-RNA binders. Conclusions Porphyrins P4 and C14 impair the clonogenic growth and migration of B78-H1 melanoma cells and inhibit melanoma tumour growth in vivo. Evidence is provided that C14 acts through light-dependent (mRNA photocleavage) and light-independent (translation inhibition) mechanisms. Keywords: Melanoma B78-H1 cells; Cationic porphyrins; Biodistribution; C57/BL6 mice; Ras genes; G4-RNA; ERK pathwa

Potent Virucidal Activity In Vitro of Photodynamic Therapy with Hypericum Extract as Photosensitizer and White Light against Human Coronavirus HCoV-229E

The emergent human coronavirus SARS-CoV-2 and its high infectivity rate has highlighted the strong need for new virucidal treatments. In this sense, the use of photodynamic therapy (PDT) with white light, to take advantage of the sunlight, is a potent strategy for decreasing the virulence and pathogenicity of the virus. Here, we report the virucidal effect of PDT based on Hypericum extract (HE) in combination with white light, which exhibits an inhibitory activity of the human coronavirus HCoV-229E on hepatocarcinoma Huh-7 cells. Moreover, despite continuous exposure to white light, HE has long durability, being able to maintain the prevention of viral infection. Given its potent in vitro virucidal capacity, we propose HE in combination with white light as a promising candidate to fight against SARS-CoV-2 as a virucidal compound

Purine twisted-intercalating nucleic acids: a new class of anti-gene molecules resistant to potassium-induced aggregation

Sequence-specific targeting of genomic DNA by triplex-forming oligonucleotides (TFOs) is a promising strategy to modulate in vivo gene expression. Triplex formation involving G-rich oligonucleotides as third strand is, however, strongly inhibited by potassium-induced TFO self-association into G-quartet structures. We report here that G-rich TFOs with bulge insertions of (R)-1-O-[4-(1-pyrenylethynyl)-phenylmethyl] glycerol (called twisted intercalating nucleic acids, TINA) show a much lower tendency to aggregate in potassium than wild-type analogues do. We designed purine-motif TINA–TFOs for binding to a regulatory polypurine-polypyrimidine (pur/pyr) motif present in the promoter of the KRAS proto-oncogene. The binding of TINA–TFOs to the KRAS target has been analysed by electrophoresis mobility shift assays and DNase I footprinting experiments. We discovered that in the presence of potassium the wild-type TFOs did not bind to the KRAS target, differently from the TINA analogues, whose binding was observed up to 140 mM KCl. The designed TINA–TFOs were found to abrogate the formation of a DNA–protein complex at the pur/pyr site and to down-regulate the transcription of CAT driven by the murine KRAS promoter. Molecular modelling of the DNA/TINA–TFO triplexes are also reported. This study provides a new and promising approach to create TFOs to target in vivo the genome

Role of NF-kB/Snail/RKIP loop in photodynamic therapy

2nonenoneRapozzi V; Xodo LERapozzi, Valentina; Xodo, Luig

Role of RKIP in the tumor response to photooxidative damage

The Raf-kinase inhibitor protein (RKIP) plays a role in the regulation of different processes, through its interaction with several signaling pathways. These pathways include the mitogen activated protein kinase (MAPK), nuclear factor kappa-light chain enhancer of activated B cells (NF-\u3baB), G protein-coupled receptors (GPCR) and glycogen synthase kinase 3 beta (GSK 3\u3b2). RKIP negatively affects tumor survival and proliferation, acting as a metastasis suppressor. Moreover, RKIP overexpression has been reported to reverse tumor chemo/immuno/radio-resistance and support the anticancer host immuno-surveillance. The aim of this work is to evaluate the role of RKIP in cancer cells during a photooxidative damage induced by photodynamic therapy (PDT). PDT treatment is based on three components: a photosensitizer, light and oxygen. Their combined action produces singlet oxygen (1O2) and/or reactive oxygen species (ROS) leading to an oxidative insult in tumor cells. Dependently on the PDT dose, the response of tumor cells can have a double outcome: stimulation of tumor cell proliferation with a low PDT dose (IC50), or tumor growth arrest in the case of a high PDT dose (IC50). We evaluated RKIP expression within its complex network, correlating with other factors involved in the tumor response to oxidative stress. We found a link between the expression of RKIP and NF-\u3baB, MAPK, Snail and Nrf2 according to the type of the oxidative insult. In the presence of low PDT, RKIP is downregulated, while NF-\u3baB, Snail and Nrf2 are upregulated: an expression profile that stimulates tumor proliferation and resistance. Conversely, RKIP is overexpressed in the case of high PDT, thus allowing an arrest of tumor growth.Considering that many antitumor drugs develop ROS/RNS causing disease recurrence and drug resistance, RKIP could be a good prognostic marker to follow patients' response to anticancer therapies