Gaia Giuberti

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.

Alternative therapy of earth elements increases the chondroprotective effects of chondroitin sulfate in mice

The administration of mineral sulphur water is an alternative experimental approach for the treatment of rheumatic diseases, such as osteoarthritis (OA), that cause the degeneration of bone and cartilage and sufferance to the patients. Chondroitin sulfate (CS) is a symptomatic slow acting nutropeucital agent currently used in molecular therapy of OA. Therefore, we have studied the role and efficacy of the selective soil paste from the mineral sulphur enriched spring (mud)-therapy alone or in combination with CS in the treatment of OA. The study was performed on 40 C57 Black 6N mice, an experimental model which spontaneously develop an osteoarthritic process. The animals were divided in 4 groups and were treated with the single agents or with the combination. After 30 days of treatment all the mice were sacrificed and right knees and blood were collected. It was found that CS determined a reduction of radiological and histological features of chondrodegeneration and that mud-therapy increased the effects of CS in the animal group treated with the combination. However, the effects of thermal therapy alone were not statistically significant. Since OA is characterized by an increase of the production of nitric oxide (NO) by chondrocytes in extracellular matrix with its consequent elevation in serum and synovial fluid, we have evaluated the effects of the treatments on serum NO levels. CS alone induced a statistically significant reduction of NO serum levels (90±13 µM vs 219±60 µM of control group, P< 0.05) while mud-therapy alone induced a not statistically significant reduction of serum NO (170 ±62µM, P>0.05). However, the latter strongly potentiated the decrease of serum NO induced by CS (31±1.5 µM) with a high statistical significance if compared to both the control group (P <0.01) and the CS-treated group (P<0.05). In conclusion, this study demonstrates that mud-therapy with sulphur mineral water could represent an important phase of the therapeutic strategy of OA. This experimental strategy could integrate and potentiate the standard pharmacological tools. Moreover, we have set a valid experimental in vivo model for the study of the thermal effects on the development of OA.

Ubiquitination of tissue transglutaminase is modulated by interferon alpha in human lung cancer cells.

The addition of 2500 i.u./ml interferon alpha (IFNalpha) for 48 h induced apoptosis, and caused an approx. 4-fold increase in the activity and expression of tissue transglutaminase (tTG), in human lung cancer H1355 cells. However, the increase in mRNA levels for tTG was just 1.6-fold. On the basis of these data, we investigated whether tTG levels may be regulated through regulation of its degradation via ubiquitination. It was found that 2500 i.u./ml IFNalpha induced a time-dependent decrease in tTG ubiquitination. On the other hand, addition of the proteasome inhibitor lactacystin led to accumulation of the ubiquitinated form of the enzyme and to a consequent increase in its expression. Treatment of the cells with the two agents combined antagonized the accumulation of the ubiquitinated isoforms of tTG induced by lactacystin and caused a potentiation of tTG expression. Moreover, the tTG inducer retinoic acid was also able to cause increased expression and ubiquitination of tTG in H1355 cells. The addition of monodansylcadaverine (a tTG inhibitor) to IFNalpha-treated H1355 cells completely antagonized growth inhibition and apoptosis induced by the cytokine. In conclusion, we demonstrate for the first time that tTG is ubiquitinated and degraded by a proteasome-dependent pathway. Moreover, IFNalpha can, at least in part, induce apoptosis through the modulation of this pathway.

Pharmacological inhibition of HSP90 and ras activity as a new strategy in the treatment of HNSCC

Advanced head and neck squamous cell cancer (HNSCC) is currently treated with taxane‐based chemotherapy. We have previously shown that docetaxel (DTX) induces a ras‐dependent survival signal that can be antagonized by farnesyl transferase inhibitors (FTI) such as tipifarnib (TIP). Here we show that the synergistic TIP/DTX combination determines synergistic apoptotic conditions but, at the same time, it modulates the expression of the components of the multichaperone complex that is, in turn, involved in the regulation of the stability of members of the ras‐mediated pathway. Therefore, we have stably transfected HNSCC KB and Hep‐2 cells with a plasmid encoding for HSP90. The expression of the protein was increased in both transfected cell lines but its activation status was increased in Hep‐2 clones and decreased in KB clones. On the basis of these results, we have treated both parental and HSP90‐transfected cells with a HSP90 inhibitor geldanamycin (GA). We have found that the antiproliferative activity of GA is dependent upon the activation status of HSP90 and that it is strongly synergistic when added in combination with TIP but not with DTX in cells overexpressing HSP90 and even more in cells with increased HSP90 activity. These data were paralleled by the decreased expression and activity of the components belonging to the ras→mediated signal transduction pathway. The present results suggest that multichaperone complex activation could be a resistance mechanism to the anti‐proliferative and apoptotic effects induced by TIP and that the combination of FTIs such as TIP with GA could be a suitable therapeutic strategy in the treatment of HSP90‐overexpressing HNSCC. J. Cell. Physiol. 228: 130–141, 2013.

W7FW14F apomyoglobin amyloid aggregates-mediated apoptosis is due to oxidative stress and AKT inactivation caused by Ras and Rac

We have previously reported that addition of prefibrillar aggregates (PFAs) derived from W7FW14F apomyoglobin mutant to NIH‐3T3 cells affects their viability. In this article, we have found that cytotoxicity induced by PFAs in NIH 3T3 and SH‐SY5Y human neuroblastoma cells was due to early activation of apoptotic cell death dependent from a caspase‐3‐ and ‐9‐mediated mitochondrial pathway. A time‐dependent increase of intracellular ROS and an about twofold decrease of mitochondrial localization of scavenger protein MnSOD was found. The use of the anti‐oxidant agent N‐acetyl‐cysteine (NAC) antagonized both the increase of intracellular ROS and apoptosis induced by PFAs. PFAs caused an about 60% increase of the activity of both Ras and Erk‐1/2 at 30 and 45 min while they were restored to basal levels at later time points. This effect was paralleled by a time‐dependent decrease of the activity of the survival enzyme Akt. Effects similar to those on Ras activity were also recorded on the activity of the stress involved small GTP binding protein Rac that was about 75% increased after 30 min but resumed to basal levels at later time points. This effect was paralleled by a time‐dependent activation of p38 kinase activity and HSP‐70 expression. The use of both the ras farnesyltransferase inhibitor tipifarnib and the Rac geranyl‐geranyltransferase GGTI‐298, but not of the MEK‐1 inhibitor U0126 partially antagonized the effects of PFAs on apoptosis occurrence. On the other hand, the PI3K/Akt inhibitor LY 294002 potentiated apoptosis induced by PFAs. Our results indicate a role for Ras and Rac in the induction of both intracellular ROS increased levels and apoptosis mediated by PFAs and disclose a new scenario of intervention in neurodegenerative diseases. J. Cell. Physiol. 221: 412–423, 2009.

Bovine serum amine oxidase and spm potentiate docetaxel and interferon-α effects in inducing apoptosis on human cancer cells through the generation of oxidative stress

It was previously demonstrated that bovine serum amine-oxidase (BSAO) and SPM (SPM) addition to cancer cells induces cell growth inhibition and over-run the multi-drug resistance (MDR) phenotype through the oxidative stress caused by polyamine metabolites. In this study, it is reported that BSAO/SPM enzymatic system antagonizes the survival pathway induced by either docetaxel (DTX) or interferon alpha (IFNalpha) in human epidermoid cancer KB cells. The combination of BSAO/SPM with either DTX or IFNalpha had a synergistic effect on cell growth inhibition through apoptosis in both human epidermoid KB and breast cancer MCF-7 cell lines. The effects of the BSAO/SPM-DTX combination on apoptosis were caspase 3 and 9-dependent and were paralleled by the enhancement of intracellular O(2-), nitric oxide levels and of lipo-oxidation. The scavenger moiety N-acetyl-cysteine antagonized the effects on apoptosis and cell growth inhibition induced by the combination suggesting a role of the oxidative products of SPM. These effects occurred together with a decrease of the physiological scavenger MnSOD and an increase of both p38 kinase activity and DNA damage. The results suggest that DTX and IFNalpha could sensitize tumour cells to the oxidative stress and apoptosis induced by BSAO/SPM through the induction of a survival ras-dependent pathway and the consequent elevation of the intracellular polyamine pool. These data allow the design of new therapeutic strategy based on the use of this combination in human neoplasms.

Anticancer drugs and hyperthermia enhance cytotoxicity induced by polyamine enzymatic oxidation products

Summary.A correlation between regulation of cell proliferation and polyamine metabolism is described. The latter can enter protein synthesis through the modification of eukaryotic initiation factor 5A (eIF5A) and the formation of the peculiar amino acid hypusine. Specific inhibitors of hypusine formation induce apoptosis that can be potentiated by the combination with cytokines such as interferonα (IFNα) that itself decreases hypusine synthesis. We have also demonstrated that the concomitant treatment of cancer cells with IFNα and the protein synthesis inhibitor fusion protein TGFα/Pseudomonas Aeruginosa toxin synergize in inducing cancer cell growth inhibition. Another way used by polyamines to induce apoptosis is the generation of intracellular oxidative stress through the interaction with bovine serum amine oxidase (BSAO). This enzyme used simultaneously to spermine induces apoptosis, necrosis, inhibition of cell proliferation and inhibition of DNA and protein synthesis in several cell types. The enzymatic oxidation products of polyamine, H2O2 and aldehyde(s) cause these effects. We have recently found that the cytotoxicity of anti-cancer agents, either etoposide or docetaxel, in cancer cells is potentiated in the presence of BSAO/Spermine. In conclusion, polyamine metabolites could be useful in the design of new therapeutic strategies.

Experimental study on vasoactive intestinal peptide (VIP) and its diaminopropane bound (VIP-DAP) analog in solution

Summary.Bioactive peptides represent an exciting area of research in the fields of biochemistry and medicine and in particular the VIP/PACAP network appears to be of interest. Vasoactive intestinal peptide (VIP) is a pleiotropic factor that exerts a physiological regulatory influence and is involved in the pathogenesis of several human disorders. In this paper we have reported structural characterization of VIP by experimental and computational methods as well as a comparative analysis of the peptide with its transglutaminase catalyzed analog VIP-Diaminopropane (VIP-DAP).

Effects of VIP and VIP-DAP on proliferation and lipid peroxidation metabolism in human KB cells.

Abstract:  In the present study, we have utilized the transglutaminase (TGase) enzyme to modify the primary structure of VIP with diamminopropane (DAP) at the level of the Gln16. We have investigated the conformational stability of VIP and VIP–DAP in solution by limited proteolysis experiments. The VIP–DAP appears to be more resistant to the proteolytic attack of trypsin, thus indicating that the derivatization in position 16 is able to stabilize the structure of the peptide. However, we have studied their role in cell cycle modulation and antioxidant activity in the oropharyngeal epidermoid carcinoma KB cells.

The N-terminal 1-16 peptide derived in vivo from protein seminal vesicle protein IV modulates α-thrombin activity: potential clinical implications

We have previously shown that seminal vesicle protein IV (SV-IV) and its 1-70 N-terminal fragment have anti-inflammatory activity and modulate anti-thrombin III (AT) activity. Moreover, mass spectrometry analysis of purified SV-IV has shown that the protein was found to be highly heterogeneous and 14% of the total SV-IV molecules are truncated forms, of particular interest the 1-16, 1-17, and 1-18 peptides. In this work we report experimental data which demonstrate that the 1-16 peptide (P1-16) possesses a marked effect on the AT activity by preventing the formation of the thrombin-AT complex. We found that the formation of thrombin-AT complex is markedly decreased in the presence of P1-16 used at equimolar concentration with thrombin as evaluated with SDS-PAGE. We also monitored the conformational changes of thrombin in the presence of different P1-16 concentrations, and calculated the Kd of thrombin/P1-16 system by circular dichroism technique. The probable interaction sites of P1-16 with thrombin have been also evaluated by molecular graphics and computational analyses. These results have potential implications in the treatment of sterility and thrombotic diseases.