Bioactivity Screening of Antarctic Sponges Reveals Anticancer Activity and Potential Cell Death via Ferroptosis by Mycalols

Sponges are known to produce a series of compounds with bioactivities useful for human health. This study was conducted on four sponges collected in the framework of the XXXIV Italian National Antarctic Research Program (PNRA) in November-December 2018, i.e., Mycale (Oxymycale) acerata, Haliclona (Rhizoniera) dancoi, Hemimycale topsenti, and Hemigellius pilosus. Sponge extracts were fractioned and tested against hepatocellular carcinoma (HepG2), lung carcinoma (A549), and melanoma cells (A2058), in order to screen for antiproliferative or cytotoxic activity. Two different chemical classes of compounds, belonging to mycalols and suberitenones, were identified in the active fractions. Mycalols were the most active compounds, and their mechanism of action was also investigated at the gene and protein levels in HepG2 cells. Of the differentially expressed genes, ULK1 and GALNT5 were the most down-regulated genes, while MAPK8 was one of the most up-regulated genes. These genes were previously associated with ferroptosis, a programmed cell death triggered by iron-dependent lipid peroxidation, confirmed at the protein level by the down-regulation of GPX4, a key regulator of ferroptosis, and the up-regulation of NCOA4, involved in iron homeostasis. These data suggest, for the first time, that mycalols act by triggering ferroptosis in HepG2 cells.     

Stress-induced biosynthesis of dicaffeoylquinic acids in globe artichoke

Leaf extracts from globe artichoke ( Cynara cardunculus L. var. scolymus) have been widely used in medicine as hepatoprotectant and choleretic agents. Globe artichoke leaves represent a natural source of phenolic acids with dicaffeoylquinic acids, such as cynarin (1,3-dicaffeoylquinic acid), along with its biosynthetic precursor chlorogenic acid (5-caffeoylquinic acid) as the most abundant molecules. This paper reports the development of an experimental system to induce caffeoylquinic acids. This system may serve to study the regulation of the biosynthesis of (poly)phenolic compounds in globe artichoke and the genetic basis of this metabolic regulation. By means of HPLC-PDA and accurate mass LC-QTOF MS and MS/MS analyses, the major phenolic compounds in globe artichoke leaves were identified: four isomers of dicaffeoylquinic acid, three isomers of caffeoylquinic acid, and the flavone luteolin 7-glucoside. Next, plant material was identified in which the concentration of phenolic compounds was comparable in the absence of particular treatments, with the aim to use this material to test the effect of stress application on the regulation of biosynthesis of caffeoylquinic acids. Using this material, the effect of UV-C, methyl jasmonate, and salicylic acid treatments on (poly)phenolic compounds was tested in different globe artichoke genotypes. UV-C exposure consistently increased the levels of dicaffeoylquinic acids in all genotypes, whereas the effect on compounds from the same biosynthetic pathway, for example, chlorogenic acid and luteolin-7-glucoside, was much less pronounced and was not statistically significant. No effect of methyl jasmonate or salicylic acid was found. Time-response experiments indicated that the level of dicaffeoylquinic acids reached a maximum at 24 h after UV radiation. On the basis of these results a role of dicaffeoylquinic acids in UV protection in globe artichoke is hypothesized.     

Adenocarcinoma of apocrine sweat glands in a mouflon (Ovis musimon).

A free-living mouflon (Ovis musimon) was presented with a mass on the left shoulder. At necropsy, multifocal, slightly protruding whitish spots were noted on the kidneys, and several lymph nodes were abnormal. Histologically, the mass was composed of epithelial cells arranged in tubular and tubulopapillary structures. The cytoplasm of the epithelial cells had numerous periodic acid-Schiff-positive and diastase-resistant granules. Ultrastructurally, the cytoplasm of the neoplastic cells contained numerous pleomorphic secretory granules and microvilli, which partially covered the luminal surface of the tumor cells. Metastatic foci were present in prescapular and mediastinal lymph nodes and kidneys. On the basis of histological and ultrastructural findings, this tumor was diagnosed as a tubulopapillary adenocarcinoma, arising from apocrine sweat glands of the skin.     

Fusarium head blight evaluation in wheat transgenic plants expressing the maize <Emphasis Type="Italic">b-32</Emphasis> antifungal gene

The maize gene b-32, normally expressed in the maize (Zea mays) endosperm, encodes for a RIP (Ribosome Inactivating Protein) characterised by antifungal activity. Transgenic wheat plants were obtained via biolistic transformation, in which the b-32 gene is driven by the 35SCaMV promoter in association with the bar gene as a selectable marker. Plants were brought to homozygosity through genetic analysis of progeny and pathogenicity tests were performed on the fourth generation. Six homozygous b-32 wheat lines were characterised. All plants had a normal phenotype, not distinguishable from the control cv. Veery except for slightly smaller size, flowered and set seeds. Western blot analyses confirmed b-32 expression during the plant life cycle in the various tissues. Each line differed in the b-32 content in leaf protein extracts and the transgenic protein expression level was maintained at least up to 10 days after anthesis. Pathogenicity tests for Fusarium head blight (FHB) were performed on the b-32 transgenic wheat lines in comparison to the parental cv. Veery. Resistance to FHB was evaluated by the single floret injection inoculation method on immature spikes with spores of Fusarium culmorum. In all the transgenic lines, a similar reduction in FHB symptoms, not dependent on the level of b-32 protein, has been observed (20% and 30% relative to the control, respectively 7 and 14 days after inoculation). Percentage of tombstone kernels at maturity was also recorded; in all transgenic lines disease control for this parameter was around 25%. The data obtained indicate that maize b-32 was effective as in vivo antifungal protein reducing FHB symptoms in wheat lines expressing the maize RIP protein.     

Detection of pathogens in ovine and caprine abortion samples from Sardinia, Italy, by PCR.

During 2003-2005, 399 abortion samples (315 fetuses and 84 placentae) were collected from 107 ovine and caprine farms in northern Sardinia. Tissues from aborted fetuses and placentae were examined by PCR assay to detect DNA from Coxiella burnetii, Chlamydophila abortus, Salmonella enterica Serovar abortusovis, Toxoplasma gondii, and Neospora caninum. The DNA from at least 1 of these 5 infectious agents was amplified in 41% of ovine fetuses, while only 17% of the caprine fetuses yielded a positive amplification result for at least 1 of the 5 agents. Out of a total of 366 ovine aborted samples, T. gondii DNA was detected most frequently (18.1% of fetuses and 13.1% of placentae), followed by S. abortusovis (13% of fetuses and 14.4% of placentae), C. burnetii (10.9% of fetuses, of 9.2% placentae), C. abortus (2.4% of fetuses, 6.5% of placentae), and N. caninum (2% of placentae). In 33 fetuses and 9 placentae, the simultaneous presence of pathogens with different associations was detected. Out of a total of 31 caprine aborted samples, T. gondii was detected most frequently (13% of fetuses and 25% of placentae), followed by C. abortus (12.5% of placentae), C. burnetii (12.5% of placentae), and N. caninum (8.6%).     

Chemical composition and antimicrobial activity of the essential oil from Ferula glauca L. (F. communis L. subsp. glauca) growing in Marche (central Italy)

The essential oil obtained from different parts of Ferula glauca L. (formerly considered as a subspecies of F. communis) growing in Marche (central Italy), was analyzed for the first time by GC-FID and GC-MS. The major volatiles were (E)-caryophyllene and caryophyllene oxide in leaves, alpha-pinene, myrcene and germacrene D in flowers, alpha- and beta-pinene in fruits, (E)-beta-farnesene, myristicin and elemicin in roots, respectively. The differences in composition detected with respect to F. communis, made the volatile fraction a reliable marker to distinguish between them, and confirm the botanical data at the base of their discrimination. Furthermore, the oil was assayed for its antimicrobial activity by the broth microdilution method. B. subtilis was found to be the most sensitive microorganism, with the lowest MIC values.