Downregulation of survivin by siRNA inhibits invasion and promotes apoptosis in neuroblastoma SH-SY5Y cells

Neuroblastoma is a solid tumor that occurs mainly in children. Malignant neuroblastomas have a poor prognosis because conventional chemotherapeutic agents are not very effective. Survivin, a member of the inhibitor of the apoptosis protein family, plays a significant role in cell division, inhibition of apoptosis, and promotion of cell proliferation and invasion. Previous studies found that survivin is highly expressed in some malignant neuroblastomas and is correlated with poor prognosis. The aim of this study was to investigate whether survivin could serve as a potential therapeutic target of human neuroblastoma. We employed RNA interference to reduce survivin expression in the human neuroblastoma SH-SY5Y cell line and analyzed the effect of RNA interference on cell proliferation and invasion in vitro and in vivo. RNA interference of survivin led to a significant decrease in invasiveness and proliferation and increased apoptosis in SH-SY5Y cells in vitro. RNA interference of survivin inhibited tumor growth in vivo by 68±13% (P=0.002) and increased the number of apoptotic cells by 9.8±1.2% (P=0.001) compared with negative small interfering RNA (siRNA) treatment controls. Moreover, RNA interference of survivin inhibited the formation of lung metastases by 92% (P=0.002) and reduced microvascular density by 60% (P=0.0003). Survivin siRNA resulted in significant downregulation of survivin mRNA and protein expression both in vitro and in vivo compared with negative siRNA treatment controls. RNA interference of survivin was found to be a potent inhibitor of SH-SY5Y tumor growth and metastasis formation. These results support further clinical development of RNA interference of survivin as a treatment of neuroblastoma and other cancer types.

Ropinirole alters gene expression profiles in SH-SY5Y cells: a whole genome microarray study

Ropinirole (ROP) is a dopamine agonist that has been used as therapy for Parkinson's disease. In the present study, we aimed to detect whether gene expression was modulated by ROP in SH-SY5Y cells. SH-SY5Y cell lines were treated with 10 µM ROP for 2 h, after which total RNA was extracted for whole genome analysis. Gene expression profiling revealed that 113 genes were differentially expressed after ROP treatment compared with control cells. Further pathway analysis revealed modulation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway, with prominent upregulation of PIK3C2B. Moreover, batches of regulated genes, including PIK3C2B, were found to be located on chromosome 1. These findings were validated by quantitative RT-PCR and Western blot analysis. Our study, therefore, revealed that ROP altered gene expression in SH-SY5Y cells, and future investigation of PIK3C2B and other loci on chromosome 1 may provide long-term implications for identifying novel target genes of Parkinson's disease.

Selegiline increases heme oxygenase-1 expression and the cytotoxicity produced by dopamine treatment of neuroblastoma SK-N-SH cells

Increased dopamine catabolism may be associated with oxidative stress and neuronal cell death in Parkinson's disease. The present study was carried out to examine the effect of dopamine on the expression of heme oxygenase-1 and -2 (HO-1 and HO-2) in human neuroblastomas (SK-N-SH cell line) and the effects of selegiline and antioxidants on this expression. Cells were kept with close control of pH and were incubated with varying concentrations of dopamine (0.1-100 µM) for 24 h. HO-1 and HO-2 cDNA probes were prepared by reverse transcription-polymerase chain reaction amplification. The mRNA expression of HO-1 and HO-2 was measured by Northern blot analysis. The levels of HO-1 mRNA increased after dopamine treatment, in a dose-dependent manner, in all cell lines studied, whereas levels of the two HO-2 transcripts did not. The HO-1 and HO-2 protein expression was analyzed by Western blotting. HO-1 protein was undetectable in untreated SK-N-SH cells and increased after treatment with dopamine. In contrast, the HO-2 protein (36 kDa) was detected in untreated cells and the levels did not change as a result of treatment. alpha-Tocopherol (10-100 µM) and ascorbic acid (100 µM) did not attenuate the effects of dopamine. Selegiline (10 µM) produced significant increase (P < 0.01) in the induction of HO-1 by dopamine (more than six times the control values). The increased expression of HO-1 following dopamine treatment indicates that dopamine produces oxidative stress in this cell line.

Dexamethasone blocks the migration of the human neuroblastoma cell line SK-N-SH

Glucocorticoids (Gc) influence the differentiation of neural crest-derived cells such as those composing sympathoadrenal tumors like pheochromocytomas, as well as neuroblastomas and gangliomas. In order to obtain further information on the effects of Gc on cells evolving from the neural crest, we have used the human neuroblastoma cell line SK-N-SH to analyze: 1) the presence and the binding characteristics of Gc receptors in these cells, 2) the effect of dexamethasone (Dex) on the migration of SK-N-SH cells, and 3) the effect of Dex on the organization of the cytoskeleton of SK-N-SH cells. We show that: 1) receptors that bind [³H]-Dex with high affinity and high capacity (Kd of 9.6 nM, Bmax of 47 fmol/mg cytosolic protein, corresponding to 28,303 sites/cell) are present in cytosolic preparations of SK-N-SH cells, and 2) treatment with Dex (in the range of 10 nM to 1 µM) has an inhibitory effect (from 100% to 74 and 43%, respectively) on the chemotaxis of SK-N-SH cells elicited by fetal bovine serum. This inhibition is completely reversed by the Gc receptor antagonist RU486 (1 µM), and 3) as demonstrated by fluorescent phalloidin-actin detection, the effect of Dex (100 nM) on SK-N-SH cell migration is accompanied by modifications of the cytoskeleton organization that appear with stress fibers. These modifications did not take place in the presence of 1 µM RU486. The present data demonstrate for the first time that Dex affects the migration of neuroblastoma cells as well as their cytoskeleton organization by interacting with specific receptors. These findings provide new insights on the mechanism(s) of action of Gc on cells originating in the neural crest.

Study on the growth promoting capacity of calf and fetal bovine serum for animal cells "in vitro" II: electrophoretic study and survey on the antiproteolytic activity of pools of calf and fetal bovine serum

Calf serum and fetal bovine serum present great variability as to its growth promoting efficiency (GPE). As supplement of culture media to cultivate cells of animal origin they stimulate the "in vitro" multiplication and maintain cell viability. When fourteen lots of calf sera of variable GPE had the total protein contents as well as the percentages of serum fractions determined, no significant differences that could possibly explain the variability of the GPE were observed. Evaluation of the antiproteolytic activity of nineteen lots of calf serum and eighteen serum lots of younger calves showed that the former exhibited lower antiproteolytic titers (1:40 to 1:80) than the latter (1:80 to 1:160). Twelve lots of fetal bovine serum studied in parallel, showed the highest concentration of antiproteolytic factors, with titers equal to 1:320. Sera of bovine origin, but not fetal sera, are usually heat-inactivated, what was demonstrated to be responsible for the decrease of the antiproteolytic activity of 75% of the lots tested. This could explain the inability of certain heat-inactivated sera in promoting multiplication of some cells "in vitro", as verified with primary monkey kidney cells. The results obtained in this study indicated the convenience of submiting each lot of serum to be introduced in cell culture to previous determination of its characteristics, such as growth promoting efficiency, antiproteolytic activity and also toxicity, absence of extraneous agents, etc., in order to minimize the possibility of using serum lots of questionable quality, thus preventing not only the loss of cell lines, but also undesirable and sometimes expensive delays.

Glucose-6-phosphate dehydrogenase and glutathione reductase activity in methemoglobin reduction by methylene blue and cyst amine: study on glucose-6-phosphate dehydrogenase-deficient individuals, on normal subjects and on riboflavin-treated subjects

The authors have standardized methods for evaluation of the activity of the glucose-6-phosphate dehydrogenase and of glutathione reductase. The general principle of the first method was based on methemoglobin formation by sodium nitrite followed by stimulation of the glucose-6-phosphate dehydrogenase with methylene blue. Forty six adults (23 males and 23 females) were studied. Subjects were not G6PD deficient and were aged 20 to 30 years. The results showed that methemoglobin reduction by methylene blue was 154.40 and 139.90 mg/min (p<0.05) for males and females, respectively, in whole blood, and 221.10 and 207.85 mg/min (n.s.), respectively, in washed red cells. These data showed that using washed red cells and 0.7g% sodium nitrite concentration produced no differences between sexes and also shortened reading time for the residual amount of methemoglobin to 90 minutes. Glutathione reductase activity was evaluated on the basis of the fact that cystamine (a thiol agent) binds to the SH groups of hemoglobin, forming complexes. These complexes are reversed by the action of glutathione reductase, with methemoglobin reduction occurring simultaneously with this reaction. Thirty two adults (16 males and 16 females) were studied. Subjects were not G6PD deficient and were aged 20 to 30 years. Methemoglobin reduction by cystamine was 81.27 and 91.13 mg/min (p<0.01) for males and females, respectively. These data showed that using washed red cells and 0.1 M cystamine concentration permits a reading of the residual amount of methemoglobin at 180 minutes of incubation. Glutathione reductase activity was evaluated by methemoglobin reduction by cystamine in 14 females before and after treatment with 10 mg riboflavin per day for 8 days. The results were 73.69 and 94.26 jug/min (p<0.01) before and after treatment, showing that riboflavin treatment increase glutathione reductase activity even in normal individuals. Three Black G6PD-deficient individuals (2 males and 1 female) were also studied. The G6PD and glutathione reductase were partially activated, the change being more intense in males. On the basis of race and of the laboratory characteristics observed, it is possible to suggest that the G6PD deficiency of these individuals is of the African type and that the female is heterozygous for this deficiency. Analysis of the results as a whole permitted us to conclude that the methods proposed here were efficient for evaluating the activity of the glucose-6-phosphate dehydrogenase and of glutathione reductase. The latter is dependent on the pentose pathway, which generates NADPH, and on riboflavin, a FAD precursor vitamin.

Effect of scorpion toxin on the enterochromaffin-like cells in normal and Trypanosoma cruzi-infected rats: a morphological study

Intravenous injection of scorpion toxin (Tityus serrulatus) in normal and Trypanosoma cruzi infected rats did not cause ultrastructural morphologic changes on enterochromaffin-like (ECL) cells of the stomach, although it induced a significant increase of the gastric secretion. Our data seem to indicate that gastric ECL cells structure is not affected by stimulation with scorpion toxin or by acute infection with T. cruzi in the rat.

OBTENTION OF RABIES ANTIGEN THROUGH BHK21 CELLS ADHERED TO MICROCARRIERS

Four rabies antigen batches were produced from virus suspensions resulting from BHK21 cells adhered to microcarriers (Cytodex 1), inoculated and cultured in a bioreactor. In parallel the methodology of production of rabies virus through cultures of BHK21 cells in monolayers in bottles was used. The results obtained showed that infecting titles were 106.69 DL50/mL and 107.28 DL50/mL for suspensions cultured in bottles and in the bioreactor, respectively. The viral suspension volumes collected were on average 11,900 per batch from the bioreactor and 800mL per bottle. Ten horses were immunized with the antigen produced in the bioreactor. The means of antirabies antibody titers found were 240 and 212 IU/mL after the initial and the first booster doses, respectively. Rabies antigen with satisfactory infecting titers can be obtained on a large scale by culturing in a bioreactor inoculated BHK21 cells adhered to microcarriers.

IL-2 AND IFN-g, BUT NOT IL-4 SECRETION BY PERIPHERAL BLOOD MONONUCLEAR CELLS (PBMC) ARE RELATED TO CD4+ T CELLS AND CLINICAL STATUS IN BRAZILIAN HIV-1-INFECTED SUBJECTS

It has been reported that production of IL-2 and IFN-g, known as T-helper type 1 cytokines, by peripheral mononuclear cells (PBMC) decreases with progression of HIV infection. In contrast, IL-4 and IL-10 production, Th2 cytokine profile, increases with HIV disease progression. PBMC were evaluated from 55 HIV-infected subjects from Divisão de Imunologia, Hospital das Clínicas, Faculdade de Medicina da Universidade de São Paulo, to "in vitro" cytokines production after 24 hours of stimulation with PHA. Low levels of IL-4 production in both HIV- infected patients and normal subjects, were detected. The patients with CD4+ T cell counts <200 showed a significant decrease of IL-2 and IFN-g production compared to controls. Patients with higher counts of CD4+ T cells (either between 200-500 or >500 cells/mm3) also showed decreased production of IL-2 that was not statistically significant. There was a correlation between IL-2 and IFN-g release with CD4+ T cells counts. HIV-1-infected individuals with CD4+ T cells >500 cells/mm3 showed increased levels of IL-2 and IFN-g, than individuals with CD4+ T cells <500 cells/mm3. In conclusion, we observed a decline of IL-2 and IFN-g production at advanced HIV disease. IL-4 production was not affected during HIV infection. Taken together, these findings suggest that the cytokine profile might be influenced by the HIV infection rather than the cause of disease progression.

Failure of nitric oxide production by macrophages and decrease in CD4+ T cells in oral paracoccidioidomycosis: possible mechanisms that permit local fungal multiplication

Paracoccidioidomycosis is a chronic granulomatous disease that induces a specific inflammatory and immune response. The participation of nitric oxide (NO), a product of the inducible nitric oxide synthase enzyme (iNOS), as an important fungicidal molecule against Paracoccidioides brasiliensis has been demonstrated. In order to further characterize the Oral Paracoccidioidomycosis (OP), we undertook an immunohistochemical study of iNOS+, CD45RO+, CD3+, CD8+, CD20+, CD68+ cells and mast cells. The samples were distributed in groups according to the number of viable fungi per mm². Our results showed weak immunolabeling for iNOS in the multinucleated giant cells (MNGC) and in most of the mononuclear (MN) cells, and the proportion of iNOS+ MN/MNGC cells in the OP were comparable to Control (clinically healthy oral tissues). Additionally, our analysis revealed a similarity in the number of CD4+ cells between the Control and the OP groups with higher numbers of fungi. These findings suggest that a low expression of iNOS and a decrease in the CD4+ T cells in OP may represent possible mechanisms that permit the local fungal multiplication and maintenance of active oral lesions.

T CD4+ cells count among patients co-infected with human immunodeficiency virus type 1 (HIV-1) and human T-cell leukemia virus type 1 (HTLV-1): high prevalence of tropical spastic paraparesis/HTLV-1-associated myelopathy (TSP/HAM)

INTRODUCTION: HIV positive patients co-infected with HTLV-1 may have an increase in their T CD4+ cell counts, thus rendering this parameter useless as an AIDS-defining event. OBJECTIVE: To study the effects induced by the co-infection of HIV-1 and HTLV-1 upon CD4+ cells. MATERIAL AND METHODS: Since 1997, our group has been following a cohort of HTLV-1-infected patients, in order to study the interaction of HTLV-1 with HIV and/or with hepatitis C virus (HCV), as well as HTLV-1-only infected asymptomatic carriers and those with tropical spastic paraparesis/HTLV-1 associated myelopathy (TSP/HAM). One hundred and fifty HTLV-1-infected subjects have been referred to our clinic at the Institute of Infectious Diseases "Emílio Ribas", São Paulo. Twenty-seven of them were also infected with HIV-1 and HTLV-1-infection using two ELISAs and confirmed and typed by Western Blot (WB) or polymerase chain reaction (PCR). All subjects were evaluated by two neurologists, blinded to the patient's HTLV status, and the TSP/HAM diagnostic was based on the World Health Organization (WHO) classification. AIDS-defining events were in accordance with the Centers for Disease Control (CDC) classification of 1988. The first T CD4+ cells count available before starting anti-retroviral therapy are shown compared to the HIV-1-infected subjects at the moment of AIDS defining event. RESULTS: A total of 27 HIV-1/HTLV-1 co-infected subjects were identified in this cohort; 15 already had AIDS and 12 remained free of AIDS. The median of T CD4+ cell counts was 189 (98-688) cells/mm³ and 89 (53-196) cells/mm³ for co-infected subjects who had an AIDS-defining event, and HIV-only infected individuals, respectively (p = 0.036). Eight of 27 co-infected subjects (30%) were diagnosed as having a TSP/HAM simile diagnosis, and three of them had opportunistic infections but high T CD4+ cell counts at the time of their AIDS- defining event. DISCUSSION: Our results indicate that higher T CD4+ cells count among HIV-1/HTLV-1-coinfected subjects was found in 12% of the patients who presented an AIDS-defining event. These subjects also showed a TSP/HAM simile picture when it was the first manifestation of disease; this incidence is 20 times higher than that for HTLV-1-only infected subjects in endemic areas.