Andrea Santulli

Differentiative pathway activated by 3-aminobenzamide, an inhibitor of PARP, in human osteosarcoma MG-63 cells.

This study describes the molecular mechanism by which treatment with 3‐AB, a potent inhibitor of PARP, allows human osteosarcoma MG‐63 cells to restrict growth and enter differentiation. Our findings show that in MG‐63 cells, aberrant gene expression keeps Rb protein constitutively inactivated through hyperphosphorylation and this promotes uncontrolled proliferation of the cells. After 3‐AB‐treatment, the poly(ADP‐ribosyl)ation of nuclear proteins markedly decreases and this results in an increase in both the hypophosphorylated active form of Rb and pRb/E2F complexes. These effects are accompanied by G1 arrest, downregulation of gene products required for proliferation (cyclin D1, β‐catenin, c‐Jun, c‐Myc and Id2) and upregulation of those implicated in the osteoblastic differentiation (p21/Waf1, osteopontin, osteocalcin, type I collagen, N‐cadherins and alkaline phosphatase). Our study suggests that use of PARP inhibitors may induce a remodeling of chromatin with the reprogramming of gene expression and the activation of differentiation.

Identification and expansion of human osteosarcoma-cancer-stem cells by long-term 3-aminobenzamide treatment

A novel cancer stem‐like cell line (3AB‐OS), expressing a number of pluripotent stem cell markers, was irreversibly selected from human osteosarcoma MG‐63 cells by long‐term treatment (100 days) with 3‐aminobenzamide (3AB). 3AB‐OS cells are a heterogeneous and stable cell population composed by three types of fibroblastoid cells, spindle‐shaped, polygonal‐shaped, and rounded‐shaped. With respect to MG‐63 cells, 3AB‐OS cells are extremely smaller, possess a much greater capacity to form spheres, a stronger self‐renewal ability and much higher levels of cell cycle markers which account for G1‐S/G2‐M phases progression. Differently from MG‐63 cells, 3AB‐OS cells can be reseeded unlimitedly without losing their proliferative potential. They show an ATP‐binding cassette transporter ABCG2‐dependent phenotype with high drug efflux capacity, and a strong positivity for CD133, marker for pluripotent stem cells, which are almost unmeasurable in MG‐63 cells. 3AB‐OS cells are much less committed to osteogenic and adipogenic differentiation than MG‐63 cells and highly express genes required for maintaining stem cell state (Oct3/4, hTERT, nucleostemin, Nanog) and for inhibiting apoptosis (HIF‐1α, FLIP‐L, Bcl‐2, XIAP, IAPs, and survivin). 3AB‐OS may be a novel tumor cell line useful for investigating the mechanisms by which stem cells enrichment may be induced in a tumor cell line. The identification of a subpopulation of cancer stem cells that drives tumorigenesis and chemoresistance in osteosarcoma may lead to prognosis and optimal therapy determination. Expression patterns of stem cell markers, especially CD133 and ABCG2, may indicate the undifferentiated state of osteosarcoma tumors, and may correlate with unfavorable prognosis in the clinical setting. J. Cell. Physiol. 219: 301–313, 2009.

The histone deacetylase inhibitor suberoylanilide hydroxamic acid sensitises human hepatocellular carcinoma cells to TRAIL-induced apoptosis by TRAIL-DISC activation

This paper shows that the histone deacetylase inhibitor SAHA sensitised at sub-toxic doses human hepatocellular carcinoma cells (HepG2, Hep3B and SK-Hep1) to TRAIL-induced apoptosis, while it was ineffective in primary human hepatocytes (PHHs). In particular in HCC cells SAHA increased the expression of death receptor 5 (DR5) and caused a decrement of c-Flip. These two modifications provoked in the presence of TRAIL the rapid production of TRAIL-DISC and the activation of caspase-8. Consequently SAHA/TRAIL combination induced many apoptotic events, such as a cleavage of Bid into tBid, dissipation of mitochondrial membrane potential, activation of caspase-3 with the consequent cleavage of both NF-kB and Akt. The decrease in NF-kB level seemed to be responsible for the reduction in the content of IAP family antiapoptotic proteins while the decrease in Akt level caused a reduction in phospho-Bad. These events led to the activation of caspase-9, which contributed to the strong apoptotic activity of TRAIL. Sensitisation of human hepatocellular carcinoma cells to TRAIL-induced apoptosis by SAHA may suggest new strategies for the treatment of liver tumours.

Effect of sodium dodecyl sulfate (SDS) on stress response in the Mediterranean mussel (Mytilus Galloprovincialis): regulatory volume decrease (Rvd) and modulation of biochemical markers related to oxidative stress.

In this study the effects of an anionic surfactant, sodium dodecyl sulfate (SDS), are assessed on the Mediterranean mussel (Mytilus galloprovincialis), exposed for 18 days at a concentration ranging from 0.1 mg/l to 1 mg/l. The effects are monitored using biomarkers related to stress response, such as regulatory volume decrease (RVD), and to oxidative stress, such as reactive oxygen species (ROS), endogenous antioxidant systems and Hsp70 levels. The results demonstrate that cells from the digestive gland of M. galloprovincialis, exposed to SDS were not able to perform the RVD owing to osmotic stress. Further, SDS causes oxidative stress in treated organisms, as demonstrated by the increased ROS production, in comparison to the controls (p<0.05). Consequently, two enzymes involved in ROS scavenging, superoxide dismutase (SOD) and catalase (CAT) have higher activities and the proportion of oxidized glutathione (GSSG) is higher in hepatopancreas and mantle of treated animals, compared to untreated animals (p<0.05). Furthermore Hsp70 demonstrates an up-regulation in all the analyzed tissues of exposed animals, attesting the stress status induced by the surfactant with respect to the unexposed animals. The results highlight that SDS, under the tested concentrations, exerts a toxic effect in mussels in which the disruption of the osmotic balance follows the induction of oxidative stress.

Parthenolide sensitizes hepatocellular carcinoma cells to trail by inducing the expression of death receptors through inhibition of STAT3 activation

This article shows that HepG2, Hep3B, and SK‐Hep1 cells, three lines of human hepatocellular carcinoma (HCC) cells, are resistant to apoptosis induced by tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL). Parthenolide, a sesquiterpene lactone found in European feverfew, has been shown to exert both anti‐inflammatory and anti‐cancer activities. This article demonstrates that co‐treatment with parthenolide and TRAIL‐induced apoptosis with synergistic interactions in the three lines of HCC cells. In order to explain these effects we ascertained that parthenolide increased either at protein or mRNA level the total content of death receptors TRAIL‐R1 and ‐R2 as well as their surface expression. These effects were found in the three cell lines in the case of TRAIL‐R2, while for TRAIL‐R1 they were observed in HepG2 and SK‐Hep1 cells, but not in Hep3B cells. We suggest that the effects of parthenolide on death receptors depend on the decrease in the level of phosphorylated and active forms of STAT proteins, an event which could be a consequence of the inhibitory effect exerted by parthenolide on the activation of JAK proteins. In agreement with this hypothesis treatment with STAT3 siRNA increased in HCC cells the effect of parthenolide on the expression of death receptors. Sensitization by parthenolide to TRAIL stimulated in the three cell lines the extrinsic mechanism of apoptosis with the activation of both caspases 8 and 3, whereas mitochondria were not involved in the process. Our results suggest that co‐treatment with parthenolide and TRAIL could represent a new important therapeutic strategy for hepatic tumors. J. Cell. Physiol. 226: 1632–1641, 2011.

Apoptosis induced in HepG2 cells by the synthetic cannabinoid WIN: involvement of the transcription factor PPARγ.

It has recently been shown that cannabinoids induce growth inhibition and apoptosis in different tumour cell lines. In the current study, the effects of WIN 55,212-2 (WIN), a synthetic and potent cannabinoid receptor agonist, are investigated in hepatoma HepG2 cells and a possible signal transduction pathway is proposed. In these cells, WIN induces a clear apoptotic effect which was accompanied by up-regulation of the death-signalling factors Bax, Bcl-X(S), t-Bid and down-regulation of the survival factors survivin, phospho-AKT, Hsp72 and Bcl-2. Moreover, WIN-induced apoptosis is associated with JNK/p38 MAPK pathway activation and mitochondrial depolarisation demonstrated by a cytofluorimetric assay. The results also show that in HepG2 cells WIN markedly increases the level of the transcription factor PPARgamma in a dose- and time-dependent manner. The addition of the PPARgamma antagonists GW9662 and T0070907 significantly reduces the effects of the drug on both cell viability and the levels of survivin, phospho-AKT and phospho-BAD, suggesting that PPARgamma plays a key role in WIN-induced apoptosis. Altogether, the results seem to indicate a potential therapeutic role of WIN in hepatic cancer treatment.

Research paperApoptosis induced in HepG2 cells by the synthetic cannabinoid WIN: Involvement of the transcription factor PPARγ

It has recently been shown that cannabinoids induce growth inhibition and apoptosis in different tumour cell lines. In the current study, the effects of WIN 55,212-2 (WIN), a synthetic and potent cannabinoid receptor agonist, are investigated in hepatoma HepG2 cells and a possible signal transduction pathway is proposed. In these cells, WIN induces a clear apoptotic effect which was accompanied by up-regulation of the death-signalling factors Bax, Bcl-X(S), t-Bid and down-regulation of the survival factors survivin, phospho-AKT, Hsp72 and Bcl-2. Moreover, WIN-induced apoptosis is associated with JNK/p38 MAPK pathway activation and mitochondrial depolarisation demonstrated by a cytofluorimetric assay. The results also show that in HepG2 cells WIN markedly increases the level of the transcription factor PPARgamma in a dose- and time-dependent manner. The addition of the PPARgamma antagonists GW9662 and T0070907 significantly reduces the effects of the drug on both cell viability and the levels of survivin, phospho-AKT and phospho-BAD, suggesting that PPARgamma plays a key role in WIN-induced apoptosis. Altogether, the results seem to indicate a potential therapeutic role of WIN in hepatic cancer treatment.

The Synthetic Cannabinoid WIN 55,212-2 Sensitizes Hepatocellular Carcinoma Cells to Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL)-Induced Apoptosis by Activating p8/CCAAT/Enhancer Binding Protein Homologous Protein (CHOP)/Death Receptor 5 (DR5) Axis

In this article, we demonstrate that the synthetic cannabinoid R-(+)-(2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrol[1,2,3-de]-1,4-benzoxazin-6-yl)-(1-naphthalenyl) methanone mesylate (WIN 55,212-2) sensitizes human hepatocellular carcinoma (HCC) cells to apoptosis mediated by tumor necrosis-related apoptosis inducing ligand (TRAIL). The apoptotic mechanism induced by treatment with WIN/TRAIL combination involved the loss of the mitochondrial transmembrane potential and led to the activation of caspases. In HCC cells, WIN treatment induced the up-regulation of TRAIL death receptor DR5, an effect that seemed to be related to the increase in the level of p8 and CHOP, two factors implicated in cellular stress response and apoptosis. This relationship was suggested by the observation that the down-regulation of p8 or CHOP by specific small interfering RNAs attenuated both WIN-mediated DR5 up-regulation and the cytotoxicity induced by WIN/TRAIL cotreatment. Moreover, WIN induced a significant decrease in the levels of some survival factors (survivin, c-inhibitor of apoptosis protein 2, and Bcl-2) and in particular in that of the active phosphorylated form of AKT. This event seemed to be dependent on the transcription factor peroxisome proliferator-activated receptor-γ whose level significantly increased after WIN treatment. Therefore, both the induction of DR5 via p8 and CHOP and the down-regulation of survival factors seem to be crucial for the marked synergistic effects induced by the two drugs in HCC cells. Taken together, the results reported in this article indicate that WIN/TRAIL combination could represent a novel important tool for the treatment of HCC.

Paclitaxel and beta-lapachone synergistically induce apoptosis in human retinoblastoma Y79 cells by downregulating the levels of phospho-Akt.

Paclitaxel (PTX) and beta‐lapachone (LPC) are naturally occurring compounds that have shown a large spectrum of anticancer activity. In this article we show for the first time that PTX/LPC combination induces potent synergistic apoptotic effects in human retinoblastoma Y79 cells. Combination of suboptimal doses of PTX (0.3 nM) and LPC (1.5 µM) caused biochemical and morphological signs of apoptosis at 48 h of treatment. These effects were accompanied by potent lowering in inhibitor of apoptosis proteins and by activation of Bid and caspases 3 and 6 with lamin B and PARP breakdown. PTX/LPC combination acted by favoring p53 stabilization through a lowering in p‐Akt levels and in ps166‐MDM2, the phosphorylated‐MDM2 form that enters the nucleus and induces p53 export and degradation. Treatment with wortmannin or transfection with a dominant negative form of Akt anticipated at 24 h the effects induced by PTX/LPC, suggesting a protective role against apoptosis played by Akt in Y79 cells. In line with these results, we demonstrated that Y79 cells contain constitutively active Akt, which forms a cytosolic complex with p53 and MDM2 driving p53 degradation. PTX/LPC treatment induced a weakness of Akt–MDM2–p53 complex and increased nuclear p53 levels. Our results suggest that phospho‐Akt lowering is at the root of the apoptotic action exerted by PTX/LPC combination and provide strong validation for a treatment approach that targets survival signals represented by phospho‐Akt and inhibitor of apoptosis proteins. J. Cell. Physiol. 222: 433–443, 2010.

Effects of dietary administration of fenugreek seeds, alone or in combination with probiotics, on growth performance parameters, humoral immune response and gene expression of gilthead seabream (Sparus aurata L.)

ABSTRACT The use of immunostimulants is considered a promising preventive practice that may help to maintain animal welfare and a healthy environment, while increasing production and providing higher profits. The purpose of this study was to evaluate the effects on gilthead seabream (Sparus aurata L.) of the dietary administration of fenugreek (Trigonella foenum graecum) seeds, alone or combined with one of the following probiotic strains: Bacillus licheniformis (TSB27), Lactobacillus plantarum or Bacillus subtilis (B46). Gilthead seabream were fed a control or one of the supplemented diets for 3 weeks. The effects of these supplemented diets on growth performance parameters and the humoral immune response (natural haemolytic complement, peroxidase, total IgM levels, proteases and antiproteases activities) were evaluated after 2 and 3 weeks of feeding. Simultaneously, the expression levels of some immune‐relevant genes (igm, tcr‐&bgr;, csfr1 and bd) were measured in the head‐kidney. Interestingly, all probiotic supplemented diets increased seabream growth rates, especially the B. licheniformis supplemented diet. Generally, humoral immune parameters were enhanced by the dietary supplementation at the different time points measured. The results showed a significant increases in the immune parameters, principally in fish fed only fenugreek or fenugreek combined with B. subtilis. Furthermore, real time qPCR revealed that dietary supplementation significantly enhances the expression of immune‐associated genes in the head‐kidney, particularly igm gene expression. These results suggest that fenugreek alone or combined with one of the probiotic strains mentioned enhances the immune response of gilthead seabream, a species with one of the highest rates of production in marine aquaculture. HIGHLIGHTSInnate immune response was enhanced by the fenugreek dietary supplementation.Humoral immune parameters were stimulates mainly by fenugreek + B. subtilis diet.Supplemented diets induced an important up‐regulation of igm gene expression.Fenugreek and probiotic tested could be considered as a good aqua feed supplements.The use of immunostimulants is a promising alternative to maintain animal welfare.