Effect Of Co-treatment Research Articles

Characterization and Regulation of Carrier Proteins of Mitochondrial Glutathione Uptake in Human Retinal Pigment Epithelium Cells.

PurposeTo characterize two mitochondrial membrane transporters 2-oxoglutarate (OGC) and dicarboxylate (DIC) in human RPE (hRPE) and to elucidate their role in the regulation of mitochondrial glutathione (mGSH) uptake and cell death in oxidative stress.MethodsThe localization of OGC and DIC proteins in confluent hRPE, polarized hRPE monolayers and mouse retina was assessed by immunoblotting and confocal microscopy. Time- and dose-dependent expression of the two carriers were determined after treatment of hRPE with H2O2, phenyl succinate (PS), and butyl malonate (BM), respectively, for 24 hours. The effect of inhibition of OGC and DIC on apoptosis (TUNEL), mGSH, and mtDNA was determined. Silencing of OGC by siRNA knockdown on RPE cell death was studied. Kinetics of caspase 3/7 activation with OGC and DIC inhibitors and effect of cotreatment with glutathione monoethyl ester (GSH-MEE) was determined using the IncuCyte live cell imaging.ResultsOGC and DIC are expressed in hRPE mitochondria and exhibited a time- and dose-dependent decrease with stress. Pharmacologic inhibition caused a decrease in OGC and DIC in mitochondria without changes in mtDNA and resulted in increased apoptosis and mGSH depletion. GSH-MEE prevented apoptosis through restoration of mGSH. OGC siRNA exacerbated apoptotic cell death in stressed RPE which was inhibited by increased mGSH from GSH-MEE cotreatment.ConclusionsCharacterization and mechanism of action of two carrier proteins of mGSH uptake in RPE are reported. Regulation of OGC and DIC will be of value in devising therapeutic strategies for retinal disorders such as AMD.

Combined treatment with the phenolics (−)-epigallocatechin-3-gallate and ferulic acid improves cognition and reduces Alzheimer-like pathology in mice

"Nutraceuticals" are well-tolerated natural dietary compounds with drug-like properties that make them attractive as Alzheimer's disease (AD) therapeutics. Combination therapy for AD has garnered attention following a recent National Institute on Aging mandate, but this approach has not yet been fully validated. In this report, we combined the two most promising nutraceuticals with complementary anti-amyloidogenic properties: the plant-derived phenolics (-)-epigallocatechin-3-gallate (EGCG, an α-secretase activator) and ferulic acid (FA, a β-secretase modulator). We used transgenic mice expressing mutant human amyloid β-protein precursor and presenilin 1 (APP/PS1) to model cerebral amyloidosis. At 12 months of age, we orally administered EGCG and/or FA (30 mg/kg each) or vehicle once daily for 3 months. At 15 months, combined EGCG-FA treatment reversed cognitive impairment in most tests of learning and memory, including novel object recognition and maze tasks. Moreover, EGCG- and FA-treated APP/PS1 mice exhibited amelioration of brain parenchymal and cerebral vascular β-amyloid deposits and decreased abundance of amyloid β-proteins compared with either EGCG or FA single treatment. Combined treatment elevated nonamyloidogenic soluble APP-α and α-secretase candidate and down-regulated amyloidogenic soluble APP-β, β-C-terminal APP fragment, and β-secretase protein expression, providing evidence for a shift toward nonamyloidogenic APP processing. Additional beneficial co-treatment effects included amelioration of neuroinflammation, oxidative stress, and synaptotoxicity. Our findings offer preclinical evidence that combined treatment with EGCG and FA is a promising AD therapeutic approach.

Memantine potentiates cytarabine-induced cell death of acute leukemia correlating with inhibition of Kv1.3 potassium channels, AKT and ERK1/2 signaling

BackgroundTreatment of acute leukemia is challenging and long-lasting remissions are difficult to induce. Innovative therapy approaches aim to complement standard chemotherapy to improve drug efficacy and decrease toxicity. Promising new therapeutic targets in cancer therapy include voltage-gated Kv1.3 potassium channels, but their role in acute leukemia is unclear. We reported that Kv1.3 channels of lymphocytes are blocked by memantine, which is known as an antagonist of neuronal N-methyl-D-aspartate type glutamate receptors and clinically applied in therapy of advanced Alzheimer disease. Here we evaluated whether pharmacological targeting of Kv1.3 channels by memantine promotes cell death of acute leukemia cells induced by chemotherapeutic cytarabine.MethodsWe analyzed acute lymphoid (Jurkat, CEM) and myeloid (HL-60, Molm-13, OCI-AML-3) leukemia cell lines and patients’ acute leukemic blasts after treatment with either drug alone or the combination of cytarabine and memantine. Patch-clamp analysis was performed to evaluate inhibition of Kv1.3 channels and membrane depolarization by memantine. Cell death was determined with propidium iodide, Annexin V and SYTOX staining and cytochrome C release assay. Molecular effects of memantine co-treatment on activation of Caspases, AKT, ERK1/2, and JNK signaling were analysed by Western blot. Kv1.3 channel expression in Jurkat cells was downregulated by shRNA.ResultsOur study demonstrates that memantine inhibits Kv1.3 channels of acute leukemia cells and in combination with cytarabine potentiates cell death of acute lymphoid and myeloid leukemia cell lines as well as primary leukemic blasts from acute leukemia patients. At molecular level, memantine co-application fosters concurrent inhibition of AKT, S6 and ERK1/2 and reinforces nuclear down-regulation of MYC, a common target of AKT and ERK1/2 signaling. In addition, it augments mitochondrial dysfunction resulting in enhanced cytochrome C release and activation of Caspase-9 and Caspase-3 leading to amplified apoptosis.ConclusionsOur study underlines inhibition of Kv1.3 channels as a therapeutic strategy in acute leukemia and proposes co-treatment with memantine, a licensed and safe drug, as a potential approach to promote cytarabine-based cell death of various subtypes of acute leukemia.

Oxytocin Administration Alleviates Acute but Not Chronic Leptin Resistance of Diet-Induced Obese Mice

Whereas leptin administration only has a negligible effect on the treatment of obesity, it has been demonstrated that its action can be improved by co-administration of leptin and one of its sensitizers. Considering that oxytocin treatment decreases body weight in obese animals and humans, we investigated the effects of oxytocin and leptin cotreatment. First, lean and diet-induced obese (DIO) mice were treated with oxytocin for 2 weeks and we measured the acute leptin response. Second, DIO mice were treated for 2 weeks with saline, oxytocin (50 μg/day), leptin (20 or 40 µg/day) or oxytocin plus leptin. Oxytocin pre-treatment restored a normal acute leptin response, decreasing food intake and body weight gain. Chronic continuous administration of oxytocin or leptin at 40 µg/day decreased body weight in the presence (leptin) or in the absence (oxytocin) of cumulative differences in food intake. Saline or leptin treatment at 20 µg/day had no impact on body weight. Oxytocin and leptin cotreatments had no additional effects compared with single treatments. These results point to the fact that chronic oxytocin treatment improves the acute, but not the chronic leptin response, suggesting that this treatment could be used to improve the short-term satiety effect of leptin.

Cotinine Plus Krill Oil Decreased Depressive Behavior, and Increased Astrocytes Survival in the Hippocampus of Mice Subjected to Restraint Stress.

Restraint stress (RS) is a condition affecting millions of people worldwide. The investigation of new therapies to alleviate the consequences of prolonged RS is much needed. Cotinine, a nicotine-derivative, has shown to prevent the decrease in cerebral synaptic density, working memory deficits, anxiety, and depressive-like behavior after prolonged restraint stress (RS) in mice. Furthermore, post-treatment with cotinine reduced the adverse effects of chronic RS on astrocyte survival and architecture. On the other hand, the nutritional supplement krill oil (KO), has shown to be beneficial in decreasing depressive-like behavior and oxidative stress. In this study, in the search for effective preventative treatments to be used in people subjected to reduced mobility, the effect of co-treatment with cotinine plus KO in mice subjected to prolonged RS was investigated. The results show that cotinine plus KO prevented the loss of astrocytes, the appearance of depressive-like behavior and cognitive impairment induced by RS. The use of the combination of cotinine plus KO was more effective than cotinine alone in preventing the depressive-like behavior in the restrained mice. The potential use of this combination to alleviate the psychological effects of reduced mobility is discussed.

Effects of carbendazim and astaxanthin co-treatment on the proliferation of MCF-7 breast cancer cells.

There has been a controversy in the oncology field about the use of antioxidants along with chemotherapeutics in cancer treatment. This study aimed to investigate the effects of a potent antioxidant (astaxanthin) co-treatment with a promising anti-cancer drug (carbendazim), which is in phase I clinical trials, on MCF-7 breast cancer cell proliferation. MCF-7 cells were treated with carbendazim, astaxanthin, or their combinations and incubated for 24h. After the incubation, each treatment group was evaluated for proliferation, cell cycle progression, and production of reactive oxygen species (ROS) using WST-1, flow cytometry, and CM-H2DCFDA, respectively. All tested carbendazim and astaxanthin combinations increased the anti-proliferative effect of Carb treatment alone and increased the G2/M phase cell cycle arrest compared to the DMSO-treated control. Astaxanthin, at all concentrations tested, reduced the elevated intracellular ROS levels induced by the carbendazim treatment. Our data suggest that astaxanthin and carbendazim co-treatment enhances the anti-proliferative effect of carbendazim as a single agent, while alleviating the carbendazim treatment-associated ROS production in MCF-7 cells. These findings may contribute to the current debate on the use of antioxidants along with anti-cancer drugs in cancer chemotherapy.

Synergistic Anticancer Effects of Curcumin and Hinokitiol on Gefitinib Resistant Non-Small Cell Lung Cancer Cells

This study evaluated the synergistic effect of curcumin (diferuloylmethane) and hinokitiol (β-thujaplicin), natural product derived phytochemicals, on gefitinib (Iressa) resistant non-small cell lung cancer (NSCLC) cells. Gefitinib, a tyrosine kinase inhibitor targeting epidermal growth factor receptor (EGFR), is widely used for lung cancer treatment. However, gefitinib resistance is easily acquired by NSCLC and followed by the development of progressive disease. Curcumin and hinokitiol are well-known bioactive compounds demonstrating anti-inflammation, anti-bacteria and anticancer effects. However, the effects of co-treatment of curcumin and hinokitiol on cancer cells have not been reported. Here, we postulated, for the first time, the possibility of combination therapy with curcumin and hinokitiol for treatment of gefitinib resistant NSCLC via increment of apoptosis and lysosomal enlargement.

96 - Retino-protective effect of Bucida buceras against oxidative stress induced by H2O2 in human retinal pigment epithelial cells line

Background Reactive Oxygen Species (ROS) impair the physiological functions of Retinal Pigment Epithelial (RPE) cells, which are known as one major cause of age-related macular degeneration and retinopathy diseases. The purpose of this study is to explore the cytoprotective effects of the antioxidant Bucida buceras extract in co-treatment with hydrogen peroxide (H2O2) delivery as a single addition or with continuous generation using glucose oxidase (GOx) in ARPE-19 cell cultures. The mechanism of Bucida buceras extract is believed to be associated with their antioxidant capacity to protect cells against oxidative stress. Material and methods A comparative oxidative stress H2O2-induced was performed by addition and enzymatic generation using glucose oxidase on human retinal pigment epithelial cells line. H2O2-induced injury was measured by toxic effects (cell death and apoptotic pathway) and intracellular redox status: glutathione (GSH), antioxidant enzymes (catalase and glutathione peroxidase) and reducing power (FRAP). The retino-protective effect of co-treatment with Bucida buceras extract on H2O2-induced human RPE cell injury was investigated by cell death (MTT assay) and oxidative stress biomarkers (H2O2, GSH, CAT, GPx and FRAP). Results Bucida buceras L. extract is believed to be associated with the ability to prevent cellular oxidative stress. When added as a pulse, H2O2 is rapidly depleted and the cytotoxicity analyses show that cells can tolerate short exposure to high peroxide doses delivered as a pulse but are susceptible to lower chronic doses. Co-treatment with Bucida buceras was able to protect the cells against H2O2-induced injury. In addition to preventing cell death treatment with antioxidant plant could also reverse the significant decrease in GSH level, catalase activity and reducing power caused by H2O2. Conclusion These findings suggest that Bucida buceras could protect RPE against ocular pathogenesis associated with oxidative stress induced by H2O2-delivered by addition and enzymatic generation.

Enhanced efficacy of histone deacetylase inhibitor combined with bromodomain inhibitor in glioblastoma

BackgroundGlioblastoma (GBM) is the most common and most malignant primary brain cancer in adults. Despite multimodality treatment, the prognosis is still poor. Therefore, further work is urgently required to discover novel therapeutic strategies for GBM treatment.MethodsThe synergistic effects of cotreatment with the histone deacetylase (HDAC) inhibitor panobinostat and bromodomain inhibitor JQ1 or OTX015 were validated using cell viability assays in GBM cell lines. Furthermore, the inhibitory mechanisms were investigated via an EdU proliferation assay, an apoptosis assay, qPCR, Western blot and RNAseq analyses.ResultsWe found that the cotreatment with panobinostat and JQ1 or OTX015 synergistically inhibited cell viability in GBM cells. The cotreatment with panobinostat and JQ1 or OTX015 markedly inhibited cell proliferation and induced apoptosis in GBM cells. Compared with treatment with each drug alone, the cotreatment with panobinostat and JQ1 induced more profound caspase 3/7 activation and cytotoxicity. Mechanistic investigation showed that combination of panobinostat with JQ1 or OTX015 results in stronger repression of GBM-associated oncogenic genes or pathways as well as higher induction of GBM-associated tumor-suppressive genes.ConclusionOur study demonstrated that HDAC inhibitor and bromodomain inhibitor had synergistical efficacy against GBM cells. The cotreatment with HDAC inhibitor and bromodomain inhibitor warrants further attention in GBM therapy.

Leukemia inhibitory factor inhibits erythropoietin-induced myelin gene expression in oligodendrocytes

BackgroundThe pro-myelinating effects of leukemia inhibitory factor (LIF) and other cytokines of the gp130 family, including oncostatin M (OSM) and ciliary neurotrophic factor (CNTF), have long been known, but controversial results have also been reported. We recently overexpressed erythropoietin receptor (EPOR) in rat central glia-4 (CG4) oligodendrocyte progenitor cells (OPCs) to study the mechanisms mediating the pro-myelinating effects of erythropoietin (EPO). In this study, we investigated the effect of co-treatment with EPO and LIF.MethodsGene expression in undifferentiated and differentiating CG4 cells in response to EPO and LIF was analysed by DNA microarrays and by RT-qPCR. Experiments were performed in biological replicates of N ≥ 4. Functional annotation and biological term enrichment was performed using DAVID (Database for Annotation, Visualization and Integrated Discovery). The gene-gene interaction network was visualised using STRING (Search Tool for the Retrieval of Interacting Genes).ResultsIn CG4 cells treated with 10 ng/ml of EPO and 10 ng/ml of LIF, EPO-induced myelin oligodendrocyte glycoprotein (MOG) expression, measured at day 3 of differentiation, was inhibited ≥4-fold (N = 5, P < 0.001). Inhibition of EPO-induced MOG was also observed with OSM and CNTF. Analysis of the gene expression profile of CG4 differentiating cells treated for 20 h with EPO and LIF revealed LIF inhibition of EPO-induced genes involved in lipid transport and metabolism, previously identified as positive regulators of myelination in this system. In addition, among the genes induced by LIF, and not by differentiation or by EPO, the role of suppressor of cytokine signaling 3 (SOCS3) and toll like receptor 2 (TLR2) as negative regulators of myelination was further explored. LIF-induced SOCS3 was associated with MOG inhibition; Pam3, an agonist of TLR2, inhibited EPO-induced MOG expression, suggesting that TLR2 is functional and its activation decreases myelination.ConclusionsCytokines of the gp130 family may have negative effects on myelination, depending on the cytokine environment.

Protective effects of combined Losartan and Nilotinib on carbon tetrachloride (CCl4)-induced liver fibrosis in rats

Tyrosine kinase inhibitors (TKIs) have been developed as therapeutic compounds for inhibiting the progression of liver fibrosis. In the present study, the simultaneous treatment of Nilotinib (TKIs) and Losartan was studied. Forty rats were divided into eight groups of fibrosis induced by carbon tetrachloride (CCl4) and therapeutics (Nilotinib, Losartan, and combination therapy). In the end, serum parameters of the liver and gene expression analysis of transforming growth factor-β1, its receptors (TβRII), platelet-derived growth factor, its receptors (PDGFRβ), matrix metalloproteinases (MMP-2 and MMP-9), tumor necrosis factor-α, cytochrome P450 2E1, and collagen1 type 1 were performed. The oxidant/antioxidant factors were also analyzed. Histopathology analysis along with α-SMA immunohistochemistry and hydroxyproline evaluation was also conducted for a more in-depth study. The overall results indicated a better therapeutic effect of co-treatment of Nilotinib–Losartan in comparison with the treatment of each of them alone. Interestingly, some gene and protein factors and fibrotic indices were reduced even to the normal levels of the control group. The results of this study suggest that co-administration of these two combinations, strengthens their anti-fibrotic properties and, due to the routine use of these compounds against AML and blood pressure, these compounds can be used with caution against human liver fibrosis.

Synergistic antimetastatic effect of cotreatment with licochalcone A and sorafenib on human hepatocellular carcinoma cells through the inactivation of MKK4/JNK and uPA expression.

To improve the clinical outcome of tumor chemotherapy, more effective combination treatments against tumor metastasis and recurrence are required. Licochalcone A (LicA) is the root of Glycyrrhiza inflata and has been reported to possess anti-inflammatory, antimicrobial, and antitumor effects. Sorafenib (Sor), a multikinase inhibitor, is used to treat patients with solid tumors such as advanced hepatocellular carcinoma (HCC). However, the synergistic effects of LicA and Sor on the metastasis of human HCC cells have not been reported. We found that LicA and Sor did not have cytotoxic effects or arrest growth in human SK-Hep-1 and Huh-7 cells. In addition, treatment with LicA or Sor alone inhibited migration and invasion in human SK-Hep-1 and Huh-7 HCC cells. Furthermore, cotreatment with LicA and Sor synergistically inhibited the migration and invasion of HCC cells and significantly inhibited uPA protein expression. Notably, cotreatment of LicA and Sor synergistically and significantly downregulated MKK4-JNK expression. Through tail vein injection in nude mice, the aforementioned cotreatment synergistically suppressed SK-Hep-1 cell-mediated lung metastasis. These findings first revealed the synergistic effects of LicA and Sor cotreatment against human HCC cells, further suggesting that beneficial effects on tumor regression could be confirmed through prospective clinical trials.

Oleoylethanolamide and Palmitoylethanolamide Protect Cultured Cortical Neurons Against Hypoxia.

Introduction: Perinatal hypoxic–ischemic (HI) encephalopathy is defined as a neurological syndrome where the newborn suffers from acute ischemia and hypoxia during the perinatal period. New therapies are needed. The acylethanolamides, oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), possess neuroprotective properties, and they could be effective against perinatal HI. These lipid mediators act through peroxisome proliferator-activated receptors subtype α (PPARα), or transient receptor potential vanilloid (TRPV), such as TRPV subtype 1 and 4.Materials and Methods: The objectives of this study were to discern: (1) the neuroprotective role of OEA and PEA in parietotemporal cortical neurons of newborn rats and mice subjected to hypoxia, and (2) the role of the receptors, PPARα, TRPV1, and TRPV4, in neuroprotective effects. Cell culture of cortical neurons and the lactate dehydrogenase assay was carried out. The role of receptors was discerned by using selective antagonist and agonist ligands, as well as knockout (KO) PPARα mice.Results: The findings indicate that OEA and PEA exert neuroprotective effects on cultured cortical neurons subjected to a hypoxic episode. These protective effects are not mediated by the receptors, PPARα, TRPV1, or TRPV4, because neither PPARα KO mice nor receptor ligands significantly modify OEA and PEA-induced effects. Blocking TRPV4 with RN1734 is neuroprotective per se, and cotreatment with OEA and PEA is able to enhance neuroprotective effects of the acylethanolamides. Since stimulating TRPV4 was devoid of effects on OEA and PEA-induced protective effects, effects of RN1734 cotreatment seem to be a consequence of additive actions.Conclusion: The lipid mediators, OEA and PEA, exert neuroprotective effects on cultured cortical neurons subjected to hypoxia. Coadministration of OEA or PEA, and the TRPV4 antagonist RN1734 is able to enhance neuroprotective effects. These in vitro results could be of utility for developing new therapeutic tools against perinatal HI.

Influence of hesperidin and vitamin C on glycemic parameters, lipid profile, and DNA damage in rats treated with sucrose overload.

We evaluated the influence of hesperidin and vitamin C (VitC) on glycemic parameters, lipid profile, and DNA damage in male Wistar rats treated with sucrose overload. Rats were divided into six experimental groups: I-water control; II-sucrose control; III-hesperidin control; IV-VitC control; V-co-treatment of sucrose plus hesperidin; VI-co-treatment of sucrose plus VitC. We measured the levels of triglycerides, total cholesterol, HDL-c, LDL-c, fasting glucose, and glycated hemoglobin (A1C). DNA damage was evaluated in blood and brain cells using the comet assay and the micronucleus test was used to evaluate chromosomal damages in the rat bone marrow. Co-treatment with VitC, but not with hesperidin, normalized the serum glucose. No effect of co-treatments was observed on A1C. The co-treatment with VitC or hesperidin did not influence the lipid profile (p>0.05). Rats co-treated with hesperidin had a significantly lower DNA damage level in blood (p<0.05) and brain (p<0.05). Rats treated with VitC only, but not those co-treated with VitC plus sucrose, had significantly higher DNA damage in brain (p<0.05). No significant differences were observed in the results of micronucleus test (p>0.05). Hesperidin and VitC showed different effects on sucrose and DNA damage levels. While VitC lowered the serum glucose, hesperidin reduced the DNA damage.

Effects of cotreatment with omega-3 polyunsaturated fatty acids and anticancer agents on oxidative stress parameters: a systematic review of in vitro, animal, and human studies.

Omega-3 (n-3) polyunsaturated fatty acids (PUFAs), especially docosahexaenoic acid and eicosapentaenoic acid, demonstrate possible beneficial effects as adjuvants in cancer treatment. One mechanism seems to be related to alterations in the redox status of cancer cells. Such alterations are thought to act in synergy with conventional anticancer agents. This review examines published data on the effects of cotreatment with anticancer agents and n-3 PUFAS on oxidative stress parameters to determine whether any patterns of oxidative stress alterations can be identified. A systematic search of MEDLINE (via PubMed) was conducted to identify articles published in English, Spanish, or Portuguese until November 2017. The following inclusion criteria were applied: (1) individuals or animals with cancer or malignant cell lines supplemented with some source of n-3 PUFAs; (2) concomitant use of anticancer treatment; and (3) evaluation of oxidative stress-related variables. A standardized outline was used to extract the following data: study type, supplement used, type of cells, tumor or patient characteristics, study design, anticancer treatment used, and oxidative stress-related outcomes. After the literature search and screening of 1563 citations, 28 studies were included for data extraction and evaluation: 16 in vitro studies (2 of which also used in vivo studies), 8 animal studies, and 4 human studies (3 clinical trials and 1 case series). In most in vitro and animal studies, intervention groups receiving cotreatment with n-3 PUFAs showed enhanced lipid peroxidation and cytotoxicity compared with groups receiving anticancer treatment alone. Eleven of the 12 studies that investigated the effect of vitamin E on the sensitivity of cancer cells to the oxidative stress caused by n-3 PUFAs showed that vitamin E abolished the positive effects of cotreatment. Alterations in oxidative stress caused by cotreatment with anticancer agents and n-3 PUFAs can exert positive effects on the efficacy of conventional treatment. This seems to occur in most cells and tumors tested thus far, but not all. Identifying tumors that are sensitive to these oxidative effects may provide support for the rational use of n-3 PUFAs as an adjuvant treatment in specific types of cancer.

PO-003 Metformin restores nickel-reduced glutathione S-transferase Mu2 expression by promoter methylated alteration

IntroductionGlutathione S-transferases M2 (GST-M2) is a family of inducible enzymes that are important in carcinogen detoxification. GST-M2 has been reported as a great potential tumour suppressor and low expression of GST-M2 has associated with promoter hypermethylation in lung cancer cells. Nickel compounds have been identified as human carcinogens. Our objective is to clarify the expression of GST-M2 in lung cells under nickel exposure. Moreover, we attempted to investigate the effects of the antidiabetic drug metformin on GST-M2 expression and lung cancer chemoprevention.Material and methodsGST-M2 mRNA level and transcription activity were measured by real-time PCR and reporter assay. CHIP assay, bisulfite sequencing and methylation specific PCR (MSP) and site-directed reporter assay were used to evaluate the effects of nickel and metformin co-treatments, including silencing of Sp1 by shRNA, on promoter epigenetic alteration in BEAS-2B cells.Results and discussionsGST-M2 mRNA level but not GST-M1 was significantly reduced in lung bronchial epithelial cells BEAS-2B and fibroblast WI-38 after nickel treatment. Metformin restored nickel-decreased GST-M2 mRNA expression and promoter activity in BEAS-2B cellsGST-M2 exhibited high frequency of promoter hypermethylation in nickel-exposure cells and metformin reversed. After nickel and metformin co-treatment, the methylation of specificity protein 1 (Sp1) was revealed binding to GST-M2 promoter and regulating gene transcription.ConclusionOur results demonstrated that metformin rescues nickel-inhibited GST-M2 expression via Sp1-binding and promoter methylated status alteration.

Immune Modulatory Effect of Thalidomide and Dexamethasone Co-Treatment on Immune Cells

Immune Modulatory Effect of Thalidomide and Dexamethasone Co-Treatment on Immune Cells

Abstract 3531: Glycolysis inhibition and its effect in doxorubicin resistance in neuroblastoma

Abstract Most cancer cells exhibit increased glycolysis and use this metabolic pathway to generate ATP (Adenosine triphosphate) as the main source of their energy supply. This phenomenon is known as the Warburg effect and is considered a fundamental metabolic alteration during malignant transformation. Increased glycolysis results in elevated production of lactate, which leads to acidification of tumor tissue and provides a microenvironment that promotes and selects cellular resistance to drugs. Thus, inhibition of glycolysis may lead to more effective treatment strategies for dealing with drug resistance. 3-Bromopyruvate (3-BrPA), an inhibitor of hexokinase II and an inhibitor of glycolysis, has been shown to be a potent chemotherapeutic agent. Despite many studies showing the efficacy of 3-BrPA against various cancer cells, there have been relatively few studies examining the effects of 3-BrPA on the anti-cancer effects of doxorubicin (Dox) in doxorubicin-resistant (DoxR) human neuroblastoma. The purpose of this study is to examine the effects of 3-BrPA upon DoxR human neuroblastoma cells to treatment with doxorubicin. In present study, we have found that 3-BrPA enhances the response of DoxR neuroblastoma to treatment with Dox and this enhancement exists in both normoxic conditions as well as hypoxic conditions. The results also suggested that this increased response of DoxR cells to treatment with Dox is mediated through decreased intracellular levels of ATP. Additionally the lactate assay revealed that intracellular lactate levels were decreased in neuroblastoma lines when glycolysis was inhibited with 3-BrPA. In order to determine if 3-BrPA, either alone or in combination with cytotoxic drugs, will lead to maximal efficacy of treating of drug-resistant tumor xenografts in vivo. Animal experiments were carried out to study the effects of 3-BrPA in drug-resistant neuroblastoma in nude mice. We have found that 3-BrPA combined with doxorubicin provided significant reduction in tumor size and the rate of tumor growth in mice. The efficacy of doxorubicin itself (88% reduction in tumor volume) overshadowed any effect of cotreatment with 3-BrPA on the Wild type tumors. However, in the DoxR tumors, doxorubicin alone had no efficacy as expected, but co-treatment with 3-BrPA suppressed tumor growth with an 80% reduction in tumor volume. These studies pave the way to determine whether inhibition of glycolysis may be an effective strategy to kill neuroblastoma cells and overcome drug resistance associated with hypoxic conditions. This novel approach may have broad applications in cancer treatment, considering the prevalent Warburg effect observed in a wide array of human cancers. Citation Format: Fei Chu, Yiyong Qiu, Sandra Clark, Kathryn Solka, Mary Beth Madonna. Glycolysis inhibition and its effect in doxorubicin resistance in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3531.

Abstract 885: Anthracycline-induced apoptosis through caspase3/7 activity is p53-dependent in bladder cancer cells

Abstract Doxorubicin (Dox) is a chemotherapy drug widely used for treatment of several cancers including bladder cancer. However, cardiovascular side effects caused by doxorubicin-induced cardiotoxicity has necessitated the synthesis and validation of next generation chemotherapy drugs that are as effective but not cardiotoxic. AD 312 (N-Nitrosureidodaunorubicin; Daunomustine®, Paradox Pharmaceuticals, Inc.) and AD 198 (N- benzyladriamycin-14-valerate; Benzarubicin®, Paradox Pharmaceuticals, Inc.) are novel anthracyclines with confirmed non-cardiotoxicity in mice. Compared to Dox, AD 312 and AD 198 induce apoptosis in cells through distinct mechanisms. The tumor suppressor gene p53 is a key regulator of cell-cycle arrest and apoptosis. Dysregulation of p53 expression and function was identified in over 70% of the bladder cancers and reported as a potential target for therapy. As a result, small molecule compounds that can restore mutated p53 function to wild-type, including PRIMA-1, are under intensive investigation. In this study, we have compared the efficacy of AD 312 and AD 198 to Dox to induce apoptosis in human bladder transitional cell carcinoma (TCC) cells by MTT assay and assessed the possible mechanisms of anthracyclines-induced apoptosis in TCC cells expressing wild-type (wt) p53 (RT4, SW780) and mutated (mt) p53 (UM-UC-3, 5637, J82, and TCCSUP) protein. We have also investigated the effects of co-treatment of anthracyclines with PRIMA-1 in J82 cells, as a representative mt-p53 TCC cell line, and compared the co-treatment effects in RT4, as a representative wt-p53 cell line. Our results demonstrated that anthracyclines inhibited cell viability of all tested TCC cells in vitro, with AD 198 demonstrating highest efficacy. Furthermore, anthracyclines upregulated p53 protein expression in wt-p53 RT4 and SW780 cells, but not in tested mt-p53 UM-UC-3, 5637, J82, and TCCSUP cells. A strong correlation was observed between p53 mutation status and activation of caspase 3/7 and cleavage of PARP. The upregulated caspase 3/7 activity and PARP cleavage was observed in wt-p53, but not in mt-p53 TCC cells. AD198 blocked c-myc expression in mt-p53 TCC cells and no expression of c-myc was detected in wt-p53 TCC cells. Furthermore, sensitivity to AD 198 treatment in mt-p53 J82 cells was significantly improved by a co-treatment with PRIMA-1. However, PRIMA-1 had no effect on cell viability in RT4, the wt-p53 TCC cells. Overall, our results demonstrated that AD 312 and AD 198 are potent inhibitors of bladder cancer cells in vitro and are promising candidates for new therapeutic strategies to replace Dox for bladder cancer treatment. PRIMA-1 even further sensitized mt-p53 cells to AD 198 treatment and thereby this co-treatment improved the inhibition of bladder cancer in vitro. Citation Format: Sony Pandey, Leonard Lothstein, Maria Cekanova. Anthracycline-induced apoptosis through caspase3/7 activity is p53-dependent in bladder cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 885.

Hypermethylated CD36 gene affected the progression of lung cancer

ObjectiveOur study aimed to explore the relationship between CD36 methylation and the development of lung cancer and investigate the effect of combine treatment of Decitabine and Chidamide in lung cancer. MethodsThe differentially expression genes in tumor samples and normal samples were determined by microarray analysis. Weighted gene co-expression network analysis (WGCNA) was utilized to analyze gene expression data of lung cancer and the hub genes were screened in the modules. Methylation-specific PCR (MSP) was conducted to detect the methylation level of CD36 in lung cancer cells. QRT-PCR analysis and western blotting were performed to explore the relative mRNA expression and protein level. MTT assays, wound healing assay, Transwell assays and flow cytometry were conducted to clarify the cell proliferation, migration, invasion and cell cycle of lung carcinoma cell lines in vitro respectively. By xenograft and immunohistochemistry, the effect of co-treatment of Decitabine and Chidamide was further verified in vivo. ResultsThe heatmap displayed that the top 20 differential mRNA-expression gene in lung cancer tissues and normal tissues in which CD36 was low expressed in the tumor samples and high expressed in the normal samples. By WGCNA, CD36 was selected to be the hub gene in the brown module. And then CD36 was confirmed to be differential expressed and hypermethylated in lung cancer through qRT-PCR and western blotting. CD36 inhibited the lung cancer cell proliferation, promoted cell apoptosis, blocked cell cycle at G0/G1 phase, and inhibited cell migration. What's more, we found that Decitabine (DCTB) and Chidamide (CDM) co-treatment induced de-methylation and re-expression of silenced CD36 by conducting the vivo experiments. DCTB + CDM co-treatment synergistically suppressed tumor growth. ConclusionOur results showed that the high methylation of CD36 in lung cancer played an important role in the procession of lung cancer and Decitabine joint Chidamide had obvious effect of inhibiting the growth of lung tumor.