New Insights in Purinergic Therapy: Novel Antagonists for Uridine 5'-Triphosphate-Activated P2Y Receptors from Brazilian Flora.

P2Y2 and P2Y4 receptors are physiologically activated by uridine 5'-triphosphate (UTP) and are widely expressed in many cell types in humans. P2Y2 plays an important role in inflammation and proliferation of tumor cells, which could be attenuated with the use of antagonists. However, little is known about the physiological functions related to P2Y4, due to the lack of selective ligands for these receptors. This can be solved through the search for novel compounds with antagonistic activity. The aim of this study was to discover new potential antagonist candidates for P2Y2 and P2Y4 receptors from natural products. We applied a calcium measurement methodology to identify new antagonist candidates for these receptors. First, we established optimal conditions for the calcium assay using J774.G8, a murine macrophage cell line, which expresses functional P2Y2 and P2Y4 receptors and then, we performed the screening of plant extracts at a cutoff concentration of 50 µg/mL. ATP and ionomycin, known intracellular calcium inductors, were used to stimulate cells. The calculated EC50 were 11 µM and 103 nM, respectively. These cells also responded to the UTP stimulation with an EC50 of 1.021 µM. Screening assays were performed and a total of 100 extracts from Brazilian plants were tested. Joannesia princeps Vell. (stem) and Peixotoa A. Juss (flower and leaf) extracts stood out due to their ability to inhibit UTP-induced responses without causing cytotoxicity, and presented an IC50 of 32.32, 14.99, and 12.98 µg/mL, respectively. Collectively, our results point to the discovery of potential antagonist candidates from Brazilian flora for UTP-activated receptors.

Nucleotide excision repair activity on DNA damage induced by photoactivated methylene blue.

The nucleotide excision repair (NER) mechanism is well known to be involved in the removal of UV-induced lesions. Nevertheless, the involvement of this pathway in the repair of lesions generated after DNA oxidation remains controversial. The effects of visible-light-excited methylene blue (MB), known to generate reactive oxygen species (ROS), were examined directly in xeroderma pigmentosum (XP)-A and XP-C NER-deficient human fibroblasts. Initially, MB was confirmed as being incorporated in similar amounts by the cells and that its photoexcitation induces the generation of (1)O2 within cells. The analysis of cell survival indicated that NER-deficient cells were hypersensitive to photoactivated MB. This sensitivity was confirmed with cells silenced for the XPC gene and by host-cell reactivation (HCR) of plasmid exposed to the photosensitizing effects of photoexcited MB. The sensitivity detected by HCR was restored in complemented cells, confirming the participation of XPA and XPC proteins in the repair of DNA lesions induced by photosensitized MB. Furthermore, DNA damage (single- and double-strand breaks and alkali-sensitive sites) was observed in the nuclei of treated cells by alkaline comet assay, with higher frequency of lesions in NER-deficient than in NER-proficient cells. Likewise, NER-deficient cells also presented more γ-H2AX-stained nuclei and G2/M arrest after photoactivated MB treatment, probably as a consequence of DNA damage response. Notwithstanding, the kinetics of both alkali- and FPG-sensitive sites repair were similar among cells, thereby demonstrating not only that MB photoexcitation generates nuclear DNA damage, but also that the removal of these lesions is NER-independent. Therefore, this work provides further evidence that XPA and XPC proteins have specific roles in cell protection and repair/tolerance of ROS-induced DNA damage. Moreover, as XPC-deficient patients do not present neurodegeneration, premature aging, or developmental clinical symptoms, the results indicate that defects in the repair/tolerance of oxidatively generated DNA lesions are not sufficient to explain these severe clinical features of certain XP patients.

Dynamics of a supramolecular capsule assembly with pyrene.

Water-soluble octaacid cavitands (OAs) form dimeric capsules suitable for guest incorporation. Our studies reveal that the mechanism of pyrene (Py) binding involves the rapid (<1 ms) formation of the Py·OA complex followed by slower binding with the second OA. The dissociation of the capsular OA·Py·OA complex occurs with a lifetime of 2.7 s, which is 5 orders of magnitude slower than the microsecond opening/closing ("breathing") previously observed to provide access of small molecules to the encapsulated guest. These different dynamics of the capsules have a potential impact on how the chemistry of included guests could be altered.

Methylene blue in photodynamic therapy: From basic mechanisms to clinical applications.

Methylene blue (MB) is a molecule that has been playing important roles in microbiology and pharmacology for some time. It has been widely used to stain living organisms, to treat methemoglobinemia, and lately it has been considered as a drug for photodynamic therapy (PDT). In this review, we start from the fundamental photophysical, photochemical and photobiological characteristics of this molecule and evolved to show in vitro and in vivo applications related to PDT. The clinical cases shown include treatments of basal cell carcinoma, Kaposi's Sarcoma, melanoma, virus and fungal infections. We concluded that used together with a recently developed continuous light source (RL50(®)), MB has the potential to treat a variety of cancerous and non-cancerous diseases, with low toxicity and no side effects.