Antonella Minutolo

The novel proapoptotic activity of nonnatural enantiomer of Lentiginosine

D-(-)-Lentiginosine [(-)-4], the nonnatural enantiomer of the iminosugar indolizidine alkaloid L-(+)-lentiginosine, acts as apoptosis inducer on tumor cells of different origin, in contrast to its natural enantiomer. Although D-(-)-4 exhibited a proapoptotic activity towards tumor cells at level lower than the chemotherapeutic agent, SN38, it was less proapoptotic towards normal cells and less cytotoxic. Apoptosis induced by D-(-)-4 was caspase-dependent, as shown by the increased expression and activity of caspase-3 and -8 in treated cells, and by inhibition following treatment with the pan caspase inhibitor, ZVAD-FMK. This study highlighted how a natural iminosugar alkaloid and its synthetic enantiomer, which were simply known for their inhibition against a fungal glucoamylase, could behave in a complete different way when tested towards cell growth and death of cells of different origin.

D (−)lentiginosine-induced apoptosis involves the intrinsic pathway and is p53-independent

We have recently found that D(−)lentiginosine, a synthetic iminosugar exerting glucosidase inhibitory activity, but not its natural enantiomer lentiginosine, is endowed with an unexpected, pro-apoptotic activity. Here, we investigated mechanisms involved in apoptosis induced by D(−)lentiginosine in MOLT-3, HT-29 and SH-SY5Y tumour cell lines. The results showed that D(−)lentiginosine increased caspase 9 expression at 18 h in all the cell lines from 1.5–3.1 folds. Cytochrome c in the cytoplasm was found to be increased from 2.3–2.6 folds in treated cells with respect to control cells. These effects were accompanied by a remarkable collapse of the mitochondrial membrane potential and by the downregulation of anti-apoptotic genes, as well as the upregulation of pro-apoptotic genes of the Bcl-2 family. U937Bcl-2 transfectants, highly expressing Bcl-2, were reluctant to undergo apoptosis even following treatment with 500 μM D(−)lentiginosine, whereas apoptosis by D(−)lentiginosine was induced also in U937 cells, naturally deficient in P53. Thus, our study establishes that the enantiomer of a natural iminosugar is endowed with a possible anti-tumorigenic effect that might be ascribed not only to their capacity to inhibit glycosidases but also to other unknown mechanisms. These data encourage further investigation on similar compounds to make them an interesting platform for the generation of new anticancer drugs.

Dysregulated NF-κB pathway in peripheral mononuclear cells of Alzheimer's disease patients.

Diagnosis and therapeutic strategies in Alzheimers disease (AD) might greatly benefit of the present multidisciplinary approach for studying the molecular pathogenesis of the disorder. Gene expression profile at peripheral level could be a promising tool for pathogenic studies as well as for early diagnosis of AD. A dysregulated inflammatory response, as well as other systemic disorders, have been described in AD. Therefore, we investigated the expression, at peripheral level, of a number of genes involved in the inflammatory, oxidative stress and proliferative response of a well defined, small cohort of sporadic AD patients. Firstly, the mRNA expression of inflammatory, stress and proliferation/ differentiation genes were evaluated, using SuperArray, in mitogen-stimulated peripheral blood mononuclear cells (PBMC) from a group of 12 well-characterized, sporadic AD patients with various levels of dementia, by comparison with aged-matched controls. Real-time RT-PCR confirmed the trend of alteration in 16 genes out of the 36 supposed to be dysregulated in AD patients, by the preliminary screening. The expression level of the NFKB1(p105/50Kd) gene was significantly higher in AD with respect to adult age-matched controls (AA) and was related to the Mini-Mental State Examination (MMSE) score of the same patients. In addition, the expression of various NF-κB target genes and of both NF-κBp50 and NF-κBp65 DNA-binding activity were increased in PBMC from AD patients in comparison with those from AA. Our results suggest that NF-κB activation at peripheral blood cell level could be a potential new hallmark of AD progression and sustain a rationale to more deeply investigate the therapeutic potential of specific NF-κB inhibitors in AD.

Inhibition of NF-κB activation sensitizes U937 cells to 3'-azido-3'-deoxythymidine induced apoptosis.

In this study, we investigated molecular mechanisms underlying low susceptibility to apoptosis induced by the nucleoside analog azidothymidine (AZT) and the role of nuclear factor-κB (NF-κB) activation in these phenomena. A preliminary screening in different cell lines indicated U937 monocytic cell line as suitable to this purpose. Treatment of U937 cells even with suprapharmacological concentrations of AZT induced only moderate levels of apoptosis. Surprisingly, SuperArray analysis showed that AZT induced the transcriptional activity of both pro- and anti-apoptotic genes. Interestingly, moreover, several genes upregulated by AZT were NF-κB related. In fact, AZT, after an initial inhibition of NF-κB activation with respect to control, induced a transient, but consistent, increase in NF-κB-binding activity. Inhibition of NF-κB activation in U937 cells, stably transfected with a dominant-negative IκBα or by pharmacological treatment, sensitized them to apoptosis induced by AZT and impaired the upregulation of anti-apoptotic genes in response to AZT treatment, with respect to control cells. These results indicate that NF-κB activation by AZT has a role in protecting target cells from apoptotic cell death, improving our understanding of the toxicology and the therapeutic usage of this drug.

Platinum-(IV)-derivative satraplatin induced G2/M cell cycle perturbation via p53-p21waf1/cip1-independent pathway in human colorectal cancer cells

Aim:Platinum-(IV)-derivative satraplatin represents a new generation of orally available anti-cancer drugs that are under development for the treatment of several cancers. Understanding the mechanisms of cell cycle modulation and apoptosis is necessary to define the mode of action of satraplatin. In this study, we investigate the ability of satraplatin to induce cell cycle perturbation, clonogenicity loss and apoptosis in colorectal cancer (CRC) cells.Methods:CRC cells were treated with satraplatin, and the effects of satraplatin on apoptosis and the cell cycle were evaluated by flow cytometry. Western blot analysis was used to investigate the effects of satraplatin on cell cycle and apoptosis-related proteins. RT-qPCR was used to evaluate p53-related mRNA modulation.Results:Satraplatin induced an accumulation of CRC cells predominantly in the G2/M phase. Increased p53 protein expression was observed in the p53 wild-type HCT116 and LoVo cells together with p21waf1/cip1 protein up-regulation. However, p21waf1/cip1 protein accumulation was not observed in the p53 mutant HCT15, HT29, and WiDr cells, even when p53 protein expression was compromised, suggesting that the cell cycle perturbation is p53-p21waf1/cip1 independent. Following a candidate approach, we found an elevated expression of 14-3-3σ protein levels in CRC cells, which was independent of the status of p53, further supporting the role of satraplatin in the perturbation of the G2/M cell cycle phase. Moreover, satraplatin treatment induced apoptosis along with Bcl-2 protein down-regulation and abrogated the clonogenic formation of CRC cells in vitro.Conclusion:Collectively, our data suggest that satraplatin induces apoptosis in CRC cells, which is preceded by cell cycle arrest at G2/M due to the effect of 14-3-3σ and in a p53-p21waf1/cip1–independent manner. Taken together, these findings highlight the potential use of satraplatin for CRC treatment.

Transcription profile of human lymphocytes following in vitro treatment with thymosin alpha-1

Thymosin alpha‐1 (Tα1) is a molecule retaining pleiotropic effects toward several pathological conditions, especially acting as a modulator of immune response and inflammation. However, molecular mechanisms underlying the peculiar, wide‐range activity of Tα1 have not been completely elucidated. To get information on the mechanism of action of Tα1 and to find new potential applications, we performed the analysis of the transcription profile of 8300 genes in human peripheral blood mononuclear cells in vitro treated with Tα1. Together with a variety of genes already known to be modulated by Tα1, our analysis revealed a remarkable number of genes not yet described before to be modulated by this thymic peptide, revealing a complex scenario that underlines the role of this peptide as a regulator of lymphocyte functions. Here, we report the list of the more representative modulated genes and discuss their implications on the modulation of the innate and adaptive responses. Our results open new perspectives on the use of Tα1 for the treatment of diseases associated with immune‐disregulation.

MicroRNA from Moringa oleifera: Identification by High Throughput Sequencing and Their Potential Contribution to Plant Medicinal Value

Moringa oleifera is a widespread plant with substantial nutritional and medicinal value. We postulated that microRNAs (miRNAs), which are endogenous, noncoding small RNAs regulating gene expression at the post-transcriptional level, might contribute to the medicinal properties of plants of this species after ingestion into human body, regulating human gene expression. However, the knowledge is scarce about miRNA in Moringa. Furthermore, in order to test the hypothesis on the pharmacological potential properties of miRNA, we conducted a high-throughput sequencing analysis using the Illumina platform. A total of 31,290,964 raw reads were produced from a library of small RNA isolated from M. oleifera seeds. We identified 94 conserved and two novel miRNAs that were validated by qRT-PCR assays. Results from qRT-PCR trials conducted on the expression of 20 Moringa miRNA showed that are conserved across multiple plant species as determined by their detection in tissue of other common crop plants. In silico analyses predicted target genes for the conserved miRNA that in turn allowed to relate the miRNAs to the regulation of physiological processes. Some of the predicted plant miRNAs have functional homology to their mammalian counterparts and regulated human genes when they were transfected into cell lines. To our knowledge, this is the first report of discovering M. oleifera miRNAs based on high-throughput sequencing and bioinformatics analysis and we provided new insight into a potential cross-species control of human gene expression. The widespread cultivation and consumption of M. oleifera, for nutritional and medicinal purposes, brings humans into close contact with products and extracts of this plant species. The potential for miRNA transfer should be evaluated as one possible mechanism of action to account for beneficial properties of this valuable species.

Are Hedgehog and Wnt/β-catenin pathways involved in hepatitis C virus-mediated EMT?

To the Editor: We read with great interest the article by Akkari et al. [1], who evaluated the EMT induction and correlated oncogenic transformation induced by HCV protein NS5A in primary hepatocyte precursors. Epithelial–mesenchymal transition (EMT) is a biological process occurring during tissue development and regeneration, moreover, it is known to be involved in tumor progression and metastasis. The EMT is a phenomenon, which well represents the cellular plasticity, i.e., the ability of polarized epithelial cells, which through multiple biochemical changes acquire a mesenchymal cell phenotype. These cells exhibit loss of cell adhesion, enhanced migratory capacity, invasiveness, elevated resistance to apoptosis and a great ability to produce extracellular matrix (ECM) components. Several molecular processes are involved in EMT onset and progression. Among them we have to mention: activation of transcription factors (e.g., Snail, Twist, etc.), expression of specific cell-surface proteins, remodelling of cytoskeletal components, production of ECM-degrading enzymes and changes in the expression of specific microRNAs (e.g., microRNA 200 family). Most of them are commonly used as biomarkers to highlight a cellular EMT [2,3]. Akkari et al. have evaluated how a viral protein can alter the typical epithelial architecture of hepatic cells. In particular, they demonstrated that the ectopic expression of hepatitis C virus (HCV) non-structural protein 5a (NS5A) is able to induce EMT in bipotential mouse embryonic liver (BMEL) cells which acquire mesenchymal phenotype, increased motility and invasiveness, and are featured by the downregulation of epithelial markers (e.g., E-Cadherin) and the upregulation of mesenchymal markers (like Vimentin and Twist2). They also confirmed their results in an in vivo xenograft mouse model. To unravel the molecular mechanisms involved in EMT occurrence, they focused their experimental investigation on transforming growth factor (TGF)-b, a main cytokine involved in EMT induction. They have shown that NS5A is not responsible for TGF-b pathway activation but it acts in a synergistic manner with the cytokine in inducing EMT. Furthermore, they also screened, in their in vitro model, the modulation of expression of EMT biomarkers, such as Snail, Slug, Zeb and Twist 1/2. They demonstrated that only Twist2 expression was specifically increased by NS5A protein, and that, accordingly, through short hairpin RNA against Twist2, they were able to counteract EMT occurrence. Interestingly, the TGF-b treatment of cells lacking of Twist2 is able to promote EMT in any case, suggesting that NS5A and TGF-b trigger EMT by distinct pathways and thus identifying Twist2 as an NS5A EMT specific effector.

Effector caspase activation, in the absence of a conspicuous apoptosis induction, in mononuclear cells treated with azidothymidine

In the present study we focused our attention on the effect of AZT, at pharmacological and suprapharmacological concentrations, on some apoptosis-related key events and, particularly, on caspase activation in fresh human peripheral blood mononuclear cells (PBMCs). The main results can be summarized as follows: (i) AZT induced a strong, dose-dependent antiproliferative effect in mitogen-stimulated PBMCs, but low levels of cytotoxicity. in comparison with 5FU; (ii) low levels of cytotoxicity were coupled with a poor increase of apoptosis after AZT treatment in PBMCs; (iii) despite low levels of apoptosis, remarkable signs of both initiator and effector caspase enhanced expression with respect to control were detected by immunoblot analysis in AZT-treated PBMCs; (iv) enhanced caspase expression was associated with an increased expression of both anti-apoptotic Bcl-2 and pro-apoptotic Fas and p53 proteins, as detected by flow cytometry analysis; (v) combination treatment in vitro with AZT and anti-Fas significantly increased apoptosis in PBMCs with respect to single treatments. Overall, these results suggest that AZT treatment activates a complex, and apparently contrasting apoptosis-related signaling activity in PBMCs and that additional events are necessary to disrupt the balance induced by AZT towards apoptosis, on these cells.

Characterization of the enhanced apoptotic response to azidothymidine by pharmacological inhibition of NF-kB.

AIMS The present study addresses the issue of enhanced apoptotic response to AZT following co-treatment with an NF-kB inhibitor. MAIN METHODS To investigate this issue, different cell lines were assayed for susceptibility to AZT-mediated apoptosis without or with the addition of the NF-kB inhibitor Bay-11-7085. For further investigation, U937 cells were selected as good-responder cells to the combination treatment with 32 or 128 μM AZT, and 1 μM Bay-11-7085. Inhibition of NF-kB activation by Bay-11-7085 in cells treated with AZT was assayed through Western blot analysis of p65 expression and by EMSA. Involvement of the mitochondrial pathway of apoptosis in mechanisms underlying the improved effect of AZT following Bay-11-7085 co-treatment, was evaluated by assaying the cytochrome c release and the mitochondrial membrane potential (MMP) status using the JC-1 dye. Moreover, the transcriptional activity of both anti- and pro-apoptotic genes in U937 cells after combination treatment was quantitatively evaluated through real-time PCR. KEY FINDINGS We found that the combined treatment induced high levels of cytochrome c release and of MMP collapse in association with evident changes in the expression of both anti- and pro-apoptotic genes of the Bcl-2 family. Overexpression of Bcl-2 significantly suppressed the sensitization of U937 cells to an enhanced apoptotic response to AZT following co-treatment with the NF-kB inhibitor. SIGNIFICANCE The new findings suggest that a combination regimen based on AZT plus an NF-kB inhibitor could represent a new chemotherapeutic tool for retrovirus-related pathologies.