Insulin in Combination with N-Acetylcysteine Protects Hypoxia-Induced Toxicity in 661W Cells

Background: Proliferative diabetic retinopathy (PDR) is the leading cause of blindness among working-age adults. Photoreceptors are the most numerous and metabolically demanding cells in the retina thus oxygen is essential for retinal function. It has been reported that photoreceptors found in rat retina are specifically vulnerable to hypoxia. Hypoxia-induced metabolic stress leads to photoreceptor atrophy and retinopathy. Furthermore, photoreceptor cell death is known to occur mainly through apoptosis. However, the protection of hypoxia-induced-cytotoxicity in cone photoreceptor cells has not been investigated extensively. The aim of this study was to determine whether co-treatment of insulin and the N-Acetyl-L-Cysteine (NAC) (a free radical scavenger) efficiently protects against hypoxia-induced cytotoxicity in 661W cells. Methods: 661W, an immortalized mouse cone photoreceptor cells, were cultured at 5% CO2 at 37˚C in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% FBS, penicillin (100 units/mL), and streptomycin (100μg/mL). Cobalt (II) Chloride hexahydrate (CoCl2) was used to induce hypoxia. Insulin was suspended in sterile water, and NAC was diluted in the culture medium. For recovery experiment, cells were pretreated with CoCl2 for 24hrs, and then followed by replacing of medium with insulin (100nM) and NAC (3mM) alone, or with a combination of the two reagents for another 24hrs. Cell viability was determined by MTT assay in a 96 well culture plate. Morphological changes of the cells were observed and photographed under phase-contrast microscope and protein expression was measured by Western blot analysis. Statistical analysis was undertaken using independent two-tailed Students’ t-test and determined with SPSS Statistics software. Results: Treatment with CoCl2 significantly inhibited cell proliferation, reduced the number of viability cells, and induced apoptosis, initiated (poly (ADP-ribose) polymerase (PARP) cleavage, and increased caspase 3 activation. In addition, CoCl2 treatment led to oxidative stress, autophagy, and ubiquitination in the 661W cells. All of these effects, including cell proliferations were significantly reversed by the combination treatment of Insulin and NAC. In contrast, treatment with Insulin alone did not result in a similar protective effect and NAC partially protects against hypoxia induced toxicity. Conclusion: Hypoxia induces significant apoptosis, oxidative stress, and protein ubiquitination in 661W cone photoreceptors. A combination treatment of Insulin and NAC completely reversed such hypoxia-induced cytotoxicity. Additional research on a combination therapy employing insulin and NAC may provide a novel and promising therapeutic strategy for hypoxia-mediated cone photoreceptor cell damage

N-Acetylcysteine in Combination with IGF-1 Enhances Neuroprotection against Proteasome Dysfunction-Induced Neurotoxicity in SH-SY5Y Cells

Ubiquitin proteasome system (UPS) dysfunction has been implicated in the development of many neuronal disorders, including Parkinson’s disease (PD). Previous studies focused on individual neuroprotective agents and their respective abilities to prevent neurotoxicity following a variety of toxic insults. However, the effects of the antioxidant N-acetylcysteine (NAC) on proteasome impairment-induced apoptosis have not been well characterized in human neuronal cells. The aim of this study was to determine whether cotreatment of NAC and insulin-like growth factor-1 (IGF-1) efficiently protected against proteasome inhibitor-induced cytotoxicity in SH-SY5Y cells. Our results demonstrate that the proteasome inhibitor, MG132, initiates poly(ADP-ribose) polymerase (PARP) cleavage, caspase 3 activation, and nuclear condensation and fragmentation. In addition, MG132 treatment leads to endoplasmic reticulum (ER) stress and autophagy-mediated cell death. All of these events can be attenuated without obvious reduction of MG132 induced protein ubiquitination by first treating the cells with NAC and IGF-1 separately or simultaneously prior to exposure to MG132. Moreover, our data demonstrated that the combination of the two proved to be significantly more effective for neuronal protection. Therefore, we conclude that the simultaneous use of growth/neurotrophic factors and a free radical scavenger may increase overall protection against UPS dysfunction-mediated cytotoxicity and neurodegeneration

Combined treatment with niclosamide and camptothecin enhances anticancer effect in U87 MG human glioblastoma cells

Glioblastoma multiforme (GBM) is one of the deadliest cancers of the brain. Its ability to infiltrate healthy brain tissues renders it difficult to remove surgically. Furthermore, it exhibits high rates of radio- and chemoresistance, making the survival rates of patients with GBM poor. Therefore, novel effective therapies for GBM remain urgently in demand. Niclosamide is an anti-helminthic drug and recently it has been receiving attention due to its reported anticancer effects in cancer models, including GBM. Furthermore, camptothecin (CPT) is a naturally-occurring alkaloid and has been previously reported to be a potential chemotherapeutic agent by targeting the nuclear topoisomerase I. In the present study, the possible combined chemotherapeutic effects of niclosamide and CPT on the human glioblastoma cell line U87 MG was investigated by MTT assay and western blot analysis. Niclosamide exhibited synergistic activities with CPT to suppress the proliferation of U87 MG cells. Additionally, niclosamide suppressed cell proliferation and induced cell death mainly by triggering ER stress and autophagy, whilst CPT induced cell apoptosis mainly through p53-mediated mitochondrial dysfunction and activation of the MAPK (ERK/JNK) pathways. Overall, these findings suggest that co-administration of niclosamide and CPT may provide a novel therapeutic treatment strategy for GBM

Anti-cancer effect of Cissus quadrangularis on human glioblastoma cells

Objectives Glioblastoma multiforme (GBM) is a common and fatal brain tumour in the central nervous system with a poor survival rate and a median survival time of 15 months only. The standard treatment is aggressive surgical resection followed by radiotherapy and chemotherapy. However, effective drugs available in chemotherapy are limited. This study was designed to evaluate, for the first time, the potential therapeutic effect of Cissus quadrangularis (CQ) in human glioblastoma cells and to investigate its possible mechanisms of action. Methods In this study, we examined the anticancer activity of CQ in human glioblastoma U87 MG cells by cell viability assay, cell migration assay, immunofluorescence staining and Western blot. Results Our results demonstrated that CQ treatment induced U87 cytotoxicity, cell cycle arrest and cell death. The cytotoxicity of CQ mediates ER stress, autophagy and mitochondrial apoptosis by suppressing pro-survival signalling pathways (extracellular signal-regulated kinase and signal transducer and activator of transcription 3 pathways). Conclusions The findings of this study imply that CQ is a promising anti-cancer candidate for the treatment of GBM. Highlights The anticancer effect of Cissus quadrangularis (CQ) was studied in human glioblastoma U87 MG cells. It was demonstrated that CQ treatment induced cytotoxicity, cell cycle arrest and cell death in U87 MG cells. CQ may become a potential chemotherapy component for the treatment of glioblastoma multiforme

Niclosamide induces protein ubiquitination and inhibits multiple pro-survival signaling pathways in the human glioblastoma U-87 MG cell line

<div><p>Glioblastoma is the most common and lethal malignant primary brain tumor for which the development of efficacious chemotherapeutic agents remains an urgent need. The anti-helminthic drug niclosamide, which has long been in use to treat tapeworm infections, has recently attracted renewed interest due to its apparent anticancer effects in a variety of <i>in vitro</i> and <i>in vivo</i> cancer models. However, the mechanism(s) of action remains to be elucidated. In the present study, we found that niclosamide induced cell toxicity in human glioblastoma cells corresponding with increased protein ubiquitination, ER stress and autophagy. In addition, niclosamide treatment led to down-regulation of Wnt/β-catenin, PI3K/AKT, MAPK/ERK, and STAT3 pro-survival signal transduction pathways to further reduce U-87 MG cell viability. Taken together, these results provide new insights into the glioblastoma suppressive capabilities of niclosamide, showing that niclosamide can target multiple major cell signaling pathways simultaneously to effectively promote cell death in U-87 MG cells. Niclosamide constitutes a new prospect for a therapeutic treatment against human glioblastoma.</p></div

Niclosamide treatment reduces U-87 MG cell viability.

<p>U-87 MG cells were treated with the indicated concentrations of niclosamide for 24 h. Cell viability was determined by MTS assay. Data represent the mean ± S.E.M of at least three independent experiments. (***) <i>p</i><0.001.</p

Niclosamide promotes protein ubiquitination and apoptosis in U-87 MG cells.

<p>(A) U-87 MG cells were treated with 5 μM niclosamide and cells were collected at the indicated time points. Total cell lysates were resolved by SDS-PAGE and immunoblotted with antibody specific to ubiquitin. (B) Total cell lysates were isolated from U-87 MG cells treated with the indicated concentrations of niclosamide for 24 h, resolved by SDS-PAGE and then immunoblotted with antibody specific for ubiquitin and PARP, an apoptotic protein. β-Actin was utilized as a loading control. The image is representative of at least three independent experiments. Relative expression levels of ubiquitinated proteins and cleaved PARP protein from cells with and without niclosamide treatment were quantified by densitometry (C and D, respectively). Data represent the mean ± S.E.M of at least three independent experiments. (***) <i>p</i><0.01 and (*) <i>p</i><0.05, respectively; NS = no significant difference.</p

Niclosamide triggers ER stress and the autophagic response in U-87 MG cells.

<p>(A) U-87 MG cells were treated with 5 μM niclosamide and collected at the indicated time points. Total cell lysates were resolved on SDS-PAGE and immunoblotted with antibodies specific for CHOP and LC3. (B) U-87 MG cells were treated with 2.5 or 5 μM niclosamide and total protein was isolated from lysed cells after 24 h. Lysates were resolved on SDS-PAGE and immunoblotted with LC3-specific antibody. (C) Representative image of U-87 MG cells incubated with 5 μM niclosamide for 24 h and then stained with MDC (0.05 mM). Fluorescence particles in the cytoplasm indicate autophagic vacuoles.</p

Niclosamide inhibits STAT3 expression.

<p>Total protein was isolated from U-87 MG cells treated with 5 μM niclosamide at the times indicated in the figure. Lysates were resolved by SDS-PAGE and immunoblotted with either P-STAT3 or STAT3-specific antibodies. β-Actin was used as loading control.</p

Niclosamide represses expression of β-catenin and its downstream effectors.

<p>(A) U-87 MG cells were treated with two levels of niclosamide as indicated in the figure for 24 h. Total protein isolated from cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies specific for β-catenin and proteins from two target genes, cyclin D1 and survivin. β-Actin was used as a loading control. (B) Time course effect of niclosamide on the expression of cyclin D1 and survivin.</p