Potent virucidal activity in vitro of photodynamic therapy with Hpericum 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 compoundThis research was funded by Fundación Universidad Autónoma de Madrid, grant number PI21/00315 and by Instituto de Salud Carlos III, grant number PI21/00953. Institutional Review Board Statement: Not applicabl

Metformin as an Adjuvant to Photodynamic Therapy in Resistant Basal Cell Carcinoma Cells

© 2020 by the authors.Photodynamic Therapy (PDT) with methyl-aminolevulinate (MAL-PDT) is being used for the treatment of Basal Cell Carcinoma (BCC), although resistant cells may appear. Normal differentiated cells depend primarily on mitochondrial oxidative phosphorylation (OXPHOS) to generate energy, but cancer cells switch this metabolism to aerobic glycolysis (Warburg effect), influencing the response to therapies. We have analyzed the expression of metabolic markers (β-F1-ATPase/GAPDH (glyceraldehyde-3-phosphate dehydrogenase) ratio, pyruvate kinase M2 (PKM2), oxygen consume ratio, and lactate extracellular production) in the resistance to PDT of mouse BCC cell lines (named ASZ and CSZ, heterozygous for ptch1). We have also evaluated the ability of metformin (Metf), an antidiabetic type II compound that acts through inhibition of the AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway to sensitize resistant cells to PDT. The results obtained indicated that resistant cells showed an aerobic glycolysis metabolism. The treatment with Metf induced arrest in the G0/G1 phase and a reduction in the lactate extracellular production in all cell lines. The addition of Metf to MAL-PDT improved the cytotoxic effect on parental and resistant cells, which was not dependent on the PS protoporphyrin IX (PpIX) production. After Metf + MAL-PDT treatment, activation of pAMPK was detected, suppressing the mTOR pathway in most of the cells. Enhanced PDT-response with Metf was also observed in ASZ tumors. In conclusion, Metf increased the response to MAL-PDT in murine BCC cells resistant to PDT with aerobic glycolysis.This research was funded by Spanish grants from Instituto de Salud Carlos III MINECO and Feder Funds (FIS PI15/00974 and PI18/00708) and Ministerio de Ciencia, Innovación y Universidades (SAF2016-75916-R).Peer reviewe

Le Peuple : organe quotidien du syndicalisme

29 juillet 19341934/07/29 (A14,N4940)-1934/07/29

Impact of GSK1016790A on cell proliferation/survival.

<p>A) Upper panel on left: Concentration-dependent reduction of cell proliferation/survival of A375 as measured by Janus Green-Assay. Upper panel on right: Note that half-maximal inhibition was achieved at ca. 1 nM GSK1016790A for all time intervals, except day-1. Lower panel on left: HC067047 antagonized the response to GSK1016790A. Lower panel on right: The negative gating-modulator of KCa3.1, the 13b derivate, RA-2 (10 μM) reduced cell proliferation/survival and potentiated the response to GSK1016790A. The positive-gating modulator of KCa3.1, SKA-121, had no effects. Data points are means ± SEM (n = 6–36 from n = 2–6 independent experiments). B) GSK1016790A impaired proliferation/survival of HaCaT cells. HC067047 partially antagonized the response. The negative and positive KCa3.1-gating modulators, RA-2 and SKA-121, respectively, did not modulate the response. Data points are means ± SEM (n = 18; number of independent experiments, n = 3). *P<0.05 vs. DMSO, #P<0.05 vs. GSK1016790A; Student’s T test.</p

GSK1016790A-induced TRPV4-currents and inhibition by HC067047 in the melanoma lines, MKTBR and SK-MEL-28, and the human non-cancer keratinocyte line, HaCaT.

<p>Data points are means ± SEM (cells, n = 4–6 each). Panel on right: Quantitative RT-PCR analysis of TRPV4 and KCa3.1 gene expression in HaCaT as percentage of GAPDH expression (replicates, n = 3). Data points are means ± SEM.</p

Characterization of TRPV4 channels in A375 melanoma cells.

<p>A) Upper panel: Exemplary whole-cell recordings showing activation of TRPV4 channels by GSK1016790A (200 nM) and inhibition of currents by HC067047 (1 μM). The arrow indicates a positive reversal potential of GSK1016790A-activated currents. Baseline currents were not considerable inhibited by HC067047. Lower panel: Co-activation of K_Ca-currents. The right arrow indicates a negative reversal potential (E_rev) of ca. -35 mV of the mixed TRPV4 and K_Ca current and the left arrow indicates an E_rev of ca. -75 mV of the isolated K_Ca-current after inhibition of TRPV4 currents by HC067047. The K_Ca-current was fully blocked by the negative-gating modulator of KCa3.1 channels, 13b (1 μM). B) Upper panel: Mean normalized currents at clamp potentials of -80 and +80 mV before and after addition GSK1016790A (n = 8, experiments) and after addition of HC067047 (n = 8). Lower panel: Mean mixed TRPV4/KCa3.1 currents at a clamp potential of 0 mV after addition of GSK1016790A (n = 5) and inhibition of TRPV4 currents by HC067047 (n = 4) and of KCa3.1 currents by 13b (n = 5). C) Quantitative RT-PCR analysis of TRPV4 and KCa3.1 gene expression as percentage of GAPDH expression (replicates, n = 3). Data points are means ± SEM.</p

FACS analysis of apoptosis.

<p>A) Representative flow cytometry dot plots with double Annexin V-FITC/PI staining for control cells (DMSO 0,2%), cells exposed to GSK1016790A (10 nM), and cells exposed to GSK1016790A and HC067047 (1 μM) at 1 h, 24 h, and 72 h. B) Summary data. C) Induction of apoptosis in HaCaT cells and protective effects of HC067047. D) Summary data. *P<0.05 vs. Control, #P<0.05 vs. GSK1016790A, ANOVA, n = 3). Data are means ± SEM (number of independent experiments, n = 3).</p

Alterations of cell morphology, cell detachment and cell death induced by GSK1016790A.

<p>A) Upper two panels: Exemplary light microscopic images illustrating time course of cell retraction, membrane blebbing (indicated by arrows), and cell detachment during the first hour of exposure to GSK1016790A (1 μM). HC067047 (1 μM) prevented visibly GSK1016790A-induced changes. HC067047 or vehicle (DMSO) had no visible effect. Lower panel: Giemsa-stained A375 cells after 1 h exposure to GSK1016790A, in combination with HC067047, or DMSO. Note the densification of nuclei (dark-grey dots indicated by arrows) in GSK1016790A-treated cells. B) Counts of non-viable, “death” cells in supernatant. Data points are means ± SEM (number of independent experiments, n = 3). *P<0.05 vs. DMSO, #P <0.05 vs. GSK1016790A; Student’s T test.</p