Sabera Ruždijić

Potential mechanism of cell death in the developing rat brain induced by propofol anesthesia.

Commonly used general anesthetics can have adverse effects on the developing brain by triggering apoptotic neurodegeneration, as has been documented in the rat. The rational of our study was to examine the molecular mechanisms that contribute to the apoptotic action of propofol anesthesia in the brain of 7‐day‐old (P7) rats. The down‐regulation of nerve growth factor (NGF) mRNA and protein expression in the cortex and thalamus at defined time points between 1 and 24 h after the propofol treatment, as well as a decrease of phosphorylated Akt were observed. The extrinsic apoptotic pathway was induced by over‐expression of tumor necrosis factor (TNF) which led to the activation of caspase‐3 in both examined structures. Neurodegeneration was confirmed by Fluoro‐Jade B staining. Our findings provide direct experimental evidence that the anesthetic dose (25 mg/kg) of propofol induces complex changes that are accompanied by cell death in the cortex and thalamus of the developing rat brain.

Behavioral and biochemical effects of various food-restriction regimens in the rats.

In this paper we describe the effects of six different food restriction (FR) regimens on amphetamine (AMPH)-induced locomotor and nonlocomotor activities in male rats. Changes in serum corticosterone (CORT), insulin and glucose levels were also examined. Each regimen was implemented through different daily food allowance (50%, 25% and 12.5% of the daily food intake, referred to as 50%, 75% and 87.5% FR groups, respectively) and by a specific feeding regimen - either every day (ED) or every other day (EOD). AMPH injection led to a significant increase of locomotor activity in all rats subjected to FR compared to ad libitum fed rats. A significant increase of nonlocomotor activity was observed only in the 75% FR and 87.5% FR groups. The serum CORT levels were significantly elevated and the serum insulin and glucose levels were significantly decreased in all of the FR groups in comparison to the AL rats. The results presented in this paper suggest that the ED regimens produced changes in motor activity and biochemical parameters, which were more-or-less dependent on the degree of FR. In contrast, the EOD regimens induced very similar changes irrespective of the degree of FR degree. Our data support the possible mechanistic roles of CORT and insulin in the effect of FR on locomotor activity, since the most pronounced increase of serum CORT and more pronounced decrease in serum insulin concentration was observed in the groups that also exhibited the highest locomotor activities.

New anti-cancer characteristics of jatrophane diterpenes from Euphorbia dendroides.

Jatrophane diterpenes were shown to be inhibitors of P-glycoprotein (P-gp). There are also evidences on their microtubule-interacting activity in cancer cells. We evaluated new anti-cancer characteristics of two jatrophane type compounds from Euphorbia dendroides. For that purpose, the model system of sensitive non-small cell lung cancer cell line (NCI-H460) and its resistant counterpart (NCI-H460/R) was used. Although both jatrophanes showed inhibitory effect on cancer cell growth, they were non-toxic for peripheral blood mononuclear cells (PBMC). We examined their effects in combination with paclitaxel (PTX), a well-known mitotic spindle interacting chemotherapeutic. Jatrophanes overcome PTX resistance in concentration-dependent manner in MDR cancer cell line (NCI-H460/R). We observed that this synergistic effect is not caused merely by P-gp inhibition. In combination with PTX, jatrophanes induce cell killing and change cell cycle distribution leading to G2/M arrest. Furthermore, they exert an anti-angiogenic effect by decreasing the vascular endothelial growth factor (VEGF) secretion. The reduction of the level of mdr1 mRNA expression in sensitive cells, suggests that these compounds could not contribute to the development of resistance. In conclusion, present study provides a rational basis for the new cancer treatment approach with jatrophanes that are non-toxic to normal cells and have new favorable anti-cancer characteristics.

Changes in markers of neuronal and glial plasticity after cortical injury induced by food restriction

The regenerative capacity of the adult central nervous system is limited. We investigated whether short-term food restriction (FR; 50% of the daily food intake lasting 3 months) modulates processes of brain plasticity after cortical injury. Quantitative changes of growth-associated protein 43 (GAP-43) and synaptophysin (SYP) mRNA levels in the ipsilateral cortex of the adult rat during the recovery period (from 2 to 28 days) after injury were investigated by real-time RT-PCR. Using Western blot and immunohistochemical analyses we examined the levels and localization of proteins involved in neuronal plasticity, SYP and GAP-43, as well as glial fibrillary acidic protein (GFAP), a marker of glial plasticity. A marked rise in GAP-43 and SYP immunoreactivity observed in the FR group on the 7th day after injury pointed to increases in axonal branching and synapses in the cortex surrounding the lesion. The appearance of reactive astrocytes was accompanied by the absence of immunoreactivity for GAP-43 and SYP in ad libitum fed animals. This finding supports the hypothesis that morphological hypertrophy of astrocytes associated with GFAP synthesis is responsible either directly or indirectly for the inhibitory role of activated glia on axonal regeneration. Examination of the effects of FR on serum corticosterone and glucose concentrations and GAP-43, SYP and GFAP expression revealed that FR facilitated recovery of the injured region by attenuating reactive astrogliosis and enhancing the expression of neuronal plasticity markers.

Propofol anesthesia induces proapoptotic tumor necrosis factor-α and pro-nerve growth factor signaling and prosurvival Akt and XIAP expression in neonatal rat brain.

Previously we observed that prolonged exposure to propofol anesthesia causes caspase‐3‐ and calpain‐mediated neuronal death in the developing brain. The present study examines the effects of propofol anesthesia on the expression of tumor necrosis factor‐α (TNFα), pro‐nerve growth factor (NGF), and their receptors in the cortex and the thalamus. We also investigated how propofol influences the expression of Akt and X‐linked inhibitor of apoptosis (XIAP) expression, proteins that promote prosurvival pathways. Seven‐day‐old rats (P7) were exposed to propofol anesthesia lasting 2, 4, or 6 hr and killed 0, 4, 16, or 24 hr after anesthesia termination. The relative levels of mRNA and protein expression were estimated by RT‐PCR and Western blot analysis, respectively. The treatments caused marked activation of TNFα and its receptor TNFR‐1 and pro‐NGF and p75NTR receptor expression. In parallel with the induction of these prodeath signals, we established that propofol anesthesia promotes increased expression of the prosurvival molecules pAkt and XIAP during the 24‐hr postanesthesia period. These results show that different brain structures respond to propofol anesthesia with a time‐ and duration of exposure‐dependent increase in proapoptotic signaling and with concomitant increases in activities of prosurvival proteins. We hypothesized that the fine balance between these opposing processes sustains homeostasis in the immature rat brain and prevents unnecessary damage after exposure to an injurious stimulus. The existence of this highly regulated process provides a time frame for potential therapeutic intervention directed toward suppressing the deleterious component of propofol anesthesia.

The effect of MK-801 on motor activity and c-Fos protein expression in the brain of adolescent Wistar rats

The changes that occur during adolescence have a profound impact on the brain and behavior later in life. In this work we examined changes in motor activity during habituation to a novel environment and after treatment with MK-801 (0.025, 0.05, 0.1mg/kg) in peripubertal, pubertal and adult Wistar rats. The involvement of the motor cortex and striatum in motor activity was assessed by analyzing changes in c-Fos protein levels that served as an indicator of neuronal activity. During the habituation period, locomotor activity in peripubertal rats was higher during the first 10 min than in other groups. The same amount of stereotypy-like movements was detected in all three groups. MK-801 induced dose- and age-dependent changes in motor activity. Peripubertal rats were the most sensitive to treatment with MK-801. We also report a surprising finding that systemic application of MK-801 induced a similar age-related profile of changes in motor activity and c-Fos protein expression in the motor cortex but no c-Fos induction in the striatum. Our results demonstrate that, depending on the phase of adolescence the same dose of MK-801 affected behavioral functions in a different manner and that activity of the motor cortex rather than striatal activity was linked to drug-motor activity interactions.

Sulfinosine-induced cell growth inhibition and apoptosis in human lung carcinomas in vitro

In spite of tremendous effort for improvedtherapy, lung cancer remains the leadingcause of cancer-related deaths worldwide.In the present study, we used the novelpurine ribunocleoside sulfinosine andevaluated its antiproliferative andapoptotic outcome on the non-small celllung carcinoma cell line (NSCLC) and thesmall cell lung carcinoma cell line (SCLC).Using a BrdU incorporation-test sulfinosineinhibited cell growth in a dosedependent-manner. ID50 values were4.65 ± 0.17 μM in the case of NSCLCcells, and 3.59 ± 0.81 μM in thecase of SCLC cells. MTT testing revealedthat IC50 values were 6.24 ±0.77 μM for NSCLC and 5.68 ±0.58 μM for SCLC. Inhibitoryconcentrations (IC50 and ID50)for sulfinosine were nonsignificantly lowerin SCLC cells compared to NSCLC cells,indicating similar susceptibility of thecells. Flow-cytometric analysis, TUNELstaining, DNA laddering and cell deathELISA test were used to investigateapoptotic cell death. Our resultsdemonstrated that high concentrations ofsulfinosine can cause typical DNAladdering, a hallmark for apoptosis.Evidence of free nucleosomes and enzymaticlabeling of fragmented DNA confirmedapoptosis involvement in sulfinosinecytotoxicity. In addition, flow-cytometricanalysis showed that 25 μM sulfinosinearrested cell cycle progression atthe G2M phase and induction ofapoptosis in both cell lines. From theseresults, we concluded that sulfinosine mayact as an anticancer agent and furtherstudies may prove its efficacy in lungcancer cells. Thus the biological effectsof sulfinosine may be due to modulation ofcell growth, cell death, and cell cycleregulatory molecules.

Molecular and cytogenetic changes in multi-drug resistant cancer cells and their influence on new compounds testing

PurposeMulti-drug resistance (MDR) is a major obstacle to successful cancer treatment. Therefore, in vitro models are necessary for the investigation of the phenotypic changes provoked by cytotoxic agents and more importantly for preclinical testing of new anticancer drugs.MethodsWe analyzed chromosomal, numerical, and structural changes after development of MDR, alterations in p53 and PTEN, single nucleotide polymorphisms (SNPs) in the mdr1 gene and corresponding protein expression of P-glycoprotein (P-gp) in three human MDR cancer cell lines: non-small cell lung carcinoma NCI-H460/R, colorectal carcinoma DLD1-TxR, and glioma U87-TxR. In addition, we explored how these molecular and phenotypic alterations influence the anticancer effect of new drugs.ResultsCytogenetic analysis showed polyploidy reduction after development of MDR in U87-TxR. Losses of 6q in all resistant cancer cell lines and inactivation of p53 in U87-TxR and PTEN in DLD1-TxR were also revealed. Overexpression of P-gp was observed in all MDR cancer cell lines. We evaluated the anticancer activities and MDR reversal potential of Akt inhibitor GSK690693, Ras inhibitor Tipifarnib, and two P-gp inhibitors (jatrophane diterpenoids). Their effects vary due to the cell-type differences, existence of MDR phenotype, presence of mdr1 SNP, and tumor suppressors’ alterations. Tipifarnib and jatrophane diterpenoids significantly sensitized MDR cancer cells to paclitaxel.ConclusionIn conclusion, investigated MDR cancer cells obtained new molecular and cytogenetic characteristics that may serve as potential clinical prognostic markers. In addition, these MDR cancer cell lines present a valuable model for preclinical evaluation of new anticancer agents.

The combination of gamma ionizing radiation and 8-Cl-cAMP induces synergistic cell growth inhibition and induction of apoptosis in human prostate cancer cells.

SummaryThe antiproliferative and cytotoxic potential of the nucleotide analog 8-Cl-cAMP was tested in PC-3 and DU145 metastatic human prostate cancer cells. The drug was examined as the only therapeutic agent and in combination with ionizing irradiation (IR). Highly synergistic effects of IR and 8-Cl-cAMP were observed in both cell lines when examined by the MTT viability and BrdU proliferation assays. The combination of IR and 8-Cl-cAMP at clinically relevant doses exerted substantial growth inhibition. The combination of IR and 8-Cl-cAMP caused a significant disturbance in the distribution of cell cycle phases. Cell cycle arrest in the sub-G0/G1 phase predominated in both cell lines. The most striking observation was a significant increase in apoptotic PC-3 and DU145 cells. The DU145 cells were three times more sensitive to the combined treatment than PC-3 cells. The initial resistance to IR-induced apoptosis in these p53-deficient prostate cancer cell lines was overcome through an alternative proapoptotic pathway induced by 8-Cl-cAMP. Considering the low effective doses of treatments, improved tumor eradication rates and minimal undesirable side effects, the combination of IR and 8-Cl-cAMP could be the therapy of choice in treating prostate cancer.

Induction of TNF-α signaling cascade in neonatal rat brain during propofol anesthesia

Propofol anesthesia can trigger pro‐ and anti‐apoptotic signaling pathways in the rat brain. In our previous work, we demonstrated that propofol causes widespread apoptotic neurodegeneration in 7‐postnatal‐day‐old (PND7) but not in PND14 rat neurons. The mechanism responsible for these opposing outcomes is unknown, apparently linked to the specific stage of brain development. The present study aims to elucidate the anti‐apoptotic process that is activated in the cortex and thalamus of PND14 Wistar rats during the first 48 h after the onset of propofol anesthesia. We showed that the expression of tumor necrosis factor‐α (TNF‐α) and several components of its pathway, TNFR1 and caspase‐8, was significantly increased in the cortex and thalamus. Nuclear factor kappa B (NF‐κB) p65 was downregulated in the cortex and upregulated in the thalamus. The expression of c‐Fos was upregulated only in the cortex, showing opposed profile compared to NF‐κB p65. Double immunofluorescence staining revealed the colocalization of NF‐κB p65 with neuronal marker (NeuN), but with predominantly cytoplasmic localization. Finally, X‐linked inhibitor of apoptosis protein (XIAP) was upregulated in both examined structures. Immunohistochemical staining with Iba‐1 revealed that the treatment did not induce changes in microglial morphology. Our results (i) reveal that the simultaneous activation of pro‐ and anti‐apoptotic signaling occurs after propofol anesthesia, and (ii) pinpoint the potential neuroprotective role of XIAP in anesthesia‐induced neurotoxicity.