Monica Marra

Molecular targets and oxidative stress biomarkers in hepatocellular carcinoma: an overview

Hepatocellular carcinoma (HCC) is a complex and heterogeneous tumor with multiple genetic aberrations. Several molecular pathways involved in the regulation of proliferation and cell death are implicated in the hepatocarcinogenesis. The major etiological factors for HCC are both hepatitis B virus (HBV) and hepatitis C virus infection (HCV).Continuous oxidative stress, which results from the generation of reactive oxygen species (ROS) by environmental factors or cellular mitochondrial dysfunction, has recently been associated with hepatocarcinogenesis. On the other hand, a distinctive pathological hallmark of HCC is a dramatic down-regulation of oxido-reductive enzymes that constitute the most important free radical scavenger systems represented by catalase, superoxide dismutase and glutathione peroxidase.The multikinase inhibitor sorafenib represents the most promising target agent that has undergone extensive investigation up to phase III clinical trials in patients with advanced HCC. The combination with other target-based agents could potentiate the clinical benefits obtained by sorafenib alone. In fact, a phase II multicenter study has demonstrated that the combination between sorafenib and octreotide LAR (So.LAR protocol) was active and well tolerated in advanced HCC patients.The detection of molecular factors predictive of response to anti-cancer agents such as sorafenib and the identification of mechanisms of resistance to anti-cancer agents may probably represent the direction to improve the treatment of HCC.

The translation elongation factor 1A in tumorigenesis, signal transduction and apoptosis: Review article

Summary.An increasing number of evidences suggest the involvement of the eukaryotic elongation factor 1A, a core component of the protein synthesis machinery, at the onset of cell transformation. In fact, eEF1A is shown to be up-regulated in cell death; moreover, it seems to be involved in the regulation of ubiquitin-mediated protein degradation. In addition, eEF1A undergoes several post-translational modifications, mainly phosphorylation and methylation, that generally influence the activity of the protein. This article summarizes the present knowledges on the several extra-translational roles of eEF1A also in order to understand as the protein synthesis regulatory mechanisms could offer tools for cancer intervention.

Alpha‐interferon and its effects on signal transduction pathways

Interferon‐α (IFNα) is a recombinant protein widely used in the therapy of several neoplasms such as myeloma, renal cell carcinoma, epidermoid cervical and head and neck tumors, and melanoma. IFNα, the first cytokine to be produced by recombinant DNA technology, has emerged as an important regulator of cancer cell growth and differentiation, affecting cellular communication and signal transduction pathways. However, the way by which tumor cell growth is directly suppressed by IFNα is not well known. Wide evidence exists on the possibility that cancer cells undergo apoptosis after the exposure to the cytokine. Here we will review the consolidate signal transducer and activator of transcription (STAT)‐dependent mechanism of action of IFNα. We will discuss data obtained by us and others on the triggering of the stress‐dependent kinase pathway induced by IFNα and its correlations with the apoptotic process. The regulation of the expression of proteins involved in apoptosis occurrence will be also described. In this regard, IFNα is emerging as a post‐translational controller of the intracellular levels of the apoptosis‐related protein tissue transglutaminase (tTG). This new way of regulation of tTG occurs through the modulation of their proteasome‐dependent degradation induced by the cytokine. Until today, inconsistent data have been obtained regarding the clinical effectiveness of IFNα in the therapy of solid tumors. In fact, the benefit of IFNα treatment is limited to some neoplasms while others are completely or partially resistant. The mechanisms of tumor resistance to IFNα have been studied in vitro. The alteration of JAK‐STAT components of the IFNα‐induced signaling, can be indeed a mechanism of resistance to IFN. However, we have recently described a reactive mechanism of protection of tumor cells from the apoptosis induced by IFNα dependent on the epidermal growth factor (EGF)‐mediated Ras/extracellular signal regulated kinase (Erk) signaling. The involvement of the Ras→Erk pathway in the protection of tumor cells from the apoptosis induced by IFNα is further demonstrated by both Ras inactivation by RASN17 transfection and mitogen extracellular signal regulated kinase 1 (Mek‐1) inhibition by exposure to PD098059. These data strongly suggest that the specific disruption of the latter could be a useful approach to potentiate the antitumour activity of IFNα against human tumors based on the new mechanistic insights achieved in the last years.

Electromagnetic Fields at Mobile Phone Frequency Induce Apoptosis and Inactivation of the Multi-chaperone Complex in Human Epidermoid Cancer Cells

The exposure to non‐thermal microwave electromagnetic field (MW‐EMF) at 1.95 MHz, a frequency used in mobile communication, affects the refolding kinetics of eukaryotic proteins (Mancinelli et al., 2004 ). On these basis we have evaluated the in vivo effect of MW‐EMF in human epidermoid cancer KB cells. We have found that MW‐EMF induces time‐dependent apoptosis (45% after 3 h) that is paralleled by an about 2.5‐fold decrease of the expression of ras and Raf‐1 and of the activity of ras and Erk‐1/2. Although also the expression of Akt was reduced its activity was unchanged likely as a consequence of the increased expression of its upstream activator PI3K. In the same experimental conditions an about 2.5‐fold increase of the ubiquitination of ras and Raf‐1 was also found and the addition for 12 h of proteasome inhibitor lactacystin at 10 μM caused an accumulation of the ubiquitinated isoforms of ras and Raf‐1 and counteracted the effects of MW‐EMF on ras and Raf‐1 expression suggesting an increased proteasome‐dependent degradation induced by MW‐EMF. The exposure of KB cells to MW‐EMF induced a differential activation of stress‐dependent pathway with an increase of JNK‐1 activity and HSP70 and 27 expression and with a reduction of p38 kinase activity and HSP90 expression. The overexpression of HSP90 induced by transfection of KB cells with a plasmid encoding for the factor completely antagonized the apoptosis and the inactivation of the ras → Erk‐dependent survival signal induced by MW‐EMF. Conversely, the inhibition of Erk activity induced by 12 h exposure to 10 mM Mek‐1 inhibitor U0126 antagonized the effects induced by HSP90 transfection on apoptosis caused by MW‐EMF. In conclusion, these results demonstrate for the first time that MW‐EMF induces apoptosis through the inactivation of the ras → Erk survival signaling due to enhanced degradation of ras and Raf‐1 determined by decreased expression of HSP90 and the consequent increase of proteasome dependent degradation.

Percutaneous Ethanol Injection Efficacy in the Treatment of Large Symptomatic Thyroid Cystic Nodules: Ten-Year Follow-Up of a Large Series

We present a prospective study on the long-term efficacy of percutaneous ethanol injection (PEI) treatment of a large series of symptomatic thyroid cystic nodules (STCN). Ninety-eight patients (72 females and 26 males) were treated. The mean basal volume of the STCN was 35.3 mL. In 92 of 98 patients PEI treatment induced a greater than 50% nodule shrinkage, only 6 of 92 responder patients relapsed at a follow-up of 9 years. Moreover, all the patients had a significant clinical benefit because a significant reduction of the cyst-associated symptoms was recorded. Furthermore, a limited number of sessions was required for the treatment of cysts larger than 40 mL (mean +/- standard deviation [SD]: 2.7 +/- 0.75) demonstrating the feasibility of the procedure also in the treatment of large cysts. In conclusion, PEI is an effective and inexpensive procedure with a high patient compliance and long-lasting effects in the treatment of cysts larger than 40 mL.

The farnesyl transferase inhibitor R115777 (Zarnestra) synergistically enhances growth inhibition and apoptosis induced on epidermoid cancer cells by Zoledronic acid (Zometa) and Pamidronate.

Pamidronate (PAM) and zoledronic acid (ZOL) are aminobisphosphonates (BPs) able to affect the isoprenylation of intracellular small G proteins. We have investigated the antitumor activity of BPs and R115777 farnesyl transferase inhibitor (FTI) against epidermoid cancer cells. In human epidermoid head and neck KB and lung H1355 cancer cells, 48 h exposure to PAM and ZOL induced growth inhibition (IC50 25 and 10 μM, respectively) and apoptosis and abolished the proliferative and antiapoptotic stimuli induced by epidermal growth factor (EGF). In these experimental conditions, ZOL induced apoptosis through the activation of caspase 3 and a clear fragmentation of PARP was also demonstrated. A strong decrease of basal ras activity and an antagonism on its stimulation by EGF was recorded in the tumor cells exposed to BPs. These effects were paralleled by impaired activation of the survival enzymes extracellular signal regulated kinase 1 and 2 (Erk-1/2) and Akt that were not restored by EGF. Conversely, farnesol induced a recovery of ras activity and antagonized the proapoptotic effects induced by BPs. The combined treatment with BPs and R115777 resulted in a strong synergism both in growth inhibition and apoptosis in KB and H1355 cells. The synergistic activity between the drugs allowed BPs to produce tumor cell growth inhibition and apoptosis at in vivo achievable concentrations (0.1 μmolar for both drugs). Moreover, the combination was highly effective in the inhibition of ras, Erk and Akt activity, while farnesol again antagonized these effects. In conclusion, the combination of BPs and FTI leads to enhanced antitumor activity at clinically achievable drug concentrations that resides in the inhibition of farnesylation-dependent survival pathways and warrants further studies for clinical translation.

Nanotechnologies to use bisphosphonates as potent anticancer agents: the effects of zoledronic acid encapsulated into liposomes.

UNLABELLED Zoledronic acid (ZOL) is a potent amino-bisphosphonate used for the treatment of bone metastases with recently reported antitumor activity. However, the short plasma half-life and rapid accumulation in bone limits the use of ZOL as an antitumor agent in extraskeletal tissues. Therefore, we developed stealth liposomes encapsulating ZOL (LipoZOL) to increase extraskeletal drug availability. Compared to free ZOL, LipoZOL induced a stronger inhibition of growth of a range of different cancer cell lines in vitro. LipoZOL also caused significantly larger inhibition of tumor growth and increased the overall survival in murine models of human prostate cancer and multiple myeloma, in comparison with ZOL. Moreover, a strong inhibition of vasculogenetic events without evidence of necrosis in the tumor xenografts from prostate cancer was recorded after treatment with LipoZOL. We demonstrated both antitumor activity and tolerability of LipoZOL in preclinical animal models of both solid and hematopoietic malignancies, providing a rationale for early exploration of use of LipoZOL as a potential anticancer agent in cancer patients. FROM THE CLINICAL EDITOR The short plasma half-life and rapid accumulation in bone limits the use of zoledronic acid as an antitumor agent in extraskeletal tissues. Therefore, stealth liposomes encapsulating ZOL (LipoZOL) have been developed to increase extraskeletal drug availability.

Mitogen-activated protein kinases and asthma

Mitogen‐activated protein kinases (MAPKs) are evolutionary conserved enzymes which play a key role in signal transduction mediated by cytokines, growth factors, neurotransmitters and various types of environmental stresses. In the airways, these extracellular stimuli elicit complex inflammatory and structural changes leading to the typical features of asthma including T cell activation, eosinophil and mast cell infiltration, as well as bronchial hyperresponsiveness and airway remodelling. Because MAPKs represent an important point of convergence for several different signalling pathways, they affect multiple aspects of normal airway function and also significantly contribute to asthma pathophysiology. Therefore, this review focuses on the crucial involvement of MAPKs in asthma pathogenesis, thus also discussing their emerging role as molecular targets for anti‐asthma drugs.

EGF activates an inducible survival response via the RAS-> Erk-1/2 pathway to counteract interferon- α -mediated apoptosis in epidermoid cancer cells

AbstractThe mechanisms of tumor cell resistance to interferon-α (IFNα) are at present mostly unsolved. We have previously demonstrated that IFNα induces apoptosis on epidermoid cancer cells and EGF antagonizes this effect. We have also found that IFNα-induced apoptosis depends upon activation of the NH2-terminal Jun kinase-1 (Jnk-1) and p38 mitogen-activated protein kinase, and that these effects are also antagonized by EGF. At the same time, IFNα increases the expression and function of the epidermal growth factor receptor (EGF-R). Here we report that the apoptosis induced by IFNα occurs together with activation of caspases 3, 6 and 8 and that EGF also antagonizes this effect. On the basis of these results, we have hypothesized that the increased EGF-R expression and function could represent an inducible survival response that might protect tumor cells from apoptosis caused by IFNα via extracellular signal regulated kinase 1 and 2 (Erk-1/2) cascades. We have found an increased activity of Ras and Raf-1 in IFNα-treated cells. Moreover, IFNα induces a 50% increase of the phosphorylated isoforms and enzymatic activity of Erk-1/2. We have also demonstrated that the inhibition of Ras activity induced by the transfection of the dominant negative Ras plasmid RASN17 and the inhibition of Mek-1 with PD098059 strongly potentiates the apoptosis induced by IFNα. Moreover, the selective inhibition of this pathway abrogates the counteracting effect of EGF on the IFNα-induced apoptosis. All these findings suggest that epidermoid tumor cells counteract the IFNα-induced apoptosis through a survival pathway that involves the hyperactivation of the EGF-dependent Ras->Erk signalling. The selective targeting of this pathway appears to be a promising approach in order to enhance the antitumor activity of IFNα.

The PPAR-γ agonist troglitazone antagonizes survival pathways induced by STAT-3 in recombinant interferon-β treated pancreatic cancer cells.

We have previously shown that cancer cells can protect themselves from apoptosis induced by type I interferons (IFNs) through a ras→MAPK-mediated pathway. In addition, since IFN-mediated signalling components STATs are controlled by PPAR gamma we studied the pharmacological interaction between recombinant IFN-β and the PPAR-γ agonist troglitazone (TGZ). This combination induced a synergistic effect on the growth inhibition of BxPC-3, a pancreatic cancer cell line, through the counteraction of the IFN-β-induced activation of STAT-3, MAPK and AKT and the increase in the binding of both STAT-1 related complexes and PPAR-γ with specific DNA responsive elements. The synergism on cell growth inhibition correlated with a cell cycle arrest in G0/G1 phase, secondary to a long-lasting increase of both p21 and p27 expressions. Blockade of MAPK activation and the effect on p21 and p27 expressions, induced by IFN-β and TGZ combination, were due to the decreased activation of STAT-3 secondary to TGZ. IFN-β alone also increased p21 and p27 expression through STAT-1 phosphorylation and this effect was attenuated by the concomitant activation of IFNbeta-induced STAT-3-activation. The combination induced also an increase in autophagy and a decrease in anti-autophagic bcl-2/beclin-1 complex formation. This effect was mediated by the inactivation of the AKT→mTOR-dependent pathway. To the best of our knowledge this is the first evidence that PPAR-γ activation can counteract STAT-3-dependent escape pathways to IFN-β-induced growth inhibition through cell cycle perturbation and increased autophagic death in pancreatic cancer cells.