WIN-induced vesiculation cooperates to the inhibition of osteosarcoma cell migration

Introduction. Animal cells release vesicles that mediate the secretion of a variety of factors in the surrounding environment affecting neighboring cells. There is increasing evidence that secreted vesicles play an important role as vehicle of intercellular communication in different biological systems and are able to influence both physiological and pathological processes. Recently, we have reported that the synthetic cannabinoid WIN55,512 is able to induce osteosarcoma MG63 cell death and negatively affect cell migration. Here, we study the effects of WIN on the induction of vesicle secretion and their possible role in WIN-dependent reduction of osteosarcoma cell migratory ability. Methods. Vesicles from MG63 cells were obtained by ultracentrifuging at 140,000g media derived from cell cultures untreated and treated for 24 h with 5 uM WIN. Purified vesicles were quantified by cytofluorimetry and by detecting acetilcholinesterase activity according to established criteria. Scratch wound healing assay was employed to monitor cell migration toward the center of a gap created in a cell monolayer. Zymographic analysis was used to evaluate metalloproteinase activities in the vesicles. Results. WIN treatment induced a significant increase (about 4-fold) in the number of vesicles released by osteosarcoma cells. Wound healing assay showed that in the presence of vesicles from WIN-treated cultures, cells only partially filled the gap with respect to those conditioned with vesicles isolated from control cells which closed the gap within about 24 h. Furthermore, zymography assay showed a reduced activity of MMP-2 and MMP-9 in the vesicles obtained from WIN-treated cells. Conclusion. Data indicate that the increase in the number of vesicles released after WIN treatment and/or their probable different composition can be responsible for the relevant inhibition of MG63 cell migration induced by the cannabinoid

New organotin(IV) complexes with L-Arginine,Nα-t-Boc-L-Arginine and L-Alanyl-L-Arginine.Synthesis, structural investigations and cytotoxic activity

Novel diorganotin(IV) derivatives of L-Arginine (HArg), Nα-(tert-Butoxycarbonyl)-L-Arginine (Boc-Arg-OH) and L-Ala-L-Arg (H2Ala-Arg), H2NC(=NH)NH(CH2)3CH(NHR)CO2H, where R = H in HArg, R = C(O)OC(CH3)3 in Boc-Arg-OH, R = H2NCH(CH3)CO in H2Ala-Arg and triorganotin(IV) derivatives of Boc-Arg-OH have been synthesized and structurally characterized. The complexes were investigated by FT-IR and 119Sn Mössbauer in the solid state and by 1H, 13C, 119Sn and 1H-1H COSY NMR spectroscopy, in solution. The spectroscopic characterization leading to the proposed molecular structures was accomplished on the basis of these experiments. L-Arginine appears to behave as a chelating ligand through carboxylate and -NH2 groups in Me2Sn(Arg)2, while in N-t-Boc-L-Arginine complex, the N-protected amino group being exempted from coordination, only the carboxylate groups are effectors of bonding to the organometallic moieties. FT-IR spectra give a clear indication that guanidino groups in all the complexes are not involved in coordination, since (C=N-H) frequency of the terminal guanidino group is fairly constant and unshifted relative to the free ligand. The biological activity of organotin(IV)-complexes was also investigated by use of human HT29 colorectal carcinoma cells. The cytotoxic activity of the compounds was determined by the MTT quantitative colorimetric assay, capable of detecting viable cells in comparison with that exerted by cisplatin. A marked cytotoxic activity for nearly all complexes, is evident being higher than that exerted by cisplatin, while no significant improvement of activity was observed for Me2Sn(Arg)2 and Me2Sn(Ala-Arg), which was confirmed by IC50 values. Then, we assessed whether the cytotoxicity induced by organotin(IV) complexes was associated with the induction of apoptosis. Light microscopy analysis, performed to study the morphological changes induced in HT29 cells, confirmed the results obtained with MTT test. No significant morphological alterations were observed in HT29 cells after treatment with Me2Sn(Ala-Arg) and Me2Sn(L-Arg)2. Cells treated with nBu2Sn(Boc-Arg)2, nBu2Sn(Ala-Arg), nBu3Sn(Boc-Arg) and Me3Sn(Boc-Arg), appeared rounded, isolated and detached from culture substrate, indicating the commitment to apoptotic cell death

ER+-derived breast cancer stem cells reveal a high expression of the serpin protease inhibitor PI-9.

Introduction: Breast cancers (BC) are the major cause of death in women. More than 70% of BCs express high levels of estrogen receptor-α (ERα) and are sustained for their growth by the hormone. Estrogens seem to protect BC cells from apoptosis mediated by immunosurveillance associated with cytotoxic T lymphocytes and NK cells granzyme B release. However, the production of granzyme B inhibitor PI-9 by tumor cells causes a short-circuit in immunosurveillance’s signalling. Although it has been shown the role of PI-9 in BC cells, its presence has not been investigated in tumor stem cells so far. Methods: Cell viability was evaluated by MTT, cell cycle by propidium iodide staining; mRNA and protein levels by qPCR and western blotting. Tumorspheres from ERα+BC MCF7 cells were isolated in ultra-low attachment conditions. The higher expression of stemness markers (Nanog, Oct3/4 and Sox2) was found in tertiary tumorspheres which were used in our study. Results: Low doses (10 nM-10 μM) of 17-β estradiol consistently increased the number of MCF7 cells more than tumorspheres, while higher doses (50-100 μM) reduced cell number as a consequence of G2/M cell cycle arrest. The analysis of ERα disclosed the presence of three different isoforms (66, 46 and 36 kDa) in MCF7 cells. In contrast, tumorspheres exhibited an increase in ERα36, which lacks transcriptional activity, while the level of ERα66 was undetectable. Then, we analyzed the level of PI-9, which is transcriptionally regulated by ERα66. Surprisingly, we found that tertiary tumorspheres, express higher levels of both PI-9 protein and mRNA than MCF7 cells. Conclusions: Our data provided evidence that the high level of PI-9 in ER+ tertiary tumorspheres could supply a selective advantage to BC stem cells by interfering with immune-surveillance systems. Ongoing studies aim to elucidate the relationship between the levels of different ERα isoforms and PI-9 high expression in BC-stem cells

NT pro BNP plasma level and atrial volume are linked to the severity of liver cirrhosis.

BACKGROUND AND AIMS: Plasma levels of NT-pro-BNP, a natriuretic peptide precursor, are raised in the presence of fluid retention of cardiac origin and can be used as markers of cardiac dysfunction. Recent studies showed high levels of NT pro BNP in patients with cirrhosis. We assessed NT pro-BNP and other parameters of cardiac dysfunction in patients with cirrhosis, with or without ascites, in order to determine whether the behaviour of NT pro BNP is linked to the stage of liver disease or to secondary cardiac dysfunction. METHODS: Fifty eight consecutive hospitalized patients mostly with viral or NAFLD-related cirrhosis were studied. All underwent abdominal ultrasound and upper GI endoscopy. Cardiac morpho-functional changes were evaluated by echocardiography and NT-pro-BNP plasma levels determined upon admission. Twenty-eight hypertensive patients, without evidence of liver disease served as controls. RESULTS: Fifty eight cirrhotic patients (72% men) with a median age of 62 years (11% with mild arterial hypertension and 31% with type 2 diabetes) had a normal renal function (mean creatinine 0.9 mg/dl, range 0.7-1.06). As compared to controls, cirrhotic patients had higher NT pro-BNP plasma levels (365.2±365.2 vs 70.8±70.6 pg/ml; p<0.001). Left atrial volume (LAV) (61.8±26.3 vs 43.5±14.1 ml; p = 0.001), and left ventricular ejection fraction (62.7±6.9 vs. 65.5±4%,; p = 0.05) were also altered in cirrhotic patients that in controls. Patients with F2-F3 oesophageal varices as compared to F0/F1, showed higher e' velocity (0.91±0.23 vs 0.66±0.19 m/s, p<0.001), and accordingly a higher E/A ratio (1.21±0.46 vs 0.89±0.33 m/s., p = 0.006). CONCLUSION: NT-pro-BNP plasma levels are increased proportionally to the stage of chronic liver disease. Advanced cirrhosis and high NT-pro-BNP levels are significantly associated to increased LAV and to signs of cardiac diastolic dysfunction. NT pro-BNP levels could hence be an useful prognostic indicators of early decompensation of cirrhosis

Synthesis, structural characterisation and biological studies of new mononuclear platinum(II) complexes with sterically hindered heterocyclic ligands

Three novel cisplatin analogues were synthesized, designed according to an approach which violates the ‘‘classical’’ structure–activity relationship, by replacing the diamine ligands with a planar N donor heterocycle giving a sterically hindered complex. Moreover, the sterical hindrance of antitumor drug candidates potentially makes them less susceptible to deactivation by sulphur-containing proteins and helping to overcome resistance mechanisms. The resulting mononuclear complexes of sterically hindered polidentate heterocyclic N ligands [PtCl(bbp)]Cl (1) [bbp = 2,6-bis(2-benzimidazolyl)pyridine], [PtCl2(dptdn)](H2O) (2) [dptdn = sodium 5,6-diphenyl-3-(20-pyridyl)-1,2,4-triazine-400,400 0-disulfonate] and [(dptdn)(dpt)Pt]Cl2(H2O) (3) [dpt = 5,6-diphenyl-3-(20-pyridyl)-1,2,4-triazine] have been prepared and structurally characterised. Both neutral and ionic complexes are present, with monofunctional (1) and bifunctional Pt(II) moieties (2) and coordinatively saturated Pt(II) ions in the mixed ligand complex (3), whose size and shape enable them to behave as novel scaffolds for DNA binding. All complexes were tested ‘‘in vitro’’ for their biological activity on human HT29 colorectal carcinoma and HepG2 hepatoma cells. The complexes (1) and (3), endowed with a positive charge, showed a potent cytotoxic activity and reduced cell viability with an efficacy higher than that of cisplatin; whilst the neutral bifunctional compound (2) was inactive. IC50 values have been calculated for the active compounds. The cytotoxic effects were confirmed by the accumulation of treated cells in subG0/G1 phase of cell cycle, by the loss of mitochondrial potential (Dwm) and by the chromatin condensation or fragmentation observed by means of fluorescence microscopy after Hoechst 33258 nuclear staining. A study on intracellular platinum uptake in HT29 cell line has been also performed and data obtained strongly suggest that the cytotoxicity of new tested complexes reported in this work is based on a different pharmacodynamic pattern with respect to cisplatin

Molecular Mechanisms of AKI in the Elderly: From Animal Models to Therapeutic Intervention

Acute kidney injury (AKI), a critical syndrome characterized by a sudden reduction of renal function, is a common disorder among elderly patients particularly in Intensive Care Unit (ICU). AKI is closely associated with both short- and long-term mortality and length of hospital stay and is considered a predictor of chronic kidney disease (CKD). Specific hemodynamic, metabolic, and molecular changes lead to increased susceptibility to injury in the aged kidney; therefore, certain causes of AKI such as the prerenal reduction in renal perfusion or vascular obstructive conditions are more common in the elderly; moreover, AKI is often multifactorial and iatrogenic. Older patients present several comorbidities (diabetes, hypertension, heart failure) and are exposed to multiple medical interventions such as the use of nephrotoxic contrasts media and medications, which can also trigger AKI. Considering the emerging relevance of this condition, prevention and treatment of AKI in the elderly should be crucial in the internist and emergency setting. This review article summarizes the incidence, the risk factors, the pathophysiology, the molecular mechanisms and the strategies of prevention and treatment of AKI in elderly patients

Involvement of PAR-4 in Cannabinoid-Dependent Sensitization of Osteosarcoma Cells to TRAIL-Induced Apoptosis

The synthetic cannabinoid WIN 55,212-2 is a potent cannabinoid receptor agonist with anticancer potential. Experiments were performed to determine the effects of WIN on proliferation, cell cycle distribution, and programmed cell death in human osteosarcoma MG63 and Saos-2 cells. Results show that WIN induced G2/M cell cycle arrest, which was associated with the induction of the main markers of ER stress (GRP78, CHOP and TRB3). In treated cells we also observed the conversion of the cytosolic form of the autophagosome marker LC3-I into LC3-II (the lipidated form located on the autophagosome membrane) and the enhanced incorporation of monodansylcadaverine and acridine orange, two markers of the autophagic compartments such as autolysosomes. WIN also induced morphological effects in MG63 cells consisting in an increase in cell size and a marked cytoplasmic vacuolization. However, WIN effects were not associated with a canonical apoptotic pathway, as demonstrated by the absence of specific features, and only the addition of TRAIL to WIN-treated cells led to apoptotic death probably mediated by up-regulation of the tumor suppressor factor PAR-4, whose levels increased after WIN treatment, and by the translocation of GRP78 on cell surface

Parthenolide and Its Soluble Analogues: Multitasking Compounds with Antitumor Properties

Due to its chemical properties and multiple molecular effects on different tumor cell types, the sesquiterpene lactone parthenolide (PN) can be considered an effective drug with significant potential in cancer therapy. PN has been shown to induce either classic apoptosis or alternative caspase-independent forms of cell death in many tumor models. The therapeutical potential of PN has been increased by chemical design and synthesis of more soluble analogues including dimethylaminoparthenolide (DMAPT). This review focuses on the molecular mechanisms of both PN and analogues action in tumor models, highlighting their effects on gene expression, signal transduction and execution of different types of cell death. Recent findings indicate that these compounds not only inhibit prosurvival transcriptional factors such as NF-κB and STATs but can also determine the activation of specific death pathways, increasing intracellular reactive oxygen species (ROS) production and modifications of Bcl-2 family members. An intriguing property of these compounds is its specific targeting of cancer stem cells. The unusual actions of PN and its analogues make these agents good candidates for molecular targeted cancer therapy

Redox Imbalance and Mitochondrial Release of Apoptogenic Factors at the Forefront of the Antitumor Action of Mango Peel Extract

Today, an improved understanding of cancer cell response to cellular stress has become more necessary. Indeed, targeting the intracellular pro-oxidant/antioxidant balance triggering the tumor commitment to cell demise could represent an advantageous strategy to develop cancer-tailored therapies. In this scenario, the present study shows how the peel extract of mango—a tropical fruit rich in phytochemicals with nutraceutical properties—can affect the cell viability of three colon cancer cell lines (HT29, Caco-2 and HCT116), inducing an imbalance of cellular redox responses. By using hydro-alcoholic mango peel extract (MPE), we observed a consistent decline in thiol group content, which was accompanied by upregulation of MnSOD—a mitochondrial scavenger enzyme that modulates the cellular response against oxidative damage. Such an effect was the consequence of an early production of mitochondrial superoxide anions that appeared after just 30 min of exposure of colon cancer cells to MPE. The effect was accompanied by mitochondrial injury, consisting of the dissipation of mitochondrial membrane potential and a decrease in the level of proteins localized in the mitochondrial membrane—such as voltage-dependent anion-selective channel (VDAC1), mitofilin, and some members of Bcl-2 family proteins (Mcl-1, Bcl-2 and Bcl-XL)—with the mitochondrial release of apoptogenic factors (cytochrome C and AIF). The analysis of the cytotoxic effects exerted by the different constituents of MPE (gallic acid, mangiferin, citric acid, quinic acid, pentagalloyl glucose, and methyl gallate) allowed us to identify those phytochemicals responsible for the observed anticancer effects, sustaining their future employment as chemopreventive or therapeutic agents