Browsing by Author "Singh, Meharvan PhD"
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Item Activation of a putative membrane androgen receptor increases the efficacy of the chemotherapeutic agent, temozolomide, in a human glioblastoma cell line(2016-03-23) Badeaux-McGilvray, Adrienne; Singh, Meharvan PhD; Brock, CourtneyGlioblastoma Multiforme (GBM) is a form of brain cancer with very poor prognosis such that the life expectancy of a person with this disease is about one year after diagnosis. Moreover, current treatment regimens are only able to extend the life span by mere months. Based on recent studies from our lab that identified a putative membrane androgen receptor (mAR), which when activated is capable of promoting cell death, we investigated whether exploitation of this receptor could increase the efficacy of current chemotherapeutic agents to combat this deadly and invariably lethal cancer. Using the human glioblastoma cell lines, A172 and T98G, our studies have shown that activation of the mAR (using testosterone or dihydrotestosterone conjugated to bovine serum albumin) not only sensitized the glioblastoma cells to temozolomide (TMZ), the current standard chemotherapeutic agent for GBM, but also suppressed the phosphorylation of Akt, a known survival-promoting factor. Further, in T98G cells that express high levels of O6-methylguanine DNA methyltransferase (MGMT), a DNA repair protein, activation of the mAR suppressed the expression of MGMT. Our data also suggest that these mechanisms may not be mutually exclusive such that inhibition of Akt phosphorylation in and of itself led to a reduction in MGMT expression. Collectively, our data support the targeting of a putative membrane androgen receptor as complementary treatment for glioblastoma.Item Androgen Receptor-Independent Mechanisms for Dihydrotestosterone (DHT)-induced Protection in the C6 Glioma Model of Astrocytes(2016-03-23) Rybalchenko, Nataliya; Singh, Meharvan PhD; Kubelka, NicholasTestosterone and dihydrotestosterone (DHT) exert protection through the activation of the intracellular androgen receptor (AR). However, studies suggest DHT may also exert protective effects by way of alternate mechanisms, including through prior conversion to 3beta-diol, a metabolite that can bind and activate estrogen receptors. Using the AR-deficient C6 glioma, a model of astrocytes, we found DHT was protective against iodoacetic acid (IAA) toxicity. The protective effects of DHT, as assessed by the Calcein-AM viability assay (which is a surrogate measure of cell number), were blocked by the co-application of the non-selective estrogen receptor antagonist, ICI-182,780. Using a complementary viability assay, the MTT assay, which is a surrogate for mitochondrial respiration/activity, we reproduced DHT protection and extended our results to find that 3beta-diol was also protective against IAA-induced reduction in mitochondrial activity. Interestingly, while the effects of 3beta-diol, the presumptive mediator of the effects of DHT, were blocked by ICI 182,780, they were not blocked by the estrogen receptor isoform-selective antagonists MPP (against ERa) and PHTPP (against ERb). Collectively, these data support our hypothesis that DHT is protective against cytotoxicity in a cell line devoid of the classical/intracellular androgen receptor, and that the metabolite of DHT, 3beta-diol, may be an important mediator of DHT’s effects in the central nervous system. Our results also suggest that the capacity to convert DHT to 3beta-diol may be relevant to the protective influence of androgens and estrogens in the postmenopausal women, a time when estrogen and progesterone levels decline significantly, but androgen levels persist.Item Connexin 43 as a Mediator of Estrogen-induced Protection against Oxidative Stress(2017-03-14) Rybalchenko, Nataliya; Singh, Meharvan PhD; Kubelka, NicholasHypothesis: 17-β estradiol (E2) and the estrogenic metabolite of dihydrotestosterone (DHT), 5-α-androstane-3,17-β-diol (3βdiol), protect against oxidative stress by increasing the expression and function of Connexin 43 (Cx43) – containing gap junctions in cortical astrocytes. Methods: In order to assess the expression of Cx43 and cell viability, real time RTPCR and the MTT assay was used, respectively. Cerebral cortical astrocytes derived from postnatal day 2 female C57/Bl6 mice were treated with physiologically relevant concentrations of E2, DHT, 3βdiol, or the vehicle control, DMSO, and evaluated for Cx43 mRNA expression. For the cell viability assays, astrocytes were pre-treated with either E2, DHT or DMSO vehicle control and then exposed to iodoacetic acid (IAA) oxidative insult. To further determine the role of Cx43 gap junctions, either Gap19 (inhibitor of the Cx43 hemichannel) or Gap26 (inhibitor of the dimeric Cx43 containing gap junction) were co-applied with the insult. Results: E2 treatment (3 hr) significantly increased Cx43 mRNA expression relative to DMSO control, while both DHT and 3βdiol (also applied for 3 hr) did not. Longer treatment with E2 (18 hr) yielded a non-significant trend to increase Cx43 mRNA expression. In the viability assays, neither E2 or DHT alone (18 hours pre-treatment) nor the Cx43 hemichannel selective inhibitor peptide (Gap19) alone protected against IAA toxicity. However, Gap19 did increase the protective efficacy of not only E2, but DHT as well. Interestingly, inhibition of both Cx43 hemichannels and the dimeric Cx43-containing gap junctions using Gap26, in and of itself, conferred protection against IAA toxicity. Like Gap19, Gap26 also significantly enhanced the protective efficacy of E2. Conclusions: Both E2 and DHT showed some regulatory interaction with astrocyte Cx43 that impacted protection against oxidative stress. This is the first evidence that E2 or DHT regulate Cx43 in the brain and the mechanisms underlying these interactions remain to be further characterized.Item The role of miRNA in regulating Progesterone's neuroprotective function in the ischemic brain(2016-03-23) Singh, Meharvan PhD; Su, Chang Phd, MD; Nguyen, TrinhAbstract: Stroke has been reported as the fourth leading cause of death for Americans and it is a leading cause of adult disability. The risk of ischemic stroke increases significantly with aging. Gender appears to play a profound role, with the incidence being higher in women. A large body of studies has suggested that women in postmenopausal state are at greater risk of ischemic stroke and are likely to experience much more severe impacts. A considerable amount of research has supported that progesterone (P4) is a potent neuroprotectant that may exert beneficial effects in various neurodegenerative diseases and stroke. Our laboratory has reported that Brain-derived neurotrophic factor (BDNF) is a critical mediator for P4 neuroprotective actions. BDNF has well-defined roles in synaptogenesis and neuronal survival. We recently reported that P4 enhances BDNF release from glia, but not from neurons, by acting via a novel membrane-associated progesterone receptor, Pgrmc1. Here, we identified a member of the Let-7 microRNA (miRNA) family as a potential negative regulator of Pgrmc1. Our data demonstrated an inverse association between the expression levels of Let-7 and the transcripts of Pgrmc1 and BDNF in post-ischemic mouse cortex. Literature supports the antagomir (synthetic inhibitor) of Let-7 miRNA significantly reduced infarct volume and improved neurological deficits in a rodent ischemic stroke model. When combined, these lines of evidence have strongly supported our hypothesis that in the stroked brain, Let-7 negatively regulates Pgrmc1 gene expression, which disrupts P4-induced BDNF release from glia and ultimately leads to the attenuation of P4’s positive effect on synaptogenesis. Methods: 1. Cell culture: Mouse primary cortical neurons and primary cortical astrocytes were derived from postnatal day 1 male pups. Primary astrocytes were maintained in DMEM with sodium pyruvate/10% FBS + 1% penicillin/streptomycin. Primary neurons were maintained in Neurobasal A media with 10% FBS + 1% penicillin/streptomycin. 2.Treatment of primary astrocytes: Primary astrocytes were transfected with either Let-7 mimics or inhibitors. qPCR was used to confirm the overexpression and knock down of Let-7, as well as expression of their potential targets. Western blotting was used the determine the protein levels of the miRNA potential targets. 3.Treatment of neuronal cultures with conditioned media (CM) from glia: Primary astrocytes were transfected with miRNA mimics/inhibitors. Then cultures were treated with vehicle control (0.1% DMSO ) or 10 nM P4 for 18 hrs. The CM was concentrated by Protein Concentrators with 9 KDa molecular-weight cutoff (Pierce) to remove P4. Concentrated CM was then applied to neuronal cultures. 4. Animal treatment: Female C57BL/6J mice were bilaterally ovariectomized at 4.5 months old to eliminate endogenous ovarian production of P4. One week afterward, P4 pellets or cholesterol pellets were implanted in these animal subcutaneously. 2 days after pellets implantation, ischemic stroke was induced in these mice using the middle cerebral arteries occlusion method. 24hrs after stroke induction, the antagomir of let-7or scrambled control were delivered to the penumbra via intracerebral ventricular (ICV) injection.