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Historically, Mangifera indica L. cultivations have been widely rooted in tropical areas of India, Africa, Asia and Central America. However, at least 20 years ago its spreading allowed the development of some cultivars, also in Sicily, the South of Italy, where the favorable subtropical climate and adapted soils represent the perfect field to create new sources of production for Sicilian agricultural supply chain. Currently, cultivations of Kensington Pride, Keitt, Klenn, Maya and Tommy Atkins varieties are active in Sicilian island and their products meet the requirements of local and European markets. Mango plants produce fleshy stone fruits rich in phytochemicals with an undisputed nutritional value for its high content of flavonoids, vitamins, micro- and macro-elements, vital for maintaining health. This review provides an overview of the antioxidant, anti-inflammatory and anticancer properties of Mango, a fruit that should be included in everyone’s diet for its multifaceted biochemical actions and nutraceutical potential.

ITF2357 (Givinostat) is a potent antineoplastic histone deacetylase inhibitor which is currently used in clinical trials for leukemias and myelomas and in the therapy for systemic juvenile idiopathic arthritis. Here evidence is provided that ITF2357 reduces the viability of human melanoma SK-Mel28 cells thereby inducing cell death. This compound was more efficacious than SAHA, another well known HDAC inhibitor belonging to the same class of hydroxamic acids. Moreover, we demonstrated for the first time that ITF2357 determines in SK-Mel28 cells a remarkable reduction in the level of oncogenic B-Raf, the product of the BRAF V600E mutated gene in melanoma. Western blot analysis showed that the decrease of oncogenic B-Raf induced by ITF2357 is dose and time dependent. This effect was accompanied with a decrease in the level of phosho-ERK confirming the blockage of the B-Raf mitogenic pathway. To potentiate the inhibition of this pathway, the MEK inhibitor UO126 was used in combination with ITF2357. The results indicated that this compound increases the effect of ITF2357 on cell death. Intriguingly, UO126 not only reduced ERK phosphorylation, as a confirmation of MEK inhibition, but also consistently reduced the level of B-Raf when combined with ITF2357. These preliminary results suggest that ITF2357, either alone or in combination with UO126, can be considered a good candidate in melanoma targeted therapy and ongoing studies will further clarify the mechanism of oncogenic B-Raf inhibition.

Litchi chinensis is a tropical fruit which cultivation has been recently introduced in Sicily. Some findings have shown that Litchi extracts display antitumor effects but the underlying mechanisms have not been elucidated. This study focuses on the effects of Litchi hydro-alcoholic extracts in colorectal cancer cells. The results indicated that Litchi exocarp (peel), mesocarp (pulp) and endocarp (seeds) extracts reduce the viability of HT-29 colon cancer cells in a dose dependent manner. This effect was accompanied with G2/M arrest of the cell cycle followed by cell death. Interestingly, exocarp and endocarp extracts triggered an autophagic response in the first phase of treatment (16-24h) with increase in the protein levels of ATG1/ Ulk kinase, p62 and LC3-II accumulation. In particular, time point evaluation indicated that with exocarp extracts the autophagic form LC3II appeared at 16h treatment whereas with endocarp extracts accumulated at 48h. ATG1/Ulk and p62 increased early at 16 h treatment in both cases and p62 remained elevated until 48h. On the other hand, autophagic effects were not observed with Litchi mesocarp extracts which directly induced caspase 3-dependent apoptosis. Polyphenol colorimetric assay showed that the Litchi extracts contain significant amounts of these compounds, particularly in exocarp and endocarp. Accordingly, treatment of cells with gallic acid, one bioactive component of polyphenols, mimicked the effects of the Litchi extracts. Taken together, these preliminary results provides in vitro evidence that litchi extracts activate the autophagic process preceding cell death and can thus be considered as potential chemopreventive agents for colorectal cancer.

The chaperonin Hsp60 has multiple functions, among which that of supporting the growth of some type of tumours. HDACi (histone-deacetylase inhibitors) are drugs that regulate gene expression via modulation of epigenetic mechanisms, and induce tumor-cell death. Here, we show that in the tumor cells H292 the HDACi SAHA decreases the intracellular level of Hps60 and promotes its extracellular trafficking by exosomal vesicles. SAHA caused a time- and dose-dependent decrease in cell viabil- ity with a G/2M cell-cycle arrest at 24 h and cell death at 48 h. These effects were accompanied by production of reactive oxygen species and mitochondrial membrane- potential dissipation. The marked decrease in Hsp60 level in SAHA-treated cells was not related to proteasomal degradation since it was not affected by the addition of the proteasome inhibitor MG132. Moreover, the analysis of post-translational modifica- tions of Hsp60 revealed that SAHA treatment induced a modest reduction in the ubiq- uitination of the protein, with no effect on its acetylation state, but did cause a marked increase in tyrosine-nitrated Hsp60. This effect was related to oxidative stress since it was prevented by the anti-oxidant N-acetylcysteine. Most importantly, we showed for the first time that SAHA markedly increases extracellular Hsp60 export via exosomes, which might explain the concomitant decrease of the intracellular chaperonin. Our results suggest that SAHA modifies Hsp60 by nitration and stimulates its extracellu- lar export via exosomes. Since Hsp60-bearing exosomes have been implicated in effec- tive anti-tumour responses, and since elevated intracellular levels of Hsp60 have been related to the arrest of tumour-cell death, our data offer clues to explore what might be as yet uncharacterized mechanisms by which SAHA works as antitumor drug., Italian Journal of Anatomy and Embryology, Vol. 120, No. 1 (Supplement) 2015

The chaperonin Hsp60 has multiple functions, among which that of supporting the growth of some type of tumours (1). HDACi (histone-deacetylase inhibitors) are drugs that regulate gene expression via modulation of epigenetic mechanisms, and induce tumor-cell death (2). Here, we show that in the tumor cells H292 the HDACi SAHA decreases the intracellular level of Hps60 and promotes its extracellular trafficking by exosomal vesicles. SAHA caused a time- and dose-dependent decrease in cell viability with a G/2M cell-cycle arrest at 24 h and cell death at 48 h. These effects were accompanied by production of reactive oxygen species and mitochondrial membrane-potential dissipation. The marked decrease in Hsp60 level in SAHA-treated cells was not related to proteasomal degradation since it was not affected by the addition of the proteasome inhibitor MG132. Moreover, the analysis of post-translational modifications of Hsp60 revealed that SAHA treatment induced a modest reduction in the ubiquitination of the protein, with no effect on its acetylation state, but did cause a marked increase in tyrosine-nitrated Hsp60. This effect was related to oxidative stress since it was prevented by the anti-oxidant N-acetylcysteine. Most importantly, we showed for the first time that SAHA markedly increases extracellular Hsp60 export via exosomes, which might explain the concomitant decrease of the intracellular chaperonin. Our results suggest that SAHA modifies Hsp60 by nitration and stimulates its extracellular export via exosomes. Since Hsp60-bearing exosomes have been implicated in effective anti-tumour responses, and since elevated intracellular levels of Hsp60 have been related to the arrest of tumour-cell death, our data offer clues to explore what might be as yet uncharacterized mechanisms by which SAHA works as antitumor drug. 1. Rappa et al. (2012) HSP-molecular chaperones in cancer biogenesis and tumour therapy: an overview. Anticancer Res. 32, 5139-5150. 2. Lauricella et al. (2012) SAHA/TRAIL combination induces detachment and anoikis of MDA-MB231 and MCF-7 breast cancer cells. Biochimie 94, 287-299.

The sesquiterpene lactone Parthenolide (PN) exerted a cytotoxic effect on MDA-MB231 cells, a triple-negative breast cancer (TNBC) cell line, but its effectiveness was scarce when employed at low doses. This represents an obstacle for a therapeutic utilization of PN. In order to overcome this difficulty we associated to PN the suberoylanilide hydroxamic acid (SAHA), an histone deacetylase inhibitor. Our results show that SAHA synergistically sensitized MDA-MB231 cells to the cytotoxic effect of PN. It is noteworthy that treatment with PN alone stimulated the survival pathway Akt/mTOR and the consequent nuclear translocation of Nrf2, while treatment with SAHA alone induced autophagic activity. However, when the cells were treated with SAHA/PN combination, SAHA suppressed PN effect on Akt/mTOR/Nrf2 pathway, while PN reduced the prosurvival autophagic activity of SAHA. In addition SAHA/PN combination induced GSH depletion, fall in m, release of cytochrome c, activation of caspase 3 and apoptosis. Finally we demonstrated that combined treatment maintained both hyperacetylation of histones H3 and H4 induced by SAHA and down-regulation of DNMT1 expression induced by PN. Inhibition of the DNA-binding activity of NF-kB, which is determined by PN, was also observed after combined treatment. In conclusion, combination of PN to SAHA inhibits the cytoprotective responses induced by the single compounds, but does not alter the mechanisms leading to the cytotoxic effects. Taken together our results suggest that this combination could be a candidate for TNBC therapy. J. Cell. Physiol. 230: 1276-1289, 2015. (c) 2014 Wiley Periodicals, Inc., A Wiley Company

We showed that the sesquiterpene lactone Parthenolide (PN) exerts strong cytotoxic effects on triple negative breast cancer MDA-MB231 cells. Our recent results suggest that PN exerts in these cells a cytoprotective effect, which is due to the activation of mTOR pathway. To inhibit this protective response we employ the HDAC inhibitor SAHA, which is known to prevent AKT/mTOR pathway. We show that PN activates Akt, mTOR, p70S6kinase and NRF2 while SAHA abolishes these effects. Further cell pretreatment with SAHA synergistically sensitizes the cells to the cytotoxic effect of PN. Moreover SAHA alone activates the autophagic process. The addition of PN to SAHA reduces this effect and induces apoptosis. SAHA/PN combination also inhibits DNMT1 and produces hyperacetylation of histones. Epigenetic changes caused by these effects are responsible for the increased expression of oncosuppressor gene products, such as p21 and p27 and for the decrease of Bcl2 and p65 levels. In conclusion SAHA suppresses protective response exerted by PN; PN inhibits SAHA effect on autophagy and finally SAHA/PN combination induces epigenetic modifications with changes in gene expression.

Parthenolide, a sesquiterpene lactone found in European feverfew, is used in traditional medicine for its anti-inflammatory activity. In addition, parthenolide has been considered as a novel and effective anti-tumor agent because it induces cytotoxic effects in several tumor cell lines. Our studies demonstrated that parthenolide exerted strong cytotoxic effects in osteosarcoma MG63 and melanoma SK-Mel28 cells in culture. Staining with Hoechst 33342 revealed in most cells after brief periods of treatments (3-5h) chromatin condensation and fragmentation, while only few cells were PI-positive. Prolonging the treatment (5-14h) PI-positive cells strongly augmented, denouncing the increase of necrotic effects. All these effects were prevented by NAC, while caspase inhibitors were ineffective, thus suggesting a caspase-independent cell death. The study of the mechanism of action provided evidence that treatment with parthenolide rapidly stimulated (1-2 h) ROS generation, in particular by inducing activation of extracellular signal-regulated kinase1/2 and NADPH oxidase. This event caused depletion of thiol groups and glutathione, NF-κB inhibition, JNK activation and cell detachment from the matrix. ROS generation together with mitochondrial accumulation of Ca2+ favoured dissipation of Δψm, which appeared primarily determined by the opening of the permeability transition pore (PTP), since Δψm loss was partially prevented by cyclosporin A, an inhibitor of PTP opening. Recently, we focused our attention on MDA-MB231 cells, a very aggressive and poorly differentiated breast cancer cell line, which is negative for estrogen receptor alpha. Preliminary results suggested that parthenolide induced cell death in these cells with a mechanism similar to that demonstrated in osteosarcoma and melanoma cells. Interestingly, we demonstrated that in MDA-MB231 cells the effect of parthenolide was potentiated by the addition of z-VAD-fmk, a general inhibitor of caspases. Studies are in progress to elucidate the mechanism of this interaction which could suggest new strategies for the treatment of ER-α negative breast cancer.

Parthenolide, the major bioactive sesquiterpene lactone present in Feverfew (Tanacetum parthenium), has recently attracted considerable attention because of its complex pharmacological action involving anti-microbial, anti-inflammatory and anti-cancer effects. However, the mechanism of its cytotoxic effect on tumor cells still remains scarcely defined today. The aim of this study was to analyse the mechanism of parthenolide action on two lines of cancer cells, the human osteosarcoma MG63 and the melanoma SK-MEL-28 cells, on which parthenolide exerted its action inducing similar effects. Staining with Hoechst 33342 showed that parthenolide induced in the first phase of treatment (0-5 h) in most of cells of both the lines condensation of chromatin while only few cells were PI-positive. Moreover, cells assumed a rounded shape, detached from substrate and showed a reduction of their volume. In the second phase of treatment (5-15 h) a progressive increase in the percentages of PI-positive cells was observed, suggesting that extensive damage of cellular plasma membranes occurred only after long periods of treatment. All these events were not counteracted by z-VAD-fmk and other caspase inhibitors, but were dependent on oxidative stress. In fact the study of mechanism of action of partenolide revealed that all cytotoxic effects were prevented by NAC and after a short period of time (1-2 h) ROS production occurred by inducing activation of extracellular signal-regulated kinase1/2 (ERK1/2) and NADPH oxidase. This event caused depletion of thiol groups and glutathione, NF-kB inhibition, JNK activation, cell detachment from the matrix and cellular shrinkage. The increase of ROS generation together with the mitochondrial accumulation of Ca2+ also favoured dissipation of ∆ψm, which seemed primarily determined by PTP opening, since ∆ψm loss was partially prevented by the inhibitor cyclosporin A. In addition, immunofluorescence analysis revealed that at this stage AIF translocated from mitochondria to the nucleus and co-localized with areas of condensed chromatin. Prolonging the treatment (5-15h) ATP content declined while PI-positive cells strongly augmented, denouncing the increase of necrotic effects. All these effects were prevented by NAC, while caspase inhibitors were ineffective. We suggest that AIF exerts a crucial role in parthenolide action. In accordance, down-regulation of AIF markedly inhibited parthenolide effect on the production of cells with apoptotic or necrotic signs. Taken together our results demonstrate that parthenolide causes in the two cell lines a caspase-independent cell death, which is mediated by AIF.