Brevi note in tema di "lesione del diritto di credito da parte di terzi" e di "responsabilità extracontrattuale da contratto"

<i>Artocarpus heterophyllus</i> is an evergreen tree distributed in tropical regions, and its fruit (jackfruit) is well-known as the world’s largest tree-borne fruit. Although <i>A. heterophyllus</i> has been widely used in folk medicines against inflammation, its potential in cancer chemoprevention remains unclear. Herein we identified artocarpin from <i>A. heterophyllus</i> as a promising colorectal cancer chemopreventive agent by targeting Akt kinase. Phenotypically, artocarpin exhibited selective cytotoxicity against human colon cancer cells. Artocarpin impaired the anchorage-independent growth capability, suppressed colon cancer cell growth, and induced a G1 phase cell cycle arrest which was followed by apoptotic as well as autophagic cell death. Mechanistic studies revealed that artocarpin directly targeted Akt 1 and 2 kinase activity evidenced by in vitro kinase assay, ex vivo binding assay as well as Akt downstream cellular signal transduction. Importantly, oral administration of artocarpin attenuated colitis-associated colorectal tumorigenesis in mice. Taken together, artocarpin, a bioactive component of <i>A. heterophyllus</i>, might merit investigation as a potential colorectal cancer chemopreventive agent

The Ginsenoside 20-O-β-D-Glucopyranosyl-20(S)-Protopanaxadiol Induces Autophagy and Apoptosis in Human Melanoma via AMPK/JNK Phosphorylation

<div><p>Studies have shown that a major metabolite of the red ginseng ginsenoside Rb1, called 20-O-β-D-glucopyranosyl-20(S)-protopanaxadiol (GPD), exhibits anticancer properties. However, the chemotherapeutic effects and molecular mechanisms behind GPD action in human melanoma have not been previously investigated. Here we report the anticancer activity of GPD and its mechanism of action in melanoma cells. GPD, but not its parent compound Rb1, inhibited melanoma cell proliferation in a dose-dependent manner. Further investigation revealed that GPD treatment achieved this inhibition through the induction of autophagy and apoptosis, while Rb1 failed to show significant effect at the same concentrations. The inhibitory effect of GPD appears to be mediated through the induction of AMPK and the subsequent attenuation of mTOR phosphorylation. In addition, GPD activated c-Jun by inducing JNK phosphorylation. Our findings suggest that GPD suppresses melanoma growth by inducing autophagic cell death and apoptosis via AMPK/JNK pathway activation. GPD therefore has the potential to be developed as a chemotherapeutic agent for the treatment of human melanoma.</p></div

GPD activates the AMPKα and/or JNK pathways in SK-MEL-28 human melanoma cells.

<p>(A) Phosphorylation of AMPKα is increased by GPD in a dose-dependent manner. (B) GPD upregulates phosphorylated JNK1/2 in a dose-dependent manner. (C) Levels of phosphorylated c-Jun, a well-known downstream of JNK, peak at 24 h after GPD treatment. (D) The phosphorylation of c-Jun after GPD treatment was dramatically decreased by co-treatment with the JNK inhibitor SP600125, whereas it decreased slightly in the presence of SB202180, a p38 inhibitor. (E) Activated AMPKα negatively regulated the phosphorylation of mTOR in a time-dependent manner. Cells (5×10⁵/100 mm dish) were incubated with various concentrations of GPD for the indicated durations. (F) GPD induces the phosphorylation of Bcl-2 and the dissociation of Beclin-1 from Bcl-2. Western blot analysis was performed using specific antibodies as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104305#s2" target="_blank">Materials and Methods</a>. β-actin was used to verify equal protein leading.</p

Discovery of the Novel mTOR Inhibitor and Its Antitumor Activities In Vitro and In Vivo

Abstract The phosphoinositide 3-kinase (PI3-K)/Akt and mTOR signaling pathway plays a critical role in cell survival and proliferation and is often aberrantly activated in many types of cancer. The mTOR kinase protein, one of the key molecules in this pathway, has been shown to be an important target for cancer therapy. In the present study, a ligand docking method was used to screen for novel scaffold mTOR inhibitors. Sixty thousand compounds in the Natural Product Database were screened against the mTOR homologous structure, and 13 commercially available compounds listed in the top-ranked 100 compounds were selected for further examin- -2-oxobut-3-en-1-yl)-3-hydroxyindolin-2-one; designated herein as 3HOI-BA-01] was then selected for further study of its antitumor activity. An in vitro study has shown that 3HOI-BA-01 inhibited mTOR kinase activity in a dose-dependent manner by directly binding with mTOR. In a panel of non-small cell lung cancer cells, the compound also attenuated mTOR downstream signaling, including the phosphorylation of p70S6K, S6, and Akt, resulting in G 1 cell-cycle arrest and growth inhibition. Results of an in vivo study have shown that intraperitoneal injection of 3HOI-BA-01 in A549 lung tumor-bearing mice effectively suppressed cancer growth without affecting the body weight of the mice

GPD induces apoptosis in SK-MEL-28 human melanoma cells.

<p>(A) GPD induces apoptosis in SK-MEL-28 cells. Cells (1.2×10⁵/60 mm dish) were incubated with or without 20 µM, 40 µM of GPD or 40 µM Rb1 for 48 h and apoptotic cells were stained with annexin V and propidium iodide (PI). (B) Apoptosis was determined by Fluorescence Activated Cell Sorting (FACS). The results are shown as mean of total apoptosis % ±SE (n = 3). The asterisk (**) indicates a significant difference (p<0.005) compared with the untreated control group. (C) GPD increases DNA fragmentation in SK-MEL-28 human melanoma cells. Cells were incubated with or without 20 µM, 40 µM of GPD or 40 µM Rb1 for 72 h and fragmented DNA was detected using a fragmentation assay as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104305#s2" target="_blank">Materials and Methods</a>. (D) The expression of the apoptotic markers cleaved PARP and cleaved caspases 3, 7 and 9, increased after 40 µM of GPD treatment, as confirmed by western blot analysis.</p

GPD induces autophagic cell death in a dose-dependent manner.

<p>(A) GPD induces autophagy in SK-MEL-28 cells whereas Rb1 cannot. GFP-LC3-transfected SK-MEL-28 cells were incubated with or without 40 µM of GPD and compared with Rb1 treatment for 48 h. Cells were fixed with 4% formaldehyde and then incubated with anti-p62/SQSTM1 antibody as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104305#s2" target="_blank">Materials and Methods</a>. Immunofluorescent staining was observed by confocal microscopy. (B) LC3 aggregations in transfected cells increase in the presence of GPD treatment in a dose-dependent manner. GFP-LC3 aggregations were counted by microscopic (×400) observation of the immunofluorescently stained cells. Results are shown as means ±SD. The asterisks (*) and (**) indicate statistical significance (p<0.05 and p<0.001, respectively) compared with untreated control groups. (C) The expression of LC3-II, p62/SQSTM1 and Beclin-1, which are critical autophagic markers, increased in the presence of GPD treatment, but not Rb1 treatment. Cells were treated with or without various concentration of GPD or 40 µM of Rb1 for 48 h, and protein expression was determined by western blot analysis. (D) GPD increased the translocation of cytochrome c from the mitochondria to the cytosol. Mitochondrial fractionation was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104305#s2" target="_blank">Materials and Methods</a>.</p

Hypothetical model for the anticancer effect of GPD in human melanoma.

<p>Hypothetical model for the anticancer effect of GPD in human melanoma.</p

GPD inhibits both anchorage-dependent and -independent growth in SK-MEL-28 human melanoma cells.

<p>(A) GPD inhibits the proliferation of SK-MEL-28 cells whereas Rb1 exhibits no significant effect. BrdU-incorporated cells were treated with various concentrations of GPD (20, 40, 60 µM) or Rb1 (60 µM). Results are shown as % of control ±SD (n = 4). The asterisks (**) and (***) indicate statistical significance (p<0.001 and p<0.0001, respectively) compared with the untreated control groups. (B) GPD markedly inhibits the anchorage-independent cell growth of SK-MEL-28 human melanoma cells, while Rb1 does not. A soft agar assay was performed with or without 40 µM of GPD or 40 µM of Rb1 and the number of colonies was counted under a microscope with the aid of Image-Pro Plus software (Version 6.2). Results are shown as means ±SE (n = 3). The asterisks (***) indicates a significant difference (p<0.001) compared with untreated control groups. (<b>C</b>) The effect of GPD on anchorage-independent cell growth inhibition is dose-dependent. The soft agar assay was repeated with various concentrations of GPD. Results are shown as means ±SE (n = 3). The asterisk (*) indicates statistical significance (p<0.05).</p