Studies of the mechanism of daunorubicin carcinogenesis: Modulation by vitamin E

Daunorubicin (DNR) is an anthracycline antibiotic used as a cancer chemotherapeutic agent. However, it causes mammary adenocarcinomas in female Sprague-Dawley (SD) rats. Vitamin E (E) has been found to reduce DNR carcinogenicity. I investigated the mechanism of DNR carcinogenicity and its interaction with E in SD rats by studying DNR-DNA adduct formation and the influence of E status on DNR clearance and free radical producing and detoxifying enzymes.^ The hypothesis was that DNR exerts its tumorigenic effect via free radicals generated during redox cycling and production of reactive intermediates capable of forming DNA adducts. E was postulated to act as a protective agent through a combination of its antioxidant property, modulation of drug clearance and levels of free radical producing and detoxifying enzymes.^ DNA adduct formation was measured by the nuclease P1 $\sp{32}$P-post labeling assay. In vitro, DNR was activated by rat liver microsomes and either NADPH or cumene hydrogen peroxide (CuOOH). Rat liver DNA incubated with this mixture formed two adducts when the cofactor was NADPH and three adducts when CuOOH was used. In vivo, SD rats were treated with i.v. doses of DNR. No detectable DNR-DNA adducts were formed in liver or mammary DNA in vivo, although there was an intensification of endogenous DNA adducts.^ Groups, 1, 2, 3 and 4 of weanling female SD rats were fed 0, 100, 1,000 and 10,000 mg $\alpha$-tocopheryl acetate/kg diet respectively. A comparison of Groups 1 and 4 showed no effect of E status on clearance of 10 mg tritiated DNR/kg body weight over 72 hours. However, liver cleared DNR at a faster rate than mammary epithelial cells (MEC).^ Xanthine oxidase, which catalyzes DNR redox cycling, was significantly decreased in liver and MEC of rats in group 4 compared to groups 1, 2, and 3. Detoxifying enzymes were not dramatically affected by E supplementation. Quinone reductase in MEC was significantly increased in group 4 compared to other groups. Overall, the liver had higher levels of free radical detoxifying enzymes compared to MEC.^ These data support a role of free radicals in DNR carcinogenicity because (1) endogenous DNA adducts formed due to free radical insult are further intensified by DNR treatment in vivo, (2) MEC, the specific target of DNR carcinogenicity, cannot rapidly clear DNR and have a lower free radical detoxifying capability than liver, (3) E supplementation caused lowering of free radical generating potential via xanthine oxidase, and increased DNR detoxification due to elevation of quinone reductase in MEC. ^

Gene by BMI Interactions Influencing C-Reactive Protein Levels in European-Americans

C-Reactive Protein (CRP) is a biomarker indicating tissue damage, inflammation, and infection. High-sensitivity CRP (hsCRP) is an emerging biomarker often used to estimate an individual’s risk for future coronary heart disease (CHD). hsCRP levels falling below 1.00 mg/l indicate a low risk for developing CHD, levels ranging between 1.00 mg/l and 3.00 mg/l indicate an elevated risk, and levels exceeding 3.00 mg/l indicate high risk. Multiple Genome-Wide Association Studies (GWAS) have identified a number of genetic polymorphisms which influence CRP levels. SNPs implicated in such studies have been found in or near genes of interest including: CRP, APOE, APOC, IL-6, HNF1A, LEPR, and GCKR. A strong positive correlation has also been found to exist between CRP levels and BMI, a known risk factor for CHD and a state of chronic inflammation. We conducted a series of analyses designed to identify loci which interact with BMI to influence CRP levels in a subsample of European-Americans in the ARIC cohort. In a stratified GWA analysis, 15 genetic regions were identified as having significantly (p-value < 2.00*10-3) distinct effects on hsCRP levels between the two obesity strata: lean (18.50 kg/m2 < BMI < 24.99 kg/m2) and obese (BMI ≥ 30.00 kg/m2). A GWA analysis performed on all individuals combined (i.e. not a priori stratified for obesity status) with the inclusion of an additional parameter for BMI by gene interaction, identified 11 regions which interact with BMI to influence hsCRP levels. Two regions containing the genes GJA5 and GJA8 (on chromosome 1) and FBXO11 (on chromosome 2) were identified in both methods of analysis suggesting that these genes possibly interact with BMI to influence hsCRP levels. We speculate that atrial fibrillation (AF), age-related cataracts and the TGF-β pathway may be the biological processes influenced by the interaction of GJA5, GJA8 and FBXO11, respectively, with BMI to cause changes in hsCRP levels. Future studies should focus on the influence of gene x bmi interaction on AF, age-related cataracts and TGF-β.

Upregulation of Reactive Oxygen Species During the Retrovirus Life Cycle and Their Roles in a Mutant of Moloney Murine Leukemia Virus, ts1-Mediated Neurodegeneration

Viral invasion of the central nervous system (CNS) and development of neurological symptoms is a characteristic of many retroviruses. The mechanism by which retrovirus infection causes neurological dysfunction has yet to be fully elucidated. Given the complexity of the retrovirus-mediated neuropathogenesis, studies using small animal models are extremely valuable. Our laboratory has used a mutant moloney murine leukemia retrovirus, ts1-mediated neurodegneration. We hypothesize that astrocytes play an important role in ts1-induced neurodegeneration since they are retroviral reservoirs and supporting cells for neurons. It has been shown that ts1 is able to infect astrocytes in vivo and in vitro. Astrocytes, the dominant cell population in the CNS, extend their end feet to endothelial cells and neuronal synapse to provide neuronal support. Signs of oxidative stress in the ts1-infected CNS have been well-documented from previous studies. After viral infection, retroviral DNA is generated from its RNA genome and integrated into the host genome. In this study, we identified the life cycle of ts1 in the infected astrocytes. During the infection, we observed reactive oxygen species (ROS) upregulations: one at low levels during the early infection phase and another at high levels during the late infection phase. Initially we hypothesized that p53 might play an important role in ts1-mediated astrocytic cell death. Subsequently, we found that p53 is unlikely to be involved in the ts1-mediated astrocytic cell death. Instead, p53 phosphorylation was increased by the early ROS upregulation via ATM, the protein encoded by the ataxia-telangiectasia (A-T) mutated gene. The early upregulation of p53 delayed viral gene expression by suppressing expression of the catalytic subunit of NADPH oxidase (NOX). We further demonstrated that the ROS upregulation induced by NOX activation plays an important role in establishing retroviral genome into the host. Inhibition of NOX decreased viral replication and delayed the onset of pathological symptoms in ts1-infected mice. These observations lead us to conclude that suppression of NOX not only prevents the establishment of the retrovirus but also decreases oxidative stress in the CNS. This study provides us with new perspectives on the retrovirus-host cell interaction and sheds light on retrovirus-induced neurodegeneration as a result of the astrocyte-neuron interaction.

Resting and reactive blood pressure as predictors of ambulatory blood pressure in older hypertensive adults

Background. A review of the literature suggests that Hypertension (HTN) in older adults is associated with sympathetic stimulation that results in increasing blood pressure (BP) reactivity. If clinical assessment of BP captured sympathetic stimulation, it would be valuable for hypertension management. ^ Objectives. The study examined whether reactive change scores from a short BPR protocol, resting blood pressure (BP), or resting pulse pressure (PP) is a better predictor of 24 hour ambulatory BP and BP load in cardiac patients. ^ Method. The study used a single-group design, with both an experimental clinical component and an observational field component. Both components used repeated measurement methods. The study population consisted of 45 adult patients with a mean age of 64.6 ± 8.5 years who were diagnosed with cardiac disease and who were taking anti-hypertensive medication. Blood pressure reactivity was operationalized with a speech protocol. During the speech protocol, BP was measured with an automatic device (Dinamap 825XT) while subjects talked about their health and about their usual day. Twenty-four hour ambulatory BP measurement (Spacelabs 90207 monitor) followed the speech protocol. ^ Results. Resting SBP and resting PP were significant predictors of 24-hour SBP, and resting SBP was a significant predictor of SBP load. No predictors were significant of 24-hour DBP or DBP load. ^ Conclusions. Initial resting BP and PP may be used in clinical settings to assess hypertension management. Future studies are necessary to confirm the ability of resting BP to predict ABP and BP load in older, medicated hypertensives. ^

Reactive oxygen species-dependent mechanisms of N-(4-hydroxyphenyl)retinamide (4HPR)-induced apoptosis in human carcinoma cells

4HPR is a synthetic retinoid that has shown chemopreventive and therapeutic efficacy against premalignant and malignant lesions including oral leukoplakia, ovarian and breast cancer, and neuroblastoma. 4HPR induces apoptosis in various cancer cells and production of reactive oxygen species (ROS) has been suggested as a possible cause underlying these effects. However, the mechanisms governing these effects by 4HPR are not fully elucidated. In this study, we explored the mechanisms of 4HPR-induced ROS increase and apoptosis in human cancer cells. ^ First, we identified genes modulated by 4HPR using oligonucleotide gene expression arrays and found that they fall into specific functional canonical pathways and gene networks using Ingenuity Pathways Analysis®. Further analysis has shown that 4HPR induced up-regulation of Endoplasmic Reticulum (ER)-related genes such as Heat shock proteins 70 and 90 and the transcriptional factor, GADD153. These findings were validated using quantitative real-time PCR. ^ Second, we found that 4HPR induced extensive ER stress evidenced by dilation of the ER and endoribonuclease-mediated splicing and activation of the transcriptional factor, XBP-1. In addition, 4HPR induced the up-regulation of various ER stress-related genes and their protein products, as well as cleavage and activation of the ER specific Caspase-4. Concomitantly with XBP-1 splicing, all of these effects were dependent on ROS generation by 4HPR. Furthermore, chemical inhibition and RNA interference studies revealed a novel pro-apoptotic role for HSP70/A1A in 4HPR-mediated apoptosis. ^ Third, we observed rapid activation of the small GTPase Rac by 4HPR which was upstream of ROS generation. Inhibition of Rac activity or silencing of its expression by RNA interference inhibited ROS generation and apoptosis induction by 4HPR. siRNA targeting PAK1 and expression of a dominant negative Rac, decreased 4HPR-mediated ROS generation, while expression of a constitutive active Rac increased basal and 4HPR-induced ROS generation and PARP cleavage. Furthermore, metastatic cancer cells exhibited higher Rac activation, ROS generation, and cell growth inhibition due to 4HPR exposure compared to their primary cancer cell counterparts. ^ These findings provide novel insights into 4HPR-mediated ROS generation and apoptosis induction and support the use of ROS inducing agents such as 4HPR against metastatic cancer cells. ^

The role of reactive astrocytes in the resistance of melanoma brain metastasis to chemotherapy

Brain metastasis is resistant to chemotherapy while the leaky blood-brain-barrier in brain metastasis can not be the underlying reason. Metastatic tumor cells (“seed”) exploit the host microenvironment (“soil”) for survival advantages. Astrocytes which maintain the homeostasis of the brain microenvironment become reactive subsequent to brain damages and protect neurons from various injuries. We observed reactive astrocytes surrounding and infiltrating into brain metastasis in both clinical specimen and experimental animal model, thus raising a possibility that reactive astrocytes may protect tumor cells from cytotoxic chemotherapeutic drugs. ^ To test this hypothesis, we first generated an immortalized astrocyte cell line from H-2Kb-tsA58 mice. The immortal mouse astrocytes expressed specific markers including GFAP. Scanning electron microscopy demonstrated that astrocytes formed direct physical contact with tumor cells. Moreover, the expression of GFAP by astrocytes was up-regulated subsequent to co-culture with tumor cells, indicating that the co-culture of astrocytes and tumor cells may serve as a model to recapitulate the pathophysiological situation of brain metastasis. ^ In co-culture, astrocytes dramatically reduced apoptosis of tumor cells produced by various chemotherapeutic drugs. This protection effect was not because of culturing cells from different species since mouse fibroblasts did not protect tumor cells from chemotherapy. Furthermore, the protection by astrocytes was completely dependent on a physical contact. ^ Gap junctional communication (GJC) served as this physical contact. Tumor cells and astrocytes both expressed the major component of gap junctional channel—connexin 43 and formed functional GJC as evidenced by the “dye transfer” assay. The blockage of GJC between tumor cells and astrocytes by either specific chemical blocker carbenoxolone (CBX) or by genetically knocking down connexin 43 on astrocytes reversed the chemo-protection. ^ Calcium was the signal molecule transmitted through GJC that rescued tumor cells from chemotherapy. Accumulation of cytoplasmic calcium preceded the progress of apoptosis in tumor cells treated with chemotherapeutic drugs. Furthermore, chelation of accumulated cytoplasmic calcium inhibited the apoptosis of tumor cells treated with chemotherapeutic drugs. Most importantly, astrocytes could “shunt” the accumulated cytoplasmic calcium from tumor cells (treated with chemotherapeutic drug) through GJC. We also used gene expression micro-array to investigate global molecular consequence of tumor cells forming GJC with astrocytes. The data demonstrated that astrocytes (but not fibroblasts), through GJC, up-regulated the expressions of several well known survival genes in tumor cells. ^ In summary, this dissertation provides a novel mechanism underlying the resistance of brain metastasis to chemotherapy, which is due to protection by astrocytes through GJC. Interference with the GJC between astrocytes and tumor cells holds great promise in sensitizing brain metastasis to chemotherapy and improving the prognosis for patients with brain metastasis. ^

GMZ27 is a new organic arsenic derivative with anti-leukemic activity, via the induction of reactive oxygen species through NADPH oxidase and mitochondrial pathway that lead to leukemia cell apoptosis

Arsenic trioxide (ATO) is an inorganic arsenic derivative that is very effective against relapsed acute promyelocytic leukemia. It is being investigated as therapy for other cancers, but the risk/benefit ratio is questionable due to significant side effects. In contrast, organic arsenic derivatives (OAD) are known to be much less toxic than ATO. Based on high activity, we selected GMZ27 (dipropil-s-glycerol arsenic) for further study and have confirmed its potent activity against human acute leukemia cell lines. This anti-leukemic activity is significantly higher than that of ATO. Both in vivo and in vitro tests have shown that GMZ27 is significantly less toxic to normal bone marrow mononuclear cells and normal mice. Therefore, further study of the biological activity of GMZ27 was undertaken. ^ GMZ27, in contrast to ATO, can only marginally induce maturation of leukemic cells. GMZ27 has no effect on cell cycle. The anti-leukemic activity of GMZ27 against acute myeolocytic leukemia cells is not dependent upon degradation of PML-RARα fusion protein. GMZ27 causes dissipation of mitochondrial transmembrane potential, cleavage of caspase 9, caspase 3 activation. Further studies indicated that GMZ27 induces intracellular reactive oxygen species (ROS) production, and modification of intracellular ROS levels had profound effect on its potential to inhibit proliferation of leukemic cells. Therefore ROS production plays a major role in the anti-leukemic activity of GMZ27. ^ To identify how GMZ27 induces ROS, our studies focused on mitochondria and NADPH oxidase. The results indicated that the source of ROS generation induced by GMZ27 is dose dependent. At the low dose (0.3 uM) GMZ27 induces NADPH oxidase activity that leads to late ROS production, while at the high dose (2.0 uM) mitochondria function is disrupted and early ROS production is induced leading to dramatic cell apoptosis. Therefore, late, ROS production can be detected in mitochondria are depleted Rho-0 cells. Our work not only delineates a major biologic pathway for the anti-leukemic activity of GMZ27, but also discusses possible ways of enhancing the effect by the co-application of NADPH oxidase activator. Further study of this interaction may lead to achieving better therapeutic index.^

LOW REACTIVE OXYGEN SPECIES AND HIGH GLYCOLYSIS IN GLIOBLASTOMA STEM CELLS:MECHANISMS AND THERAPEUTIC IMPLICATIONS

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor with poor prognosis due in part to drug resistance and high incidence of tumor recurrence. The drug resistant and cancer recurrence phenotype may be ascribed to the presence of glioblastoma stem cells (GSCs), which seem to reside in special stem-cell niches in vivo and require special culture conditions including certain growth factors and serum-free medium to maintain their stemness in vitro. Exposure of GSCs to fetal bovine serum (FBS) can cause their differentiation, the underlying mechanism of which remains unknown. Reactive oxygen species (ROS) play an important role in normal stem cell differentiation, but their role in affecting cancer stem cell fate remains unclear. Whether the metabolic characteristics of GSCs are different from other glioblastoma cells and can be targeted are also unknown. In this study, we used several stem-like glioblastoma cell lines derived from clinical tissues by typical neurosphere culture system or orthotopic xenografts, and showed that addition of fetal bovine serum to the medium induced an increase of ROS, leading to aberrant differentiation and decreases of stem cell markers such as CD133. We found that exposure of GSCs to serum induced their differentiation through activation of mitochondrial respiration, leading to an increase in superoxide (O2-) generation and a profound ROS stress response manifested by upregulation of oxidative stress response pathway. This increase in mitochondrial ROS led to a down-regulation of molecules including SOX2, and Olig2, and Notch1 that are important for stem cell function and an upregulation of mitochondrial superoxide dismutase SOD2 that converts O2- to H2O2. Neutralization of ROS by antioxidant N-acetyl-cysteine in the serum-treated GSCs suppressed the increase of superoxide and partially rescued the expression of SOX2, Olig2, and Notch1, and prevented the serum-induced differentiation phenotype. Additionally, GSCs showed high dependence on glycolysis for energy production. The combination of a glycolytic inhibitor 3-BrOP and a chemotherapeutic agent BCNU depleted cellular ATP and inhibited the repair of BCNU-induced DNA damage, achieving strikingly synergistic killing effects in drug resistant GSCs. This study uncovers the metabolic properties of glioblastoma stem cells and suggests that mitochondrial function and cellular redox status may profoundly affect the fates of glioblastoma stem cells via a ROS-mediated mechanism, and that the active glycolytic metabolism in cancer stem cells may provide a biochemical basis for developing novel therapeutic strategies to effectively eliminate GSCs.

The relationship between obesity, autoimmune disease, and C-reactive protein: A secondary data analysis of the National Health and Nutrition Examination Survey (NHANES) 2009--2010

Autoimmune diseases are a group of inflammatory conditions in which the body's immune system attacks its own cells. There are over 80 diseases classified as autoimmune disorders, affecting up to 23.5 million Americans. Obesity affects 32.3% of the US adult population, and could also be considered an inflammatory condition, as indicated by the presence of chronic low-grade inflammation. C-reactive protein (CRP) is a marker of inflammation, and is associated with both adiposity and autoimmune inflammation. This study sought to determine the cross-sectional association between obesity and autoimmune diseases in a large, nationally representative population derived from NHANES 2009–10 data, and the role CRP might play in this relationship. Overall, the results determined that individuals with autoimmune disease were 2.11 times more likely to report being overweight than individuals without autoimmune disease and that CRP had a mediating affect on the obesity-autoimmune relationship. ^

Regulation of the heat shock response by thiol-reactive compounds in the yeast Saccharomyces cerevisiae

Cells govern their activities and modulate their interactions with the environment to achieve homeostasis. The heat shock response (HSR) is one of the most well studied fundamental cellular responses to environmental and physiological challenges, resulting in rapid synthesis of heat shock proteins (HSPs), which serve to protect cellular constituents from the deleterious effects of stress. In addition to its role in cytoprotection, the HSR also influences lifespan and is associated with a variety of human diseases including cancer, aging and neurodegenerative disorders. In most eukaryotes, the HSR is primarily mediated by the highly conserved transcription factor HSF1, which recognizes target hsp genes by binding to heat shock elements (HSEs) in their promoters. In recent years, significant efforts have been made to identify small molecules as potential pharmacological activators of HSF1 that could be used for therapeutic benefit in the treatment of human diseases relevant to protein conformation. However, the detailed mechanisms through which these molecules drive HSR activation remain unclear. In this work, I utilized the baker's yeast Saccharomyces cerevisiae as a model system to identify a group of thiol-reactive molecules including oxidants, transition metals and metalloids, and electrophiles, as potent activators of yeast Hsf1. Using an artificial HSE-lacZ reporter and the glucocorticoid receptor system (GR), these diverse thiol-reactive compounds are shown to activate Hsf1 and inhibit Hsp90 chaperone complex activity in a reciprocal, dose-dependent manner. To further understand whether cells sense these reactive compounds through accumulation of unfolded proteins, the proline analog azetidine-2-carboxylic acid (AZC) and protein cross-linker dithiobis(succinimidyl propionate) (DSP) were used to force misfolding of nascent polypeptides and existing cytosolic proteins, respectively. Both unfolding reagents display kinetic HSP induction profiles dissimilar to those generated by thiol-reactive compounds. Moreover, AZC treatment leads to significant cytotoxicity, which is not observed in the presence of the thiol-reactive compounds at the concentrations sufficient to induce Hsf1. Additionally, DSP treatment has little to no effect on Hsp90 functions. Together with the ultracentrifugation analysis of cell lysates that detected no insoluble protein aggregates, my data suggest that at concentrations sufficient to induce Hsf1, thiol-reactive compounds do not induce the HSR via a mechanism based on accumulation of unfolded cytosolic proteins. Another possibility is that thiol-reactive compounds may influence aspects of the protein quality control system such as the ubiquitin-proteasome system (UPS). To address this hypothesis, β-galactosidase reporter fusions were used as model substrates to demonstrate that thiol-reactive compounds do not inhibit ubiquitin activating enzymes (E1) or proteasome activity. Therefore, thiol-reactive compounds do not activate the HSR by inhibiting UPS-dependent protein degradation. I therefore hypothesized that these molecules may directly inactivate protein chaperones, known as repressors of Hsf1. To address this possibility, a thiol-reactive biotin probe was used to demonstrate in vitro that the yeast cytosolic Hsp70 Ssa1, which partners with Hsp90 to repress Hsf1, is specifically modified. Strikingly, mutation of conserved cysteine residues in Ssa1 renders cells insensitive to Hsf1 activation by cadmium and celastrol but not by heat shock. Conversely, substitution with the sulfinic acid and steric bulk mimic aspartic acid led to constitutive activation of Hsf1. Cysteine 303, located in the nucleotide-binding/ATPase domain of Ssa1, was shown to be modified in vivo by a model organic electrophile using Click chemistry technology, verifying that Ssa1 is a direct target for thiol-reactive compounds through adduct formation. Consistently, cadmium pretreatment promoted cells thermotolerance, which is abolished in cells carrying SSA1 cysteine mutant alleles. Taken together, these findings demonstrate that Hsp70 acts as a sensor to induce the cytoprotective heat shock response in response to environmental or endogenously produced thiol-reactive molecules and can discriminate between two distinct environmental stressors.

The effects of omega -3 fatty acid -enriched fish on C -reactive protein levels in postmenopausal women

Coronary heart disease (CHD) is the leading cause of death in women and rates markedly increase among women after 65 years of age. C-reactive protein (CRP) is a new clinical indicator of atherosclerotic-related inflammation with a direct pathogenic role. Studies show lifestyle factors can modulate CRP. Omega-3 fatty acids have anti-inflammatory properties and studies suggest that eating fish high in omega-3 fatty acids may lower CHD risk in women. This study sought to assess the possible role of omega-3 fatty acids in the reduction of CHD-related inflammation by investigating the effect of fish consumption on CRP levels. Methods. Twenty-four healthy postmenopausal women were randomly assigned to a fish group (usual diet plus two servings per week of enriched fish) or control group (usual diet with no fatty fish) for eight weeks. Omega-3 fatty acid-enriched fish developed by the West Virginia University Aquaculture Division was used. Serum CRP, serum interleukin-6 (IL-6), and the fatty acid content of red blood cells (RBC) were measured before and after the study. Women also completed food records. RESULTS: Baseline levels of CRP were low (85% of the fish group had normal levels) and few changes in CRP risk category were observed. Mean IL-6 levels were reduced by 27% and 35% in the fish and control groups, respectively (p for between-group difference = 0.60). Changes in RBC fatty acid composition were not statistically significant. Compared to control women, women in the fish group had greater reductions in mean triglycerides (p = 0.08), total cholesterol (P = 0.04), and LDL cholesterol levels (p = 0.06). Baseline dietary intake of total and monounsaturated fatty acids tended to be positively associated with baseline CRP, while vitamin E intake was inversely related. Saturated fat intake tended to have a positive association with IL-6. Conclusions. Findings regarding the effect of two servings of fish on CRP and IL-6 levels are inconclusive due to low baseline levels of CRP and IL-6. However, results indicate two servings of fatty fish have favorable effects on blood lipids. The relationship of dietary components with CRP and IL-6 is complex and further research is needed to determine the varying roles of diet on the inflammatory process. ^