The expression of CD73 on pathological B-cells is associated with shorter overall survival of patients with CLL.

CD73 is a membrane-bound enzyme that catalyzes the extracellular conversion of adenosine monophosphate to adenosine. Adenosine is thought to play a role in promoting tumor growth and survival together with suppressing the host immune responses, which contribute to the multistep process of tumorigenesis. Here, we studied the expression of this antigen in chronic lymphocytic leukemia (CLL). The expression of CD73 was analyzed by multiparametric flow cytometry on normal and pathological B-cells from peripheral blood and bone marrow samples from 71 patients with CLL. Pathological B-cells expressed significantly lower levels of CD73 than normal B-cells (p<0.01). Patients with splenomegaly showed a higher expression of CD73 on pathological B-cells than patients without splenomegaly (p<0.05). The expression of CD73 also correlated with beta-2-microglobulin levels (p<0.05). Clinically, patients with higher levels of CD73 versus those with lower expression presented with shorter overall survival (median OS of 65 vs. 113 months, p<0.05). Our data indicate that CD73 may play a role in CLL pathophysiology, is correlated with poor clinical and biological prognostic factors and may be of potential value as a prognostic marker and therapeutic target.

Isolation and characterization of synovial mesenchymal stem cells.

Synovial membrane and synovial fluid represent a good source of mesenchymal stem cells. They have been regarded as a promising therapeutic tool for musculoskeletal regeneration. Synovium-derived mesenchymal stem cells have higher expression of CD44 and better chondrogenic potential in vitro than mesenchymal stem cells from other tissues. In this study we compared mesenchymal stem cells from synovium and synovial fluid on the base of morphological, immunophenotype and differentiation features. A heterogeneous population of cells with different morphology was obtained after isolation and 4-day cultivation. The mesenchymal stem cell immunophenotype was confirmed by positive expression of CD105, CD90, and CD44 by flow cytometry and cells were negative for CD45. CD105+ cells were selected by immunomagnetic separation after 2-4 weeks of cultivation. The percentage of CD105+ cells in the mesenchymal stem cell population from synovia was between 40-50 % before immunomagnetic separation and increased to 95 % following the immunomagnetic separation. Von Kossa, Alcian blue and Oil Red O staining was used to assess the differentiation potential of synovial mesenchymal stem cells. Long-term cultivation did not affect the morphology and immunophenotype of synovial mesenchymal stem cells. Our results confirmed that immunomagnetic separation based on CD 105 antigen is a suitable method to enrich the subpopulation of CD105+ synovial mesenchymal stem cells.

Mesenchymal stem cells rescue the Alzheimer's disease cell model from cell death induced by misfolded truncated tau.

We have developed a stably transfected human cell model for Alzheimer's disease with doxycycline-inducible expression of human misfolded truncated tau protein (AT tau). We have showed that AT tau reduced the metabolic activity of the AT tau cells, slowed down cell proliferation, and induced caspase-3-independent apoptosis-like programmed cell death, tauoptosis. The aim of this study was to test the possible capability of rat mesenchymal stem cells (MSCs) to interfere with AT tau protein-induced cell death. AT tau cells after treatment with 10 µM all-trans retinoic acid were either co-cultivated with MSCs or supplemented with MSC secretome for 6 and 9 days. We found that both MSCs and MSC secretome promoted survival and increased the metabolic activity of the cells. Moreover stem cells induced cell differentiation and formation of neurites with numerous varicosities. Strikingly, treatment had no effect on tau expression suggesting that MSC induced self-protecting mechanism that prevented AT tau cells from tauoptosis. Our results showed that mesenchymal stem cells and their secretome are able to rescue the Alzheimer's disease cell model from cell death induced by misfolded truncated tau. We suggest that cell therapy may represent an alternative therapeutic avenue for treatment of human Alzheimer's disease and related tauopathies.

Cytogenetic abnormalities predict treatment-free interval and response to therapy in previously untreated chronic lymphocytic leukemia patients.

We evaluated the prognostic impact of chromosomal abnormalities as detected by interphase fluorescence in situ hybridization (iFISH) in 86 chronic lymphocytic leukemia (CLL) patients. Overall, 39 of 86 (45%) patients displayed one (35%) or more (10%) chromosomal abnormalities, del13q (31%) being more frequently detected than trisomy 12 (19%) followed by del11q (17%), del17p (6%) and del6q (5%). Significant differences in the treatment free intervals (TFIs) were observed among individual cytogenetic subgroups (p=0.027) with the shortest mean TFIs in subgroups with del17p, del11q and trisomy 12 (10, 12 and 14 months, respectively) as compared to subgroups with normal cytogenetics (38 months) and del13q (68 months). Poor response to therapy was observed in subgroups with del11q (p=0.044) and trisomy 12 (p=0.047) while patients with normal cytogenetics had good response (p=0.003). Furthermore, del17p and del11q were associated with highest tumor burden and disease activity as reflected by corresponding laboratory data.

MDR1 (C3435T) polymorphism: relation to the risk of breast cancer and therapeutic outcome.

P-glycoprotein (PGP), the product of the MDR1 gene, is a transmembrane active efflux pump for a variety of carcinogens and cytostatics. It has been suggested that MDR1 polymorphisms contribute to the variability in cancer risk and therapeutic outcome. We examined the relevance of C3435T polymorphism in relation to breast cancer susceptibility, clinical and pathological characteristics of breast carcinoma, the therapeutic response and hematologic toxicities after anthracycline-based chemotherapy. A significant association between allele frequencies and histological type, stage and histological grade was observed (P=0.024, 0.014, 0.006, respectively, chi(2)-test or Fisher's exact test). We also found significantly higher (P=0.019, chi(2)-test) T allele frequency in breast cancer patients (n=221) than in controls (n=113). A significantly enhanced therapeutic outcome after neoadjuvant therapy (n=38; P=0.021, Fisher's exact test) and longer time to progression after anthracycline-based chemotherapy (n=102; P=0.049, log-rank test) were observed in CC homozygotes. However, no significant association between hematologic toxicities and C3435T polymorphism was detectable.

In vitro antiproliferative and antiangiogenic effects of Flavin7.

Flavin7 (F7) is a nutritional supplement often taken by cancer patients in Central Europe during chemo- and radiation therapy. In this study, investigation of the antiproliferative and antiangiogenic activities of this supplement were performed. Flavin7 showed antiproliferative activity in Jurkat as well as in HeLa cells. It significantly reduced the growth of both cancer cell lines at the doses of 200 microg/ml to 20 microg/ml (p<0.001 and p<0.01, respectively). In F7-treated Jurkat cells we found a significant increase in the fraction of cells with sub-G(0)/G(1) DNA content, which is considered to be a marker of apoptotic cell death. Apoptosis was also confirmed by annexin V staining and DNA fragmentation. Furthermore, F7 at the doses of 100 microg/ml to 4 microg/ml inhibited endothelial cell migration and capillary tube formation what indicates its potential antiangiogenic properties. Flavin7 also inhibited the activity of matrix metalloproteinases (MMPs), preferentially MMP-9, at the doses of 100 microg/ml to 4 microg/ml. Our data suggest that F7 possesses marked antiproliferative and antiangiogenic properties in vitro. Further research is needed to elucidate also its in vivo activities.

Meningioma 40 years after radiation therapy for retinoblastoma: genetic and phenotypic analysis, and minireview of literature.

The authors present a case of 44-year-old Caucasian female diagnosed with meningothelial meningioma 40 years after radiotherapy for sporadic unilateral retinoblastoma. The genetic analysis of DNA from the meningioma revealed no oncogenic mutation in the RB1 gene. The analysis of meningioma cells by flow cytometry revealed the following immunophenotype: vimentin++ CD56+ GFAP- EGFR-. Intermediate intensities of Her-2/neu and Pgp expression were detected in a small percentage of tumour cells. Data suggest that the tumour was most likely induced by radiotherapy and did not arise as a second tumour as there was no hereditary predisposition to retinoblastoma.

Diazepam enhances hypericin-induced photocytotoxicity and apoptosis in human glioblastoma cells.

Glioblastoma multiforme (GBM) is neoplasm which is resistant to all currently used treatment modalities including surgery, radiation therapy and chemotherapy. Photodynamic therapy (PDT) has been suggested as a novel therapeutical approach to the treatment of malignant gliomas. Here, we attempted to enhance hypericin-induced photocytotoxicity and apoptosis by diazepam, a non-selective ligand of peripheral benzodiazepine receptors (PBR) which seem to play an important role in apoptosis regulation. For the study, we used U-87 MG and U373 MG glioma cell lines and primary cultures of GBM cells prepared from peroperatively obtained tumor specimens. The patients included 7 histologically confirmed GBMs. Colorimetric MTT assay was employed to study the photocytotoxic effects of hypericin and diazepam. Flow cytometry was used to detect apoptosis and assess the proapoptotic effects of diazepam. We found that hypericin upon photoactivation exerts strong cytotoxic effects against U-87 MG and U373 MG cells as well as primary GBM cell cultures. No cytotoxic effect of hypericin was observed under dark conditions. Diazepam inhibited cell growth in U-87 MG cells and primary cultures whereas proliferation of U373 MG cells remained unaffected. When hypericin was combined with diazepam, photocytotoxicity was increased in U-87 MG cells and primary cultures unlike U373 MG cells. Flow cytometric analysis revealed photoactivated hypericin-induced apoptosis in both cell lines. Apoptosis was significantly enhanced by diazepam in U-87 MG cells. However, no such effect was observed in U373 MG cells. In the present study, we showed that photocytotoxic effect of hypericin in glioma cells can be potentiated by diazepam. This effect is underlied by the ability of diazepam to facilitate hypericin-induced apoptosis. This work provides support to performe clinical studies involving diazepam in the antiglioma treatment regimens as an apoptosis-modulating agent.

Cruciferous phytoalexins: antiproliferative effects in T-Jurkat leukemic cells.

We tested antiproliferative activity of selected cruciferous phytoalexins including brassinin, 1-methoxybrassinin, (+/-)-spirobrassin, (+/-)-1-methoxyspirobrassinin and (+/-)-1-methoxyspirobrassinol, in leukemic Jurkat cell. The most effective of the tested phytoalexins was 1-methoxybrassinin with IC(50) 10 micromol l(-1). However, significant effect of all phytoalexines was also determined at concentration 1 micromol l(-1). In 1-methoxybrassinin-treated Jurkat cells, we found significant increase in the fraction of cells with a sub-G(0)/G(1) DNA content, which is considered to be a marker of cell death by apoptosis. Apoptosis was also confirmed by the annexin V staining. In summary, 1-methoxybrassinin exerted potent antiproliferative activity probably due to cell cycle arrest and apoptosis induction.

Effects of static magnetic field on human leukemic cell line HL-60.

A number of structures with magnetic moments exists in living organisms that may be oriented by magnetic field. While most experimental efforts belong to the area of effects induced by weak and extremely low-frequency electromagnetic fields, we attempt to give an attention to the biological effects of strong static magnetic fields. The influence of static magnetic field (SMF) on metabolic activity of cells was examined. The metabolic activity retardation is observed in human leukemic cell line HL-60 exposed to 1-T SMF for 72 h. The retardation effect was observed as well as in the presence of the mixture of the antineoplastic drugs 5 fluorouracil, cisplatin, doxorubicin and vincristine.

Modulation of the phototoxic effect of hypericin in human leukemia CEM cell line by N-ethylmaleimide, amiloride and omeprazole.

Hypericin is a photosensitizing plant pigment from Hypericum perforatum with multiple modes of light-induced biological activities due to production of singlet oxygen and/or excited-state proton transfer with consequent pH drop in the hypericin environment. In the present work, we studied the effects of three inhibitors of crucial mechanisms responsible for intracellular pH (pHi) regulation on hypericin phototoxicity: N-ethylmaleimide (NEM), an inhibitor of H+-ATPase, 5'-(N,N-dimethyl)-amiloride (DMA), an inhibitor of Na+/H+ exchanger, and omeprazole (OME), an inhibitor of H+K+-ATPase. Our experiments show that the effect of hypericin at 1 x 10(-5) and 1 x 10(-6) mol x l(-1) was significantly potentiated by NEM (1 x 10(-7)-1 x 10(--9) mol x l(-1)) and DMA (1 x 10(-6) and 1 x 10(-7) mol x l(-1)) in leukemic CEM cell line. On the other hand, OME had no significant effect on hypericin cytotoxicity. Our results support the hypothesis that the excited-state proton transfer and the consequent acidification of hypericin environment could play a role in the biological activity of hypericin.

Changes of gastric lipase activity after ethanol and indomethacin administration: influence of pretreatment with allopurinol, pentoxifylline and L-DOPA.

Gastric lipase (GL) plays an important role in emulsification and digestion of food fat. Lipids are components of the hydrophobic mucus and mucosa barrier. Damage of the gastric mucosa may therefore be related to changes in the lipid content and GL activity. In the present paper, we studied the effect of administration of a single dose of 96 % ethanol (E) and indomethacin 20 mg x kg(-1) (IND) on the activity of GL and on the concentrations of nonesterified fatty acids (NEFA) and triacylglycerols (TG) in the gastric mucosa of rats. Furthermore, we studied how these changes are affected by allopurinol (ALO), pentoxifylline (PX) and L-DOPA pretreatment 30 min before administration of E or IND. The effect of sialoadenectomy (SA) on these parameters was also evaluated. We found: 1) significant (p < 0.01) inhibition of GL activity after administration of E and IND and also ALO, as well as after pretreatment with ALO before E and PX before IND. L-DOPA administered alone stimulated GL activity, but its administration before IND significantly (p < 0.01) inhibited this enzymatic activity. GL activity was decreased to the threshold values in SA rats and after administration of E to SA animals. 2) NEFA concentrations were decreased after E and increased significantly (p < 0.01) after IND administration. A marked significant (p < 0.01) decrease in NEFA was found after PX and L-DOPA administration. The administration of ALO also lowered the concentration of NEFA. Pretreatment by drugs before E and IND resulted in a significant increase of NEFA in comparison with the drugs given alone (p < 0.05 for ALO + E; p < 0.01 for PX + IND). 3) TG were also decreased in all experimental groups in comparison with the control group, i.e. after E and IND, after ALO and SA and also after pretreatment by ALO before E. The concentration of TG decreased after PX, significantly (p < 0.05) after L-DOPA and after pretreatment by PX before IND. Pretreatment by ALO before E and L-DOPA before IND resulted in the increase of TG in comparison with drugs alone. Thus, these results suggest certain protective effect of pretreatment with ALO, PX and L-DOPA against the E- and IND-induced decrease in NEFA and TG during injury of the gastric mucosa. On the other hand, inhibition of GL activity was also apparent after administration of these drugs before E and IND, which suggest presence of a persisting impairment of lipid digestion in the stomach.

Diazepam enhances etoposide-induced cytotoxicity in U-87 MG human glioma cell line.

Various approaches might be employed in an effort to increase efficacy of the chemotherapeutic treatment of cancer. Recently, various modulators of anticancer therapy effectiveness have been studied. Antiproliferative effects of peripheral benzodiazepine receptor (PBR) ligands might be exploited to enhance cytotoxic effect of a chemotherapeutic drug towards cancer cells. In this work, we sought to enhance cytotoxic effect of etoposide (VP-16) by a PBR ligand, diazepam (DZ) in U-87 MG human glioma cells. Cytotoxicity of VP-16, DZ and their combinations was assessed by using the microculture MTT assay. Cell survival, effective concentrations (EC) and the onset of cytotoxic effect were determined. After 72 h of cultivation, survival of U-87 MG cells was reduced to 57 +/- 7% in the presence of VP-16 at 12.5 microg/mL alone, whereas DZ at 10-4 mol/L alone caused 28 +/- 6% reduction in cell survival. Coincubation of VP-16 at 12.5 microg/mL with DZ at 10-4 mol/L led to a further decrease in cell survival to 45 +/- 6%. Furthermore, DZ at 10-4 mol/L significantly decreased effective concentrations, EC10, EC30 and EC50, of VP-16 and the dose-response curves were shifted to the left. Addition of DZ at 10-4 mol/L to VP-16 also facilitated the onset of its cytotoxic effect. The same decrease in survival was thus achieved approximately 30 h earlier in comparison with VP-16 alone. However, DZ at 10-9 mol/L failed both to exert any effect on glioma cells survival and enhance cytotoxic effect of VP-16. DZ at 10-4 mol/L was capable of both reducing U-87 MG glioma cells survival when applied alone and also enhancing the cytotoxic effect of VP-16. No such observation was made for the lower concentrations of DZ. Potential implementation of diazepam in the antiglioma/anticancer armamentarium awaits further experimentation but phase I and phase II clinical trials could be suggested.

The effect of quercetin on light-induced cytotoxicity of hypericin.

Protective effect of quercetin, a natural antioxidant compound, on hypericin-induced cytotoxicity was studied in human promyelocytic leukemia cells (HL-60). Hypericin (10(-5) mol x l(-1)) alone significantly decreased cell survival to 21% that found in the controls, whereas in combination with quercetin (10(-5) mol x l(-1)) this decrease was diminished to 46%. Lower concentrations of quercetin had no protective effect. These findings indicate that oxygen radicals can play an important role in hypericin-induced phototoxic effects.