Maja Jovanovic

The protective role of AMP-activated protein kinase in alpha-synuclein neurotoxicity in vitro.

In the present study, we investigated the role of the main intracellular energy sensor, AMP-activated protein kinase (AMPK), in the in vitro neurotoxicity of α-synuclein (ASYN), one of the key culprits in the pathogenesis of Parkinsons disease. The loss of viability in retinoic acid-differentiated SH-SY5Y human neuroblastoma cells inducibly overexpressing wild-type ASYN was associated with the reduced activation of AMPK and its activator LKB1, as well as AMPK target Raptor. ASYN-overexpressing rat primary neurons also displayed lower activity of LKB1/AMPK/Raptor pathway. Restoration of AMPK activity by metformin or AICAR reduced the in vitro neurotoxicity of ASYN overexpression, acting independently of the prosurvival kinase Akt or the induction of autophagic response. The conditioned medium from ASYN-overexpressing cells, containing secreted ASYN, as well as dopamine-modified or nitrated recombinant ASYN oligomers, all inhibited AMPK activation in differentiated SH-SY5Y cells and reduced their viability, but not in the presence of metformin or AICAR. The RNA interference-mediated knockdown of AMPK increased the sensitivity of SH-SY5Y cells to the harmful effects of secreted ASYN. AMPK-dependent protection from extracellular ASYN was also observed in rat neuron-like pheochromocytoma cell line PC12. These data demonstrate the protective role of AMPK against the toxicity of both intracellular and extracellular ASYN, suggesting that modulation of AMPK activity may be a promising therapeutic strategy in Parkinsons disease.

The prognostic relevance of tumor associated macrophages in advanced stage classical Hodgkin lymphoma

Although the treatment of Hodgkin lymphoma (HL) has been improved, distinguishing reliable prognostic biomarkers could better stratify patients for more effective treatment. We analyzed the prognostic relevance of CD68+ tumor-associated macrophages (TAMs) by immunohistochemical analysis at diagnosis and standard clinical parameters in 52 ABVD (doxorubicin, bleomycin, vinblastine, dacarbazine)-treated patients with advanced stage classical HL (cHL). Patients with >25% CD68+ TAMs compared to those with ≤25% had worse 5-year overall survival (45% vs. 77%, log-rank p = 0.019) and showed a trend toward shorter 5-year event-free survival (51% vs. 71%, log-rank p = 0.19). Additionally, no significant correlation with selected clinical features was found. Significantly shorter 5-year overall survival was associated with International Prognostic Score (IPS) >2, bulky disease, elevated erythrocyte sedimentation rate (log-rank test, p = 0.003, p = 0.049, p = 0.007, respectively). In multivariate analysis, increased CD68+TAMs, IPS >2, and bulky disease were identified as independent prognostic factors for overall survival (Cox multivariate model, p = 0.006, p = 0.007, p = 0.013, respectively). Tumor-associated macrophages represent a potential prognostic biomarker which could contribute to better risk stratification of patients with cHL.

Autophagy-dependent and -independent involvement of AMP-activated protein kinase in 6-hydroxydopamine toxicity to SH-SY5Y neuroblastoma cells.

The role of the main intracellular energy sensor adenosine monophosphate (AMP)-activated protein kinase (AMPK) in the induction of autophagic response and cell death was investigated in SH-SY5Y human neuroblastoma cells exposed to the dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA). The induction of autophagy in SH-SY5Y cells was demonstrated by acridine orange staining of intracellular acidic vesicles, the presence of autophagosome- and autophagolysosome-like vesicles confirmed by transmission electron microscopy, as well as by microtubule-associated protein 1 light-chain 3 (LC3) conversion and p62 degradation detected by immunoblotting. 6-OHDA induced phosphorylation of AMPK and its target Raptor, followed by the dephosphorylation of the major autophagy inhibitor mammalian target of rapamycin (mTOR) and its substrate p70S6 kinase (S6K). 6-OHDA treatment failed to suppress mTOR/S6K phosphorylation and to increase LC3 conversion, p62 degradation and cytoplasmatic acidification in neuroblastoma cells in which AMPK expression was downregulated by RNA interference. Transfection of SH-SY5Y cells with AMPK or LC3β shRNA, as well as treatment with pharmacological autophagy inhibitors suppressed, while mTOR inhibitor rapamycin potentiated 6-OHDA-induced oxidative stress and apoptotic cell death. 6-OHDA induced phosphorylation of p38 mitogen-activated protein (MAP) kinase in an AMPK-dependent manner, and pharmacological inhibition of p38 MAP kinase reduced neurotoxicity, but not AMPK activation and autophagy triggered by 6-OHDA. Finally, the antioxidant N-acetyl cysteine antagonized 6-OHDA-induced activation of AMPK, p38 and autophagy. These data suggest that oxidative stress-mediated AMPK/mTOR-dependent autophagy and AMPK/p38-dependent apoptosis could be valid therapeutic targets for neuroprotection.

Synthesis and in vitro Anticancer Activity of Ruthenium–Cymene Complexes with Cyclohexyl-Functionalized Ethylenediamine-N,N′-diacetate-Type Ligands

Herein we describe the synthesis, characterization, and anticancer activity of novel p‐cymeneruthenium(II) complexes containing methyl, ethyl, n‐propyl, and n‐butyl esters of (S,S)‐ethylenediamine‐N,N′‐di‐2‐(3‐cyclohexyl)propanoic acid. The results of IR, UV/Vis, ESIMS, 1H, and 13C NMR characterization reveal that ligand coordination occurs through nitrogen donor atoms of the ester ligands, with the organoruthenium moiety being kept in complex. These ruthenium(II) complexes are cytotoxic toward various cancer cell lines including leukemic HL‐60, K562, and REH cells (IC50: 1.0–20.2 μM), with the n‐butyl ester complex being the most effective. It causes apoptotic cell death associated with mitochondrial depolarization, caspase activation, phosphatidylserine exposure, and DNA fragmentation. Importantly, the n‐butyl ester complex is more effective against leukemic patients′ blood mononuclear cells relative to those from healthy control subjects, thus indicating a fairly selective antileukemic action of RuII‐based compounds.

The number of lymphoma-associated macrophages in tumor tissue is an independent prognostic factor in patients with follicular lymphoma.

The clinical course of patients with follicular lymphoma is variable from a slowly progressive disease to a progressive disease with a survival time of approximately 1 year. Many prognostic models have been suggested to identify high-risk patients. Recent gene profiling analysis showed that the clinical behavior of follicular lymphoma is determined by the properties of the nonmalignant tumor microenvironment. We investigated the role of lymphoma-associated macrophages (LAMs) in tumor tissue in patients with newly diagnosed follicular lymphoma. The LAM was determined immunohistochemically in lymph node tissue sections by anti-CD68 PG-M1 and analyzed through high-power field (HPF) magnification intrafollicularly (IF) and extrafollicularly. In our study, the patients who had an IF LAM count equal to or more than 10/HPF had significantly shorter overall survival (P=0.018) and 3 years of progression-free survival (P=0.034) compared with patients with <10 LAM/HPF. Multivariate analysis indicated that IF LAM/HPF ≥10 and Eastern Cooperative Oncology Group performance status >1 are independent prognostic factors for a poor outcome.

Palladium(II) complexes with N-heteroaromatic bidentate hydrazone ligands: the effect of the chelate ring size and lipophilicity on in vitro cytotoxic activity.

Novel Pd(II) complex with N‐heteroaromatic Schiff base ligand, derived from 8‐quinolinecarboxaldehyde (q8a) and ethyl hydrazinoacetate (haOEt), was synthesized and characterized by analytical and spectroscopy methods. The structure of novel complex, as well as structures of its quinoline and pyridine analogues, was optimized by density functional theory calculations, and theoretical data show good agreement with experimental results. A cytotoxic action of the complexes was evaluated on cultures of human promyelocytic leukemia (HL‐60), human glioma (U251), rat glioma (C6), and mouse fibrosarcoma (L929) cell lines. Among investigated compounds, only complexes with quinoline‐based ligands reduce the cell numbers in a dose‐dependent manner in investigated cell lines. The observed cytotoxic effect of two isomeric quinoline‐based complexes is predominantly mediated through the induction of apoptotic cell death in HL‐60 cell line. The cytotoxicity of most efficient novel Pd(II) complex is comparable to the activity of cisplatin, in all cell lines investigated.

Unusual presentation of gastric plasmablastic lymphoma in HIV-negative patient

Plasmablastic lymphoma (PBL) has initially been described as a rapidly progressive and almost invariably fatal diffuse large-cell lymphoma with plasmablastic features, exclusively involving the jaw and oral mucosa in HIV-positive patients. Although its clinical features may help in differential diagnosis, an extra-oral localization in a patient without HIV makes it more difficult to suspect clinically. We describe a very rare case of gastric PBL primarily involving stomach in a middle age man without an HIV infection. A biopsy was performed and its findings revealed a diffuse, monomorphous proliferation of the tumor cells with features of immunoblasts, MUM-1, EMA, and lambda light chains positive. Serology was negative for the human immunodeficiency virus (HIV), HBsAg, and hepatitis C virus infection. The patient started treatment with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) chemotherapy, but unfortunately died before the second cycle was given. To our knowledge, this is the second case of gastric PBL presented in HIV-negative patients. The findings in this case suggest that PBL should be included in the differential diagnosis of gastrointestinal tumors.

Increased angiogenesis-associated poor outcome in acute lymphoblastic leukemia: a single center study.

Angiogenesis in solid tumors is important for tumor growth, invasion, and metastasis. However, angiogenesis plays also an important role in hematological malignancies. We have analyzed the expression of vascular endothelial growth factor (VEGF) in the leukemic blast cells and microvessel density (MVD) in the bone marrow biopsy samples of the patients with acute lymphoblastic leukemia (ALL). Bone marrow MVD of the patients with ALL was significantly higher compared with normal controls and complete remission (P<0.001), but slightly lower than in patients with relapsed ALL (P>0.05). The bone marrow blast VEGF expression was significantly higher in newly diagnosed ALL patients, with predominant strong VEGF expression as compared with complete remission patients (who had negative or weak VEGF expression) (P<0.05), whereas initial values were slightly lower than in relapsed patients. There was a strong positive correlation between VEGF expression and MVD at presentation of ALL. Stronger expression of VEGF on blast cells indicates shorter overall survival in ALL. Furthermore, initial values of MVD had positive correlation with overall survival and leukemia-free survival (P=0.024 and P=0.017, respectively). Our data suggest that increased angiogenesis (confirmed by immunohistochemical expression of VEGF in leukemic blasts), and MVD may play an important role in the pathophysiology of ALL with prognostic implications. Thus, targeting VEGF pathway may bring the new approach for ALL treatment—using antiangiogenic drugs and tyrosine kinase inhibitors in combination with standard chemotherapy regimens.

Assessment of bone marrow microvessel density in chronic lymphocytic leukemia.

IntroductionAngiogenesis is a physiologic process of new blood vessels formation mediated by various cytokines called proangiogenic and antiangiogenic factors. Enhancement of angiogenesis in chronic lymphocytic leukemia (CLL) has been recognized more recently. Our study assesses CD34 and von Willebrand factor (vWf) expression and microvessel density (MVD) in the bone marrow of patients with CLL. Aims(1) To assess bone marrow MVD in CLL using 2 different monoclonal antibodies, CD34 and vWf; and (2) To examine the possible association of marrow MVD and clinical course, pattern of marrow infiltration, Rai stage, CD38 positivity, and cytogenetic abnormalities detected by fluorescence in situ hybridization. Materials and MethodsBone marrow specimens from 33 patients with CLL and 10 controls were studied. A single microvessel was defined as any vessel with a clear lumen. The screening of the slides was carried out by hotspot method. The slides were initially screened at low power to identify the areas with highest number of microvessel or vascularity hotspot. The count of microvessel in a sufficiently extended field (40× objective lens, 10× ocular lens) was then performed. The mean value of 10 most vascularized areas at 400× field was considered as MVD for a sample. ResultsThere was a significant difference between MVD counts according to the antibody used. MVD was higher using CD34 versus vWF (CD34: mean ± SD, 35.91±15.7; 95% confidence interval of mean, 30.34-41.48 vessels/field versus vWF: 8.15±4.65; 95% confidence interval of mean, 4.11-12.44 vessels/field; P<0.0001]. Bone marrow MVD detected by CD34 was significantly higher in patients with CD38 expression more than 30% (P=0.006) and in patients with unfavorable cytogenetic abnormalities. However, no significant MVD differences were detected between CLL subgroups with regard to clinical course, pattern of marrow infiltration, and Rai stage. Bone marrow MVD in patients with CLL was significantly higher than that in controls (P<0.0001). ConclusionsMVD assessment using anti-CD34 resulted in higher MVD counts than when using anti-vWF antibody. However, no MVD differences were detected between CLL subgroups subdivided according to the above-mentioned prognostic factors except CD38 expression and genetic abnormalities.

Solvent and structural effects in tautomeric 3-cyano-4-(substituted phenyl)-6-phenyl-2(1H)-pyridones: experimental and quantum chemical study

The tautomeric equilibria between 2-pyridone and 2-hydroxypyridine forms of methoxy, chloro, and nitro derivatives of 3-cyano-4-(2-, 3-, and 4-substituted phenyl)-6-phenyl-2(1H)-pyridones were evaluated from UV/Vis spectral data. Linear solvation energy relationships of Kamlet–Taft and Catalán-rationalized solvent have influence on tautomeric equilibria. Transmission of substituent effect was analyzed by the Hammett equation. Quantum chemical calculations were performed by density functional theory (B3LYP). The experimental data were interpreted with the aid of time-dependent density functional method. Electron density distribution was analyzed by Bader’s analysis. It was found that substituents of different electronic properties change the extent of conjugation, and affect intramolecular charge transfer character. Theoretical calculations and experimental results gave insight into the influence of the molecular conformation on the transmission of substituent effects, as well as on contribution of different solvent–solute interactions.