Purpose: The present study was designed to investigate the effect of hydrostatic pressure on CELL viability, apoptosis induction, morphology and CELL-substrate interactions of PC12 CELLs.Materials and Methods: PC12 as a neuronal CELL LINE maintained in RPMI 1640 culture medium supplemented with 10% fetal bovine serum. PC12 CELLs were subjected to hydrostatic pressure. Experimental pressure condition was 100mmHg set above atmospheric pressure for 2 h. Controls were treated identically except for the application of pressure. Dye exclusion was used for viability assay, TUNEL staining was used for apoptosis detection. CELL area was assessed as morphometry and then CELL adhesion, extension and migration were investigated. Results: Hydrostatic pressure had not changed viability of CELLs. It induced apoptosis in PC12 CELLs. In addition, hydrostatic pressure reduced CELL area, adhesion, extension and migration ability of these CELLs (P<0.05). Conclusion: Hydrostatic pressure may induce apoptosis in PC12 CELLs as a result of inappropriate CELL to substrate adhesion. Thus it is suggest that occurring apoptosis in these CELLs be an anoikis CELL death induced by loss of attachment to the substrate.

Background: Opioid drugs are considered as important members of drugs of abuse. Opioid abusers are more likely to be infected which may be due to apoptotic effects of the drugs on immune CELLs. Furthermore, there are some reports on the apoptotic effect of morphine on neural CELLs. In the present study, the effect of morphine and lithium on apoptosis in PC12 CELL LINE (as a model of neural CELLs) was examined.Methods: We used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, annexin Vfluorescein isothiocyanate test and quantitative real time RT-polymerase chain reaction for detection of necrosis and apoptosis (programmed CELL death).Results: PC12 CELLs were exposed to different concentrations of morphine for six, 12, 24, 48, and 96 hours. Quantitative real-time RT-polymerase chain reaction revealed that mRNA expression of BAX (proapoptotic element) increased while a decrement in the mRNA expression of BCL-2 (protective element) was observed after six hours (but not after 12 or 24 hours) exposure to morphine. Furthermore, the results of MTT assay and annexin V-fluorescein isothiocyanate test indicated that morphine exposure causes an increase in thepercentage of apoptotic and necrotic CELLs, respectively. Interestingly, the results of MTT assay and annexin V-fluorescein isothiocyanate test were observed 12 and 24 hours after morphine exposure. Thus, it can be concluded that alteration in mRNA expression is an early event rather than as a consequence of apoptosis or necrosis. On the other hand, lower concentrations of lithium elicit protective effect against apoptosis in some of mammalian CELLs while the higher concentrations are toxic. Despite large body of evidences on the protective effect of lithium, elucidations of downstream events are still unknown. In the present study, 72-hour preincubation of PC12 CELLs with 1.2mM lithium chloride reversed the effects of morphine on the mRNA expression of BAX and BCL-2. Furthermore, the results of real time RT-polymerase chain reaction were supported by annexin V-fluorescein isothiocyanate test and MTT assay.Conclusion: The protective effect of lithium on the morphine-induced cytotoxicity is mediated via down-regulation of BAX and up-regulation of BCL-2 mRNA expression.

CELL damage induced by free radicals has been implicated as causal factor in a wide variety of human diseases such as cancer, neurodegenerative disorders. Scientific recognition of natural antioxidants may be helpful in preventing many diseases. Aqueous Date fruit extract (ADFE)- PC12 CELL LINE. ADFE at concentrations of 0.1-10% containing 0.005% H2O2 and H2O2 at concentrations of 0.01-1% containing 10% ADFE were prepared. 100ml from a CELL suspension equal to 106 CELLs/ml of PC12 CELLs were added to centrifuge tubes and incubated for 24 hours. CELL suspension rinsed twice with medium and incubated in 96-multidish for 72 hrs. Then, CELL growth was assayed by XTT-assay. Aqueous Date fruit extract (ADFE) at concentrations of 0.1, 1 and 10% comparing to 0.005 H2O2 showed significant increase in PC12 CELL growth (by 11, 57 and 74% respectively) (P<0.05 to 0.001). ADFE at concentration of 10% showed protective effects against different concentrations of H2O2 (0.01-1%) (P<0.05 to P<0.001). Date fruit contains compounds with potent antioxidant activity and daily consumption may be helpful in preventing many diseases, especially neurodegenerative disorders such as Alzheimer’s disease and Parkinson’s disease.

Objective: Parkinson's disease (PD) is known as a degenerative disorder of the central nervous system. In this disease, the death of dopaminergic neurons in the substantianigra, a region of the midbrain is occurred. Annexin A1 (AnxA1) is reported as an anti-inflammatory factor. ANXA1 might play a neuroprotective role in PD by acting as an anti-inflammatory mediator. The aim of this study was isolation of PC12 CELL LINE overexpressing ANXA1. Materials and Methods: At first step, cDNA of ANXA1was prepared from neutrophils and then inserted to PGL268 vector to produce recombinant PGL/ANXA1 vector. The accuracy of cloning was confirmed by further sequencing. Transfection was carried out in the dopaminergic neuronal CELL LINE, PC12 (a CELL LINE derived from a pheochromocytoma of the rat adrenal medulla), using of lipofectamin LTX and after 48 hours EGFP reporter was detected. Furthermore, PC12 CELLs expressing ANXA1 gene were selected followed by G418 treatment after 2 weeks. Expression of ANXA1 was evaluated by real time PCR, western blot and flow cytometry techniques.Results: The results indicated strong expression of AnXA1 in PC12 CELLs. Conclusion: PC12 CELLs containing ANXA1 DNA could be further used as an in vitro model for evaluation of ANXA1 Anti-inflammatory effects.

Recently use of endosulfan has aroused a great concern among clinical and basic neuroscientists as besides adult population even the children residing in some areas of touristic state of Kerala, India which are under constant spraying of endosulfan had shown symptoms of CNS dysfunctioning including cerebral palsy, retardation of mental and physical growth, epilepsy and various congenital anomalies. In spite of several studies demonstrating neurotoxic potential of endosulfan, very little is known about the mechanism of its neurotoxicity. In the present study an attempt has been made to deLINEate the mechanism by which endosulfan a chlorinated hydrocarbon insecticide exerts its neurotoxic potential at CELLular level using PC12, a dopaminergic CELL LINE. CELLs exposed to endosulfan (0-100 mM) for 1, 24 and 48 h showed significant loss in CELL viability /mitochondrial functioning in a dose- and time- dependent manner following MTT assay and LDH leakage assessment. An oxidative stress mediated neurotoxicity was evident where endosulfan (1uM, a non cytotoxic concentration) exposure is accompanied by increase in lipid peroxidation and lowering in antioxidant defense as malonaldehyde (MDA), a product of lipid peroxidation was enhanced by 49 and 62% following 24 and 48 hours endosulfan exposure. A decrease in levels of GSH (41 & 68%) along with catalase (39 and 52%) and SOD (26 & 44%) activities was also evident 24 & 48 h post exposure. Induced expression of early response gene protein (c-Fos, P<0.001; c-Jun P<0.05 and GAP-43, P<0.05) during 1-24 h endosulfan (1 mM) exposure was accompanied by PKC activation. The present results suggest possible role of PKC/early response gene mediated oxidative injury and mitochondrial dysfunction in endosulfan neurotoxicity.

Background: The rat pheochromocytoma CELL LINE, (PC12) will differentiate through in vitro condition by some factors such as nerve growth factor (NGF) and convert into neuron-like CELLs. Bee venom (BV) contain different components such as phospholipase 2 (PLA2) which has differentiational effect on PC12 CELLs by itself but the effect of BV as a whole biological product has not been well known, So, in this study, the effects of bee venum on differentiation of PC12 was studied.Materials and Methods: In this experimental study, the PC12 CELLs were seeded in the culture medium (RPMI-1640) at 5×103 CELL/well in poly-D-lysine (0.05 mg/ml) coated 24-well culture plates, and were treated with NGF with the concentration of 50 ng/ml and the bee venom with the concentrations of 1 µg/ml and 3 µg/ml. The viability of PC12 CELLs were analyzed by using MTT assay, and CELL differentiation were surveyed morphologically and the neurite outgrowth were measured and the neuronal like CELLs were confirmed by enzymatically method (AChE activity assay).Results: The results confirmed the differentiotional effect of BV at the low dosage and this effect was increased when the BV and NGF were used together. We conclude that BV and NGF may use in CELL therapy to induce differentiation of undifferentiated CELLs.Conclusion: This study showed that BV at the low dosage lead to CELL death and at the high dosage convert PC12 CELLs to neuronal- like CELLs.Concurrent use of BV and NGF may use in CELL therapy to induce differentiation of undifferentiated CELLs.

MicroRNAs (miRNAs) are a class of small non coding regulatory RNAs that have key functions in multiple CELL processes. Deregulation of these tiny miRNAs is involved in various human diseases. MiR-155 is one of the multifunctional miRNA that its over-expression has been found to be associated with different kinds of cancer such as leukemia, breast and colon cancers. It is thought that deregulation and over-expression of this microRNA may be associated with PC12 CELL proliferation. So, the aim of this study was to investigate the role of miR-155 expression on PC12 CELL growth. For this reason, PC12 CELLs were cultured and transfected by 3 different concentration (25, 50 and 75nmol) of either LNA anti-miR-155 or scramble antisense in 24-well plate. Then, total RNA was extracted from transfected CELLs. miRNA cDNAs were synthesized from isolated total RNA. In the second step, miR-155 expression level was analyzed using the quantitative real-time polymerase chain reaction (QRT-PCR). MTT test was performed to evaluate CELL viability.In the next step, apoptosis assay was assessed to investigate anti miR-155 effect on PC12 CELLs death. Obtained results were analyzed with t-test. MTT test revealed that CELL viability of transfected CELLs with 75nM of anti-miR- 155 to be reduced by half of the control and scramble groups (0.5 vs.0.97 and 0.94). Our data suggest that miR-155 over-expression is associated with PC12 CELL growth. So, miR-155 down regulation by anti-miR-155 could open up new ways to restrain brain tumor growth, as anti-miR-155 causes PC12 CELLs to repress.