Salvatore Florio

Design, synthesis, biophysical and biological studies of trisubstituted naphthalimides as G-quadruplex ligands

A series of trisubstituted naphthalimides have been synthesized and evaluated as telomeric G-quadruplex ligands by biophysical methods. Affinity for telomeric G-quadruplex AGGG(TTAGGG)(3) binding was first screened by fluorescence titrations. Subsequently, the interaction of the telomeric G-quadruplex with compounds showing the best affinity has been studied by isothermal titration calorimetry and UV-melting experiments. The two best compounds of the series tightly bind the telomeric quadruplex with a 2:1 drug/DNA stoichiometry. These derivatives have been further evaluated for their ability to inhibit telomerase by a TRAP assay and their pharmacological properties by treating melanoma (M14) and human lung cancer (A549) cell lines with increasing drug concentrations. A dose-dependent inhibition of cell proliferation was observed for all cellular lines during short-term treatment.

Imatinib treatment inhibit IL‐6, IL‐8, NF‐KB and AP‐1 production and modulate intracellular calcium in CML patients

Imatinib (IM) is considered the gold standard for chronic myeloid leukemia (CML) treatment, although resistance is emerging as a significant problem. The proinflammatory cytokines interleukin‐6 (IL‐6) and interleukin‐8 (IL‐8) play an important role in cell proliferation, survival, and resistance to glucocorticoid‐mediated cell death. Several transcription factors such as NF‐KB and AP‐1 are activated in response to physiopathological increases and modulation of intracellular calcium levels. Our previous study demonstrated that lymphocytes from CML patients showed dysregulated calcium homeostasis and oxidative stress. Alteration in ionized calcium concentration in the cytosol has been implicated in the initiation of secretion, contraction, and cell proliferation. In this study, we hypothesized that IL‐6, IL‐8, NF‐kB, AP‐1, and intracellular calcium may be used as selective and prognostic factors to address the follow‐up in CML patients treated with imatinib. Our results demonstrated a significant down‐regulation in IL‐6 and IL‐8 release as well as NF‐kB and AP‐1 activation in lymphomonocytes from Imatinib‐treated patients, compared to samples from untreated patients. In parallel, IM treatment, in vivo and in vitro, were able to modulate the intracellular calcium concentration of peripheral blood mononuclear cells of CML patients by acting at the level of InsP3 receptor in the endoplasmic reticulum and at the level of the purinergic receptors on plasma membrane. The results of this study show that measurements of NF‐kB, AP‐1, IL‐6, IL‐8, and intracellular calcium in CML patients treated with Imatinib may give important information to the hematologist on diagnostic criteria and are highly predictive in patients with newly diagnosed CML. J. Cell. Physiol. 227: 2798–2803, 2012.

Dysregulated calcium homeostasis and oxidative stress in chronic myeloid leukemia (CML) cells

Chronic myeloid leukemia (CML) is a hematopoietic stem cell disorder caused by the oncogenic activity of the Bcr‐Abl protein, a deregulated tyrosine kinase. Calcium may act directly on cellular enzymes and in conjunction with other cellular metabolites, such as cyclic nucleotides, to regulate cell functions. Alteration in the ionized calcium concentration in the cytosol has been implicated in the initiation of secretion, contraction, and cell proliferation as well as the production of reactive oxygen species (ROS) has been correlates with normal cell proliferation through activation of growth‐related signaling pathways. In this study we evaluated in peripheral blood leukocytes from CML patients the role of the balance between intracellular calcium and oxidative stress in CML disease in order to identify possible therapeutic targets in patients affected by this pathology. Our results demonstrated that peripheral blood mononuclear cells derived from CML patients displayed decreased intracellular calcium [Ca2+]i fluxes both after InsP3 as well as ATP and ionomycin (IONO) administration. CML cells showed lower levels of superoxide dismutase (SOD) activity and significantly higher malondialdehyde levels (MDA) than peripheral blood mononuclear cells derived from control patients. Finally we showed that resveratrol is able to down‐regulate InsP3 and ATP effects on intracellular calcium [Ca2+]i fluxes as well as the effects of ATP and IONO on oxidative stress in CML cells. J. Cell. Physiol. 224: 443–453, 2010.

Hydrocortisone has a protective effect on CyclosporinA-induced cardiotoxicity.

CyclosporinA (CsA) is an immunosuppressive drug which induces severe adverse effects such as cardiotoxicity and nephrotoxicity. In several therapeutic protocols CsA is used in association with corticosteroids to obtain better therapeutic results. Recently, our studies showed that CsA increases blood pressure while inhibit Nitric Oxide (NO) production in vivo. In this study we evaluated in rat cardiomyocytes the effects of CsA, used alone or in association with Hydrocortisone (HY), on intracellular calcium concentration, NO production and lipid peroxidation (MDA level). Our results demonstrated that CsA increased intracellular calcium and such effect was dose‐dependent. HY used alone, slightly decreased intracellular calcium, while dramatically reduced CsA‐induced calcium fluxes. CsA (3.2 μM) increased lipid peroxidation and this effect was blunted by HY. Both CsA and HY inhibited NO production in rat cardiomyocytes acting on this pathway synergically. Our results demonstrated that in rat cardiomyocytes, CsA toxicity is due to a calcium overload, which in turn induce lipid peroxidation and determines oxidative stress‐induced cell injury. Treatment with HY effectively inhibits CsA‐induced toxicity, decreasing lipid peroxidation as well as calcium intracellular concentration. Our findings seem to suggest that glucocorticoids may be effective in reducing CsA‐induced cardiotoxicity at concentrations which are consistent with current therapeutic doses.

Combined effects of PI3K and SRC kinase inhibitors with imatinib on intracellular calcium levels, autophagy, and apoptosis in CML-PBL cells

Imatinib induces a complete cytogenetic regression in a large percentage of patients affected by chronic myeloid leukemia (CML) until mutations in the kinase domain of BCR-ABL appear. Alternative strategies for CML patients include the inhibition of phosphatidylinositol 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) pathway, which is constitutively activated in leukemia cells and seems important for the regulation of cell proliferation, viability, and autophagy. In this study, we verified the effect of imatinib mesylate (IM), alone or in association with LY294002 (LY) (a specific PI3K protein tyrosine kinase inhibitor) or 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine (PP1) (a Src tyrosine kinase inhibitor), on viability, intracellular calcium mobilization, apoptosis, and autophagy, in order to verify possible mechanisms of interaction. Our data demonstrated that PP1 and LY interact synergistically with IM by inducing apoptosis and autophagy in Bcr/Abl+ leukemia cells and this mechanism is related to the stress of the endoplasmic reticulum (ER). Our findings suggest a reasonable relationship between apoptotic and autophagic activity of tyrosine kinase inhibitors (TKIs) and the functionality of smooth ER Ca2+-ATPase and inositol triphosphate receptors, independently of intracellular calcium levels. Therapeutic strategies combining imatinib with PI3K and/or Src kinase inhibitors warrant further investigations in Bcr/Abl+ malignancies, particularly in the cases of imatinib mesylate-resistant disease.

2,3,7,8-Tetrachlorodibenzo-p-dioxin induced autophagy in a bovine kidney cell line

The administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to a variety of cultured cells may alter their ability to proliferate and die. In a previous study we demonstrated that TCDD induced proliferation in Madin-Darby Bovine Kidney (MDBK) cells where no signs of apoptosis were observed, but herein, analysis of MDBK cell morphology, in a large number of exposed cells, revealed some alterations, as expanded cytoplasm, an increase of intercellular spaces and many pyknotic nuclei. Hence, the aim of the current study was to elucidate the influences of dioxin on cell proliferation and cell death. We found that dioxin increased proliferation, as well as, activated cell death with autophagy, as we detected by increased amount of LC3-II, an autophagosome marker. Furthermore, formation of acidic vesicular organelles was observed by fluorescence microscopy following staining with the lysosomotropic agent acridine orange. These results were accompanied by down-regulation of telomerase activity, bTERT and c-Myc. Key tumor-suppressor protein p53 and expression of cell cycle inhibitor p21Waf1/Cip1 were activated after TCDD exposure. These changes occurred with activation of ATM phosphorylation in the presence of a decrease in Mdm2 protein levels. Taken together, these results support the idea that TCDD in MDBK cells, may exert its action, in part, by enhancing cell proliferation, but also by modulating the incidence of induced cell death with autophagy.

Interference of bovine herpesvirus 1 (BHV-1) in sorbitol-Induced apoptosis

In order to determine the ability of bovine herpesvirus type 1 (BHV‐1) to suppress apoptosis, we examined the effects of BHV‐1 infection on sorbitol‐induced apoptosis on Madin–Darby bovine kidney (MDBK) cells. BHV‐1 suppresses sorbitol‐induced apoptosis in a manner similar to that of herpes simplex virus type 1 (HSV‐1), indicating that BHV‐1 has one or more anti‐apoptotic genes. To elucidate the molecular mechanisms of apoptosis, expression of some genes encoding apoptosis‐inhibiting and ‐promoting factors were analyzed on BHV‐1 infected cells during the process of sorbitol‐induced apoptosis. Our results revealed that the expression of bcl‐2 and bcl‐xL decreased after 5 and 3 h p.i., respectively; while bax and procaspase‐3 expression increased with respect to control as a function of p.i. times and at 7 h p.i. they were not observed. We further show that the expression of p53 gene was also enhanced, suggesting that this apoptotic mechanism is p53 dependent. From these results, we propose that BHV‐1 has one or more genes encoding apoptosis‐inhibiting factors which interfere with the involvement of bcl‐2 gene family members and apoptotic pathway, depending upon caspase‐3, triggered by sorbitol. J. Cell. Biochem. 89: 373–380, 2003.

Prevention of Nephrotoxicity Induced by Cyclosporine‐A: Role of Antioxidants

Cyclosporine A (CsA) is a powerful immunosuppressive drug used to prevent allograft rejection after organ transplantation as well as in human and veterinary medicine. Unfortunately, its use is hampered by its nephrotoxic effects. The mechanisms of CsA‐induced hypertension and nephrotoxicity are not clear, but several studies suggest the possible involvement of free radicals. In this review we have summarized the effect of some antioxidants that we have used in the recent years, in combination with CsA, to better understand the exact mechanism of action of CsA and to try to open new perspectives in the treatment of CsA nephrotoxicity. J. Cell. Biochem. 116: 364–369, 2015.

2,3,7,8-Tetrachlorodibenzo-p-dioxin increases Bovine Herpesvirus type-1 (BHV-1) replication in Madin-Darby Bovine Kidney (MDBK) cells in vitro.

Dioxin—2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) is a common environmental toxin of current interest. In the last years, higher levels of TCDD than those permitted in UE [European Commission. 2002. European Commission Recommendation 2002/201/CE. Official Gazette, L 67/69] were detected in milk samples from cow, water buffalo, goat, and sheep raised on some areas of Campania Region (South Italy). Dioxin often causes immunosuppression and might render the animal liable to viral infections. In addition, viral infections are able to alter the pattern of dioxin distribution in different organs of the exposed animals. Bovine Herpesvirus type‐1 (BHV‐1) is a widespread pathogen, which causes infectious rhinotracheitis and infectious pustular vulvovaginitis in cattle. Herein, we have studied the effects of TCDD and BHV‐1 infection, in Madin‐Darby Bovine Kidney (MDBK) cells, alone as well as in association, so as cellular proliferation, apoptosis, and virus replication. We have observed an increase in cell viability of confluent monolayers at low TCDD concentrations. TCDD treated cells demonstrated increased viability compared to controls as evaluated by MTT test. TCDD exposure increased cell proliferation but induced no changes on apoptosis. Cells exposed to TCDD along with BHV‐1 showed a dose‐dependent increase in cytopathy, represented by ample syncytia formation with the elimination of the cellular sheets and increased viral titer. These results suggest that TCDD increases viral replication in MDBK cells while BHV‐1 further decreases viability of TCDD exposed cells. Since very low concentrations (0.01 pg/ml) are sufficient to augment BHV‐1 titer, TCDD may contribute to reactivate BHV‐1 from latency, leading to recurrent disease and increase virus transmission. J. Cell. Biochem. 103: 221–233, 2008.

The Protective Effect of Apocynin on Cyclosporine A-Induced Hypertension and Nephrotoxicity in Rats

Cyclosporine A (CsA) is the prototype of immunosuppressant drugs that has provided new perspectives in human and veterinary medicine to prevent organ transplant rejection and to treat certain autoimmune diseases and dermatologic diseases. Unfortunately, the treatment with CSA is often limited by severe adverse effects such as hypertension and nephrotoxicity. Some data suggest that reactive oxygen species (ROS) and the oxidative stress play an important role in its pathogenesis, in particular the superoxide (O2−) that is the most powerful free radical generated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase present mainly in the kidney. The present study has been designed to investigate the role of Apocynin a selective inhibitor of NADPH oxidase activity on cyclosporine‐induced adverse effect. In this study, we have evaluated the effect of CsA, used alone or in association with Apocynin on blood pressure (BP), on glomerular filtration rate (GFR), on absoluted fluid reabsorption (Jv) in proximal tubule (PT), on O2− concentration, and on nitric oxide (NO) production. We have demonstrated that CsA administration increases superoxide concentration in the aorta, decreases the NO concentration, reduces GFR and the Jv in PT, and induces a significant increase in BP. Moreover, we have shown that Apocynin treatment restores these hemodynamic alterations, as well as NO and superoxide productions. In conclusion, the reported data indicate that CsA induced nephrotoxicity and hypertension are related to NADPH oxidase activity, in fact Apocynin protects the kidney function and BP from toxic effects induced by CsA through the inhibition of NADPH oxidase activity. J. Cell. Biochem. 116: 1848–1856, 2015.