Marisa Piovano

Lichen metabolites prevent UV light and nitric oxide-mediated plasmid DNA damage and induce apoptosis in human melanoma cells.

In humans both UV-A and UV-B can cause gene mutations and suppress immunity, which leads to skin cancer, including melanoma. Inhibition of reactive oxygen species (ROS) and reactive nitrogen species (RNS) appears particularly promising as ROS and RNS production by both UV-A and UV-B contributes to inflammation, immunosuppression, gene mutation and carcinogenesis. We evaluated the effect of two lichen compounds, sphaerophorin (depside) and pannarin (depsidone) on pBR322 DNA cleavage induced by hydroxyl radicals (()OH), and by nitric oxide (NO), and their superoxide anion (O(2)(-)) scavenging capacity. In addition, we investigated the growth inhibitory activity of these compounds against human melanoma cells (M14 cell line). Sphaerophorin and pannarin showed a protective effect on plasmid DNA and exhibited a superoxide dismutase like effect. The data obtained in cell culture show that these lichen metabolites inhibit the growth of melanoma cells, inducing an apoptotic cell death, demonstrated by the fragmentation of genomic DNA (COMET and TUNEL Assays) and by a significant increase of caspase-3 activity, and correlated, at least in part, to the increase of ROS generation, These results confirm the promising biological properties of sphaerophorin and pannarin and encourage further investigations on their molecular mechanisms.

Cytotoxic activity and antioxidant capacity of purified lichen metabolites: an in vitro study.

The purpose of this study was to investigate the effects of six lichen metabolites (diffractaic acid, lobaric acid, usnic acid, vicanicin, variolaric acid, protolichesterinic acid) on proliferation, viability and reactive oxygen species (ROS) level towards three human cancer cell lines, MCF‐7 (breast adenocarcinoma), HeLa (cervix adenocarcinoma) and HCT‐116 (colon carcinoma). Cells were treated with different concentrations (2.5–100 μM) of these compounds for 48 h. In this comparative study, our lichen metabolites showed various cytotoxic effects in a concentration‐dependent manner, and usnic acid was the most potent cytotoxic agent, while variolaric acid did not inhibit the proliferation of any of the three cell lines used. All tested lichen compounds did not exhibit free radical scavenging activity using the 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) assay. The lichen metabolites did not significantly increase the intracellular ROS level and did not prevent oxidative injury induced by t‐butylhydroperoxide in HeLa cells. To better clarify the mechanism(s) of cytotoxic effect induced by protolichesterinic acid in HeLa cells, we investigated apoptotic markers such as condensation and fragmentation of nuclear chromatin and activation of caspase‐3, 8 and 9. Our results revealed that the antiproliferative activity of 40 μM protolichesterinic acid in HeLa cells is related to its ability to induce programmed cell death involving caspase‐3, 8 and 9 activation. Copyright

In vitro interaction of usnic acid in combination with antimicrobial agents against methicillin-resistant Staphylococcus aureus clinical isolates determined by FICI and ΔE model methods.

The in vitro antimicrobial activities of usnic acid were evaluated in combination with five therapeutically available antibiotics, using checkerboard microdilution assay against methicillin-resistant clinical isolates strains of Staphylococcus aureus. MIC₉₀, MIC₅₀, as well as MBC₉₀ and MBC₅₀, were evaluated. A synergistic action was observed in combination with gentamicin, while antagonism was observed with levofloxacin. The combination with erythromycin showed indifference, while variability was observed for clindamycin and oxacillin. Data from checkerboard assay were analysed and interpreted using the fractional inhibitory concentration index (FICI) and the response surface approach using the ΔE model. Discrepancies were found between both methods for some combinations. These could mainly be explained by the failure of FIC approach, being too much subjective and sensitive to experimental errors. These findings, beside confirm the well known antimicrobial activity of usnic acid, suggest, however, that this substance might be a good candidate for the individuation of novel templates for the development of new antimicrobial agents or combinations of drugs for chemotherapy.

Effect of vicanicin and protolichesterinic acid on human prostate cancer cells: Role of Hsp70 protein

With the aim of identifying novel agents with antigrowth and pro-apoptotic activity on prostate cancer cells, in the present study, we evaluated the effect of five lichen secondary metabolites the depsides atranorin (1), diffrattaic (2) and divaricatic (3) acids, the depsidone vicanicin (4) and the protolichesterinic acid (5) on cell growth in androgen-sensitive (LNCaP) and androgen-insensitive (DU-145) human prostate cancer cells. The cell viability was measured using MTT assay. LDH release, a marker of membrane breakdown, was also measured. For the detection of apoptosis, the evaluation of DNA fragmentation (COMET assay) and caspase-3 activity assay were employed. The expression of Bcl-2, Bax, TRAIL, COX-2, NOS2 and Hsp70 proteins was detected by western blot analysis. Generation of reactive oxygen species was measured by using a fluorescent probe. It was observed that atranorin (1), diffrattaic (2) and divaricatic (3) acids showed a lower activity inhibiting the prostate cancer cells only at more high concentrations (25 and 50μM). Whereas compounds vicanicin (4) and protolichesterinic acid (5) showed a dose-response relationship in the range of 6.25-50μM concentrations in DU-145 and LNCaP cells, activating an apoptotic process. The novel finding, in the present study, is that apoptosis induced by these compounds appears to be mediated, at least in part, via the inhibition of Hsp70 expression, that may be correlated with a modulation of redox-sensitive mechanisms. The combination of vicanicin (4) and protolichesterinic acid (5) with other anti-prostate cancer therapies could be considered a promising strategy that warrants further in vivo evaluation.

Diterpenoids from Calceolaria thyrsiflora

Abstract Three novel diterpenes, thyrsiflorin A, thyrsiflorin B, thyrsiflorin C, and the dehidroabietane derivatives, abietatrien-3β-ol and 3β-acetoxy abietatrien were isolated from the aerial parts of Calceolaria thyrsiflora . The structures of the new compounds were elucidated by spectroscopic methods, chemical transformation and X-ray analysis.

Dehydroabietane diterpenoids from Calceolaria ascendens

Abstract Two new dehydroabietane diterpenes, dehydroabietinol and 19-malonyloxy-dehydroabietinol, and the known compound 4-epi-dehydroabietic acid were isolated from the aerial parts of Calceolaria ascendens. The structures of the new compounds were elucidated by spectroscopic methods.

18-Malonyloxy-9-epi-ent-isopimarol, a diterpene from Calceolaria glandulosa

Abstract A new pimarane diterpene, 18-malonyloxy-9- epi - ent -isopimarol, was isolated from the aerial parts of Calceolaria glandulosa . The structure of the new compound was established by spectroscopic evidence, chemical transformations and X-ray analysis.

In vitro antimicrobial activity of pannarin alone and in combination with antibiotics against methicillin-resistant Staphylococcus aureus clinical isolates.

The in vitro antimicrobial activities of pannarin, a depsidone isolated from lichens, collected in several Southern regions of Chile (including Antarctica), was evaluated alone and in combination with five therapeutically available antibiotics, using checkerboard microdilution assay against methicillin-resistant clinical isolates strains of Staphylococcus aureus. MIC(90), MIC(50), as well as MBC(90) and MBC(50), were evaluated. A moderate synergistic action was observed in combination with gentamicin, whilst antagonism was observed in combination with levofloxacin. All combinations with erythromycin were indifferent, whilst variability was observed for clindamycin and oxacillin combinations. Data from checkerboard assay were analysed and interpreted using the fractional inhibitory concentration index and the response surface approach using the ΔE model. Discrepancies were found between both methods for some combinations. In order to asses cellular lysis after exposure to pannarin, cell membrane permeability assay was performed. The treatment with pannarin produces bactericidal activity without significant calcein release, consistent with lack of lysis or even significant structural damage to the cytoplasmic membrane. Furthermore, pannarin shows low hemolytic activity and moderate cytotoxic effect on peripheral blood mononuclear cells. These findings suggest that the natural compound pannarin might be a good candidate for the individualization of novel templates for the development of new antimicrobial agents or combinations of drugs for chemotherapy.

Pro-apoptotic activity of ergosterol peroxide and (22E)-ergosta-7,22-dien-5α-hydroxy-3,6-dione in human prostate cancer cells

With the aim of identifying novel agents with antigrowth and pro-apoptotic activity on prostate cancer cells, we assayed the effect of ergosterol peroxide and (22E)-ergosta-7,22-dien-5alpha-hydroxy-3,6-dione, a semisynthetic compound, against androgen-sensitive (LNCaP) and androgen-insensitive (DU-145) human prostate cancer cells. Our results indicate that after 72h of incubation, ergosterol peroxide and (22E)-ergosta-7,22-dien-5alpha-hydroxy-3,6-dione at micromolar concentrations exhibited an inhibitory effect on LNCaP and DU-145 cell growth (MTT assay), but the semisynthetic compound was the most active. In addition, our results indicate that apoptotic cell demise is induced in LNCaP and DU-145 cells. In fact, a significant increase of caspase-3 activity, not correlated to LDH release, marker of membrane breakdown, was observed in both cell lines treated with ergosterol peroxide and the semisynthetic compound. With respect to genomic DNA damage, determined by COMET and TUNEL assays, the results obtained show a significant increase in DNA fragmentation when compared with the untreated control. In conclusion, the results obtained in this study, demonstrating that ergosterol peroxide and (22E)-ergosta-7,22-dien-5alpha-hydroxy-3,6-dione attenuate the growth of prostate cells, at least in part, triggering an apoptotic process, permit to confirm the use of mushrooms as origin of compounds to be used as novel therapeutic agents for prostate cancer treatment, or as models for molecules more active and selective.

Foliosate, a bis-diterpene and 9-epi-ent-7,15-isopimaradiene· derivatives from Calceolaria foliosa

Abstract In addition to four new 9- epi -pimarane diterpenes, the aerial part of Calceolaria foliosa yielded a new bis-diterpene, foliosate, derived from the esterification of malonic acid by two 17-hydroxy-9- ent -7,15-isopimaradiene units. The structures of the new compounds were elucidated by spectroscopic methods and chemical transformations.