Browsing by Subject "Apoptosis"

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CASPASE-7 ABLATION PROTECTS THE T17M RHODOPSIN MICE FROM SEVERE RETINAL DEGENERATION THROUGH REPROGRAMING OF THE UPR AND INHIBITION OF TRAF2-JNK APOPTOSIS
(2013-04-12) Choudhury, Shreyasi
Purpose: We previously demonstrated that misfolded T17M rhodopsin (RHO) activates the Unfolded Protein Response (UPR) in mouse rod photoreceptor cells eventually leading to Autosomal Dominant Retinitis Pigmentosa. We also have shown that the ablation of the UPR-induced CASP-7 in T17M RHO transgenic mice slows down the rate of retinal degeneration measured by ERG and SD-OCT. Therefore, the goal of this study is to elucidate the molecular mechanisms involved in the preservation of vision in T17MRHO CASP7-/- retinas to validate new therapeutic targets Methods: In vitro and in vivo studies were conducted to elucidate the pathway by which CASP-7 ablation promotes the ADRP photoreceptor cell survival. RNA and protein extracts were obtained from the 661W cells co-transfected with either wt or T17M RHO plasmid and control or CASP-7 siRNA. Retinas were harvested from C57/BL6, T17MRHO, T17MRHO CASP7-/- mice at P30 to perform qRT-PCR and western blot analysis. Results: The study of the cellular signaling in T17MRHO CASP7-/- retina demonstrated that the preservation of the structure and function of ADRP photoreceptors is occurred via down-regulation of the UPR-induced gene and protein expression. The ATF4, pATF6, mTor and Hif1 proteins were down regulated by 55%, 57%, 31%, 77% correspondingly and the level of pAKT was elevated by 60% in T17MRHO CASP7-/- retina. In addition, the inhibition of PARP1 and TNFa proteins in T17MRHO CASP7-/- retina was observed. All together these modifications lead to diminishing the TRAF2 and pc-Jun by 31%, 50% correspondingly. In vitro study also confirmed the modulation of cellular signaling observed in T17M RHO CASP7-/- retina. Conclusions: Both in vivo and in vitro studies indicated that the ablation of CASP-7 in the T17MRHO retina prevents the deterioration of retinal function and structure through reprograming of the UPR and modulation of TRAF2-JNK-induced apoptosis. This reduction is believed to occur through the down-regulation of the mTOR and Hif1a proteins. The inhibition of the PARP1 and TNFa proteins is also found to be responsible for diminishing the TRAF2-JNK apoptosis. In both scenarios, the reduction in c-Jun apoptosis leads to ADRP photoreceptor survival. This study points out c-Jun as a potential therapeutic targets for ADRP treatment.
Cytotoxic Activity of the Mesoionic Compound MIH 2.4Bl in Breast Cancer Cell Lines
(Sage Publications, 2020-07-01) Amaral de Mascena Costa, Luciana; Harmon, Ashlyn C.; Aguiar Coelho Teixeira, Alvaro; Cassio Silva de Lima, Filipe; de Sousa Araujo, Silvany; Del Piero, Fabio; Diogenes da Silva Souza, Helivaldo; Filgueiras de Athayde Filho, Petronio; Alves Junior, Severino; de Mascena Diniz Maia, Maria; Wischral, Aurea; Adriao Gomes Filho, Manoel; Mathis, J. Michael
In this work, we report the synthesis of a new 1,3-thiazolium-5-thiolate derivative of a mesoionic compound (MIH 2.4Bl) and the characterization of its selective cytotoxicity on a panel of breast cancer cells lines. The cytotoxic effect of MIH 2.4Bl on breast cancer cell lines was determined by XTT and crystal violet assays, flow cytometry analysis, electron microscopy characterization, and terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) apoptosis assays. As determined using XTT cell growth and survival assays, MIH 2.4Bl exhibited growth inhibition activity on most breast cancer cell lines tested, compared with normal human mammary epithelial cells. Three breast cancer cell lines (MCF-7, T-47D, and ZR-75-1) showed a more potent sensitivity index to growth inhibition by MIH 2.4Bl than the other breast cancer cell lines. Interestingly, these 3 cell lines were derived from tumors of Luminal A origin and have ER (estrogen receptor), PR (progesterone receptor), and HER2 (human epidermal growth factor receptor 2) positive expression. Additional analysis of cytotoxicity mediated by MIH 2.4Bl was performed using the MCF-7 cell line. MCF-7 cells displayed both time- and dose-dependent decreases in cell growth and survival, with a maximum cytotoxic effect observed at 72 and 96 hours. The MCF-7 cells were also characterized for cell cycle changes upon treatment with MIH 2.4Bl. Using flow cytometry analysis of cell cycle distribution, a treatment-dependent effect was observed; treatment of cells with MIH 2.4Bl increased the G2/M population to 34.2% compared with 0.1% in untreated (control) cells. Ultrastructural analysis of MFC-7 cells treated with MIH 2.4Bl at 2 different concentrations (37.5 and 75 muM) was performed by transmission electron microscopy. Cells treated with 37.5 muM MIH 2.4Bl showed morphologic changes beginning at 6 hours after treatment, while cells treated with 75 muM showed changes beginning at 3 hours after treatment. These changes were characterized by an alteration of nuclear morphology and mitochondrial degeneration consistent with apoptotic cell death. Results of a TUNEL assay performed on cells treated for 96 hours with MIH 2.4Bl supported the observation of apoptosis. Together, these results suggest that MIH 2.4Bl is a promising candidate for treating breast cancer and support further in vitro and in vivo investigation.
Pyruvate-Enhanced Fluid Resuscitation for Hemorrhagic Shock and Hindlimb Ischemia
(2009-05-01) Flaherty, Devin C.; Mallet, Robert T.
Traumatic blood loss often necessitates the use of resuscitative fluids to replenish blood volume and stabilize blood pressure. The use of tourniquets to achieve hemostasis imposes ischemia-reperfusion on wounded limbs after release. Resuscitation with the physiological antioxidant and natural intermediary metabolite pyruvate may abrogate reperfusion injury of muscle by scavenging oxyradicals and stabilizing cytoprotective proteins. This study was designed to determine the effects of pyruvate in the setting of hemorrhagic shock with resuscitation and hindlimb ischemia-reperfusion. All experiments were conducted on isoflurane-anesthetized male goats. A controlled hemorrhage was performed to lower mean arterial pressure (MAP) to c. 50 mmHg, then the right femoral artery and vein were occluded for 90 min. Lactate Ringer’s (LR) or pyruvate Ringer’s (PR) was infused intravenously (10 ml/min) for 90 min, from 30 min occlusion until 30 min after reperfusion. At 4 h reperfusion, the right gastrocnemius muscle and left ventricular myocardium were biopsied and flash-frozen for analyses of metabolites, enzymes, pro- and anti-apoptotic proteins and markers of oxidative and inflammatory stress. During the first phase of experimentation we hypothesized that controlled resuscitation with PR vs. LR more effectively stabilizes MAP and attenuates myocardial inflammation post-resuscitation. MAP (mmHg) was increased in PR (59 ± 4) vs. LR (47 ± 3) resuscitated goats (p During the second phase of experimentation, we hypothesized that PR resuscitation would protect ischemic hindlimb muscle in the setting of hemorrhagic shock and limb reperfusion. Lactate dehydrogenase and creatine kinase activities fell by 36 and 20%, respectively in LR-resuscitated vs. sham muscle (p We conclude that 1) Systemic hypotension and hindlimb ischemia-reperfusion with conventional LR treatment imposed pro-oxidative and pro-inflammatory stress both systemically and locally, thus preventing stabilization of MAP during recovery and initiating apoptotic mechanisms in the hindlimb musculature; 2) Pyruvate-fortified Ringer’s effectively stabilized hemodynamics and dampened systemic inflammation after hemorrhagic shock with resuscitation and hindlimb ischemia-reperfusion; 3) PR-fortified resuscitation blunted oxidative and inflammatory stress within the ischemic hindlimb and suppressed pro-apoptotic signaling. These investigations demonstrate the anti-oxidative and anti-inflammatory effects of pyruvate in a system exposed to hemorrhagic shock with fluid resuscitation, as well as identify the cytoprotection pyruvate affords tissue experiencing ischemia-reperfusion.
Retinal ganglion cell death in acute and chronic models of glaucoma: Is the degeneration subtype specific?
(2018-12) Daniel. Steffi; McDowell, Colleen; Clark, Abbot F.; Pang, Iok-Hou; Krishnamoorthy, Raghu R.
Glaucoma, the world's leading cause of irreversible blindness, is a group of optic neuropathies with multifactorial etiologies that lead to optic nerve degeneration and retinal ganglion cell (RGC) death, severing the communication between the eye and the brain causing vision loss. As glaucoma is a progressive disease, many ocular changes occur before the actual vision loss and needs comprehensive eye examination to detect these changes. One of the major risk factors of glaucoma is elevated intraocular pressure (IOP), a modifiable factor which is targeted by current therapeutic strategies. None of the available therapies cure the disease but are used to manage it by slowing its progression. Because glaucoma is such a complex disease with many different cellular and molecular pathways at play, a therapy to cure or reverse the disease is not yet developed. RGC neurons are the ones affected in glaucoma. There are about 50000 RGCs per retina. Nerve fibers from these neurons combine together in an optic nerve and relay visual cues transported from other neurons of the retina to the brain. There are more than 30 different subtypes of RGCs that differ in morphology, dendritic arborization, physiological properties and molecular signatures. These RGCs detect different visual signals and project to their respective visual centers in the brain. As these RGCs differ in so many aspects, a lot of studies postulate that they their response to glaucomatous injury also differs in a way that some RGCs are more susceptible to injury than the others. We hypothesize that RGC subtypes are differentially susceptible to glaucomatous injury. To test this hypothesis, we used transgenic animals expressing GFP in individual RGC subtypes and subjected them to glaucomatous insults by using optic nerve crush model (induced model of glaucoma) or nee model (experimental glaucoma model of early onset glaucoma). Our data suggests that RGCs do have a specific pattern of degeneration in under glaucomatous insults. In our induced and inherent models, we found that melanopsin positive RGCs are the most resilient to injury. We also found that axonal degeneration in RGC subtypes after glaucomatous insult is independent of their respective somal degradation. In this study we have also discussed degeneration and regeneration in the central nervous system (CNS) as well as utilized imaging techniques to evaluate individual RGC subtype projections in the brain through its retinorecipient areas.
Sigma-1R Protects Retinal Ganglion Cells in Optic Nerve Crush Model for Glaucoma
(ARVO Journals, 2021-08-18) Li, Linya; He, Shaoqing; Liu, Yang; Yorio, Thomas; Ellis, Dorette Z.
Purpose: The purpose of this study was to determine the effects of the Sigma-1R (sigma-1r) on retinal ganglion cell (RGC) survival following optic nerve crush (ONC) and the signaling mechanism involved in the sigma-1r protection. Methods: The overall strategy was to induce injury by ONC and mitigate RGC death by increasing sigma-1r expression and/or activate sigma-1r activity in sigma-1r K/O mice and wild type (WT) mice. AAV2-sigma-1r vector was used to increase sigma-1r expression and sigma-1r agonist used to activate the sigma-1r and RGCs were counted. Immunohistochemical and Western blot analysis determined phosphorylated (p)-c-Jun, c-Jun, and Caspase-3. Pattern electroretinography (PERG) determined RGC activity. Results: RGC counts and function were similar in pentazocine-treated WT mice when compared to untreated mice and in WT mice when compared with sigma-1r K/O mice. Pentazocine-induced effects and the effects of sigma-1r K/O were only observable after ONC. ONC resulted in decreased RGC counts and activity in both WT and sigma-1r K/O mice, with sigma-1r K/O mice experiencing significant decreases compared with WT mice. The sigma-1r transgenic expression resulted in increased RGC counts and activity following ONC. In WT mice, treatment with sigma-1r agonist pentazocine resulted in increased RGC counts and increased activity when compared with untreated WT mice. There were time-dependent increases in c-jun, p-c-jun, and caspase-3 expression in ONC mice that were mitigated with pentazocine-treatment. Conclusions: These findings suggest that the apoptotic pathway is involved in RGC losses seen in an ONC model. The sigma-1r offers neuroprotection, as activation and/or transgenic expression of sigma-1r attenuated the apoptotic pathway and restored RGCs number and function following ONC.
Stress and interferon signalling-mediated apoptosis contributes to pleiotropic anticancer responses induced by targeting NGLY1
(Springer Nature, 2018-11-02) Zolekar, Ashwini; Lin, Victor J. T.; Mishra, Nigam M.; Ho, Yin Ying; Hayatshahi, Hamed S.; Parab, Abhishek; Sampat, Rohit; Liao, Xiaoyan; Hoffmann, Peter; Liu, Jin; Emmitte, Kyle A.; Wang, Yu-Chieh
BACKGROUND: Although NGLY1 is known as a pivotal enzyme that catalyses the deglycosylation of denatured glycoproteins, information regarding the responses of human cancer and normal cells to NGLY1 suppression is limited. METHODS: We examined how NGLY1 expression affects viability, tumour growth, and responses to therapeutic agents in melanoma cells and an animal model. Molecular mechanisms contributing to NGLY1 suppression-induced anticancer responses were revealed by systems biology and chemical biology studies. Using computational and medicinal chemistry-assisted approaches, we established novel NGLY1-inhibitory small molecules. RESULTS: Compared with normal cells, NGLY1 was upregulated in melanoma cell lines and patient tumours. NGLY1 knockdown caused melanoma cell death and tumour growth retardation. Targeting NGLY1 induced pleiotropic responses, predominantly stress signalling-associated apoptosis and cytokine surges, which synergise with the anti-melanoma activity of chemotherapy and targeted therapy agents. Pharmacological and molecular biology tools that inactivate NGLY1 elicited highly similar responses in melanoma cells. Unlike normal cells, melanoma cells presented distinct responses and high vulnerability to NGLY1 suppression. CONCLUSION: Our work demonstrated the significance of NGLY1 in melanoma cells, provided mechanistic insights into how NGLY1 inactivation leads to eradication of melanoma with limited impact on normal cells, and suggested that targeting NGLY1 represents a novel anti-melanoma strategy.
TETRANDRINE INDUCES ROS-DRIVEN CASPASE-DEPENDENT APOPTOSIS OF PROSTATE CANCER CELLS VIA MITOCHONDRIAL AND CELL DEATH RECEPTOR PATHWAY
(2014-03) Chaudhary, Pankaj; Vishwanatha, Jamboor K.
Androgen deprivation is still the standard systemic therapy for prostate cancer, but patients invariably relapse with a more aggressive form of prostate cancer termed hormone refractory, androgen independent, or castration resistant prostate cancer. Once prostate cancer becomes castration-resistant, metastasis is a significant problem and treatment options are limited. Therefore, identification of novel agents that can selectively kill tumor cells with no additional toxicity to normal tissue would have significant impact on prostate cancer therapy. Purpose (a): Tetrandrine, a bisbenzylisoquinoline alkaloid, isolated from the root of Stephania tetrandra is used in traditional Chinese medicine as an anti-rheumatic, anti-inflammatory, and anti-hypertensive agent for the past several years. During recent years, increasing number of studies have focused on the potential of tetrandrine in cancer therapy. Despite its great potential as an anti-cancer agent, the effect of tetrandrine in prostate cancer has not been studied. Therefore, in the present study, we demonstrate the cytotoxic efficacy of tetrandrine in human androgen-independent prostate cancer cells, PC3 and DU145, and delineate the mechanism of this effect. Methods (b): Prostate cancer cell lines, PC3 and DU145, and normal prostate PWR-1E cells were cultured in ATCC recommended medium. The toxicity of tetrandrine was analyzed by MTT assay and Vybrant Apoptosis Assay Kit. Western blotting was used to detect the expression of proteins involved in apoptosis. Results (c): Our results indicate that tetrandrine selectively inhibits the growth of PC3 and DU145 cancer cells compared to normal prostate PWR-1E cells. Treatment of cancer cells with tetrandrine caused the upregulation of Fas and Bax, downregulation of Bcl-2, cleavage of Bid, and release of cytochrome c, which were accompanied by activation of caspases-9, -3 and -8 and subsequently poly(ADP-ribose) polymerase cleavage. Pre-incubation with caspase-8 inhibitor significantly blocked the tetrandrine-induced Bid cleavage, reduction in mitochondrial membrane potential, and activation of caspase 3, and cell death. Together, these results suggest that the mitochondrial pathway is primarily involved in tetrandrine-induced apoptosis. Additionally, our results demonstrated that tetrandrine-induced apoptosis was caused by the generation of reactive oxygen species (ROS) and most of the signaling effects were attenuated with the preincubation of cells with N-acetylcysteine, thereby further confirming the involvement of ROS in these events. Conclusions (d): Our results demonstrated that treatment of prostate cancer cells with tetrandrine induces caspase-dependent apoptosis via Fas-mediated Bid cleavage and cytochrome c release.