Selective Cytotoxic Effects of 5-Trifluoromethoxy-1H-indole-2,3-dione 3-Thiosemicarbazone Derivatives on Lymphoid-originated Cells.

AIM: The present study aims to identify the anticancer effect of novel 1H-indole-2,3-dione 3- thiosemicarbazone derivatives. These compounds could be promising anticancer agents in leukemia treatment. BACKGROUND: Conventional chemotherapeutic agents accumulate in both normal and tumor cells due to nonspecificity. For effective cancer treatment, new drugs need to be developed to make chemotherapeutics selective for cancer cells. The ultimate goal of cancer treatment is to reduce systemic toxicity and improve the quality of life. METHODS: In this study, the anticancer effects of 5-trifluoromethoxy-1H-indole-2,3-dione 3-thiosemicarbazone derivatives (A-L) were investigated in chronic myelogenous leukemia K562, Burkitt's lymphoma P3HR1, acute promyelocytic leukemia HL60 cells, and vincristine-resistant sublines of K562 and P3HR1 cells. Additionally, the compounds were tested on lymphoid-derived cells from ALL patients. In order to investigate the particular mechanism of death caused by the cytotoxic effects of the compounds, immunohistochemical caspase 3 staining was performed in P3HR1 cells, and the resulting apoptotic activities were demonstrated. RESULTS: All tested compounds have been found to have cytotoxic effects against lymphoma cells at submicromolar concentrations (IC50= 0.89-1.80 µM). Most compounds show significant selectivity for the P3HR1 and P3HR1 Vin resistance. The most effective and selective compound is 4-bromophenyl substituted compound I (IC50=0.96 and 0.89 µM). Cyclohexyl and benzyl substituted compounds D and E have also been found to have cytotoxic effects against K562 cell lines (IC50=2.38 µM), while the allyl substituted compound C is effective on all cell lines (IC50=1.13-2.21 µM). 4-Fluorophenyl substituted F compound has been observed to be effective on all cells (IC50=1.00-2.41 µM) except K562 cell. Compound C is the only compound that shows inhibition of HL-60 cells (IC50= 1.13 µM). Additionally, all compounds exhibited cytotoxic effects on lymphoidderived cells at 1µM concentration. These results are in accordance with the results obtained in lymphoma cells. CONCLUSION: All compounds tested have submicromolar concentrations of cytotoxic effects on cells. These compounds hold potential for use in future treatments of leukemia.

The effects of regular swimming exercise during sodium valproate treatment on seizure behaviors and EEG recordings in pentylenetetrazole-kindled rats.

We investigated the effects of alone/combined regular swimming exercise and sodium valproate on epileptic seizure behaviors and EEG recordings, anti-oxidative mechanism, learning, and memory in pentylenetetrazole (PTZ)-kindled rats. Forty-eight healthy rats were randomly divided into eight equal groups as control (CONT), swimming exercise (EX), sodium valproate (SV), SV+EX, PTZ, EX+PTZ, SV+PTZ and SV+EX+PTZ. The rats were forced to regular swimming exercise for 60 min every other day, 13 doses of PTZ (40 mg/kg) were given to induce epileptic seizures and 200 mg/kg SV was given for 28 days. Epileptic seizures were evaluated by visual observation and EEG recordings (total spike numbers and number of epileptiform discharges). Memory and learning skills were assessed with passive avoidance test. According to our visual seizure observations, seizure latency was prolonged only in SV+EX+PTZ (p < 0.001) group, seizure severity score decreased in SV+PTZ (p < 0.05) and SV+EX+PTZ (p < 0.001) groups and seizure frequency was reduced in SV+PTZ (p < 0,001), EX+PTZ (p < 0,001), and SV+EX+PTZ (p < 0,001) groups. Total spike numbers and number of epileptiform discharges highly increased in PTZ group, whereas they decreased in swimming exercise and/or SV treatment groups. The most effective result was seen in the combined therapy group. Memory deficit was observed in PTZ -kindling group, but it didn't change with exercise or SV. Based on our results, regular swimming exercise had positive effects on PTZ-induced seizure frequency, and combined therapy of regular swimming exercise and SV is the most effective way to ameliorate visual seizure behaviors and decrease spike numbers and number of epileptiform discharges according to EEG recordings. Regular swimming exercise could be an alternative option to reduce the dose of SV and the side effects of SV can be avoided in clinical aspects.

Effects of erythropoietin pretreatment on single dose pentylentetrazole-induced seizures in rats.

Although it is accepted that prolonged and repeated seizures can cause epileptogenesis, memory deficits and neuronal death, the precise relation between epileptic seizures and neuronal death remains unclear. Erythropoietin (EPO) exhibits neuroprotective and anti-epileptic effects. We investigated the effect of a single pentylentetrazole (PTZ) induced tonic-clonic seizure on the pyramidal neurons of the cornu ammonis 1 (CA1) and CA3 regions of hippocampus. We also investigated the effects of EPO on seizure, memory and on brain-derived neurotrophic factor (BDNF), tyrosine receptor kinase-B, sirtuin-1 (SIRT1), which are important for memory. Forty male rats were divided into four groups: control, saline treated, single 60 mg/kg dose PTZ treated, 3000 IU/kg EPO treated, and 3000 IU/kg EPO treated 24 h before PTZ administration. Seizure latency and severity were assessed following PTZ injection. A passive avoidance test was performed 24 h after seizure. BDNF, TrkB and SIRT1 levels were measured in serum, hippocampus and cortex. The hippocampus was examined histologically, and neuronal nuclear antigen (NeuN) was investigated using immunohistochemistry. EPO pretreatment decreased seizure severity and prolonged seizure latency. Single dose PTZ-induced seizures did not affect memory. Numbers of cells in the CA1 region did not change, although the number of dark stained neuron increased. Both total cell numbers and percentage of dark stained cells were elevated in the CA3 region following PTZ induced seizures. EPO pretreatment decreased the number of dark cells in both CA1 and CA3 regions and the number of cells in the CA3 region. NeuN labeling was unchanged in the CA1 and CA3 regions in the PTZ group; however, EPO pretreatment increased NeuN labeling in the CA3 region. Although EPO exhibited an anticonvulsive effect, single dose EPO pretreatment did not affect memory in either animals not exposed to PTZ or animals that had been subjected to PTZ-induced seizures. EPO pretreatment prolonged seizure latency and reduced seizure severity after PTZ-induced seizures. The anti-seizure and neuroprotective effects of EPO pretreatment may be due to the protection of CA1 and CA3 neurons, possibly owing to SIRT1 and BDNF activity.

Protective effects of cytokine combinations against the apoptotic activity of glucocorticoids on CD34+ hematopoietic stem/progenitor cells.

Haematopoietic stem cells can self-renew and produce progenitor cells, which have a high proliferation capacity. Chemotherapeutic drugs are toxic to normal cells as well as cancer cells, and glucocorticoids (GCs), which are essential drugs for many chemotherapeutic protocols, efficiently induce apoptosis not only in malignant cells but also in normal haematopoietic cells. Studies have shown that haematopoietic cytokines can prevent the apoptosis induced by chemotherapy and decrease the toxic effects of these drugs. However, the apoptosis induction mechanism of GCs in CD34+ haematopoietic cells and the anti-apoptotic effects of cytokines have not been well elucidated. In this study, we investigated the apoptotic effects of GCs on CD34+, a haematopoietic stem/progenitor cell (HSPC) population, and demonstrated the protective effects of haematopoietic cytokines. We used a cytokine cocktail containing early-acting cytokines, namely, interleukin-3 (IL-3), thrombopoietin, stem cell factor and flt3/flk2 ligand, and dexamethasone and prednisolone were used as GCs. Apoptotic mechanisms were assessed by immunohistochemical staining and quantified using H-scoring. Dexamethasone and prednisolone induced apoptosis in CD34+ HSPCs. GC treatment caused a significant increase in apoptotic Fas, caspase-3, cytochrome c and Bax, but a significant decrease in anti-apoptotic Bcl-2. Furthermore, as expected, cytokines caused a significant decrease in all apoptotic markers and a significant increase in Bcl-2. Thus, our findings suggest that CD34+ HSPCs are an extremely sensitive target for GCs and that cytokines protect these cells from GC-induced apoptosis.

The effects of 17ß-estradiol on blood brain barrier integrity in the absence of the estrogen receptor alpha; an in-vitro model.

The blood-brain barrier (BBB), which saves the brain from toxic substances, is formed by endothelial cells. It is mainly composed of tight junction (TJ) proteins existing between endothelial cells. Estrogen is an important regulatory hormone of BBB permeability. It protects the BBB before menopause, but may increase BBB permeability with aging. In addition, nitric oxide modulates BBB permeability. Alcohol impairs the integrity of the BBB with oxidants and inflammatory mediators such as iNOS. We investigated the effects of estrogen on BBB integrity in an in vitro BBB model created with ERα-free HUVEC (human umbilical vein endothelial-like cells) to mimics the menopausal period. In vitro BBB model is created with HUVEC/C6 (rat glioma cells) co-culture. The effect of 17ß-estradiol on ethanol-induced BBB disruption and change/or increase of iNOS activity, which modulate BBB integrity, were evaluated. Inducibility and functionality of BBB were investigated using transendothelial electrical resistance (TEER) and the expression of proteins TJ proteins (occludin and claudin-1) and iNOS activity by immunostaining. Our results revealed that 17ß-estradiol treatment before and after ethanol decrease expression of occludin and claudin-1 and value of TEER which are BBB disrupt indicators. In addition, ethanol and 17ß-estradiol separately and pre- and post-ethanol 17ß-estradiol treatment increased iNOS expression. Thus our study suggests caution in the use of 17ß-estradiol after menopause because 17ß-estradiol at this time may both increase the inflammatory process as well as damage the BBB. We think that beneficial effects of 17ß-estradiol may be through ERα but it needs further studies.

Long term consequences on spatial learning-memory of low-calorie diet during adolescence in female rats; hippocampal and prefrontal cortex BDNF level, expression of NeuN and cell proliferation in dentate gyrus.

Calorie restriction (CR) is argued to positively affect general health, longevity and normally occurring age-related reduction of cognition. Obesity during adolescence may adversely affect cognition in adulthood but, to date effects of CR have not been investigated. We hypothesized that feeding with as low as 15% low-calorie diet (LCD) during adolescence would increase hippocampal and prefrontal BDNF (Brain-derived neurotrophic factor) levels, proliferative cells and neuron numbers in dentate gyrus (DG), thus positively affecting spatial memory in adulthood. Spatial learning-memory function was improved in adult female Sprague-Dawley rats fed with LCD during adolescence. PCNA (Proliferating cell nuclear antigen-cell proliferation marker) expressing cells and NeuN (Neuronal nuclear antigen-neuron marker) expressing cells in hippocampus DG which are critically involved in memory were increased. Hippocampus and prefrontal cortex BDNF levels were increased while serum glucose levels and level of lipid peroxidation indicator malondialdehyde in serum and hippocampus were reduced. Our unique results suggest that improved cognition in adult rats with LCD feeding during adolescence may result from the increase of neurogenesis and BDNF. These findings reveal the importance of nutrition in adolescence for cognitive function in adulthood. Our results may be useful for further studies aiming to treat age-related cognitive impairments.

Anticancer effect of a novel palladium-saccharinate complex of terpyridine by inducing apoptosis on Ehrlich ascites carcinoma (EAC) in Balb-C mice.

BACKGROUND/AIM: [Pd(sac)(terpy)](sac)•4H2O (sac=saccharinate and terpy=2,2':6',2"-terpyridine) is newly-synthesized palladium(II) (Pd) complex. We investigated the antiproliferative and apoptotic effects of this complex on Ehrlich ascites carcinoma (EAC). MATERIALS AND METHODS: EAC cells were administered to 33 Balb/c mice. Mice were divided randomly into four groups: control, cisplatin, Pd(II) complex and paclitaxel. Control group animals received 0.9% NaCl; other groups received treatments cisplatin, Pd(II) complex and paclitaxel on days 7 and 12. At day 14, animals were sacrificed. Expression of active caspase-3, p53 and proliferating cell nuclear antigen (PCNA) was investigated and apoptosis was evaluated by terminal deoxynucleotidyltransferase (TdT)-mediated nick-end labelling (TUNEL) technique. RESULTS: Expression of p53 and PCNA were found to be decreased (p<0.0001), cells with active caspase-3 and TUNEL-positive cells were found to be increased (p<0.0001) in all treatment groups. CONCLUSION: Like cisplatin and paclitaxel, this Pd(II) complex has a strong anticancer activity against EAC by inducing apoptosis and suppressing proliferation in vivo.

The evaluation of human tenon's fibroblasts and endothelial cell responses to antifibrotics alone and in combination with α-tocopherol.

PURPOSE: We aimed to evaluate the influence of current antifibrotic agents as well as the possible results obtained by combining these agents. This study included α-tocopherol, a strong antifibrotic and an efficient neuromediator of pathways used by other agents. MATERIALS AND METHODS: Mitochondrial Bcl-2, Bax, cytochrome c and cytoplasmic caspase-3 expression, as well as toxic effect patterns, mitosis and cellular reactions due to α-tocopherol alone or combined with paclitaxel, mitomycin C and 5-flurouracil (5-FU), was studied in series obtained from human endothelial and primary Tenon's fibroblast cell cultures. RESULTS: The strongest apoptotic effect in both cell groups belonged to paclitaxel, followed by mitomycin C, and despite the overall suppressive effect of the α-tocopherol combination, mitomycin C increased its efficiency on the endothelial cells. The apoptosis/necrosis ratio was highest in α-tocopherol and lowest in paclitaxel, with α-tocopherol generally decreasing necrosis. Bax was observed at a high level with mitomycin C. Cytotoxicity was the highest with paclitaxel, and the caspase-3 reaction was markedly higher with mitomycin C in both cell types. In the α-tocopherol and 5-FU slides, mitosis and a layered formation were observed. The addition of α-tocopherol reduced the cytotoxicity of all antifibrotic agents in both cell series by decreasing the cell numbers, leading to necrosis. CONCLUSIONS: Alone or in combination, the use of α-tocopherol and 5-FU is safer than other agents. By suppressing the cytotoxic effects of other antifibrotic agents, α-tocopherol is a promising drug for improving the effects of antifibrotics in many aspects of medicine. In addition, it has the potential to play a role beyond its antioxidant and antifibrotic activity in ocular surgery.

Erythropoietin pretreatment suppresses seizures and prevents the increase in inflammatory mediators during pentylenetetrazole-induced generalized seizures.

Erythropoietin (EPO) suppresses epileptic seizures, but the mechanism is unclear. The search for novel targets in the therapy of epilepsy has focused recently on brain inflammation since brain inflammation and the associated blood-brain barrier (BBB) damage appears to be an integral part of epilepsy pathophysiology. We examined the effects of EPO on proinflammatory mediators in brain and serum in PTZ-induced generalized seizure model. The inflammation markers (IL-1ß, TNF-α, IL-6, IL-10), BBB and neuron damage markers (S100B, Neuron specific enolase; NSE, respectively) in serum and brain of Sprague-Dawley male rats were examined with the ELISA method. Nitric oxide synthase (NOS) isoforms were investigated immunohistochemically in hippocampus. EPO treatment 4 h and 24 h before PTZ administration had diverse effects. EPO treatment 4 h before PTZ administration elongated the seizure latency, decreased the inflammation and damage markers in serum and brain significantly, whereas EPO treatment 24 h before PTZ administration lowered inflammation and damage markers to control levels and decreased the seizure stage. PTZ-induced seizures increased inducible NOS (iNOS) activity and decreased endothelial NOS (eNOS) activity in hippocampus. Both EPO pretreatments reversed these effects. These findings, i.e., decreased iNOS activity and increased eNOS activity by EPO suggest the first time that the favorable effect of EPO pretreatment on inflammatory mediators triggered by PTZ-induced seizures. This can provide further insight into epilepsy treatment and new prophylactic strategies against epilepsy risk.

The effects of atorvastatin on memory deficit and seizure susceptibility in pentylentetrazole-kindled rats.

Deficits in memory function have been observed in pentylentetrazole (PTZ)-kindled rats. In the present study we examined the effects of atorvastatin ((3-hydroxy-3-methylglutaryl-coenzyme A [HMG-CoA] reductase inhibitor) on PTZ kindling and related memory deficits in rats trained with the passive avoidance test. Subconvulsive PTZ doses rendered a gradual increase in seizure activity. PTZ kindling caused long-term memory to deteriorate. Atorvastatin per se and in PTZ-kindled rats improved learning and memory functions. It also prolonged latency (time to appearance of spike potentials) and diminished the amplitude and frequency of spike potentials, which indicate epileptic discharges. These novel findings suggest that the favorable effect of the atorvastatin on memory deficits provoked by PTZ kindling might be of clinical utility.

Apoptosis-inducing effects of Morinda citrifolia L. and doxorubicin on the Ehrlich ascites tumor in Balb-c mice.

Morinda citrifolia L. (Noni) is a herbal remedy with promising anti-cancer properties. However, its effects on various cancers are to be investigated to make a firm conclusion before implementing it into the clinical practice. Therefore, we investigated the cytotoxic potential of noni on Ehrlich ascites tumor grown in female Balb-c mice and also combined it with a potent anti-cancer agent, doxorubicin. One group received noni only (n = 8), another one doxorubicin (n = 8), and the other one noni + doxorubicin (n = 8) for 14 days after the inoculation of cells. The control group (n = 7) received 0.9% NaCl only. We found that short and long diameters of the tumor tissues were about 40-50% smaller, compared to those in control group. This anti-growth effect resulted from the induction of apoptosis, which was confirmed by the positive results from the Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) analysis and the active caspase-3 cells in tissues. Apoptosis also confirmed by caspase-cleaved cytokeratin 18 elevation in serum of the treated groups. Further, the proliferation was decreased, which was immunohistochemically shown by the PCNA staining. We conclude that noni may be useful in the treatment of breast cancer either on its own or in combination with doxorubicin. Further studies are warranted to assess the dosage and safety of using noni fruit juice in conjuction with anti-cancer drugs against breast cancer.

The relationship between prolactin (PRL), leptin, nitric oxide (NO), and cytokines in patients with hyperprolactinemia.

Leptin is a key mediator in the maintenance of neuroendocrine homeostasis. The aim of this study was to determine the changes in serum leptin, tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), nitric oxide (NO) levels in patients with hyperprolactinemia. The study consists of 16 consecutive patients with high prolactin (PRL) levels (group I) and a control group of 11 normoprolactinemic patients (group II). Pituitary tumor tissues of patients in groups I and II were analyzed for immunohistochemical (IHC) expression of prolactin and leptin. Group I has significantly higher levels of leptin than group II (P < 0.001). There is a strong correlation between PRL and leptin concentrations in group I. However, there were no statistically significant differences for NO, TNF-alpha, IL-6 between the two groups. IHC staining showed that there was strong immunoreactivity for leptin protein in PRL-secreting pituitary adenomas. Double immunostaining of adenoma tissues with PRL and leptin showed that the adenoma cells expressed both. These findings together are suggestive that leptin co-secretion from a prolactinoma may be the cause of increased serum leptin concentration, independently from the peripheral action of prolactin.

The relationship between nitric oxide and leptin in the lung of rat with streptozotocin-induced diabetes.

Lung structural changes and immunoreactivity of endothelial (eNOS)- and inducible nitric oxide synthase (iNOS) were investigated by light microscopy in lungs of treated and untreated diabetic rats. Diabetes was induced by a single intraperitoneal (i.p.) injection of 65 mg kg(-1) streptozotocin (STZ) in Wistar albino male rats. Diabetic rats received daily i.p. doses of dexamethasone (2 mg kg(-1)), leptin (0.5 microg kg(-1)) and intramuscular insulin (20 U kg(-1)) or a combination of these drugs for 1 week starting 4 weeks after the STZ injections. After treatment, the blood levels of glucose, leptin, insulin and nitrate/nitrite (NO(3) (-)/NO(2) (-)) were measured. Dilatation of alveoli and alveolar ducts, partial alveolar wall thickening and increased eNOS- and iNOS characterized the diabetic rat lungs. High blood glucose and nitrate/nitrite levels as well as low insulin and leptin levels were also present. Treatment with insulin, dexamethasone and a combination of these drugs resulted in improvement of the structural and immunohistochemical abnormalities. The most effective treatment was insulin therapy. Leptin administration resulted in increased relative amounts of extracellular material, which led to noticeable respiratory efficiency in the diabetic rat lungs. All treatments except leptin lowered blood glucose levels. The combination of insulin and dexamethasone increased blood leptin and insulin, while the remaining diabetic rats had blood with low leptin and insulin concentrations. These results suggest that therapy with insulin plus dexamethasone but not therapy with leptin is beneficial for diabetics.

In vitro effects of growth factors and interferon-alpha on busulfan cytotoxicity.

An experimental approach to increasing the effectiveness of leukemia treatment with S-phase-specific cytotoxics is to increase the cycling of leukemia cells with growth factors. However, growth factors may have a different relationship with non-cell-cycle-specific agents. The authors examined the effects of granulocyte colony-stimulating factor (G-CSF), granulocyte/macrophage colony-stimulating factor (GM-CSF), and interferon-alpha (INF-alpha) on the cytotoxic effects of the alkylating agent busulfan on the erythro-myeloid cell line K562. G-CSF and GM-CSF increased the proliferation and colony-forming ability of K562 cells and protected the cells from busulfan effects. INF-alpha decreased the colony-forming ability and proliferation of the K562 cells and demonstrated a possibly additive effect with busulfan. In the cell line K562, the growth factors G-CSF and GM-CSF protected the cells from the non-cell-cycle-specific alkylating agent busulfan, whereas IFN-alpha demonstrated an additive cytotoxic effect.

Sex differences in modulating blood brain barrier permeability by NO in pentylenetetrazol-induced epileptic seizures.

Susceptibility to epilepsy as well as BBB dysfunction in some pathological conditions varies depending on sex difference. It has recently been shown that systemically given NO donor and antagonists modify the nature of seizures induced by PTZ (pentylenetetrazol) differently in male and female rats. This study investigates the role of NO on BBB permeability in PTZ seizures with sex differences using NO donor, sodium nitroprusside (SNP), and NOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME). Nitrite+nitrate levels as indices of NO generation in the brain were also assessed. L-NAME prolonged seizure latency in male rats, seizure intensity and seizure duration were lessened. L-NAME depicted opposite effects in seizure nature in female rats. SNP prolonged seizure latency, while seizure intensity and duration were lessened only in female rats. L-NAME in male rats increased L-NAME use in female rats (not in male rats) which resulted in a more leaky BBB especially in midbrain, thalamus, hippocampus, corpus striatum and cerebellum whereas SNP use in male rats and not in female rats resulted in pronounced BBB opening in all brain regions studied than PTZ per se. L-NAME while decreasing nitrite+nitrate levels in male rat brains, acted in an opposite fashion in females. SNP use depicted an inverse picture with respect to L-NAME, with an opposite action in different sexes. This study reveals that NO effect on BBB in PTZ-induced seizures depends unequivocally on sex difference. The sex-dependent action of NO in seizures and in CNS pathologies warrants further investigation.

Vanadyl sulfate protects against streptozotocin-induced morphological and biochemical changes in rat aorta.

The aim of this study was to investigate the protective effects of vanadyl sulfate on aorta tissue of normal and streptozotocin (STZ)-induced diabetic rats, morphologically and biochemically. The animals were made diabetic by an intraperitoneal injection of streptozotocin (65 mg/kg) and vanadyl sulfate (100 mg/kg) that was given every day for 60 days by gavage technique to rats. Under the light and transmission electron microscopes, hypertrophy of the vessel wall, focal disruption in the elastic lamellae, an increase in thickness of total aortic wall, tunica intima, subendothelial space and adventitial layer, and a disorganization in smooth muscular cells of the tunica media were observed in diabetic animals. The aorta lipid peroxidation (LPO) levels were significantly increased and the aorta glutathione (GSH) levels were significantly reduced in STZ diabetic rats. In diabetic rats administered vanadyl sulfate for 60 days, aorta LPO levels significantly decreased and the aorta GSH level significantly increased. In conclusion, in vivo treatment with vanadyl sulfate of diabetic rats prevented the morphological and biochemical changes observed in thoracic aorta of diabetic animals.

Cytotoxic effects of leaf, stem and root extracts of Nerium oleander on leukemia cell lines and role of the p-glycoprotein in this effect.

Nerium oleander (No), is a toxic plant. In recent studies, it was determined that the extracts of this plant are effective to treat some types of cancer, but these studies are limited and do not include human leukemia. In the present study, firstly we aimed to investigate in vitro the cytotoxic effects of No on the HL60 and K562 leukemia cell lines. The cells were incubated with six different concentrations of each three extracts. MTT assay was employed as a cytotoxicity test. It was observed that concentrations of 1000, 500 and 50 microg/ml from each extract possess marked antileukemic effects. No leaf and root extracts were seen to be more cytotoxic than the stem extract according to LC50. Secondly, in order to understand the role of P-gp in cytotoxicity, P-gp levels of K562 resistant and sensitive cells were measured by flow cytometry before treatment extracts, and then, the cells were incubated with No leaf, stem and root extracts in 500 microg/ml concentrations overnight. After incubation, measurements showed decreased levels of P-gp in the cells. Hence, it is possible to think contributes to their cytotoxic effects that inhibiting of the P-gp pump by No extracts on leukemia cell lines.

Nitric oxide involvement in seizures elicited by pentylentetrazol and sex dependence.

It has been known that susceptibility to some types of epilepsy is affected by sex. In addition, the role of NO in epileptogenesis is still unclear; NO has been suggested to be either an anticonvulsive or a proconvulsive agent. In an attempt to elucidate both the role of NO and sex differences in sensitivity to seizures, male and female Wistar rats were treated intraperitoneally (i.p.) by pentylentetrazol (PTZ)(80 mg/kg) and by a nitric oxide synthase(NOS) inhibitor N-omega-nitro-L-arginine-mthylester(L-NAME)(50mg/kg) and a NO precursor sodium-nitroprusside(SNP)(2.5mg/kg)- applied 15 min. before PTZ injection. Latency, frequency, severity, and duration of generalized clonic and clonic-tonic convulsions were recorded. Furthermore, alterations in severity, latency, frequency, and duration of convulsions were observed to correlate with NO. Both sexes, injected with PTZ, showed repetitive seizure patterns. Seizures were found to be more severe in females. L-NAME and SNP pretreatment produced paradoxical effects on PTZ-induced seizures in both sexes. L-NAME completely prevented PTZ-induced seizures in male rats, whereas increased severity, frequency, duration, and significantly shortened the latency in female rats. Unexpectedly, SNP increased convulsion severity, frequency, duration, and shortened latencies in male, whereas it decreased convulsion severity, frequency, and duration and prolonged latency in females. These results indicate that endogenous NO is involved in the regulation of convulsive action suggesting a role depending on sex.

Immunocytochemical detection of effects of TRH and T4 on prolactin- and TSH-producing cells in the pituitary gland of Rana ridibunda.

Effects of synthetic thyrotropin-releasing hormone (TRH) and various doses of thyroxin (T4) on prolactin (PRL)-producing cells and thyrotropic cells in the pituitary were investigated in adult male and female Rana ridibunda frogs. Animals were given 200 microg TRH once a week for 4 weeks and 0.2-0.5 mg T4 during 3 days per week for a period of 2 weeks by injections in the groin. PRL-producing cells and thyrotropic cells were identified with light microscopical and electron microscopical immunocytochemical methods, using rabbit anti-PRL and rabbit anti-thyroid stimulating hormone (TSH) as primary antibodies. TRH caused cytological changes in both cell types, which were consistent with increased synthesis and release of both PRL and TSH. Treatment with 0.5 mg T4 activated both cell types less than TRH treatment did, whereas 0.2 and 0.4 mg T4 caused inactivation of both cell types. In conclusion, mammalian TRH is effective on both types of frog pituitary cells. Our study suggests that T4 has a positive rather than a negative effect when concentrations above a certain threshold are given.