Synergy between sublethal doses of shikonin and metformin fully inhibits breast cancer cell migration and reverses epithelial-mesenchymal transition.

BACKGROUND: Shikonin is a natural multipotent anti-tumorigenic compound. We investigated potential synergy between shikonin and anti-diabetic metformin against tumorigenic properties of breast cancer cell line MCF-7. METHODS AND RESULTS: The IC50 of shikonin and metformin was determined after a single treatment of two cell lines MCF-7 and MDA-MB-231. We then measured optimal doses of each drug, used in combination, in MCF-7 cells. These sub-IC50 doses were co-applied for all subsequent combined treatments to evaluate their synergistic effects on MCF-7 tumorigenic properties. Next, we examined expression levels of the genes crucial for apoptosis, cell growth, and EMT using RT-PCR or real-time PCR and monitored CD44/CD24 ratios using flow cytometry. Binding energies between shikonin and growth molecules were measured by in silico simulation. Shikonin caused significantly reduced cell survival that was accelerated by the synergizing presence of metformin. Drug combination induced apoptosis and ROS levels while fully blocking cell migration and reverting EMT. RT-PCR showed strong suppression of BCL-2 but induction of BAX and PTEN. Prolonged shikonin treatment caused a total loss of the nuclear membrane, whereas metformin prevented this damage while promoting apoptotic morphologies. Our real-time PCR detected reduced levels of EMT genes but increases in the anti-EMT gene CDH1. Combined treatment also reduced CD44/CD24 ratios in favor of chemosensitivity. Binding energies strongly favored shikonin interactions with growth-signaling molecules. CONCLUSIONS: Shikonin and metformin synergize in inhibiting the tumorigenic activities of MCF-7 cells including their proliferation, invasiveness, and EMT with a potential to inhibit multidrug resistance.

Complementation of dopaminergic signaling by Pitx3-GDNF synergy induces dopamine secretion by multipotent Ntera2 cells.

Human teratocarcinoma cell line Ntera2 (NT2) expresses dopamine signals and has shown its safe profile for clinical applications. Attempts to restore complete dopaminergic (DAergic) phenotype enabling these cells to secrete dopamine have not been fully successful so far. We applied a blend of gene transfer techniques and a defined medium to convert NT2 cells to fully DAergic. The cells were primarily engineered to overexpress the Pitx3 gene product and then cultured in a growth medium supplemented with knockout serum and retinoic acid to form embroid bodies (EBs). Trypsinization of EB colonies produced single cells ready for differentiation. Neuronal/DAergic induction was promoted by applying conditioned medium taken from engineered human astrocytomas over-secreting glial cell-derived neurotrophic factor (GDNF). Immunocytochemistry, reverse-transcription and real-time polymerase chain reaction analyses confirmed significantly induced expression of molecules involved in dopamine signaling and metabolism including tyrosine hydroxylase, Nurr1, dopamine transporter, and aromatic acid decarboxylase. High-performance liquid chromatography analysis indicated release of dopamine only from a class of fully differentiated cells expressing Pitx3 and exposed to GDNF. In addition, Pitx3 and GDNF additively promoted in vitro neuroprotection against Parkinsonian toxin. One month after transplantation to the striatum of 6-OHDA-leasioned rats, differentiated NT2 cells survived and induced significant increase in striatal volume. Besides, cell implantation improved motor coordination in Parkinson's disease (PD) rat models. Our findings highlight the importance of Pitx3-GDNF interplay in dopamine signaling and indicate that our strategy might be useful for the restoration of DAergic fate of NT2 cells to make them clinically applicable toward cell replacement therapy of PD.

The Superiority of Sucrose Cushion Centrifugation to Ultrafiltration and PEGylation in Generating High-Titer Lentivirus Particles and Transducing Stem Cells with Enhanced Efficiency.

Viral gene delivery is hailed as a great milestone in gene-based therapeutic approaches. The human immunodeficiency virus-derived lentiviral vectors (LVs) are advantageous in infecting both dividing and non-dividing cells leading to continuous expression of transgenes. A variety of protocols are available for concentration of LVs. We primarily generated our internal ribosome entry site (IRES)-based LVs. Virus titration and transduction efficiency were compared between various strategies that included sucrose cushion centrifugation (SCC), protein column ultrafiltration and polyethylene glycol precipitation. Among these approaches, SCC resulted in concentration of high-titer EGFP-expressing lentivirus (1.4 ± 0.3 × 109 TU/ml) with the lowest protein impurities. Further, we examined transduction strengths of our three methods on two challenging stem cells. Both human NT2 and mouse bone marrow-derived mesenchymal stem cells demonstrated high transduction using SCC of 65 ± 2.8 and 49 ± 0.8%, respectively. Finally, lentivirus particles harboring IRES-based transfer vectors of specific genes, concentrated by SCC, integrated into host genome. Taken together, development of cost-effective and efficient concentration strategies such as our SCC method is yet highly demanded to broaden the horizons of lentivirus application in clinical and translational research.

Umbilical cord: an unlimited source of cells differentiable towards dopaminergic neurons.

Cell replacement therapy utilizing mesenchymal stem cells as its main resource holds great promise for ultimate treatment of human neurological disorders. Parkinson's disease (PD) is a common, chronic neurodegenerative disorder hallmarked by localized degeneration of a specific set of dopaminergic neurons within a midbrain sub-region. The specific cell type and confined location of degenerating neurons make cell replacement therapy ideal for PD treatment since it mainly requires replenishment of lost dopaminergic neurons with fresh and functional ones. Endogenous as well as exogenous cell sources have been identified as candidate targets for cell replacement therapy in PD. In this review, umbilical cord mesenchymal stem cells (UCMSCs) are discussed as they provide an inexpensive unlimited reservoir differentiable towards functional dopaminergic neurons that potentially lead to long-lasting behavioral recovery in PD patients. We also present miRNAs-mediated neuronal differentiation of UCMSCs. The UCMSCs bear a number of outstanding characteristics including their non-tumorigenic, low-immunogenic properties that make them ideal for cell replacement therapy purposes. Nevertheless, more investigations as well as controlled clinical trials are required to thoroughly confirm the efficacy of UCMSCs for therapeutic medical-grade applications in PD.

Synergy Between Choroid Plexus Epithelial Cell-Conditioned Medium and Knockout Serum Replacement Converts Human Adipose-Derived Stem Cells to Dopamine-Secreting Neurons.

Human adipose-derived stem cells (hADSCs) have great capacity to differentiate into mesodermal origins as well as nonmesodermal lineages, including neural cells. This valuable feature paves the way for the therapeutic application of hADSCs for neurodegenerative maladies such as Parkinson's disease (PD). We tested the capacity of choroid plexus epithelial cell-conditioned medium (CPEC-CM) alone or cocktailed with knockout serum (KS) to induce dopaminergic (DAergic) differentiation of hADSCs. To this end, hADSCs from lipoaspirate were phenotypically characterized and shown to maintain mesodermal multipotency so that selected media easily differentiated them into osteoblasts, chondrocytes, and adipocytes. To begin inducing hADSC neuronal differentiation, we isolated CPECs from rat brain and expanded them in culture to obtain CPEC-CM. We then treated hADSCs with optimized quantities of collected CPEC-CM, KS, or both. The ADSCs treated with either CPEC-CM or CPEC-CM and KS displayed morphological changes typical of neuron-like phenotypes. As revealed by reverse transcription polymerase chain reaction (RT-PCR), quantitative real-time PCR (qPCR), and immunostaining analyses, hADSCs cotreated with CPEC-CM and KS expressed significantly higher levels of neuronal and DAergic markers in comparison with single-treated groups. Moreover, the hADSCs began expressing dopamine-biosynthesizing enzymes mainly after cotreatment with CPEC-CM and KS. Consequently, only cotreated hADSCs were capable of synthesizing and releasing dopamine detectable by high-performance liquid chromatography (HPLC). Finally, hADSCs growing in an ordinary medium were found positive for astrocytic marker glial fibrillary acidic protein (GFAP), but stopped GFAP expression on either single or cotreatments. These combined results suggest that CPEC-CM and KS can synergize to remarkably augment DAergic induction of hADSCs, an effect that has implications for cell replacement therapy for PD and related disorders.

GDNF induces RET-SRC-HER2-dependent growth in trastuzumab-sensitive but SRC-independent growth in resistant breast tumor cells.

We investigated the role of glial cell line-derived neurotrophic factor (GDNF) in compensating trastuzumab (TZMB)-induced apoptosis in HER2+ breast cancer (BC) cells using xenograft tumors. We generated BC xenografts in nude mice using samples from three patients selected based on their HER2 status and response to TZMB therapy. TZMB treatment resulted in shrinkage of the HER2+ TZMB-sensitive xenograft tumor but not the HER2- or HER2+ TZMB-resistant ones. GDNF neutralized TZMB activity and induced growth in all tumors. Three distinct cell lines were derived from these tumors and named, respectively, TZMB-sensitive (TSTC), HER2- (HNTC), and TZMB-resistant (TRTC). Over 50% of TRTC but 1% of TSTC cells expressed CD44, whereas 84% of TSTC were CD24+ compared to only 1% of TRTC, despite comparable levels of HER2 detected in both. TZMB induced profound morphological changes toward apoptosis in TSTC but not in TRTC or HNTC. However, GDNF significantly compensated TZMB-mediated TSTC cell loss and promoted growth by 37 and 50%, respectively, in TSTC and TRTC. Inhibition of SRC by Saracatinib (SARC) blocked GDNF function and accelerated TZMB-mediated cell death in TSTC, but GDNF continued promoting TRTC growth. These changes paralleled with expression levels of the key molecules involved in growth and apoptosis. Collectively, we found in our xenograft samples that firstly SRC mediates GDNF pro-survival functions by bridging RET-HER2 crosstalk in TZMB-responsive BC tumors. Secondly, SARC-TZMB interactions can synergistically eradicate such tumor cells; and thirdly, GDNF can support antibody resistance by acting independent from SRC in tumors with poor HER2 response to TZMB therapy.

Dopaminergic Induction of Umbilical Cord Mesenchymal Stem Cells by Conditioned Medium of Choroid Plexus Epithelial Cells Reduces Apomorphine-Induced Rotation in Parkinsonian Rats.

BACKGROUND/OBJECTIVE: Degeneration of dopaminergic neurons in Parkinson's disease (PD) implies cell replacement using potentially differentiable sources as a promising therapeutic solution. We tested the capacity of conditioned medium from choroid plexus epithelial cells (CPECs-CM) to induce the dopaminergic potential of umbilical cord matrix mesenchymal stem cells (UCMSCs). METHODS: We isolated UCMSCs from human umbilical cord and CPECs from rat brain. Following expansion and characterization, CPECs-CM were collected, tested for expression of various growth factors, and applied to UCMSCs. Differentiation was examined and UCMSCs were injected into 6-OHDA-leasioned striatum to test their survival and function. RESULTS: RT-PCR and immuno-staining demonstrated neuronal/dopaminergic signaling in UCMSCs induced by CPECs-CM and accelerated by addition of retinoic acid (RA) and fibroblast growth factor-2. Expression of ß-tubulin-3, Nestin and MAP2 confirmed neuronal differentiation whereas tyrosine hydroxylase, aromatic acid decarboxylase and dopamine transporter were expressed as signs of dopaminergic differentiation. Post-transplantation, the UCMSCs survived, showed reduced rate of apoptosis and led to animals' recovery from apomorphine-induced rotations. CONCLUSION: The combination of neurotrophic factors present in CPECs-CM and RA can synergize to maximize dopaminergic differentiation of potential cell sources including UCMSCs. Our study may have implications for PD cell replacement therapy.

SLUG and SOX9 Cooperatively Regulate Tumor Initiating Niche Factors in Breast Cancer.

Presence of tumor initiating cells and a proper niche is essential for metastatic colonization. SLUG and SOX9 transcription factors play essential roles in induction and maintenance of tumor initiating capacity in breast cancer cells. On the other hand, Tenascin-C and Periostin are crucial factors in metastatic niche that support tumor initiating capability in breast cancer. In this study, regulatory effect of SLUG and SOX9 transcription factors on the expression of Tenascin-C and Periostin was examined. SLUG and SOX9 were overexpressed and knocked-down in MCF7 and MDA-MB-231 cells, respectively. The cells as little and highly invasive breast cancer-derived cells were infected by inducing and shRNA lentivirus constructs. Then, Tenascin-C and Periostin as well as SLUG and SOX9 expression levels were measured in the cells via Real-Time PCR. Simultaneous overexpression of SLUG and SOX9 significantly induced Tenascin-C and Periostin expression. SLUG and SOX9 knock-down also significantly reduced the expression of Tenascin-C and Periostin. In this analysis Periostin showed the most deviation in both up- and down-regulation levels. This regulatory effect might shed light to a crosstalk between factors involved in the tumor initiating capacity and metastatic niche of the breast cancer.

The effect of 8-methoxypsoralen on pituitary-gonad axis and ovarian function in mice.

OBJECTIVE: 8-Methoxypsoralen (8-MOP) is a photoactive compound widely used in the treatment of proliferate disorders. The present study investigates the effects of 8-MOP on ovary function and pituitary-gonad axis in mice. MATERIALS AND METHODS: : In this experimental analytical study, 45 female Balb/C mice were divided into three groups (n=15), control, sham (olive oil injection) and experimental. The experimental group were received an intraperitoneal (i.p.) injection of the LD50 dose of 60 mg/kg 8-MOP. At 30 days after injection, the animals were sacrificed while in the proestrus stage and examined for morphological and histological changes their ovaries. Blood samples were collected and estrogen, luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels were assessed by radioimmunoassay. Data were analyzed using one-way ANOVA and the t test. RESULTS: The mean levels of estrogen and progesterone in the experimental group significantly decreased (p< 0.001). However, there was a significant increase in LH and FSH levels in this group compared to the control groups (p< 0.001). The mean number and diameter of the corpus luteum (CL) and the number of growing follicles in the experimental group significantly reduced compared to the control and sham groups (p< 0.001). The mean granulosa thickness in the experimental group also significantly decreased compared to the control and sham groups (p< 0.001). CONCLUSION: Our data indicated that 8-MOP can affect the levels of LH, FSH, estrogen and progesterone. Our findings further suggest that consecutive doses of 8-MOP may impair the female reproductive tract (or development).

Evidence for crossing the blood barrier of adult rat brain by human adipose-derived mesenchymal stromal cells during a 6-month period of post-transplantation.

BACKGROUND AIMS: Therapeutic promises of adult stem cells have been overshadowed by an elicited immune response, low maintenance of implanted cells or concerns regarding their migration to non-target sites. These problems might be lessened by the use of immune privilege cells and tissues for implantation. METHODS: In this study, human adipose-derived mesenchymal stromal cells (hADMSCs) were stably transfected with a vector containing Turbo green fluorescent protein (GFP) and JRed, which allows tracing the cells after transplantation. Labeled hADMSCs were transplanted into the adult rat brain followed by assessment of their survival and migration during 6 months after transplantation. RESULTS: Results indicate that there were no postsurgical complications, and the animals thrived after transplantation. The lesions of the surgical process were remarkable at the first weeks, and a high number of transplanted cells were accumulated around them. Cell populations declined over time as they partly migrated away from the injection sites; nonetheless, they were detectable at each examination time point. Although the cells could survive and remain at the injection site for up to 6 months, some of them drifted to spleen, which is an indication of their ability to cross the blood-brain barrier. CONCLUSIONS: Despite the high survival rate of hADMSCs in the xenogenic condition, which is an ideal criterion in cell therapy, irregular migration tendency must be handled with caution.

Human adipose-derived mesenchymal stem cells can survive and integrate into the adult rat eye following xenotransplantation.

BACKGROUND: Novel threads of discovery provide the basis for optimism for the development of a stem-cell-based strategy for the treatment of retinal blindness. Accordingly, achievement to suitable cell source with potential-to-long-term survival and appropriate differentiation can be an effective step in this direction. METHODS: After derivation of human adipose-derived mesenchymal stem cells (HAD-MSCs), they were stably transfected with a vector containing Turbo-green fluorescent protein (GFP) and JRed to be able to trace them after transplantation. Labeled HAD-MSCs were transplanted into the intact adult rat eye and their survival, integration, and migration during 6 months post-transplantation were assessed. RESULTS: The transplanted cells were traceable in the rat vitreous humor (VH) up until 90 days after transplantation, with gradual reduction in numbers, their adhesion and expansion capacity after recovery. These cells were also integrated into the ocular tissues. Nonetheless, some of the implanted cells succeeded to cross the blood-retina barrier (BRB) and accumulate in the spleen with time. CONCLUSIONS: The survival of the HAD-MSCs for a period of 90 days in VH and even longer period of up to 6 months in other eye tissues makes them a promising source to be considered in regenerative medicine of eye diseases. However, the potency of crossing the BRB by the implanted cells suggests that use of HAD-MSCs must be handled with extreme caution.

Shikonin protects dopaminergic cell line PC12 against 6-hydroxydopamine-mediated neurotoxicity via both glutathione-dependent and independent pathways and by inhibiting apoptosis.

We have investigated the mechanism of shikonin function on protection of dopaminergic neurons against 6-OHDA-induced neurotoxicity. Treatment of rat pheochromocytoma cell line PC12 by serial dilutions of shikonin determined 10 µM of the compound as its optimum concentration for protection saving nearly 70 % of the cells against toxicity. Reverse transcription-PCR analysis of shikonin-treated cells showed threefold increase in mRNA levels of glutathione peroxidase-1 (GPX-1) as a representative component of the intracellular anti-oxidant defense system. To elucidate shikonin-GPX1 relationships and maximize protection, we transduced PC12 cells using recombinant lentivirus vectors that harbored GPX-1 coding sequence. This change upregulated GPX-1 expression, increased peroxidase activity and made neuronal cells resistant to 6-OHDA-mediated toxicity. More importantly, addition of shikonin to GPX1-overexpressing PC12 cells augmented GPX-1 protein content by eightfold leading to fivefold increase of enzymatic activity, 91 % cell survival against neurotoxicity and concomitant increases in intracellular glutathione (GSH) levels. Depletion of intracellular GSH rendered all cell groups highly susceptible to toxicity; however, shikonin was capable of partially saving them. Subsequently, GSH-independent superoxide dismutase mRNA was found upregulated by shikonin. As signs of apoptosis inhibition, the compound upregulated Bcl-2, downregulated Bax, and prevented cell nuclei from undergoing morphological changes typical of apoptosis. Also, a co-staining method demonstrated GPX-1 overexpression significantly increases the percent of live cells that is maximized by shikonin treatment. Our data indicate that shikonin as an antioxidant compound protects dopaminergic neurons against 6-OHDA toxicity and enhances their survival via both glutathione-dependent and direct anti-apoptotic pathways.

Crosstalk between breast cancer stem cells and metastatic niche: emerging molecular metastasis pathway?

Metastatic colonization represents the final step of metastasis, and is the major cause of cancer mortality. Metastasis as an "inefficient" process requires the right population of tumor cells in a suitable microenvironment to form secondary tumors. Cancer stem cells are the only capable population of tumor cells to progress to overt metastasis. On the other hand, the occurrence of appropriate microenvironmental conditions within the target tissue would be critical for metastasis formation. Metastatic niche seems to be the specialized microenvironment to support tumor initiating cells at the distant organ. Master regulators not only determine cancer stem cell state, but also may have regulatory roles in metastatic niche elements. Meanwhile, both cancer stem cell and metastatic niche may function like two sides of the metastatic coin. Hypoxia inducible factors have multiple roles in regulation of both sides of this coin. TGF-ß superfamily, also, have been considered as master regulators of epithelial to mesenchymal transition and metastasis and may play crucial roles in regulation of metastatic niche as well. In this regard, we hypothesize the presence of a possible emerging molecular pathway in the biological process of breast cancer metastasis. In this process, non-Smad TGF-ß-induced metastasis connects cancer stem cell and metastatic niche formation through a central path, "Metastasis Pathway".

Upregulation of glutathione peroxidase-1 expression and activity by glial cell line-derived neurotrophic factor promotes high-level protection of PC12 cells against 6-hydroxydopamine and hydrogen peroxide toxicities.

We examined the impact of strong co-presence and function of glutathione peroxidase-1 (GPX-1) and glial cell line-derived neurotrophic factor (GDNF) on protecting the rat dopaminergic pheochromocytoma cell line PC12 against 6-hydroxydopamine (6-OHDA) and hydrogen peroxide (H2O2) toxicities. Primarily, GPX-1 over-expression by PC12 cells infected with pLV-GPX1 lentivirus vectors significantly increased cell survival against 6-OHDA toxicity (p<0.01). Addition of conditioned medium collected from growing wild-type astrocytes (Control astro-CM) increased survival rate of pLV-GPX1 infectants by 10% compared to their un-treated counterparts (p<0.05) and 20% compared to their treated empty vector control (p<0.01). Treatment of pLV-GPX1 cells with astro-CM of GDNF-over-secreting astrocytes (Test astro-CM) significantly induced GPX-1 expression, peroxidase enzymatic activity, and intra-cellular glutathione (GSH) levels. These changes paralleled with protection of 90% of GDNF⁺/GPX1⁺ PC12 cells against toxicity, a rate that was 37% up from their un-infected un-treated (GDNF⁻/GPX1⁻) controls (p<0.001), and 12% up from pLV-GPX1 cells that received only Control astro-CM (GPX⁺/GDNF⁻) (p<0.01). GPX-1 over-expression per se suppressed intra-cellular H2O2 elevation upon 6-OHDA exposure, and addition of GDNF medium significantly accelerated this suppression (p<0.01). Substitution of 6-OHDA with H2O2 induced similar intra-cellular changes and comparable protection levels. In all cell groups, increased cell survival against either compound was further confirmed by increased live cell counts measured by double staining. Following depletion of intra-cellular GSH, only 46% of pLV-GPX1 cells survived 6-OHDA toxicity, whereas over 70% of them were saved upon GDNF treatment (p<0.001). Moreover, capase-3 activation was reduced in pLV-GPX1 cells and maximized by addition of GDNF. Comparison analyses established correlations between GPX-1-GDNF co-presence and both enhanced cell protection and diminished levels of activated caspase-3. Our data collectively indicate that GDNF is capable of inducing anti-oxidant activities of intra-cellular GPX-1 and that growth-promoting potential of GDNF and anti-oxidant properties of GPX-1 can, in concert, maximize survival of dopaminergic neurons.

Increase of autophagy and attenuation of apoptosis by Salvigenin promote survival of SH-SY5Y cells following treatment with H2O2.

Oxidative stress is a major component of harmful cascades activated in neurodegenerative disorders. Here, we tried to elucidate the possible neuroprotective effect of Salvigenin, a natural polyphenolic compound, on oxidative stress-induced apoptosis and autophagy in human neuroblastoma SH-SY5Y cells. We measured cell viability by MTT test and found that 25 µM is the best protective concentration of Salvigenin. GSH and SOD assays suggested that Salvigenin activates antioxidant factors. At the same time, measurement of ER stress-associated proteins including calpain and caspase-12 showed the ability of Salvigenin to decrease ER stress. We found that Salvigenin could decrease the apoptotic factors. Salvigenin inhibited H(2)O(2)-induced caspase-3 which is a hallmark of apoptosis in addition to reducing Bax\Bcl-2 ratio by 1.45 fold. Additionally, Salvigenin increased the levels of autophagic factors. Our results showed an increase in LC3-II/LC3-I ratio, Atg7, and Atg12 in the presence of 25 µM of Salvigenin by about 1.28, 1.25, and 1.54 folds, respectively, compared to H(2)O(2)-treated cells. So it seems that H(2)O(2) cytotoxicity mainly results from apoptosis. Besides, Salvigenin helps cells to survive by inhibiting apoptosis and enhancing autophagy that opens a new horizon for the future experiments.

Target points in trastuzumab resistance.

Epidermal growth factor (EGF) family of receptors is involved in cell growth and differentiation. The human EGF2 (HER2) lacks natural ligands, and correlation between HER2 levels and carcinogenesis makes the receptor an ideal candidate for targeted therapy in breast cancer. Trastuzumab is a humanized antibody applied against HER2-positive breast tumors in clinic. Metastatic tumors respond well to trastuzumab therapy for the first year, but development of antibody resistance helps the tumors to regrow allowing the disease to progress. Trastuzumab resistance is shaped via a range of intracellular signaling pathways that are interconnected and share in key effector molecules. Identification of a common node central to these resistance pathways could provide an ultimate solution for trastuzumab resistance in breast and other cancers.

Optimized quantities of GDNF overexpressed by engineered astrocytes are critical for protection of neuroblastoma cells against 6-OHDA toxicity.

Optimized levels of glial cell line-derived neurotrophic factor (GDNF) are critical for protection of dopaminergic neurons against parkinsonian cell death. Recombinant lentiviruses harboring GDNF coding sequence were constructed and used to infect astrocytoma cell line 1321N1. The infected astrocytes overexpressed GDNF mRNA and secreted an average of 2.2 ng/mL recombinant protein as tested in both 2 and 16 weeks post-infection. Serial dilutions of GDNF-enriched conditioned medium from infected astrocytes added to growing neuroblastoma cell line SK-N-MC resulted in commensurate resistance against 6-OHDA toxicity. SK-N-MC cell survival rate rose from 51% in control group to 84% in the cells grown with astro-CM containing 453 pg secreted GDNF, an increase that was highly significant (P < 0.0001). However, larger volumes of the GDNF-enriched conditioned medium failed to improve cell survival and addition of volumes that contained 1,600 pg or more GDNF further reduced survival rate to below 70%. Changes in cell survival paralleled to changes in the percent of apoptotic cell morphologies. These data demonstrate the feasibility of using astrocytes as minipumps to stably oversecrete neurotrophic factors and further indicate that GDNF can be applied to neuroprotection studies in PD pending the optimization of its concentrations.

The effect of Carbaryl on the pituitary-gonad axis in male rats.

BACKGROUND: Carbaryl is a carbamate insecticide widely used to control pests in agriculture and farm. Carbaryl adversely affect the reproductive endocrine systems in animals. OBJECTIVE: The aim of this study was to evaluate Carbaryl effects on the pituitary-gonad axis in rats. MATERIALS AND METHODS: In this experimental - analytical study, 60 adult male rats were divided into four equal groups: control, sham and experimental (1 and 2) groups that received 10 and 30 mg/kg Carbaryl via intraperitoneally injection. The sham group was subjected to intraperitoneally injection with olive oil while the control group did not receive any injection. Animals were sacrificed 35 days after the last treatment. Tissue sections were prepared from testes to investigate possible changes occurring in spermatogenic and Leydig cells. Blood samples were collected in which the levels of testosterone, luteinizing hormones (LH) and follicle stimulating hormone (FSH) were measured. RESULTS: The results showed significant reduction in testes weight (p=0.042) and seminiferous diameters (p<0.001) within the experimental groups compared with control group. Also, the number of germ cells, spermatocyts, spermatids and Leydig cells on the testes of the experimental groups was significantly decreased (p<0.001). Accordingly, significant decline in the testosterone levels (p<0.001) and increase in LH and FSH levels were observed (p<0.05). CONCLUSION: These results demonstrated that Carbaryl has capacity to exert adverse effects on fertility. Therefore, have to be taken to account in applying Carbaryl for any studies and or commercial use.

Synergy between glutathione peroxidase-1 and astrocytic growth factors suppresses free radical generation and protects dopaminergic neurons against 6-hydroxydopamine.

The degeneration of dopaminergic neurons in the course of Parkinson disease is largely blamed on oxidative damage in the brain. This study examined the potency of glutathione peroxidase-1 (GPX-1) to protect dopaminergic neurons against toxicity induced by the parkinsonian neurotoxin 6-hydroxydopamine (6-OHDA). We generated pLV-GPX1, a recombinant lentivirus vector carrying the coding sequence for human GPX-1, into the SK-N-MC neuroblastoma cell line. The pLV-GPX1-infected neurons showed an over 3-fold increase in enzyme expression and a 2.6-fold increase in enzyme activity compared to the pLV-EGFP-infected control cells. In the pLV-GPX1-infected cells, we also detected significantly increased neuronal survival and resistance to 6-OHDA-mediated toxicity compared to our controls (75 ± 4% versus 51 ± 7%, p < 0.001). To maximize this protection, the neurons were treated with conditioned medium taken from growing primary astrocytes (astro-CM). We found the treated pLV-GPX1-infected neurons even more significantly resistant to 6-OHDA toxicity compared to their untreated counterparts (86 ± 5% versus 75 ± 4%, p < 0.001). Concomitant with increased neuroprotection, co-presence of overexpressed GPX-1 and astro-CM significantly increased glutathione (GSH) levels compared to when either of the two was present (p < 0.001). Further analysis showed nearly 2.7-fold reduction, in the presence of astro-CM, of hydrogen peroxide (H(2)O(2)) levels released from the pLV-GPX1-infected neurons compared to control groups (p < 0.001). Finally, regression analysis between H(2)O(2) levels and cell viability showed that co-presence of GPX-1 and astro-CM reduced 33% of cell death rate (p < 0.05). These data highlight the antioxidant properties of GPX-1 in protecting dopaminergic neurons and further emphasize the capacity of astrocytes in pumping growth-inducing factors that may synergize with GPX-1 to accelerate neuroprotection.

Towards ß-globin gene-targeting with integrase-defective lentiviral vectors.

We have developed an integrase-defective lentiviral (LV) vector in combination with a gene-targeting approach for gene therapy of ß-thalassemia. The ß-globin gene-targeting construct has two homologous stems including sequence upstream and downstream of the ß-globin gene, a ß-globin gene positioned between hygromycin and neomycin resistant genes and a herpes simplex virus type 1 thymidine kinase (HSVtk) suicide gene. Utilization of integrase-defective LV as a vector for the ß-globin gene increased the number of selected clones relative to non-viral methods. This method represents an important step toward the ultimate goal of a clinical gene therapy for ß-thalassemia.