Browsing by Subject "apoptosis"

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Alterations in mRNA Levels of Selected Gene Products During Hypoglycemia, Hypoxia, and Ischemia Induced Apoptosis of Cultured Rat Retinal Ganglion Cells
(2001-08-01) Vopat, Kelly S.; Agarwal, Neeraj; Wordinger, Robert J.; Pang, Iok-Hou
Vopat, K., Alterations in mRNA Levels of Selected Gene Products during Hypoglycemia, Hypoxia, and Ischemia Induced Apoptosis of Cultured Rat Retinal Ganglion Cells. Master of Science (Biomedical Science), August 2001. 54 pp., 2 tables, 10 illustrations, bibliography, 105 titles. In order to explore the mechanisms involved in the signal transduction pathways of ischemia-induced apoptosis of RGCs in glaucoma, an in vitro ischmia model of transformed rat retinal ganglion cells (RGC-5) was utilized. RGC-5 cells were exposed to hypoglycemia, hypoxia, and ischemia for six hours. Hypoxia and ischemia resulted in apoptosis of RGC-5 cells as determined by TUNEL assay. The bax mRNA levels increased significantly in cells exposed to hypoxia. The mRNA levels of hemoxygenase, c-fos HSP 70, and BDNF showed a trend of increase in both the hypoxic and ischemic conditions. These results demonstrate that retinal ganglion cells undergo apoptosis in hypoxic conditions likely via an increase in bax/bcl-2. The up-regulation of BDNF and some stress proteins may be part of a cellular rescue effort trying to overcome the damage created by hypoxic and ischemic stresses.
Artemisinin Prevents Glutamate-Induced Neuronal Cell Death Via Akt Pathway Activation
(Frontiers Media S.A., 2018-04-20) Lin, Shao-Peng; Li, Wenjun; Winters, Ali; Liu, Ran; Yang, Shaohua
Artemisinin is an anti-malarial drug that has been in use for almost half century. Recently, novel biological effects of artemisinin on cancer, inflammation-related disorders and cardiovascular disease were reported. However, neuroprotective actions of artemisinin against glutamate-induced oxidative stress have not been investigated. In the current study, we determined the effect of artemisinin against oxidative insult in HT-22 mouse hippocampal cell line. We found that pretreatment of artemisinin declined reactive oxygen species (ROS) production, attenuated the collapse of mitochondrial membrane potential induced by glutamate and rescued HT-22 cells from glutamate-induced cell death. Furthermore, our study demonstrated that artemisinin activated Akt/Bcl-2 signaling and that neuroprotective effect of artemisinin was blocked by Akt-specific inhibitor, MK2206. Taken together, our study indicated that artemisinin prevented neuronal HT-22 cell from glutamate-induced oxidative injury by activation of Akt signaling pathway.
ENDOTHELIN B (ETB) RECEPTORS CONTRIBUTE TO NEURODEGENERATION IN A RODENT MODEL OF GLAUCOMA VIA UPREGULATION OF C-JUN AND BAX
(2014-03) Minton, Alena Z.; He, Shaoqing; Ma, Hai-Ying; Krishnamoorthy, Raghu R.
Glaucoma is a group of eye conditions that, if left untreated, can result in blindness. It is commonly associated with an increased pressure inside the eye, known as intraocular pressure or simply IOP. As the pressure builds up inside the eye, it causes damage to the optic nerve, which in turn results in the death of retinal ganglion cells (RGCs). Studies from our lab and others have shown that endothelin 1 (ET-1), the potent vasoactive peptide, contributes to glaucoma. Currently, our lab is interested in understanding the role of the endothelin receptor B (ETB) in glaucoma. We are using rats that do not have ETB receptor (ETB KO rats) and those that have the receptor (WT rats). To mimic glaucoma, the high salt solution is injected into the special vein in the eye (episcleral vein). This causes the build up of pressure inside the eye within 7 to 10 days. This model of glaucoma is called the Morrison’s ocular hypertension model. Previously, we found that IOP elevation for 4 weeks in WT rats caused an appreciable loss of RGCs, which was significantly attenuated in ETB KO rats. In addition, pathological changes in the optic nerve were greatly reduced in ETB KO rats, as compared to those in WT rats. To find out the molecular mechanisms responsible for the death of RGCs, we elevated the pressure inside one eye of adult WT and ETB KO rats, while the contralateral eye served as control. After 2 weeks of IOP elevation, retinal sections were obtained and stained with specific antibodies to detect the levels of c-Jun (the member of the activator protein-1 (AP-1) family) and Bax (protein involved in cell death). We found that WT rats have higher levels of c-Jun and Bax in the retina (especially in the ganglion cell layer), as compared to ETB KO rats. Interestingly, using the Promo 3 software, we found 15 binding sites for members of the AP-1 family of proteins on the rat 1.95 kb upstream promoter region of Bax. Therefore, the transcription factor c-Jun may be an upstream regulator of Bax. In conclusion, transcription factor AP-1 could be involved in the elevation of the ETB receptor levels in the Morrison's model of glaucoma. Conversely, deletion of the ETB receptor results in the lower expression of c-Jun. Taken together, there may be a reciprocal relationship between the AP-1 and ETB receptors. Purpose (a): Previously, our lab has demonstrated that increased levels of ETB receptors contribute to the death of retinal ganglion cells (RGCs) and degeneration of optic nerve axons in the Morrison's elevated intraocular pressure (IOP) model of glaucoma in rats. Moreover, these pathological changes were greatly attenuated in ETB receptor-deficient transgenic Wistar Kyoto rats. Interestingly, an increase in ETB receptor levels in RGCs, following 2 weeks of IOP elevation in Brown Norway rats, was shown to be associated with increased expression of c-Jun, a member of the activator protein-1 (AP-1) family. The current study was aimed at investigating whether the increased expression of c-Jun observed in wild type rats is reduced in ETBreceptor-deficient Wistar Kyoto rats subjected to the Morrison’s model of glaucoma. The status of another apoptotic protein, Bax, was also assessed in these rats. Methods (b): IOP was elevated in one eye of adult wild type and ETB receptor-deficient transgenic Wistar Kyoto rats using the Morrison’s method (injection of hypertonic saline through episcleral veins), while the contralateral eye served as control. After IOP was elevated, rats were maintained for 2 weeks and sacrificed. Retinal sections were obtained and stained with specific antibodies to detect the expression of c-Jun and Bax by immunohistochemistry. In addition, retinal sections were immunostained using an antibody to βIII-tubulin, which is selectively expressed by RGCs in the retina. Images were taken using Zeiss LSM-510 confocal microscope with Z-scan. Results (c): Immunohistochemical analysis showed that IOP elevation for 2 weeks caused increased expression of c-Jun and Bax mainly in the ganglion cell layer (GCL) of wild type transgenic Wistar Kyoto rats as compared to ETB receptor-deficient transgenic Wistar Kyoto rats. Interestingly, using the Promo 3 software, we found 15 binding sites for members of the AP-1 family of proteins on the rat 1.95 kb upstream promoter region of Bax. Therefore, the transcription factor c-Jun may be an upstream regulator of Bax (pro-apoptotic factor). Conclusions (d): Transcription factor AP-1 could be involved in the elevation of the ETB receptor levels in the Morrison's model of glaucoma. Conversely, deletion of the ETB receptor results in the downregulation of c-Jun. Taken together, there may be a reciprocal feedback loop between the AP-1 and ETB receptors.
Estrogen Signaling Protects Mitochondrial Membrane Potential Integrity from Oxidative Stress in Lens Epithelial Cells
(2008-05-01) Flynn, James Martin; Cammarata, Patrick R.; Wordinger, Robert J.; Dimitrijevich, S. Dan
Flynn, James Martin, Estrogen Signaling Protects Mitochondrial Membrane Potential Integrity from Oxidative Stress in Lens Epithelial Cells. Doctor of Philosophy, (Cell Biology and Genetics) May, 2008, 265 pages, 36 figures, bibliography, 190 titles. Loss of mitochondrial membrane potential has been determined to be one of the initiating factors in activation of apoptosis after cellular damage. Estrogen and estrogen analogues have been shown to enhance cell survival in numerous tissues through rapid pro-survival cell signaling. This study was focused on elucidating mechanisms through which estrogen protects the cells by preventing the activation of mitochondrial permeability transition pores and the subsequent loss of mitochondrial membrane potential. It is hypothesized that the anti-apoptotic mitochondrial protein BAD, once phosphorylated by estrogen activated upstream kinases, can prevent the formation of the permeability transition pre via direct interaction. To address this, lens epithelial cells were used as a model system for the examination of mitochondrial depolarization during periods of either oxidative or hyperglycemic stress. Estrogen attenuated the loss of impermeability of the mitochondrial membrane, thus maintaining the cells during acute periods of stress. It was discovered that a number of the estrogen receptor isoforms are expressed in lens epithelium, and that the wild-type estrogen receptor-β1 isoform is localized to the mitochondria in lens epithelial cultures derived from both human males and females. siRNA treatment against estrogen receptor-β determined that is a required component to elicit estrogen’s protective abilities against oxidative stress induced mitochondrial depolarization. Furthermore, administration of exogenous estrogen rapidly activated signaling pathways, particularly ERK, which were shown to have influence over the loss of mitochondrial membrane potential. Studies using both pharmacological inhibitors of MAPK signaling, as well as siRNA of ERK2 kinase demonstrate a correlation between the activation of ERK and the severity of response to oxidative stress. Investigation of downstream substrates of ERK revealed that the mitochondrial protein BAD is phosphorylated after the administration of estrogen, yet it is not required for the prevention of mitochondrial depolarization as originally hypothesized. In conclusion, these studies have confirmed a mitochondrial targeted mechanism activated by estrogen which is rapid, gender independent, estrogen receptor-β mediated signal transduction pathway. The targeting of mitochondrial function to reduce oxidative or hyperglycemic stress, thereby preventing activation of the permeability transition pore, defines a novel concept which will contribute to innovative regimens for prevention or treatment of mitochondrial pathology.
FKBP51 and Methylene Blue as Neuroprotective Targets
(2011-05-01) Daudt, Donald R.; Thomas Yorio
Purpose: Neurodegenerative diseases and neurotraumas typically result in apoptosis of specific neurons leading to the pathology observed during the disease state. Existing treatments target the symptoms instead of preventing the death of these neurons. Although neuroprotective drugs should be useful as a treatment to prevent further loss of neurons, efficacious molecules are lacking. FK506 (tacrolimus), a widely used immunosuppressant drug, has significant neuroprotective and neuroregenerative properties throughout the central nervous system, including the eye. FK506 achieves these properties through interaction with FK506 binding proteins (FKBP), including FK506 binding protein 51 (FKBP51). In this study, we examine the effects of FKBP51 as a neuroprotective agent on a neuronal cell line. Methods: We cultured 661w cell cultures with or without FK506, or stably transfected them with an FKBP51 expression vector. These cells were then exposed to the apoptosis inducing agent staurosporine. Cell viability was determined using a calcein AM/propidium iodide assay. Protein levels and activation of nuclear factor kappa-light chain-enhancer of activated B cells (NF-κB) were determined by western immunoblot analysis. Results: FKBP51 overexpression significantly protected 661w cell cultures from staurosporineinduced apoptosis. FKBP51 overexpression also significantly increased NF-κB p65 protein 35 levels and activated NF-κB p65. FK506 treatment significantly protected 661w neuronal cultures from staurosporine-induced apoptosis. FK506 increased FKBP51, NF-κB p65, and levels of activated NF-κB p65 protein. Conclusions: These results suggest that FKBP51 protects 661w cell cultures from apoptosis induced by staurosporine. Additionally, FK506 protected 661w cell cultures from apoptosis and displayed a mechanism similar to that of FKBP51 overexpression. Both FK506 and FKBP51 appear to act through activation of NF-κB p65 protein, suggesting a common pathway for neuroprotection. These findings suggest that FKBP51 is a compound important to neuronal cell culture survival. FKBP51 may be a potential therapeutic drug target for preventing the neurodegeneration and neurotrauma that occur during neurodegenerative diseases.
Hypertrophic Versus Apoptotic Response of Vascular Smooth Muscle to β1 Adrenergic Receptor Stimulation
(2004-05-01) Hannon, Sherry Beth; Stephen Grant; Glenn Dillon; Robert Mallet
Hannon, Sherry Beth., Hypertrophic vs. Apoptotic Response of Vascular Smooth Muscle to β1 Adrenergic Receptor Stimulation. Master of Science (Biotechnology), May, 2004, 64 pp., 3 tables, 16 illustrations, references, 41 titles. This project explores how β1 adrenoceptor (β1-AR) stimulation affects cellular hypertrophy and apoptosis in PAC-1, a cultured rat pulmonary artery cell line. Insights into these responses may further the current understanding of vascular remodeling. Promoter-reporter activity for the hypertrophy-specific gene smooth muscle myosin heavy chain decreased as measured by a luciferase assay when PAC-1 cells were treated with the selective β1-AR agonist dobutamine (DOB) in 0.4% fetal bovine serum (FBS) supplemented media. However, activity of a β1-gal control vector also decreased, and neither response was attenuated by pre-treatment with a β1-AR selective antagonist metoprolol. A MTS [3-(4,5-dimethlythiazol-2-yl)-5-(3-carboxymethoxyphenly)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] viability assay shows that while there is a loss of cells with decreasing amounts of serum, this effect is not exacerbated by DOB in 0.4% FBS. DNA fragmentation assays were inconclusive as to the mode of cell death occurring. However, an increase in Bax/Bcl-2 ratio suggest that apoptosis is induced with DOB treatment in 10% FBS, but that this DOB treatment in 0.4% and serum-free media does not increase this apoptotic index compared to control. Both similar and conflicting cellular responses have been documented in rat neonate cardiomyocytes as well as in murine transgenic models selectively over-expressing adrenergic receptors in the heart. Comparison of the vascular smooth muscle cell response to the cardiomyoctye response may lead to a more tailored use of adrenergic agents for treatment during different stages of cardiovascular disease.
Investigation of Proteasome Chymotryptic Activities and Effects on their Inhibition in Rat and Human Natural Killer Cells
(2003-04-01) Lu, Min; Goldfarb, Ronald H.; Borejdo, Julian; Easom, Richard
Lu, Min, Investigation of Proteasome Chymotryptic Activites and Effects of Their Inhibition in Rat and Human Natural Killer Cells. Doctor of Philosophy (Biochemistry and Molecular Biology), April, 2003, 185 pp., 3 tables, 32 illustrations, bibliography, 158 titles. The proteasome is a multicatalytic proteinase complex that is involved in the major extralysosomal pathway responsible for intracellular protein degradation in mammalian cells. This dissertation focuses on investigating proteasome chymotryptic activities and the effects of selective inhibitors of these activities on the function of natural killer (NK) cells. In this dissertation, 20S proteasomes derived from rat RNK16 cells were purified and some of their biochemical and biophysical properties were investigated extensively. The results indicated that RNK16 cell-derived proteasome differ from the proteasome of other origins in many aspects including substrate selectivity, inhibitor specificity, and kinetic regulation, although they may share some common biochemical properties with others. To investigate the effects of proteasomal inhibition on the function of NK cells, several proteasome inhibitors were used including MG115, MG132, clasto-lactacystin-β-lactone, EGCG and LLnL. MG115 and MG 132 were shown to induce apoptosis of RNK16 cells, as evidenced by DNA fragmentation, caspase-3 activation and the appearance of sub-G1 cell populations. Activation of multiple caspases and increased expression of cell surface Fas (CD95) protein were also observed following the treatment of RNK16 cells by these two inhibitors. This dissertation also tested the hypothesis that different cell types could respond differentially to proteasome inhibitors. The effects of several proteasome inhibitors were determined on the purified 20S proteasomal and 26S proteasomal chymotrypsin-like activity in whole cell extracts and intact YT and Jurkat cells, human NK and T cell lines respectively. Following such treatment, caspase-3 activation occurred much earlier in Jurkat cells than YT cells; cell cycle analysis indicated a sub-G1 apoptotic cell population in Jurkat cells and G2/M arrest in YT cells. In addition, accumulation of p27 and IκB-α was detected only in Jurkat cells, but not YT cells. Therefore, proteasome inhibitors appear to act differentially in cell cycle progression and apoptosis signaling pathways between human NK and T cells. These studies indicate that the generation of ideal proteasome inhibitors for the treatment of malignancies could be screened or designed to specifically induce cancer cells to undergo programmed cell death, while having little or no apoptosis-inducing abilities for natural killer cells and other cells of the immune response, thus enhancing the selectivity and specificity of the anti-cancer, apoptosis-inducing capabilities of proteasome inhibitors.
Involvement of Caspase-2 in Cisplatin-Induced Cell Death in 2008 Ovarian Cancer Cells
(2008-04-01) Adkins, Brett T.; Basu, Alakananda; Berg, Rance E.; Gryczynski, Zygmunt
Adkins, B., Involvement of caspase-2 in cisplatin-induced cell death in 2008 ovarian cancer cells. Master of Science (Molecular Biology and Immunology) April, 2008, 59 pp., 12 illustrations, bibliography, 73 titles. Cisplatin, one of the most effective anticancer drugs in the treatment of ovarian cancer, causes DNA damage and leads to apoptosis. Caspases, a family of cysteine proteases, are essential for the induction of apoptosis. Initiator caspases activate effector caspases to trigger apoptosis. Caspase-2 can function as both an initiator and effector caspase although there are controversies regarding its role in DNA damage-induced apoptosis. Caspase-2 is the only caspase constitutively located in the nucleus, although its function there is unknown. In the present study we have investigated if caspase-2 is important during cisplatin-induced apoptosis and whether cisplatin treatment affects the localization of caspase-2. Caspase-2 depletion suggested that caspase-2 acts upstream of caspase-2 acts upstream of caspase-9 in cisplatin-induced apoptosis. We also made a novel observation that rottlerin, an inhibitor of DNA damage-induced apoptosis, specifically downregulates caspase-2 via the ubiquitin proteamose-mediated pathway. We further show that cisplatin induces caspase-2 translocation out of the nucleus. Moreover, translocation of caspase-2 is more important for cisplatin-induced cell death.
Involvement of Caspase-7 in Photoreceptor and Retinal Ganglion Cell Death
(2014-08-01) Choudhury, Shreyasi; Pang, Iok-Hou; Wordinger, Robert J.; Krishnamoorthy, Raghu R.
Apoptosis has been implicated in retinal cell death during both retinal differentiation and degeneration. In diseases such as retinitis pigmentosa, glaucoma, age-related macular degeneration, diabetic retinopathy and traumatic optic neuropathy, retinal cell apoptosis plays an important role. Caspases, a family of cysteine proteases, are major players of apoptosis. Thus, one obvious target for modulating apoptosis is the caspase family of proteins. The role of initiator caspases (caspase-1, -2, -8, -9) and effector caspases (caspase-3, -6) in retinal neuronal apoptosis has been studied previously. But the role of a unique effector caspase, caspase-7, has never been studied before. The purpose of this study was to investigate the role of caspase-7 in retinal neuronal cell apoptosis, especially in photoreceptor and retinal ganglion cell (RGC) death. We used the T17M RHO mouse, an animal model for Autosomal Dominant Retinitis Pigmentosa, to study photoreceptor cell apoptosis, and evaluate the role of caspase-7 in a corresponding caspase-7 knockout mouse. Our results show that morphological (evaluated by spectral-domain optical coherence tomography (SD-OCT) and histology) and functional (by electroretinography (ERG)) degenerations in the photo-receptor cells of the T17M RHO mouse are significantly protected by knocking out caspase-7. We further discovered that caspase-7 inhibition reprograms the unfolded-protein response and reduces JNK-induced photoreceptor cell death. To assess the role of caspase-7 in RGC apoptosis, we used the mouse optic nerve crush-induced RGC death as a study model. We found that the insult activates caspase‐7 in RGCs in a time-dependent manner, concomitant with loss of the cells. We also observed the activation of calpain-1, an upstream activator of caspase-7 and the hydrolysis of caspase-7 specific substrates, confirming the involvement of caspase-7. Most importantly, in caspase--‐7 knockout mice, significantly more RGCs survive the optic nerve injury when compared to injured wild type mice as assessed morphologically (immunohistochemistry and SD-OCT) and functionally (ERG) throughout the 28-day post crush study period. Altogether, our findings indicate that caspase-7 appears to play a critical role in photoreceptor and RGC death and inhibition of caspase-7 activity may be a novel therapeutic strategy for retinal degenerative diseases.
Involvement of p70S6K in Cisplatin-Induced Cell Death
(2008-05-01) Dhar, Rohini; Basu, Alakananda; Singh, Meharvan; Vishwanatha, Jamboor
Rohini Dhar, Involvement of p70S6K in cisplatin-induced cell death. Doctor of Philosophy (Biochemistry and Molecular Biology) May 2008, 151 pp., 23 illustration, 3 tables, 153 references. Cisplatin is used for the treatment of solid tumors; however its success is often compromised due to relapse and chemoresistance. The purpose of this dissertation is to delineate the role of p70S6K in cisplatin-induced apoptosis. A comparison of p70S6K levels in H69 cells that acquired resistance to cisplatin (H69/CP) compared to parental H69 cells revealed that levels of phosphorylated p70S6K and S6 were elevated. Cisplatin treatment resulted in the activation of p70S6K and downregulation of total p70S6K. Inhibition of the phosphoinositide 3-kinase (P13K) pathway by itself augmented cisplatin-induced PARP cleavage and also blocked the phosphorylation of p70S6K. Inhibition of extracellular signal-regulated kinase (ERK) pathway however attenuated cisplatin-induced PARP cleavage. These results reveal that phosphorylation of p70S6K is associated with increased cisplatin resistance, and inhibition of P13K/ p70S6K pathway could reverse cisplatin resistance. We have found that cisplatin caused a time- and concentration-dependent downregulation of p70S6K. While the calpain and proteasome inhibitors had no effect on the downregulation of p70S6K, the broad specificity caspase inhibitor z-VAD-fmk (z-VAD) reversed p70S6K downregulation by cisplatin. Furthermore, the caspase-3 inhibitor and knockdown of caspase-3 prevented cisplatin-induced proteolytic cleavage of p70S6K. While, cisplatin failed to induce cleavage of p70S6K in MCF-7 cells that lack functional caspase-3, overexpression of caspase-3 in these cells resulted in cisplatin-induced cleavage of p70S6K. Thus, these results demonstrate that p70S6K is a novel substrate for caspase-3. Examination of the role of p70S6K in cisplatin-induced death shows that rapamycin in a pharmacological inhibitor of p70S6K, enhanced cisplatin-induced apoptosis in A549 cells. However, knockdown of p70S6K by siRNA resulted in a decrease in cisplatin-iduced apoptosis. In addition, caspase-3 mediated cleavage of p70S6K at the aspartic acid residue at the 393 position and site-directed mutagenesis of Asp393 to Ala resulted in protection against cisplating-mediated apoptosis. Interestingly, introduction of the N-terminal cleaved fragment [∆(394-525)] resulted in potentiation of cisplatin-induced apoptosis. These results suggest that the proteolytic cleavage of p70S6K by caspase-3 is important for cisplatin-induced apoptosis.
Molecular Mechanisms of and Potential Therapies for Oxidative Damage to the Retinal Pigment Epithelium
(2007-09-01) Wang, Zhaohui; Roque, Rouel S.; Wordinger, Robert J.; Das, Hriday
Wang, Zhaohui, Molecular Mechanisms of and Potential Therapies for Oxidative Damage to the Retinal Pigment Epithelium. Doctor of Philosophy (Biomedical Sciences), September 2007, 161 pages, 34 illustrations, bibliography, 119 titles. Age-related macular degeneration (AMD), the most common cause of irreversible vision loss in the elderly, results mainly from degeneration of the retinal pigment epithelium (RPE) and loss of photoreceptor cells. Oxidative stress has been acknowledged as a leading cause of RPE degeneration and concomitant photoreceptor cell loss, but the exact role of reactive oxygen species (ROS) in RPE cell death remains to be established. Moreover, while mitogen-activated protein kinases (MAPKs) are suggested to be involved in RPE degeneration induced by oxidative stress, the precise functions and molecular mechanisms of MAPKs in RPE degeneration remain elusive. In spite of the numerous therapeutic modalities proposed for AMD, the treatment of AMD remains unsatisfactory. Recent studies suggesting stem cells as a potential source for trophic factors in damaged murine hearts led us to investigate a possible role for stem/progenitor cell-derived factors in protecting RPE cells from oxidative damage. Furthermore, human retinal progenitor cells promote RPE cell survival by regulating p42/p44 MAPK activity. When exposed to oxidative stress produced by glucose oxidase/glucose, human RPE cells exhibited membrane blebbing and cytoskeleton remodeling in the early phase of oxidative stress. Prolonged exposure to oxidative stress induced mitochondrial membrane potential depolarization, cell death and DNA condensation, but not DNA fragmentation. Furthermore, both p38 MAPK and p42/p44 MAPK were activated by oxidative injury. P38 MAPK inhibitor, but not p38 MAPK siRNA, inhibited RPE cell death induced by oxidative stress. Overexpression of constitutively active MEK1 inhibited RPE cell death exposed to oxidative damage. In contrast, interfering p42/p44 MAPK expression accelerated oxidative-stress induced RPE cell death. To investigate the effects of human retinal progenitor cells (hRPC) on RPE cells, we isolated and expanded hRPC in vitro. The hRPCs expressed markers of neuronal and retinal progenitor cells, and were capable of differentiating into neuronal phenotype in defined medium. In the presence of 10% fetal bovine serum, hPRC suppressed RPE cell death induced by oxidative damage. Furthermore, conditioned medium of hRPC induced activation of p42/p44 MAPK, and the protective effect of hRPC and conditioned medium was suppressed by p42/p44 MAPK inhibitor. Our studies increase our understanding of the molecular mechanisms that could be employed to rescue RPE cells from degeneration and support the therapeutic potential of retinal progenitor cells. It will provide further insight into molecular mechanisms of AMD and establish a foundation for the long-term prevention and treatment of AMD
Neuroprotection of Rodent and Human Retinal Ganglion Cells In Vitro/Ex Vivo by the Hybrid Small Molecule SA-2
(MDPI, 2022-12-12) Pham, Jennifer H.; Johnson, Gretchen A.; Rangan, Rajiv S.; Amankwa, Charles E.; Acharya, Suchismita; Stankowska, Dorota L.
The mechanisms underlying the neuroprotective effects of the hybrid antioxidant-nitric oxide donating compound SA-2 in retinal ganglion cell (RGC) degeneration models were evaluated. The in vitro trophic factor (TF) deprivation model in primary rat RGCs and ex vivo human retinal explants were used to mimic glaucomatous neurodegeneration. Cell survival was assessed after treatment with vehicle or SA-2. In separate experiments, tert-Butyl hydroperoxide (TBHP) and endothelin-3 (ET-3) were used in ex vivo rat retinal explants and primary rat RGCs, respectively, to induce oxidative damage. Mitochondrial and intracellular reactive oxygen species (ROS) were assessed following treatments. In the TF deprivation model, SA-2 treatment produced a significant decrease in apoptotic and dead cell counts in primary RGCs and a significant increase in RGC survival in ex vivo human retinal explants. In the oxidative stress-induced models, a significant decrease in the production of ROS was observed in the SA-2-treated group compared to the vehicle-treated group. Compound SA-2 was neuroprotective against various glaucomatous insults in the rat and human RGCs by reducing apoptosis and decreasing ROS levels. Amelioration of mitochondrial and cellular oxidative stress by SA-2 may be a potential therapeutic strategy for preventing neurodegeneration in glaucomatous RGCs.
Regulation of Autophagy by Protein Kinase C-epsilon in Breast Cancer Cells
(MDPI, 2020-06-15) Basu, Alakananda
Protein kinase C-ɛ (PKCɛ), an anti-apoptotic protein, plays critical roles in breast cancer development and progression. Although autophagy is an important survival mechanism, it is not known if PKCɛ regulates autophagy in breast cancer cells. We have shown that silencing of PKCɛ by siRNA inhibited basal and starvation-induced autophagy in T47D breast cancer cells as determined by the decrease in LC3-II, increase in p62, and decrease in autophagy puncta both in the presence and absence of bafilomycin A1. The mechanistic target of rapamycin (mTOR) associates with Raptor or Rictor to form complex-1 (mTORC1) or complex-2 (mTORC2), respectively. Knockdown of PKCɛ attenuated an increase in autophagy caused by the depletion of Raptor and Rictor. Overexpression of PKCɛ in MCF-7 cells caused activation of mTORC1 and an increase in LC3-I, LC3-II, and p62. The mTORC1 inhibitor rapamycin abolished the increase in LC3-I and p62. Knockdown of mTOR and Rictor or starvation enhanced autophagy in PKCɛ overexpressing cells. While overexpression of PKCɛ in MCF-7 cells inhibited apoptosis, it induced autophagy in response to tumor necrosis factor-ɑ. However, inhibition of autophagy by Atg5 knockdown restored apoptosis in PKCɛ-overexpressing cells. Thus, PKCɛ promotes breast cancer cell survival not only by inhibiting apoptosis but also by inducing autophagy.
Role of C/EBP Homologous Protein in Photoreceptor and Retinal Ganglion Cell Death
(2014-08-01) Nashine, Sonali R.; Pang, Iok-Hou; Clark, Abbot F.; Wordinger, Robert J.
CCAAT/ enhancer binding protein (C/EBP) homologous protein (CHOP) is a pro-apoptotic, transcription factor, and an endoplasmic reticulum (ER) stress-induced marker of the unfolded protein response. The purpose of our study was to delineate the role of CHOP in photoreceptor and RGC death. We used CHOP knockout (Chop-/-) and C57BL/6J (WT) mice and the following two mouse models of retinal degeneration: the T17M RHO model and the retinal I/R model. We found that CHOP deficiency led to a significant reduction in ERG a-wave amplitudes and the thickness of the outer nuclear layer at P30. Also, we observed significant downregulation of mouse Rho, T17M RHO, Crx, Nrl, sXBP1 and P300 whereas the expression of peIF2α and Hdac1 was significantly increased in the ADRP mouse retina. CHOP was found to be upregulated in the RGCs 3 d after I/R and deficiency of CHOP significantly increased RGC survival at 3 d, 7 d, 14 d, and 28 d after I/R. RGC function was also found to be improved at 3 d and 7 d after I/R. These results indicate that 1) CHOP plays a cytoprotective role in T17M ADRP photoreceptors and ablation of CHOP does not prevent loss of photoreceptors and 2) CHOP knockout partially protects against loss of RGC number and function after retinal I/R. In conclusion, CHOP contributes significantly to the survival and death pathways of photoreceptors and RGCs and modulation of the CHOP-mediated apoptotic pathway could help in the development of plausible treatment strategies for retinal degenerative diseases.
Serum Deprivation Induces Apoptosis of Retinal Ganglion Cells Utilizing Mitochondrial Signaling Pathways
(2003-12-01) Charles, Irma E.; Rudick, Victoria; Krishnamoorthy, Raghu; Prasnna, Ganesh
Charles, Irma E., Serum Deprivation Induced Apoptosis of Retinal Ganglion Cells Utilizing Mitochondrial Signaling Pathways. Master of Science (Biomedical Sciences), December 2003, 90 pp., 10 illustrations. Apoptosis is the genetically regulated death of retinal ganglion cells (RGC) in which there is a blockade of retrograde transport. This blockade results in the loss of neurotrophic growth factors that are essential for the survival of the RGCs. This study uses several different techniques to determine mechanisms underlying apoptosis in rat RGCs deprived of growth factors. An established line of transformed RGC was subjected to serum deprivation for 2-6 days and compared to RGC cells maintained in 10% FBS to study the cellular changes that occur as a result of the treatments. The results show that serum deprivation for 48 hours resulted in a 50% cell loss due to apoptosis. Apoptotic death was associated with activation of caspases 3, 8, and 9 along with increased levels of Bax and death receptors 3 & 4. These results indicate that serum deprivation results in RGC death via mitochondrial and also extrinsic pathways.
Sigma-1 Receptor Signaling in the Eye
(2008-03-12) Tchedre, Kissaou T.; Yorio, Thomas; Singh, Meharvan; Machu, Tina
SIGMA-1 Receptor Signaling in the Eye Kissaou T. Tchedre, Department of Biomedical Sciences, University of North Texas Health Science Center Fort Worth, TX 76107. SUMMARY The sigma-1 receptor is a discovered transmembrane protein that mediates the regulation of ion channels. Sigma-1 receptor ligands have exhibited a wide variety of actions in the central nervous system including attenuation of the neuronal death associated with glutamate excitotoxicity both in vitro and in vivo. Although the sigma-1 receptor was cloned almost a decade ago, the molecular mechanism of the neuroprotective effect remains to be elucidated. In the current proposal it was hypothesized that activation of sigma-1 receptors promotes retinal ganglion cells survival by decreased calcium signaling pathways and factors linked to cell death. We showed by the ratiometric calcium imaging and patch clamp techniques that sigma-1 receptor activation could inhibit both calcium influx and intracellular calcium mobilization. The results showed that sigma-1 receptor overexpressing RGC-5 cells also had a lower glutamate-induced intracellular calcium mobilization compared to non-overexpressing RGC-5 cells. The survival assay data showed that the sigma-1 receptor agonist, (+)-SKF10047 protected RGC-5 cells showed a significant resistance to glutamate-induced apoptosis compared to the control RGC-5 cells. The sigma-1 receptor neuroprotective mechanism also included the down regulation of Bax, and caveolin-1 protein expression levels and inhibited caspase-3 activation. We also demonstrated for the first time using a co-immunoprecipitation technique, the association between L-type calcium channels and sigma-1 receptors. Thus sigma-1 receptor ligands may indirectly influence the voltage-gated calcium channels by interacting with the sigma-1 receptor associated voltage-gated calcium channel complex. In conclusion, activation of sigma-1 receptors can regulate calcium homeostasis and signaling in retinal ganglion cells. Activation of sigma-1 receptors regulate intracellular calcium levels and pro-apoptotic gene expression to promote retinal ganglion cell survival. Sigma-1 receptor ligands may be neuroprotective and targets for potential glaucoma therapeutics.
The Enigmatic Protein Kinase C-eta
(MDPI, 2019-02-13) Basu, Alakananda
Protein kinase C (PKC), a multi-gene family, plays critical roles in signal transduction and cell regulation. Protein kinase C-eta (PKCeta) is a unique member of the PKC family since its regulation is distinct from other PKC isozymes. PKCeta was shown to regulate cell proliferation, differentiation and cell death. It was also shown to contribute to chemoresistance in several cancers. PKCeta has been associated with several cancers, including renal cell carcinoma, glioblastoma, breast cancer, non-small cell lung cancer, and acute myeloid leukemia. However, mice lacking PKCeta were more susceptible to tumor formation in a two-stage carcinogenesis model, and it is downregulated in hepatocellular carcinoma. Thus, the role of PKCeta in cancer remains controversial. The purpose of this review article is to discuss how PKCeta regulates various cellular processes that may contribute to its contrasting roles in cancer.
The Role of Lipid Rafts and Membrane Androgen Receptors in Androgen's Neurotoxic Effects
(Oxford University Press, 2022-02-21) Fadeyibi, Oluwadarasimi; Rybalchenko, Nataliya; Mabry, Steve; Nguyen, Dianna H.; Cunningham, Rebecca L.
Sex differences have been observed in multiple oxidative stress-associated neurodegenerative diseases. Androgens, such as testosterone, can exacerbate oxidative stress through a membrane androgen receptor (mAR), AR45, localized to lipid rafts in the plasma membrane. The goal of this study is to determine if interfering with mAR localization to cholesterol-rich lipid rafts decreases androgen induced neurotoxicity under oxidative stress environments. We hypothesize that cholesterol-rich caveolar lipid rafts are necessary for androgens to induce oxidative stress generation in neurons via the mAR localized within the plasma membrane. Nystatin was used to sequester cholesterol and thus decrease cholesterol-rich caveolar lipid rafts in a neuronal cell line (N27 cells). Nystatin was applied prior to testosterone exposure in oxidatively stressed N27 cells. Cell viability, endocytosis, and protein analysis of oxidative stress, apoptosis, and mAR localization were conducted. Our results show that the loss of lipid rafts via cholesterol sequestering blocked androgen-induced oxidative stress in cells by decreasing the localization of mAR to caveolar lipid rafts.
Withaferin A: A Pleiotropic Anticancer Agent from the Indian Medicinal Plant Withania somnifera (L.) Dunal
(MDPI, 2023-01-22) Kumar, Suneel; Mathew, Stephen O.; Aharwal, Ravindra P.; Tulli, Hardeep S.; Mohan, Chakrabhavi D.; Sethi, Gautam; Ahn, Kwang-Seok; Webber, Kassidy; Sandhu, Sardul S.; Bishayee, Anupam
Cancer represents the second most deadly disease and one of the most important public health concerns worldwide. Surgery, chemotherapy, radiation therapy, and immune therapy are the major types of treatment strategies that have been implemented in cancer treatment. Unfortunately, these treatment options suffer from major limitations, such as drug-resistance and adverse effects, which may eventually result in disease recurrence. Many phytochemicals have been investigated for their antitumor efficacy in preclinical models and clinical studies to discover newer therapeutic agents with fewer adverse effects. Withaferin A, a natural bioactive molecule isolated from the Indian medicinal plant Withania somnifera (L.) Dunal, has been reported to impart anticancer activities against various cancer cell lines and preclinical cancer models by modulating the expression and activity of different oncogenic proteins. In this article, we have comprehensively discussed the biosynthesis of withaferin A as well as its antineoplastic activities and mode-of-action in in vitro and in vivo settings. We have also reviewed the effect of withaferin A on the expression of miRNAs, its combinational effect with other cytotoxic agents, withaferin A-based formulations, safety and toxicity profiles, and its clinical potential.